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ScliooliBiotechnolo^^^^^^ and Biomolecular ScienoBS Faculty: Science
Title:Community analysis and physiological characterisation of bacterial isolates from a nitrifying membrane bioreactor
Abstract This thesis focuses on the identification of early colonisers on membrane surfaces used in wastewater treatment, as well as the physiological characterisation of bacterial cultures isolated from different micro- environments of a membrane bioreactor (MBR).
The bacterial community composition of early biofilms on membrane surfaces under different hydrodynamic conditions (pressurised and non-pressurised) and of the activated sludge in an MBR were examined by culture-independent, molecular-based methods of PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and PGR cloning of 16S rRNA genes. A bench-scale, nitrifying MBR treating artificial waste was employed. The hollow fibre ultrafiltration membrane was made of polypropylene with an average pore diameter of 0.04 \im. Analysis of DGGE profiles of the sessile communities on membrane surfaces revealed that Tetrasphaera elongata species were important colonisers due to their ability to bind to membrane surfaces irrespective of the hydrodynamic context and exposure time.
Interactions between isolates from the bioreactor and membrane surfaces were further investigated by characterising the physiological traits important in biofilm initiation and proliferation on membrane surfaces such as motility, auto-aggregation, co-aggregation, hydrophobicity and quorum sensing. Bacterial strains were isolated from floes and supernatant phases of the activated sludge as well as from pressurised membrane surfaces. Microbacterium sp. were prevalent in all culture collections. Physiological studies revealed Microbacterium sp. possesed high hydrophobicity and auto-aggregating activity that could contribute to their colonisation on membrane surfaces and persistence in floes.
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'I hereby declare that this submission is my own work and to the best of my l Signed Date UL^lZniQ Community analysis and physiological characterisation of bacterial isolates from a nitrifying membrane bioreactor Lan Hoang Le A thesis submitted in fulfilment of the requirements for the degree of Master of Science School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales, Sydney, Australia April 2010 TABLE OF CONTENTS ACKNOWLEDGEMENT 3 LIST OF FIGURES 4 LIST OF TABLES 5 LIST OF ABBREVIATIONS 6 ABSTRACT: 7 CHAPTER 1 LITERATURE REVIEW 8 1.1 INTRODUCTION TO MEMBRANE BIOREACTORS (MBRS) 8 1.1.1 The MBR unit configuration 11 1.1.2 Advantages of membrane bioreactors: 12 1.1.3 Limitations of MBRs 14 1.2 MEMBRANE FOULING IN MBRS 15 1.2.1 Sludge characteristics 16 1.2.2 Membrane characteristics 16 1.2.3 Operating parameters 17 1.3 BIOFILM FORMATION IN MBR 17 1.3.1 Stages of bacterial biofilm formation 18 1.3.2 Biofilms on MBR membrane surfaces 19 1.4 THESIS OBJECTIVES 25 CHAPTER 2 EARLY COLONISATION OF MEMBRANE SURFACES 27 2.1 INTRODUCTION 27 2.2 MATERIALS AND METHODS 28 2.2.1 Membrane Bioreactor Set-up 28 2.2.2 Membrane sampling 29 2.2.3 Genomic DNA extraction 30 2.2.4 PGR for Denaturing Gradient Gel Electrophoresis (DGGE) 30 2.2.5 Denaturing Gradient Gel Electrophoresis (DGGE) 31 2.2.6 Gel extraction and sequencing of bands 31 2.2.7 Construction of a 16S rRNA gene clone library from sludge 32 2.2.8 Sequencing of DGGE bands and clone inserts 32 2.3 RESULTS 33 2.3.1 Bacterial community composition in sludge 33 2.3.2 Establishing a membrane sampling regime to examine colonisation 34 2.3.3 Sequencing of gel bands of interest (A, B and C) 39 2.3.4 Phylogenetic analysis of clone library of sludge 40 2.4 DISCUSSION 42 CHAPTER 3 ISOLATION AND CHARACTERISATION OF BACTERIA ISOLATED FROM MEBRANES, FLOCS AND SUPERNATANT OF AN MBR 45 3.1 INTRODUCTION 45 3.2 MATERIALS AND METHODS 47 3.2.1 Isolation of bacteria from membrane surfaces and activated sludge 47 3.2.2 Media and growth conditions 48 3.2.3 Physiological characteristics of isolates 48 3.2.4 Genomic DMA extraction 50 3.2.5 16S rRNA gene amplification 51 3.2.6 Sequencing ofl6S rDNA 51 3.2.7 Aggregation assays 52 3.3 RESULTS 53 3.3.1 Isolation and identification of cultivable bacteria 53 3.3.2 Detection ofN-acyl-L-homoserine lactone activity 55 3.3.3 Motility assays 56 3.3.4 Cell surface hydrophobicity 57 3.3.5 Auto-aggregation 58 3.3.6 Biofilm formation on polystyrene plates 59 3.4 DISCUSSION 61 CHAPTER 4 GENERAL DISCUSSION 64 REFERENCE 70 Acknowledgement I would like to thank Professor Staffan Kjelleberg for giving me the opportunity to join the Centre for Marine Bio-innovation four years ago. It has been a great discovery journey for me and I am very grateful for it. I would like to show my deep appreciation to my supervisors and co- supervisor, Dr. Diane McDougald and Dr. Mike Manefield for your huge support and patience. I am forever in debt to you for your guidance when I was most confused and had no confidence in myself. Thank you again and again and again. I would like to thank Professor Richard Stuetz and his phD student Minh Nhat Le from the School of Civil and Environmental Engineering for their help with the reactor. I would like to thank my family for their support for the past few years, to allow myself to pursuit my research passion. Thank you for having confidence in me. This is for you. Many thanks to friends and colleagues, especially people from CMB level 6: Nidhi, Leesia, Amy, Tran, Janice, Jerry, Krager. You are the ones that keep my experience in the lab so much enjoying: coffee, chocolate, tea, jokes. What else can one ask for at one's workplace? Last but not least, a hug to my boyfriend, thank you for your love, patience and confidence in me. List of Figures Figure 1.1 Flow diagram describing conventional wastewater treatment 9 Figure 1.2 MBR unit configuration 11 Figure 1.3 Development stages of a biofilm on a static substratum 19 Figure 1.4 Model of the central components of lux regulation in V. ficheri 24 Figure 2.1 Schematic diagram of the 26 litre, bench-scale MBR 29 Figure 2.2 Bacterial community fingerprints of sludge sample fi"om the MBR taken over a period of 85 days 33 Figure 2.3 Bacterial community profiles fi-om 4 day-old pressurised membrane 35 Figure 2.4 Bacterial community profiles from 4-hour and 8-hour pressurised membrane 36 Figure 2.5 Bacterial community profile fi-om 4-hour and 8-hour static membrane 37 Figure 2.6 Bacterial community profile from pressurised and static membrane in the first 3 hours of inoculation 38 Figure 2.7 DNA yield of pressurised, static and 0 h membrane samples 38 Figure 2.8 Phylogenetic tree of the sludge bacterial community 41 Figure 3.1 Pie charts showing the proportions of disfinct phylotypes among bacterial isolates from fiocs, pressurised membrane and supernatant 54 Figure 3.2 AHL-like activity detected in isolate 56 Figure 3.3 Biomass of 1, 2, and 3 day-old biofilm on polystyrene plates of supernatant, fioc and pressurised membrane isolates 60 Figure 3.4 A schematic diagram showing the phylogenetic relationship between the Microbacterium genus and Tetrasphaera genus within the Actinobacteria phylum 62 List of Tables Table 1.1 Types of foulant interactions with the membrane surface 15 Table 2.1 BLAST analysis of DGGE band sequences 39 Table 2.2 Alignment of DGGE bands with clone library 40 Table 3.1 Phylogenetic identity of isolates 53 Table 3.2 Motility of bacterial isolates 57 Table 3.3 Cell surface hydrophobicity of bacterial isolates 57 Table 3.4 Auto-aggregation of bacterial isolates 58 List of Abbreviations AHL acylated homoserine lactone AS activated sludge ASP activated sludge process EPS extracellular polysaccharide F floes HRT hydraulic retentiontime MBR membrane bioreactor MF microfiltration MLSS mixed liquor suspended solid PAO polyphosphate accumulating organism PM pressurised membrane PS polysulfone QS quorum sensing RO reverse osmosis S supernatant SI sludge SRT sludge retention time St static membrane TMP transmembrane pressure UF ultrafiltration WWT wastewater treatment Abstract: This thesis focuses on the identification of early colonisers on membrane surfaces used in wastewater treatment, as well as the physiological characterisation of bacterial cultures isolated fi-om different micro-environments of a membrane bioreactor (MBR). The bacterial community composition of early biofilms on membrane surfaces under different hydrodynamic conditions (pressurised and non-pressurised) and of the activated sludge in an MBR were examined by culture-independent, molecular-based methods of PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and PGR cloning of 16S rRNA genes. A bench-scale, nitrifying MBR treating artificial waste was employed. The hollow fibre ultrafiltration membrane was made of polypropylene with an average pore diameter of 0.04 ^im. Analysis of DGGE profiles of the sessile communities on membrane surfaces revealed that Tetrasphaera elongata species were important colonisers due to their ability to bind to membrane surfaces irrespective of the hydrodynamic context and exposure time. Interactions between isolates from the bioreactor and membrane surfaces were fiirther investigated by characterising the physiological traits important in biofilm initiation and proliferation on membrane surfaces such as motility, auto-aggregation, co-aggregation, hydrophobicity and quorum sensing. Bacterial strains were isolated from fiocs and supemantant phases of the activated sludge as well as from pressurised membrane surfaces. Microhacterium sp. were prevalent in all culture collections. Physiological studies revealed Microbacterium sp. possessed high hydrophobicity and auto-aggregating activity that could contribute to their colonisation on membrane surfaces and persistence in fiocs. Chapter 1 Literature Review 1.1 Introduction to Membrane Bioreactors (MBRs) Even though recorded history and surviving archaeological sites show that the Babylonians, Assyrians and later Romans used quite sophisticated technology to collect and dispose of their waste; wastewater treatment as we know it only began around 100 years ago (70). Industrialisation and urbanisation in the second half of the 19^ century in Europe resulted in a huge increase in the number of people in crowded central cities. With no proper sewage disposal system in place, Europe was plagued with overbearing odour problems and polluted river systems. As a result, sanitation-related diseases such as dysentery, typhoid and cholera epidemics were responsible for the deaths of thousands of people. Fortunately, the industrial revolution resulted in significant discoveries in the field of microbiology, such as the realisation by Robert Koch that bacteria were the causative agents of wide-spread disease. Sewage-contaminated drinking water was identified as a major transmission route for disease, forcing authorities to develop systems to improve sanitation. First the separation of sewage fi-om water supply sources was introduced, followed by legislation of standards in 1912 for treatment of waste in Britain. This legislation provided important momentum for the development of wastewater treatment systems and undoubtedly played an influential role in the development of the activated sludge process. Wastewater treatment by activated sludge mimics the natural activities of soil and water micro-organisms but in a much higher concentration. These organisms utilise organic matter in the sewage as their food source, leaving the treated water suitable for discharge back into the water stream. Edward Arden and William Lockett were the first to test the activated sludge process (ASP) in a pilot-plant in Manchester in 1914 (8). The AS system was so successful that it soon spread outside of Britain, and by the beginning of Worid War 11, plants had been introduced in many European and Commonwealth countries. The ASP, which was originally developed to remove carbonaceous materials, has undergone many modifications to its design and operation since this time with the goal to increase efficiency and flexibility. Many plants built today effectively remove nitrogen and phosphorous materials in addition to carbon. Current conventional wastewater treatment (WWT) plants consist of four treatment processes: preliminary, primary, secondary and tertiary treatment (Figure 1.1) (28). Preliminary Raw waste Treated waste Figure 1.1: Flow diagram describing conventional wastewater treatment, which consists of four processes: preliminary, primary, secondary and tertiary treatments. In the preliminary treatment stage, large objects are removed by bar screen before waste is passed on to the primary treatment tank where the suspended and floating materials are also removed. Biological degradation of organic matter in wastewater occurs in the secondary treatment. The incoming waste is mixed with AS in the aeration tank, and the micro-organisms, in the presence of oxygen, degrade organic matter in the wastewater to sustain their life processes. The mixture is then transferred to the sedimentation tank where the AS is allowed to settle by gravity, leaving the treated water ready for disinfection in tertiary treatment. Stricter criteria for consumer water quality and the need for water reuse has led to the development of more efficient wastewater treatment processes that aim to overcome problems experienced by conventional WWT e.g. settle-ability of sludge (sedimentation step hinders the amount of waste that can be treated) and foaming (system can fail due to growth of filamentous bacteria that prevents sedimentation of sludge) (129). Two most notable new technological breakthroughs during the past 30 years in WWT are granulation and MBRs. Granulation was first described in 1980 for strictly anaerobic systems (68). By the late 1990s, the formation and application of aerobic granules had been reported (9, 22, 91). The granulation process consists of the production of large (up to 5 mm), round and compact cell aggregates through the alteration of operational parameters. Granular sludge is considered to be a special type of biofilm composed of self- immobilised cells. Granular sludge has a number of advantages over conventional activated sludge, such as high biomass retention, excellent settle-ability (shorter sludge-sedimentafion time means more waste can be treated), and dense microbial structures that can withstand high levels of toxic substances (2). The use of granulation technology however, requires modifications to conventional WWT plant design, which can be costly, a disadvantage for the use of granulation technology. MBRs used in WWT processes involves membrane filtration combined with biological treatment. In an MBR the activated sludge is responsible for biological degradation while membrane filtration allows solid-liquid separation, retaining bacterial floes and suspended solids inside the reactor, thereby producing permeate of high clarity. The process was first introduced by Dorr-Olivier Inc. in the late 1960s when ultrafiltration (UF) and microfiltration (MF) membranes became commercially available (121). The first MBRs consisted of an AS reactor that was combined with an external membrane unit. Polymeric flat sheet membranes were used to filter AS, which was continuously pumped through a crossflow filtration loop. These first generation MBRs failed as an attractive alternative to conventional AS treatment due to the high cost of membranes, high energy consumption as well as the rapid loss of flux due to fouling. A major improvement in MBR technology was made when Yamamoto et. al (1989) introduced the idea of submerging membrane units directly into the AS reactor. Further refinements of this MBR design together with the lowering cost of membrane material again presented MBR as an attractive option for WWT applications. The first full-scale commercial application of MBR technology was in industrial WWT in North America in 1991 (125). Since that time, MBR technology has found its way into many municipal and industrial WWT plants. By 2004, 258 fiill-scale MBR installations were operational in North America alone. Currently, the MBR market is doubling every seven years and is estimated to reach US $360 million in 2010(57). 1.1.1 The MBR unit configuration MBR configurations differ with regards to the placement of the membrane unit which is either in a side-stream or immersed in the reactor itself (Figure 1.2). (a) Recirculated stream m ^Out In^—> In Membrana Air Air —^ Bloreactor Sludge Out Sludge TRENDS in Bioimhnology Figure 1.2: MBR unit configurations used in WWT. In a side-stream unit (a), the sludge is circulated through a separate membrane unit placed outside the bioreactor. Alternatively, (b) the membrane unit is immersed in the reactor itself (57). A side-stream system (Figure 1.2 a) relies on cross-flow membrane filtration. Unlike dead-end filtration where the feed is pushed through the filter, in cross-flow filtration, the feed is passed across the surface of the membrane at high velocity. The advantage of this type of filtration is that the fast, tangential motion of the bulk of the fluid across the membrane surface reduces deposition of sludge cake, hence increasing the length of time that a membrane unit can be used. However, significant energy is required to recirculate the mixed liquor suspended solids (MLSS) through the external membrane unit at high cross-flow velocity. This configuration is believed to be more suitable for wastewater sources of high temperature, high organic strength, extreme pH, high toxicity as well as low filterability, e.g. industrial wastewater (142). In an immersed MBR (Figure 1.2 b), the membrane unit is submerged into the bioreactor itself. The filtration occurs by applying suction forces across membrane surface. Depending on the pore size of the membrane used, only water molecules and selective solute species can pass through the membrane whilst biomass is retained in the reactor. The net energy used in this configuration is much lower than that needed for the side-stream system, making the immersed system more attractive for broad applications. 1.1.2 Advantages of membrane bioreactors: As one of the most promising alternatives to conventional activated sludge wastewater treatment, membrane bioreactors present many advantages. These include: 1) a small treatment plant footprint, 2) high quality effluent, 3) uncoupling hydraulic retention time and sludge retention time, 4) being fiinctional at high MLSS, 5) lower sludge production. 1.1.2.1 Small wastewater treatment plant footprint Conventional treatment of wastewater generally occurs in three successive stages: removal of greases and solid objects, followed by aerobic degradation of organic matters and lastly a sedimentation process to remove biomass fi-om the effluent. For MBRs, a combination of a membrane unit housed in the aerobic degradation tank eliminates the need for a sedimentation tank. MBRs, therefore, occupy a smaller land area. This is a great advantage when designing municipal WWT plants because acquisition of land is one of the major expenses. 1.1.2.2 High quality effluent The ability to produce high quality effluent is the main driver for MBR applications, especially when the treated water is to be reused or discharged to bathing waters. For example, filtration using membranes with a pore size of 0.03 |im can produce effluent of high quality with disinfection in a single stage (38). Because this is a physical separation process, the quality of effluent obtained by MBR filtration depends on the membrane used. There are many chemical and physical differences between microfiltration (MF), ultrafiltration (UF) and nanofiltration membranes with the later producing effluent of higher quality than the first (147). In terms of pathogen exclusion, microfiltration processes can retain cysts and bacteria, while ultra- and nanofiltration processes can also retain viruses (20, 129), and therefore can produce water of extremely pure quality. 1.1.2.3 Uncoupling hydraulic retention time and sludge retention time Hydraulic retention time (HRT) can be defined loosely as the time it takes for waste to pass through the complete treatment process, fi-om the untreated incoming waste to the final clear effluent. In conventional ASP, treated waste is separated fi-om floes by the settling of floes by gravity in the sedimentation tank. Hence, the faster the floes setfle, the shorter the HRT is. This setfling time depends on the size of the MLSS particles or floes because the bigger floes settle quicker. The time it takes for floes to form and grow is called sludge retention time (SRT). Therefore, there is an inter-relationship between the volume of effluent that can be removed (HRT) and the incubation time for floe growth (SRT). In an MBR system, effluent is separated fi-om floes by membrane filtration, HRT is therefore, uncoupled from the limiting factor of SRT. 1.1.2.4 Functional at high mixed liquor suspended solids concentration In the ASP, the MLSS concentration is important not only for the biological treatment of waste but also for the sedimentation of biomass. Too high a concentration of MLS S can lead to bulking which disrupts the sedimentation and an MLS S concentration that is too low results in insufficient biological treatment. MBRs separate biomass solids from the liquid phase based on membrane filtration and not settling, thus MBRs can operate independently of MLSS concentrations. 1.1.2.5 Lower sludge production In the ASP, periodic removal of sludge is necessary to control effluent quality and MLSS concentration. Disposal of excess sludge is both an environmental and economical challenge. Even after further treatment to reduce sludge volumes, the amount of wasted sludge is in the millions of tons. In the United States alone, it is estimated that about 10 million dry tons of sewage sludge are produced each year (5). The cost of sludge disposal may account for up to 65% of the total plant operating costs (72). With complete sludge retention, bacteria within the reactor experience conditions of substrate concentration per unit biomass. The energy obtained by the MBR biomass, is consumed mainly for cell maintenance and not cell division (139), thus the overall sludge volume is decreased. 1.1.3 Limitations of MBRs Despite having many advantages over the conventional ASP, the widespread application of MBRs for WWT has been limited due to problems with membrane fouling. "Fouling" is defined as "a process resulting in loss of performance of a membrane due to the deposition of suspended or dissolved substances on its surface, at its pore openings, or within its pores" (89). Membrane fouling is a complex phenomenon that reduces effluent flux rate, resulting in the need for membrane cleaning and ultimately shortens membrane life. Gander et al. (2000) have shown that effluent flux declines rapidly from an initial flux of 45 1 m'^ h"' to 41.75 1 m'^ h"^ after only 3 hours. In order to maintain the system, fouling control strategies, such as periodic backwashing, chemical cleaning or air bubble sparging are mandatory (104, 143). The cost for the use of these strategies together with the cost of membrane replacement in cases where cleaning fails, are reported to contribute up to 50% of the operating costs or 30% of the total cost in a typical UF membrane system (89). 1.2 Membrane fouling in MBRs While there is a desire to process waste quickly, the fouling rate is usually proportional to the flux rate such that the higher the flux rate, the quicker the membrane fouls. Therefore, it is more desirable to have a lower but sustainable flux rate than a high flux rate which results in membrane failure. The concept of critical flux was first proposed by Field et al. (1995), where it was defined as the flux below which the increase of trans-membrane pressure (TMP) with time under constant operation does not occur, while above that level, fouling is observed (30). A large number of studies have focused on the modelling of critical flux (17, 65, 96). However, it should be noted that sub-critical flux operation does not totally eliminate fouling. Two types of fouling layers have been suggested to accumulate on membrane surfaces: sludge cake and gel layer. Sludge cake is the deposition of sludge floes that, to a great extent can be controlled by adopting sub-critical flux operation (80). Gel layer, however, is formed by macromolecules, colloids and soluble microbial products and is inevitable even under sub-critical flux. The gel layer is believed to be the main cause of the gradual increase in TMP in MBRs (135). Foulants on membrane surfaces can be classified as organic solutes or inorganic solutes, colloids or biological solids. Each of these foulants interact differently with the membrane surface, preventing constant permeate flux maintenance. These interactions are summarised in Table 1.1. Table 1.1: Fouling caused by interactions of foulants with the membrane surface (133) Fouling caused by Organic solutes Adsorptive interactions with the membrane material Inorganic solutes Precipitation of salts on membrane surface, i.e. scaling Colloids Accumulation on membrane surface and crevices Biofouling Biofihn formed by biologically active organisms, mainly bacteria and fimgi Because membrane fouling is the primary problem affecting MBR operation, more than one third of the research studies investigating MBR operations deal with issues related to membrane fouling. These studies address the causes, characterisation and modelling of membrane fouling (19, 93, 142, 146). Three engineering factors that determine the degree of fouling in MBRs are sludge composition, membrane characteristics, and operating conditions. 1.2.1 Sludge characteristics Fouling of membranes is the direct result of interactions between membrane surfaces and sludge components. Studies on the correlation of sludge characteristics and membrane permeability are therefore valuable in the design of fouling-control strategies. Parameters such as concentration of the MLSS, extracellular polymeric substances (EPS), soluble microbial products (SMP), food to micro-organisms ratio (F/M) and the presence of filamentous bacteria are often mentioned in the literature. Many studies have assessed the effect of these parameters on the formation and resistance of fouling layers (see reviews by Le-Clech et al. (2006) and Meng et al. (2009)). Generally, it is expected that high concentrations of EPS, SMP and filamentous bacteria can lead to decreases in membrane permeability. The role of SMP as an important factor resulting in MBR fouling has been supported in many studies (27, 94, 113). SMPs are defined as the pool of organic compounds that are released into solution from substrate metabolism (usually with biomass growth) and biomass decay. In MBR systems, SMPs can also be provided in the feed substrate. During filtration, SMP adsorption on the membrane surface not only reduces permeate flow but also creates a conditioning film which enhances bacterial biofilm formation (112). Polysaccharide-like substances in SMP have been shown to contribute to fouling more than protein-like substances (66). 1.2.2 Membrane characteristics Membranes can be composed of ceramic, metallic (inorganic) or polymeric (organic) material. While ceramic membranes have superior hydraulics and thermal and chemical resistances, they are not preferred for MBR applications due to their high cost (117). Recently, novel stainless steel membrane modules have been explored and shown to have good hydraulic performance and recovery of performance after cleaning, when used in anaerobic MBRs for WWT (149). However, the majority of the membranes used in MBRs are polymeric-based due to their mass production and low cost. Polymeric membranes include: polyethylene (PE), polypropylene (PP), polyvinilydene fluoride (PVF) and polysulfone (PS). In a study of polyamide and PS membranes operated under quiescent conditions, Tansel et al. (2008) showed that the fouling layer on polyamide membranes was due to entrapment of EPS, a dominant component of feed waste, in membrane surface crevices. On PS membranes, however, fouling was shown to be due to molecular adhesion forces between EPS and functional groups on the membrane surface. Attachment of EPS to the membrane surface is believed to cure the surface, creating conditions optimal for bacterial fouling, which leads to irreversible fouling and membrane performance failure. Several studies have shown that membrane fouling progresses faster on hydrophobic membranes than on hydrophilic ones (29, 36) due to the hydrophobic- hydrophobic interactions between foulant species and membrane. In addition, surface structure can influence fouling tendencies as rough surfaces are more prone to microbial attachment than smooth surfaces (45) and membranes with larger pore sizes show a higher tendency to foul irreversibly (10). 1.2.3 Operating parameters Operating parameters such as SRT, HRT and aeration rates have indirect effects on membrane fouling. Long SRT (60 days) has been linked to reduction in TMP due to proliferation of slow growing bacteria that can utilize EPS, the most abundant foulant found in feed (3). Short HRT leads to increased membrane fouling due to excessive growth of filamentous bacteria which acts as a conditioning mat for fouling on membrane surfaces (85). Aeration during operation provides effective shear stress that helps to improve permeability and membrane performance reducing the fouling of sludge cake on membrane surfaces (123,137). 1.3 Biofilm formation in MBR Membrane biofouling involves the formation and growth of a biofilm on membrane surfaces (21) that ultimately impedes the bulk-flow at the surface. In fact, microbial biofilms can be found on any surfaces associated with moist environments such as those of riverine rocks in nature, or on surfaces of indwelling medical devices (42, 106,107). Most micro-organisms can form biofilms and more than 90% of all micro- organisms on Earth are living in such aggregates (21). Living within an EPS matrix offers members of aggregates great advantages which include protection from environmental stresses (34, 81), a stable niche (53) and increased genetic exchange due to the close physical proximity of the cells (44). In water systems, biofilms are frequently observed (83). In heat exchange systems, biofilm layers on the heat exchange surface increases friction resistance, which in turn, increases energy consumption. In drinking-water reservoirs and distribution systems, biofilms are a challenge for the maintenance of water quality (99). 1.3.1 Stages of bacterial biofilm formation Biofilm formation processes can be divided into three main stages: early surface attachment, biofilm maturation, and detachment/dispersal of biofilm-derived cells to the plankton (Figure 1.3) (97, 103). The attachment process begins when bacterial cells make initial contact with the surface in a reversible interaction usually involving the cell pole. Bacterial cells will usually attach and release several times before irreversible attachment occurs. During the irreversible attachment stage, bacterial cells attach firmly onto the surface along the length of the cell. Attachment to the surface triggers changes to gene expression, resulting in adaptation to the biofilm life style. Attached cells multiply to form clusters of cells, called microcolonies, which produce the EPS matrix and give rise to the complex architecture of the mature biofilm. In the mature biofilm, there is autolysis of a subpopulation of cells in the centre of the microcolonies just prior to the activation and release of dispersal cells. Mai-Prochnow et al. (2004) have shown an autolytic protein, AlpP, is responsible for this phenomenon in Pseudoalteromonas tunicata biofilms. Cell death in Pseudomonas aeruginosa has been shown to be dependent on the activation of filamentous phage, Pf4 that exists as a pro-phage within the genome (136). The life cycle of the bacterial biofilm is completed with the detachment and dispersal of biofilm cells, which colonise new surfaces. 2 a âixà^tUÂii Figure 1.3: Development of a biofilm on a static substratum is characterized by three stages: 1) early attachment where bacteria switch from planktonic to sessile life style as they approach and attach to the surface 2) biofilm maturation where cells undergo developmental changes to adapt to biofilm life and proliferate into a mature biofilm, 3) detachment and dispersal of cells into the liquid phase to colonise new surfaces. Adapted from Biofilms: The Hypertextbook - Montana State University. 1.3.2 Bíofílms on MBR membrane surfaces Even though biofilms greatly impact industrial processes, the investigation of microbial processes related to the fouling film formation has not progressed as rapidly as chemical and structural analyses of fouling layers in MBRs. Typically, the fouling layers are extracted, either by chemical means (4) or physical scraping (50), the extracts subjected to analyses and results correlated with membrane permeability indices (i.e. TMP). Given that micro-organisms are central to the biological treatment of wastewater and are the producers of important foulants (e.g. EPS) it is surprising that they have been overlooked as important parameters in MBR fouling studies. Microbiological investigations conducted on MBRs have focused mainly on the identification of members responsible for the biodégradation of certain substrates or bacterial community dynamics of suspended sludge in response to performance of MBR processes. Advances in culture-independent approaches have provided effective tools for the study of complex microbial communities in AS MBR processes. The principle behind these techniques is to use microbial biomarkers to characterise members of the complex communities. For example, quantitative chemical analyses of the respiratory biomarker, quinones, in suspended sludge of an MBR operating with intermittent aeration revealed the abundance of bacterial species that actively contributed to the biological nitrifícation/denitrifícation process (69). Quinones are important components in the transfer of electrons in the bacterial respiratory chains. They are present in most bacterial species and are chemically unique which makes them a good biomarker for profiling bacterial communities (46). Using quinone profiling, Ahmed et al. (2007) showed that under sequential anoxic/anaerobic operation and long SRTs, specific bacterial subpopulations belonging to the 6- and z-Proteohacteria as well as members of the Cytophaga- Flavobacterium cluster proliferated better than the more dominant P-Proteobacteria. Other culture-independent fingerprinting techniques involving the 16S rRNA gene, such as denaturing gradient gel electrophoresis (DGGE) or fluorescent in situ hybridisation (FISH) are also widely used to study the microbial community structure in MBRs. Such studies have revealed that the microbial community structure in MBRs diverged fi-om the original seeded AS community over time, possibly due to operational parameters (139). In fact, fluctuation in MBR community structure has been linked to stable performance of the MBR (87). Although some microbiological studies on MBRs have revealed a correlation between community structure and MBR processes, there is still a lack of knowledge on microbial biofilm formation on membrane surfaces used in WWT. A comprehensive study of the bacterial community structure on a membrane surface and of the suspended solids by Jinhua et al. (2006) revealed that the bacterial community structure on the membrane surface was differentfi"om tha t in suspension. Bacterial isolatesfi-om membran e surfaces also exhibited differences in traits related to biofilm formation such as motility and surface hydrophobicity. Differences between microbial populations attached on flatsheet membrane surfaces and in suspension were observed as early as 4 hours of operation, indicating that attachment occurred rapidly (148). Recent developments in microscopy and image analysis for the curved surfaces of hollow fiber membranes promise to improve our investigations on membrane biofilm structural parameters such as thickness, porosity and roughness (11). Characterisation of this biofilm layer may lead to more specific, targeted biofouling control measures. Membrane surfaces used in filtration systems offer a unique environment for biofilm development, in that the permeate flux creates a constant convective force that transports both bacteria and nutrients towards the membrane surface. Accumulation of rejected species at the membrane surface generates an environment relatively high in nutrient and salt concentration (51). It is not surprising that biofilms formed at the membrane surface may be composed of different species than those in the bulk suspension. 1.3.2.1 Motility and early attachment to membrane surfaces Early attachment occurs where planktonic cells are transported to and adhere to a substratum. Bacterial surface structures such as flagella and pili play particularly important roles at this stage of biofilm development. Flagella-mediated motility may assist bacterial cells in overcoming surface repulsion forces present at the medium- surface interface in Erwinia carotovora (49), Pseudomonas aeruginosa (95) and Escherichia coli (102). Type-I pili are critical for initial attachment of E. coli to abiotic surfaces while type-IV pili of P. aeruginosa are required in later stages of biofilm maturation (16). While motility may be important for attachment of bacteria to a static surface, it may not be necessary for biofilms formed on membrane surfaces. In a study of bacterial strains isolated from a reverse osmosis (RO) membrane treating potable water, three out of four strains (Dermacoccus R012, Microbacterium R018 and Rhodopseudomonas R03) were non-motile (98). In an MBR or RO system, the permeate flux acts as an additional force assisting the cells to penetrate the hydrodynamic layer, bringing the cells into contact with the membrane surface (31, 40). Other characteristics of the cells however, have been shown to be important for adherance of the cells to the membrane surface. For example, cells with higher cell- surface hydrophobicity adhere better to membrane surfaces. Jinhua et al. (2006) reported that y-Proteobacteria membrane isolates possessed higher cell surface hydrophobicity than the suspended sludge (55). 1.3.2.2 Co-aggregation and biofilm development Co-aggregation is a process by which genetically distinct bacteria become attached to one another via specific molecules. Co-aggregation can contribute to biofilm development in two ways; via recognition and adherence of single cells in suspension to biofilm cells, or co-aggregation in suspension followed by the subsequent adhesion of this co-aggregate to the developing biofilm (108). Indeed, bacterial co-aggregation is considered as an integral process in the development of multi-species biofilms. For example, bacterial co-aggregation enhances biofilm development in the oral cavity where Fusobacteria sp. acts as a physical bridging organism, forming aggregates with both early and late colonizers (61). A larger proportion of fi-esh water biofilm isolates co-aggregated when compared to planktonic isolates (110). Similar differences between species in planktonic and coexisting biofilm populations has also been reported for bottled water isolates (56). In wastewater systems, efficient co-aggregation amongst flocculating bacteria has long been realized to be essential for proper settling of floes in AS. In fact, the ability to co-aggregate is one significant physiological characteristic that enables flocculating bacteria to maintain themselves within the floes under high shear stress (110). A study by Malik and colleagues (2003) found that non-flocculating bacteria also co-aggregate. One of these isolates Acinetobacter johnsonii S35 forms aggregates with members of several genera, suggesting it may act as a bridging organism in multigeneric co-aggregates (74). 1.3.2.3 Auto-aggregation and biofilm formation Many pathogenic bacteria can form aggregates, including Mycobacterium tuberculosis (86), Staphylococcus aureus (82) and Streptococcus pyogenes (35). Auto-aggregation is believed to be an important virulence mechanism, conferring resistance to various host defences such as phagocytosis. Microbial aggregates, both intra-species and inter-species, have been associated with bacterial biofilms causing chronic endodontic infections (59). Genes encoding proteins involved in adhesion and auto-aggregation in E. coli have been shown to be highly expressed during the transition from planktonic to biofílm growth (116). Over-expression of surface adhesin antigen 43 (Ag43) produces a dominant auto-aggregation phenotype that overrides motility (131). In contrast, an increased flagellation state prevents Ag43- mediated auto-aggregation. Bacterial auto-aggregation has been proposed to play a role in AS granulation. Microbial profiling of phenol-degrading aerobic granules identified two functionally different groups of bacteria, one which had high phenol degradation rate and the other which showed low phenol degradation rate but possessed high auto- aggregation activity (54). The study proposed a functional model of the microbial community within the aerobic granules with one responsible for phenol degradation and the other for maintaining the granules compact structure. Thus, auto-aggregation is an important physiological trait exhibited by many bacterial species either during biofilm growth on surfaces or in suspended biofilms of floes and granules. 1.3.2.4 Quorum sensing control of biofílm formation Quorum sensing (QS) is a form of bacterial communication where bacterial population express particular phenotypes only when the concentration of extracellular signal molecules, or autoinducers, has reached a particular threshold. Autoinducers diffuse freely across the cell membrane and through a positive feedback mechanism, act as regulatory signals to trigger amplification of its own production and also to activate expression of specific phenotypes. QS affects many aspect of biofilm dynamics, such as heterogeneity, architecture, stress resistance, maintenance and sloughing (60), and also regulates group responses such as antibiotic production, virulence factor production, and sporulation (7, 47, 101). A range of bacterial communication systems have been found to control group responses in both Gram-negative and Gram-positive bacteria. Some Gram-negative bacteria use acylated homoserine lactone (AHLs) as autoinducer molecules, while some Gram-positive use peptide-mediated signalling The autoinducer 2 (AI2) QS system is used by both Gram-positive and Gram-negative bacteria. Quorum sensing behaviour was first identified in Vibrio flscheri, which at high cell density in the light organs of some marine fish and squid expresses luminescence (115). The lux regulon of V. flscheri, which is responsible for luminescence, is organised in two divergently transcribed opérons (120). The luxl gene encodes a synthase for the autoinducer, 7V-3-oxohexanoyl-L-homoserine lactone (OHHL), and IwcCDABEG encodes the luminescence enzymes. The luxR gene on the divergent operon encodes a transcriptional activator that binds to the autoinducer OHHL to form a complex. Binding of the OHHL-LuxR complex to the lux box between the lux opérons, induces the transcription of both opérons, creating a positive feedback loop (52) (Figure 1.4). Figure 1.4: Model of the central components of lux regulation in V. fischeri organised in two divergently transcribed opérons, the luxR gene encodes for the receptor protein LuxR ^ and the luxl gene encodes for autoinducer Luxl ^^ that diffuses freely across the cell membrane. Once bound together, the LuxR-LuxI complex binds to the lux box and induces transcription of both opérons, creating a positive feedback loop. Similar QS systems have been identified in other bacterial species, such as the las system that uses the autoinducer A^-(3-oxododecanoyl)-L-homoserine lactone (30- C12-HSL) and the rhl system that uses the autoinducer 7V-(butanoyl)-L-homoserine lactone (C3-HSL), both of which are found in P. aeruginosa (100). Because of the central role of QS in biofilm formation, interruption of this system presents a promising target for biofilm control. One example of interference with bacterial QS systems is the use of halogenated furanones, compounds isolated from the Australian marine macroalga Delisea pulchra (39). Furanones are structurally similar to AHL signalling molecules and compete with the signalling molecules by displacing autoinducers from their cognate R-protein (75). The inhibitory activity of autoinducer antagonists has been shown in a range of bacteria, including V.fischeri, Serratia liquefaciens, E. carotovora and P. aeruginosa. The work of Yeon et al. (2009) demonstrated nicely the presence of QS signals in biofilms formed on membrane surfaces in MBRs with C8-HSL being the most abundant. Further, they showed that the accumulation of AHL signals correlated with the increase in TMP (144). The addition of acylase, an enzyme that degrades AHL via hydrolysis of the acyl-amide linkage, retarded the TMP increase. Although the presence of AHL-producing bacteria in activated sludge has been previously established (132), this study was the first to show that QS-based solutions can be an effective way to prevent biofouling on membrane surfaces in MBRs, and that furanone antagonists could be an attractive option for biofouling control in MBR. 1.4 Thesis objectives As evident from this literature review, biological fouling which affects MBR operation is a result of microbial biofilm formation, yet there are few studies addressing this phenomenon from a microbiological perspective. The work presented in this thesis aimed to identify the early bacterial colonisers on membrane surfaces used in the MBR process. A second objective was to characterise the physiological traits that are known to be important for biofilm initiation and proliferation on surfaces, expressed by attached and planktonic populations. A bench-scale, nitrifying MBR treating artificial waste was employed for these studies. The hollow fiber UF membrane was made of polypropylene with an average pore diameter of 0.04 |im. The bacterial community structure of the early biofilm on membrane surfaces under different hydrodynamic conditions (pressurised and non- pressurised) and of the activated sludge were examined by culture-independent, molecular-based methods of PCR-denaturing gradient gel electrophoresis (PCR- DGGE) and PGR cloning of 16S rRNA genes. By comparing bacterial community profiles of these samples, we determined the effects of the hydrodynamic conditions on membrane community structure. In the second component of the thesis, three bacterial culture collections fi-om planktonic, floe, and membrane-bound communities were isolated. Physiological traits, which are known to be important for biofilm initiation and proliferation, such as motility, surface hydrophobicity, aggregation, and QS, were examined for all isolates. By comparing these traits across isolates from different micro-environments of the MBR, we can determine which physiological traits determine the micro- environment they occupy. Chapter 2 Early colonisation of membrane surfaces 2.1 Introduction Fouling on surfaces of membrane modules in membrane bioreactor (MBR) systems drastically reduces effluent flux flow, which eventually leads to increased operating costs through higher energy requirements and more frequent membrane cleaning and replacement. Biofouling includes the attachment, aggregation and proliferation of bacteria, also known as biofllms, on membrane surfaces. Biofllms are more complex than other forms of membrane fouling because unlike other foulants, bacteria can reproduce and can do so rapidly. Intensive investigation of biofllm formation on static surfaces has given us insight into the process of biofilm formation. The colonisation of submerged surfaces proceeds through an orderly process that starts with the formation of a conditioning film as soon as the surface is in contact with the liquid medium (77). Pioneer colonisers interact with this conditioning film and form the initial assemblage of biomass (76). Proliferation of these early colonisers modifies the physicochemical characteristics of the surface, rendering it habitable for subsequent colonisers (13, 14). Through synergistic and competitive interactions between colonist species (90, 150), as well as through continuous recruitment of new species and loss of others, a mature biofilm community is formed (79, 138). Despite much effort having been spent on the study of fouling control on membranes, the mechanisms underlying biofilm formation on membrane surfaces in MBR systems are still largely unknown. Indeed, a better fiindamental understanding of these pioneer micro-organisms responsible for the surface colonisation that ultimately leads to biofilm formation may lead to more effective control of biofouling (148). Due to their mechanism of filtration, membrane surfaces in MBR systems exhibit complicated hydrodynamics. The constant suction pressure creates a micro- environment on the membrane surface where the nutrient concentration is high, allowing certain colonisers to attach and proliferate. These colonisers may not necessarily be the same as those on a static surface submerged in the same medium. Using mini membrane modules that were connected to suction pumps and membrane modules that were simply submerged in a bench-scale nitrifying reactor, this investigation aimed to compare the early colonisers on hollow fibre membrane surfaces with and without hydrodynamic forces that are specific for membrane modules in a bioreactor. 2.2 Materials and Methods 2.2.1 Membrane Bioreactor Set-up A bench-scale submerged MBR treating artificial wastewater was used to study membrane colonisation. Aerobic sludge was taken from a municipal WWT plant located at North Head (New South Wales) and used to seed a 26 L bioreactor (Figure 2.1), which was fed with artificial wastewater (30.9 g glucose, 25.4 g sodium acetate, 7 g peptone, 4.3 g beef extract, 2.5 g MgS04, 1.3 g KH2PO4, 0.475 g FeS04.7H20 and 7.64 g NH4CI per litre of 50 x feed). The MBR was maintained with a hydraulic retention time (HRT) of 12-14 h, a sludge retention time (SRT) of 26 days and mixed liquor suspended solid (MLSS) level of 7-9 g/L. Four ultrafiltration (MEMCOR, Australia) membrane modules, each with 120 fibres and an effective surface area of 0.01 m^, were submerged on one side of the reactor and used for reactor operation. The HRT was calculated from the flux rate of these large modules. The hollow fibre ultrafiltration (UF) membrane was made of polypropylene with an average pore diameter of 0.04 |im. Three mini membrane modules with an effective surface area of 34.9 cm^ and 10 fibres per module were used for the sampling of membrane fibres instead of the large modules used for reactor operation. Besides the effective surface area, the characteristics of the small membrane modules were the same as those of the large module. The flux of the small membrane modules was controlled similarly to the large modules and permeate was re-circulated back to the bioreactor to maintain a stable HRT. Programmable controller r ' \ Suction pump I i- / -—• Permeate Sensor Permeate recirculated back to reactor Wastewater Feedin 0 i> Suction pump pump Pressurized mini membrane modules Static mini membrane modules Main membrane modules œo Air diffuser Figure 2.1: Schematic diagram of the 26 litre, bench-scale MBR. Main membrane modules maintain the operation of bioreactor. Mini membrane modules were used to study early colonisation on pressurised (by pump suction) and static (un-pressurised) membrane surfaces. 2.2.2 Membrane sampling The reactor had been in stable operation for 7 months when the first mini modules were inserted for bacterial colonisation studies. Three types of samples were collected for the early colonisation study. Firstly, small membrane modules connected to a peristaltic pump were sampled, representing membrane surfaces that were under constant suction pressure, and hence were labelled as pressurised (P). Secondly, small membrane modules with both ends sealed, were submerged in the reactor and sampled. These modules give a static substratum for bacterial attachment, and were hence labelled static (St). The last samples were sludge biomass fi-om the reactor (SI). For any given time point, unless otherwise indicated, duplicates of the small membrane modules were collected, washed three times with sterile phosphate buffer solution (PBS) (pH7), cut into small segments of 2 cm and stored in 2 ml micro-tubes (SARSTEDT, Nümbrecht) at -20®C for extraction at a later date. For sludge samples, microfuge tubes (SARSTEDT, Nümbrecht) containing 500 fil of sample were centrifuged at 16,060 x g in a microcentrifuge (Hettich, Tuttlingen) for 5 minutes. After removal of the supernatant, the sludge pellets were stored at -20®C until extraction of DNA. 2.2.3 Genomic DNA extraction Genomic DNA of membrane and sludge samples was extracted using a bead-beating, hexadecyltrimethyl ammonium bromide (CTAB) and phenol-chloroform-isoamyl alcohol based method as previously described (41). Briefly, into each microfuge tube (SARSTEDT, Nümbrecht) with either membrane samples or sludge pellets, a 200 |iL volume of 0.1 mm glass beads (Biospec, Bartlesville), 500 [i\ of 5% (w/v) CTAB and 500 \i\ of phenol/chloroform/isoamyl alcohol (25:24:1) were added. Tubes were placed in a Fast-Prep bead-beater (Q-biogene, Montreal) and agitated for 30 seconds at a setting of 5.5, followed by centrifugation at 16,060 x g for 5 minutes at 4^C. The top aqueous layer was removed and extracted with an equal volume of chloroform/isoamyl alcohol added and centrifuged as above. The top aqueous layer was transferred to a 1.5 ml microfuge tube containing 2 volumes of 30% (w/v) polyethlyne glycol 6000 (PEG) in 1.6 M NaCl, and incubated at 4^C overnight. After centrifugation at 16,060 x g for 10 minutes, the supernatant was discarded, the nucleic acid extracts washed in 70% ice-cold ethanol, air-dried and resuspended in 50 |il of molecular-grade water. Extracted DNA was quantified using a NanodroplOOO spectrophotometer (ThermoFisher Scientific, Waltham). 2.2.4 PGR for Denaturing Gradient Gel Electrophoresis (DGGE) The V3 region of the 16S rRNA genes was amplified from DNA extracts by PGR using a Mastercycler (Eppendorf, Hamburg) and primer set of 338F-GC (5'- ACTCCTACGGGAGGCAGC-3') and 530R (5'-GTATTACCGCGCCTGCTG-3') (63). Primer 338F has a 37 nucleotide GC-rich sequence at its 5' end with the following sequence: 5'- CGCCCGCGGCGCCCCCGCCCCGGCCCGCCGCCCCCGC-3'. The final 50 reaction contained 25 |LI1 of PGR Master Mix (Promega, Madison), 1 of each primer (10 fiM), 40 - 50 ng DNA template and nuclease-free water. The conditions used for PGR were as follows: 2 minutes of initial denaturation at 95^C and 30 cycles of 30 seconds at 95^C, 30 seconds at annealing temperature 60^C, and 90 seconds at 72"C. Final extension was carried out for 10 minutes at 72®C. The PGR products were electrophoresed on a 1% (w/v) agarose gel. 2.2.5 Denaturing Gradient Gel Electrophoresis (DGGE) The PCR-amplified 16S rRNA gene fragments were separated on Polyacrylamide gels (10%, 37.5:1 acrylamide-bisacrylamide) in 1 x TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA, pH 7.4) using a denaturing gradient ranging from 40% (2.8 M Urea and 16% (v/v) formamide) to 60% (4.2 M Urea and 24% (v/v) formamide). DGGE was performed as described by Muyzer (92) using a D-Code universal mutation detection system (Bio-Rad Laboratories, Hercules). Sixteen microlitres of PGR amplicon from a standard template concentration was loaded into each well and electrophoresed at 60^C for 16 hours at 65 V (constant voltage). Following electrophoresis, the gel was stained for 15 minutes in a 1 in 10,000 dilution of SYBR Gold (Molecular Probes, Eugene) and then visualized with a UV transilluminator (Bio-Rad Laboratories, Hercules). 2.2.6 Gel extraction and sequencing of bands DGGE bands of interest were excised from the DGGE gel and placed in 1.5 ml microftige tubes containing 50 |il of molecular biology grade water for 48 hours. Band extracts were diluted ten fold with molecular biology grade water and re- amplified using the same primer set (without the GC clamp) and conditions as described above. The PGR products were purified using the Purelink PGR Purification kit (Invitrogen, San Diego) before being subjected to sequencing. 2.2.7 Construction of a 16S rRNA gene clone library from sludge A clone library of 16S rRNA genes was created from a sludge sample taken the day the early colonisation experiment was carried out. Nearly complete 16S rRNA genes were amplified by PGR as described above except that primers 27F (5'- AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'- TACGGYTACCTTGTTACGACTT-3') were used (63). The cycling conditions used for the PGR were as follows: 3 minutes of initial denaturation at 95^G and 30 cycles of 20 seconds at 95^G, 20 seconds at annealing temperature 55^G, and 2.5 minutes at 72®G. A final extension was carried out for 10 minutes at 72^G. PGR products were purified (Purelink PGR Purification kit, Invitrogen, San Diego), ligated into the pGR4-TOPO cloning vector and transformed into competent 10?\0 Escherichia coli cells supplied with the TOPO TA Gloning Kit for Sequencing (Invitrogen, San Diego). Fifty clones were isolated and re-streaked onto fresh LBio supplemented with appropriate antibiotics. PGR amplification of the 16S rRNA gene insert was performed using the Ml3 primers provided. The PGR conditions and the composition of the reaction mixtures were the same as described above, except that single colonies picked with a pipette tip were used as template instead of DNA extracts and the annealing temperature adjusted to 53^G instead of 55^G. Insert length was determined by electrophoresis of PGR products on a 1% (w/v) agarose gel. Glones that did not have a full-length insert (~1.5 kb) were eliminated. The PGR products were purified using Purelink PGR Purification kit (Invitrogen, San Diego) before being subjected to sequencing. 2.2.8 Sequencing of DGGE bands and clone inserts The 16S rRNA gene insert of the clone library and the re-amplified bands of interest were amplified for sequencing according to the manufacturers instructions for the ABI 3730 Gapillary Sequencer (Ramaciotti Gentre for Gene Function and Analysis, UNSW, Australia). Briefly, 20 reactions contain 3.5 of 5x buffer, 3.2 pmol of primer (Ml3 Forward for the clones and 530 Reverse for the cut-out bands of interest), 1 fil Big Dye Terminator, 20 - 50 ng of insert product and molecular water. The conditions used for the sequencing PGR were as follows: repeat 25 cycles of 10 seconds at 96®G, 5 seconds at 50^G, 4 minutes at 60^G. The tubes were held at 4^G before products were purified and precipitated using the phenol^utanol method (130). Briefly, the products were resuspended in 80 |il of sterile milliQ-grade water. Samples were transferred to fresh 1.5 ml microfuge tubes containing 100 \i\ of phenol and vortexed for 5 seconds, centrifuged at 16,060 x g for 4 minutes, and the aqueous phase transferred to a fresh 1.5 ml microfrige tube containing 900 of butan-l-ol. The tube was vortexed for 10 seconds, and centrifriged at 16,060 x g for 10 minutes. The supernatant was discarded and the pellet was air-dried until no visible residual butan-l-ol could be seen. The samples were sequenced by the Ramaciotti Centre for Gene Function and Analysis (UNSW) on an AB3730 DNA Analyzer (Applied Biosystem, Foster City). 2.3 Results 2.3.1 Bacterial community composition in sludge The stability of the sludge bacterial community from a bench-scale reactor was monitored over a period of more than two months by denaturing gradient gel electrophoresis (DGGE) of the V3 region of the 16S rRNA genes present. The results are presented in Figure 2.2. ' i-:;-. Hiliil-'H , nrftiia: liiiii - Mh:-} 0 4 23 28 43 Time (days) Figure 2.2: Bacterial community fingerprints of sludge samples from the reactor under investigation taken over a period of 85 days. Despite stable operation of the reactor, the sludge bacterial community appeared to be highly dynamic during the initial 62 days. The overall diversity is consistent across sampling dates. The least diverse sludge community was detected on day 4 with only seven bands appearing in the DGGE fingerprint while the most diverse community was detected on days 55 and 62 with an average of ten bands. The intensity of various bands changed over time, which reflects changes in the relative abundance of species over time. Some bands were initially dominant and decreased in relative abundance while other bands were faint initially and then increased in intensity. Some bands appeared to fluctuate in intensity over time. Such fluctuations are the result of direct and indirect interactions between microbes within the community or the result of chaotic processes. 2.3.2 Establishing a membrane sampling regime to examine colonisation To examine the early colonisation of hollow fibre membranes in the reactor, membrane modules were connected to the pressure pump for a period of 4 days, after which they were harvested. DGGE was used to compare the composition of the microbial communities attached to the membranes and present in the AS at the time of sampling. Fingerprints generated fi-om membrane samples were similar indicating that the colonisation process was reproducible with respect to microbial community composition (Figure 2.3). Additionally, Figure 2.3 shows that the composition of the communities on the membrane was very similar to the AS community in the bulk phase. ^ ^ , i' 4 day-old membranes under pressure (P) sludge Figure 2.3: From left to right: Duplicate bacterial community profiles of three 4-day-old membrane mini modules and of sludge. Arrows indicate bands that were more intense in the activated sludge sample (broken arrows) than on pressurised membrane (solid arrow). Whilst there were slight differences in band intensity between membrane and sludge fingerprints, indicating that some bacterial species colonised the membranes better than others, these data suggested that sampling before 4 days was needed in order to investigate the initial species colonising the membrane. This is in accordance with previous studies that showed stable bacterial fouling on flat-sheet membrane surfaces could be observed after only 4 hours (148) and 8 hours (51) of operation. The colonisation experiment was repeated to establish bacterial community profiles after incubation times of 4 and 8 hours for membranes under pressure (Figure 2.4). As observed with the membrane communities sampled after four days, the DGGE fingerprints of the attached community after 4 or 8 hours of operation were reproducible and similar to that of the AS community, indicating that colonisation of pressurised membranes is rapid and occurs prior to 4 hours. «wwpy ----«A•-•««•!¡..-««jfc ^ A'»/-;» - sludge 4 hour P 8 hour F Figure 2.4: Bacterial community profile jfrom sludge, and in duplicates: 4-hour pressurized, and 8-hour pressurized membrane surfaces. It is possible that the extraction of liquid from the reactor through the membranes could have a major role in transporting bacterial populations indiscriminately to the membrane surface. To investigate the role of this transport in membrane colonisation over 4 to 8 hours, the experiment was repeated using non-pressurised membranes. In this experiment, transport by bulk flow was eliminated. Figure 2.5 shows DGGE fingerprints from AS and from the static membranes after 4 and 8 hours incubation in the reactor. sludge 4 hour St 8 hour St Figure 2.5: Bacterial community profilefi-om sludg e and duplicates of 4-hour static, and 8-hour static membrane surfaces. It is clear that at the time of the experiment, the sludge community was different from that observed previously, in accordance with observations made of community instability in the reactor. Despite this shift in community composition, it was still possible to make the surprising observation that the membrane-associated community was again largely similar to the bulk sludge community after just 4 or 8 hours incubation. This experiment did however, reveal a number of bands that were more dominant in the membrane associated community. This is likely to be indicative of lower species richness on the membranes, such that each band in the fingerprint is more defined and suggests that mass transport was responsible for part of the colonisation in this time period. Ultimately, these results confirmed that to study early colonisation of membrane surfaces by bacteria, even shorter incubation times were required. The experiment was repeated with shorter incubation times of 1, 2 and 3 hours (Figure 2.6). One mini membrane module was dipped into the sludge, and then taken out immediately and labelled as 0 h sample. This sample was washed and processed in the same way as P and St membranes. The bacterial community bound on this 0 h membrane represents species that are immediately attracted to membrane surface. Differences in band intensity between membrane surfaces and sludge in all DGGE profiles so far indicated there was biomass built-up on membrane surfaces. This was investigated fiirther by quantifying DNA yield from membrane attached communities (Figure 2.7). IhP IhSt 2hP 2hSt 3hP 3hSt Oh sludge Figure 2.6: Profile of the bacterial communities on pressurized (P) and static (St) membranes after 1, 2 and 3 h of incubation. The zero time point sample (0 h) represents the community binding to membrane surface when a module was dipped into the sludge and taken out immediately. Arrows indicate bands that were present on P and St membranes. >5- 140- o> S^ 120- c o 100- sc 80- o o c 60- 8 < 40- Q 20- CO "O O' Time of incubation (hour) Figure 2.7: DNA yield of pressurised (solid bars), static (hatched bars) and 0 h (open bars) membrane samples after 0, 1, 2 and 3 h incubation. It is evident that membrane surfaces under suction pressure (P) fouled more than their corresponding static (St) samples (Figure 2.7). At least three bands (Figure 2.6 A, B, C) are visible in all pressurised membrane samples, but only one band (B) can be seen from the static membrane analysis. Interestingly, this band is also present in the 0 h sample. There is a gradual increase in biomass on the pressurised membranes with longer incubation time, while biomass yield is stable for static samples (Figure 2.7). All bands present on membrane samples are dominant bands in sludge profile. 2.3.3 Sequencing of gel bands of interest (A, B and C) Three bands (Figure 2.6 A, B, C) were excised from the DGGE gel re-amplified and sequenced for further analysis. Results of BLAST searches based on 16S rDNA sequences are provided in Table 2.1. Only BLAST hits with 100% coverage and at least 99% similarity are presented here. Wherever possible, the closest genus was listed as representative of positive hits, except for band A where there is no closest genus identified. Table 2.1: BLAST analysis of band sequences Band Most closely related genus (Accession no.) A Partial 16S rRNA gene of uncultured bacterium (AY651281) B Tetrasphaera elongata gene for 16S rRNA, strain: ASP12 (AB051430) C Zoogloea caeni gene for 16S rRNA, strain EMB43 (DQ413148) Results obtained demonstrated that band B has 100% similarity with thel6S rRNA gene of species Tetrasphaera elongata. This organism was present in all membrane samples, including the 0 h, indicating that it had the ability to attach and bind tightly when in contact with membrane surfaces. T. elongata belongs to the Actinomycetales order within the Actinobacteria phylum whose role in AS systems has been acknowledged as extensive and important for the phosphorous-removal process (92). Both bands A and C belong to P-Proteobacteria group, with band C identified as similar to a partial sequence of the 16S rRNA gene of Zoogloea caeni. 2.3.4 Phylogenetic analysis of clone library of sludge DGGE is a powerful tool for comparing bacterial communities under different hydrodynamics at membrane surfaces (pressurized vs static). However, the information obtained from the 200 bp fragment of the 1500 bp 16S rRNA gene sequences will not allow unambiguous identification of the community members. Therefore, to identify members of the sludge and attached community, a clone library of 16S rRNA genes was generated. DGGE analysis of sludge samples revealed relatively low diversity within the bacterial community, hence only fifty clones were picked for re-amplification and sequencing. Out of fifty 16S rDNA clones that were sequenced, seventeen clones were found to have unique sequences. BLAST results of these seventeen clones were used to construct a phylogenetic tree (Figure 2.8). The bacterial community in the sludge consisted of four different phyla: Proteobacteria, Bacteroidetes, Actinobacteria and Gemmatimonadetes. Fourty seven percent of the clones were affiliated with Proteobacteria and clustered with the P- Proteobacteria (6 clones) and y-Proteobacteria (2 clones). Fourty one percent of the clones (7 clones) belong to Bacteroidetes phylum. There was only one representative for each phylum of Actinobacteria and Gemmatimonadetes (6%). Alignment of the sequences of the DGGE bands (A, B and C) with these seventeen unique clones reveals and confirms their presence within sludge (Table 2.2). Table 2.2: Aligment of DGGE bands with clone library Band Clone Most closely related Bacterial division Similarity no. sequence (Accession no.) A 45 Uncultured bacterium P-Proteobacteria 100% (AF234726) B 30 Tetrasphaera elongata Actinobacteria 100% (NR_024735) C 39 Zoogloea caeni p-Proteobacteria 99% (D0413148) y-Proteobacteria •Clone27 —Cl0íie28 • p-Proteohacteria 'tóidüvorasc ieflu¥ii CT)j BSB411 "ClofieiO^ PS. Chitioophaga arveasieola Ifl i 12iS0 -CloneSa —Terrimonas ferruginea ^T) -Cloned4 ^ Bacteroidetes •Cloiie22 'Adhaeribacter aqiaaticus (T); type strain: MBSGl.i 'Clone61 "^Ronella alitfeffQ-riiie |f| Tetrasphaera elongata (T); Lp2 yActinobacteria GeBiEiatimonas aurantiaca (T)j '^-^^jGemmatimonadetes Figure 2.8: Phylogenetic tree of seventeen unique clones of 16S rRNA gene amplified from sludge collected on the day of early colonisation experiment. The sludge bacterial commur contained four different phyla {Proteobacteria, Bacteroidetes, Actinobacteria, and Gemmatimonadetes). The scale bar indicates 0.1 changes per nucleotide. 2.4 Discussion To date, research in the area of membrane biofouHng has focused on the relationship between operating parameters and the degree of biofouhng, while, surprisingly the role of bacteria in membrane fouling has not been addressed adequately, given that bacteria are accepted as ubiquitous and the source of many types of foulants. Due to the lack of research focused on the biological aspects of membrane fouling, fouling control strategies such as back-washing, air-sparging and chemical treatments have not been very successful. The development of better fouling control strategies could lie in an improved fundamental understanding of the microbiological aspects of the formation and behaviour of the fouling layer on membrane surfaces (18). Membrane surfaces in MBR systems are different from other substrata for biofilm formation in that the inwards hydrodynamic suction, forced by filtration, transport both bacteria and nutrients towards the surface, creating a micro-environment of high cell density and nutrient concentration. The results presented on early colonisation of MBR membranes further support the observation that surfaces under such hydrodynamic forces foul faster than those that are not. However, the presence of T. elongata on both P and St membrane samples shows that hydrodynamic forces do not necessarily influence the identity of initial colonisers. In fact, the presence of T. elongata on membrane samples that were dipped into the reactor sludge proves that this genus adheres rapidly to membranes independent of bulk flow. The Tetrasphaera genus was only recently proposed as a new member of the family Intrasporangiaceae, that belongs to the Actinohacteria phylum (43). They are aerobic chemoheterotrophs, oval to short rods, 0.7-1 |im in diameter, 1-1.8 |im long and occasionally form elongated linear clumps reaching 4 ^im long (hence, the name "elongata"). T. elongata are non-motile and lack surface structures. They have been shown to reduce nitrate to nitrite under anaerobic conditions but do not reduce nitrite to nitrogen. The Actinohacteria phylum to which T. elongata belongs, are Gram-positive, high G+C content that can be terrestrial or aquatic. Streptomyces species belonging to the Actinobacteria phylum are better known representatives of this group of bacteria for their naturally occurring antibiotics. However, studies on bulking and foaming in activated sludge reveals a dominant role of Actinobacteria in these processes (118, 127). Filamentous Actinobacteria are thought to provide the backbone matrix for AS floe development (78), but over-growth of these bacteria in the bulk liquid disrupts the ability of floes to settle, a phenomenon known as bulking. Persistence and proliferation of Actinobacteria in AS is attributed to their abilities to a) grow on substrates that are not utilized by most other organisms such as the preferential utilisation of hydrophobic substrates (114), b) integrate into floes, hence ensuring their retention in the system (118), and c) assimilate substrates and store them intra- cellularly for use during starvation periods (134) such as the poly-phosphate accumulating Tetrasphaera sp. Poly-phosphate accumulating organisms (PAO) are often found in high concentration in enhanced biological phosphorous removal (EBPR). PAOs are characterised for their ability to assimilate readily biodegradable substrates such as acetate under anaerobic conditions to synthesize poly-hydroxyalkanoate (PHA), using polyP and glycogen as energy sources. Conversely under aerobic conditions, glycogen is replenished and phosphate from medium is taken up for polyP storage via respiration of PHA. However, Tetrasphaera sp. does not share the typical PAO phenotype. They are not able to assimilate short chain fatty acids like acetate anaerobically (62) and do not produce PHA (71). However, they have been shown to assimilate amino acids into storage compounds so far unidentified. The bioreactor used in this project was operated under conditions favourable for the oxidation of ammonia. Oxygen was constantly supplied by an air diffuser. Within the confined space of a 26 L reactor, anaerobic zones were not expected, and therefore the Poly-phosphate accumulating ability of Tetrasphaera spp. did not give the organism a competitive advantage. The results of sequencing of the clone library reflect this observation. Out of fifty clones that were isolated for 16S rDNA insert amplification and sequencing, only one clone contained the sequence of the species T. elongata. Instead, a majority of clones were affiliated with P-Proteobacteria, to which the later colonisers belong. It is not clear how this low abundance lineage adheres to membrane surfaces and the question as to whether the attachment of this species influences subsequent colonisers remains to be investigated. However, it has been shown previously that specific bacterial populations, which were not dominant in the AS were selectively accumulated on flatsheet membrane surfaces leading to the development of irreversible biofouling (88). In this study, we reported the presence of T. elongata on the membrane surface occurs instantaneously and irrespectively of the hydrodynamics, facts which indicate that this species may be important in early fouling of MBR membranes in WWT processes. Chapter 3 Isolation and characterisation of bacteria isolated from membranes, floes and supernatant of an MBR 3.1 Introduction Most research on MBR processes has focused on the analysis of microbial communities in the suspended solids and their correlation to the performance of the MBR. Whilst biofouling, which is mainly caused by bacterial biofilms, is considered the "Achilles heel of membrane proccesses" (33), surprisingly few studies attempt to identify which bacteria affect membrane fouling or understand the mechanisms by which they do so. A study by Ivnitsky et al. (2007) showed that bacteria were indeed present in the fouling layer on nanofiltration membranes and that colonisation of the membrane surfaces by bacterial cells occurred in the very early stages of operation. Atomic force microscopy and near-field scanning optical microscopy-AFM analyses revealed initial colonisation of flatsheet membranes by bacterial cells occurred by 8 hours of operation (51). The number of colonisers on the membrane surface after 1 A 0 day of operation was found to be 3-4 x 10 CFU/cm as assessed by heterotrophic plate counts. Confocal laser scanning microscopy analyses showed this mature biofilm layer was stable and maintained at approximately 20-30 fim thickness regardless of operational conditions (51). Whilst the extraction of liquid from the reactor through the membrane could have facilitated the deposition process, attachment of bacteria on the membrane surface is likely to be the result of a combination of available nutrient concentrations, shear forces and pressure drop across the membrane surface (51). Analysis of microbial communities from membrane surfaces used in water purification processes by molecular methods have indicated that, regardless of processes, Proteobacteria are the predominant colonisers with minor contributions from Gram-positive and other Gram-negative bacteria. For example, Chen et al. (2004) showed that a-Proteobacteria comprised the largest microbial fraction on a microfiltration (MF) membrane treating secondary effluent and on a reverse osmosis (RO) membrane treating portable water (15). Similar results were obtained from an MF system treating synthetic paper mill wastewater (148), however, Horsch et al. (2005) attributed a distinctive role in the initial membrane colonisation to y- Proteobacteria as this group was more predominant than p- and a-Proteobacteria in early biofilms when compared to mature ones (48). The ability to utilise a wide range of substrates, to colonise different surfaces and to proliferate under oligotrophic conditions may give Proteobacteria a competitive advantage for colonisation of membrane surfaces. It has also been reported that there is a significant difference in microbial diversity on membrane surfaces and in the suspended solids (148). For example, Zhang et al. (2006) reported that amplified ribosomal DNA restriction analysis (ARDRA) of a bacterial community on a MF membrane surface revealed that the dominant members of this community were different from the dominant members of the activated sludge in paper mill wastewater (148). The differences in microbial populations colonising different micro-environments could be explained by their physiological traits. A study by Pang et al. (2005) showed that membrane-bound isolates exhibit characteristics that promote bacterial attachment and proliferation on membrane surfaces. For example, it was known that one isolate Rhodopseudomonas sp. strain R03 had a low surface charge while Dermacoccus sp. strain R012 and Microbacterium sp. strain R018 were hydrophobic, which facilitated cell adhesion to the membrane surface (98). In addition to surface hydrophobicity and surface charge, membrane isolates were also found to possess higher EPS concentrations and higher rafios of protein to carbohydrate within the EPS than the isolates from the suspended solids (55). We reported in the previous chapter, the bacterial community structure on ultrafiltration (UF) membrane surfaces under different hydrodynamics and nutrient concentrations as assessed by culture-independent techniques. The aim of this chapter was to isolate cultivable bacterial strains and characterise them for their physiological traits, which are known to be important for biofilm initiation and formation on membrane surfaces. Bacterial strains from supernatant and floe phases of the activated sludge (AS) and from the membrane surface were isolated and compared. Here we investigate physiological characteristics such as growth rate, quorum sensing (QS) molecule production, motility, surface hydrophobicity, co- aggregation, auto-aggregation and biofilm formation on polystyrene plates. 3.2 Materials and Methods 3.2.1 Isolation of bacteria from membrane surfaces and activated sludge Following the molecular study of early colonisers on the membrane surfaces used in an MBR treating artificial wastewater (Chapter 2) and after 9 months of stable operation, AS and mini membrane modules from the same UF MBR system were acquired for the isolation and characterisation of bacterial strains. AS was collected and separated into supernatant (S) and floe (F) phases by gravity for one hour. Three 1 ml aliquots of S and F were centrifiiged at 16,060 x g for 10 minutes. The pellets were resuspended in the same volume of phosphate buffer solution (PBS, pH7) and vigorously vortexed. The samples were serially diluted and plated onto R2A plates (Oxoid, Basingstoke). Loosely attached bacteria on one-day-old pressurised membranes (PM) were removed by rinsing the membrane three times with PBS (pH7). The PM were then cut into 2 cm segments and resuspended in PBS. Bacteria were dislodged from membrane surfaces by sonication at 80 watts for 2 minutes and vortexing for 2 minutes two times. Bacterial suspensions were serially diluted and plated onto R2A plates and incubated for at least five days at room temperature. A total of 50 single colonies were selected and restreaked onto fresh plates. 3.2.2 Media and growth conditions Unless otherwise stated, bacterial isolates were kept on R2A agar plates (per Litre of milli Q water: 0.5 g proteose peptone, 0.5 g casamino acid, 0.5 g yeast extract, 0.5 g glucose, 0.5 g soluble starch, 0.3 g K2HPO4, 0.05 g MgS04.7H20, 0.3 g sodium pyruvate, 15 g agar at pH7) and the overnight cultures were grown at room temperature (25^C) with shaking at 200 rpm using polypeptone liquid medium (per Liter of milli Q water: 10 g special peptone (Oxoid, Basingstoke), 1 g (NH4)2S04, 0.5 g NaNOs, 0.1 g NaCl, 0.2 g MgS04.7H20, 0.05 g CaCh.lHiO, 0.01 g FeCl3.6H20, and 1 g K2HPO4). The medium was adjusted to pH7 before sterilisation by autoclaving. Growth studies of bacterial isolates were conducted in duplicate using conical flasks containing 30 ml of polypeptone medium. Overnight cultures of isolates were inoculated into flasks to an initial optical density of 0.01 at 600 nm. One hundred microlitres aliquots of cell cultures were taken for optical density measurement at 600 nm with a spectrophotometer (NovaSpec® II, GE, Piscataway), and another 100 fil were serially diluted in PBS (pH7) and plated on R2A agar. The plates were incubated at room temperature for three days and the colony forming units (CPU) determined. 3.2.3 Physiological characteristics of isolates 3.2.3.1 Motility Assays Bacterial isolates were tested for three types of motility: swimming, swarming and twitching (25), on R2A agar plates. Fresh overnight cultures of isolates were used as inoculum and plates were incubated at room temperature and examined daily for a period of three days. To examine isolates for swimming motility, bacterial cells were stab-inoculated into the centre of R2A plates containing 0.3% (w/v) agar. Swimming motility was evident by the formation of an opaque zone resulting from the cells migrating away from the point of inoculation. Swarming motility was determined by the inoculation of 2 |LI1 of overnight culture onto R2A plates containing 0.5% (w/v) agar, which were examined for bacterial growth on the surface of the plate. In order to test for twitching motility, bacterial cells were stab-inoculated onto R2A plates containing 1% agar. After incubation, the agar was removed and unattached cells were removed by gently washing with sterile water. The zone of twitching motility on the plate surface was visualized by staining the attached cells with 1% crystal violet. 3.2.3.2 Cell surface hydrophobicity Surface hydrophobicity of the bacterial isolates was determined by cell adhesion to hydrocarbons as described previously (111) with some modifications. Overnight cultures (10 ml) were collected by centrifugation at 16,060 x g for 10 minutes, washed twice with PBS (pH7) and resuspended in PBS to an absorbance of 0.6 at 600 nm. One and a half millilitres of this diluted cell culture was transferred into glass tubes containing different volumes of hexadecane (0.5 ml, 1 ml or 1.5 ml). This mixture was incubated for 10 minutes and vortexed vigorously for 2 minutes. The aqueous and hydrocarbon phases were allowed to partition for 30 minutes and the decrease in the absorbance of the aqueous phase determined. Hydrophobicity was calculated by expressing this absorbance value as a percentage of the initial absorbance using the formula (Absorbance before mixing - Absorbance after mixing) x 100% Absorbance before mixing 3.2.3.3 N-acyl homoserine lactone production The production of acylated homoserine lactone (AHL) signalling molecules was detected by the well-diffusion assay (105) for Agrobacterium tumefaciens strain A136. The A. tumefaciens A136 strain carries a lacZ ftision to the tral gene and produces a blue colour in the presence of 5-bromo-4-chloro-3-indolyl-P-D - galactopyranoside (X-Gal) in response to AHLs. Cell-fi-ee supematants of overnight culture of isolates were collected by filtration through 0.2 ^im membranes (Millipore, Jaffrey). Sterile supematants were tested for the presence of AHLs by adding 200 \i\ to wells of an agar plate seeded with the monitor strain A. tumefaciens A136. The agar plates were prepared as follows: 1 ml of overnight culture of A. tumefaciens A136 (inoculated in LBio supplemented with 4.5 jig/ml tetracycline, 50 |ig/ml spectomycin and incubated at 200 rpm, 30®C for at least 16 hours) was mixed with 4 ml of soft, warm R2A agar (0.8% w/v) supplemented with 50 \ig/m\ X-Gal. The agar-culture mixture was poured onto an R2A agar plate (1.2% w/v) supplemented with 50 ^ig/ml X-Gal. When the top layer had solidified, round wells were created by punching with the inverted end of a 1 ml pipette tip. Into each well, 200 |il of culture supernatant was inoculated. The plates were incubated at 30^C for 24 hours. Two hundreds microlitres of polypeptone medium mixed with 1 jil 3-oxo-C6-HSL (OHHL, 1 mM) or 200 |il of cell-free supernatant from the AHL-hyper-producer Pseudomonas aeruginosa 18A were used as positive controls. 3.2.3.4 Biofilm formation Overnight cultures of isolates were diluted in R2A broth. Triplicate aliquots of 1ml were inoculated into wells of a 24-well flat bottom, polystyrene microtitre plate (BD Biosciences, San Jose) and incubated at room temperature with agitation (60 rpm). During the incubation, spent media was replaced with fresh media daily for three days. Biofilm biomass was determined at day 1, 2 and 3 as previously described (26). Briefly, the aqueous phase was removed, wells were rinsed three times with 1 ml, 1.25 ml, and 1.5 ml of PBS (pH7) respectively and stained with 0.3% (w/v) solution of filtered crystal violet for 15 minutes at room temperature. The wells were washed three times with 2 ml, 2.25 ml, and 2.5 ml of PBS (pH7) before the crystal violet was solubilized in 1ml of absolute ethanol. The absorbance was measured at 540 nm in a multilabel microplate reader (Wallac Victor 1420, PerkinElmer, Waltham). 3.2.4 Genomic DNA extraction The genomic DNA was extracted using the XSP buffer extraction method with fresh buffer (24). Every 50 ml of XSP buffer contained 0.5 g of potassium ethyl xanthogenate, 10 ml of 4 M ammonium acetate, 5 ml of 1 M Tris-HCl (pH7.4), 2 ml of 0.45 M EDTA, 2.5 ml of 20% (w/v) SDS and sterile milHQ-grade water. A volume of 900 ^il of XS buffer was mixed with 900 |al of phenol in a 2 ml microfuge tube and heated to 65^C for 5 minutes. To the mixture, 200 ^l of overnight culture was added, inverted several times and incubated for 15 minutes at 65®C. The tubes were vortexed briefly for 10 seconds, placed on ice for 2 minutes and centrifuged at 16,060 X g for 5 minutes at room temperature. The top layer was removed and extracted twice with 900 |LI1 of phenol ichloroform (1:1) by inverting gently. The tubes were centrifuged for 5 minutes at 16,060 x g. Fifty microlitres of 3 M sodium acetate and an equal volume of isopropanol was added. The contents were mixed gently by inversion and incubated at overnight. After centrifugation for 15 minutes at 16,060 x g, the supernatant was discarded and the nucleic acid pellets washed with 70% ice-cold ethanol, air-dried and resuspended in 50 fil of molecular- grade water. Extracted DNA was quantified using a NanodroplOOO spectrophotometer (ThermoFisher Scientific, Waltham). 3.2.5 16S rRNA gene amplification Near full length 16S rRNA gene sequences were amplified by PGR using a Mastercycler (Eppendorf, Hamburg) and primer set of 27F (5'- AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'- TACGGYTACCTTGTTACGACTT-3') (63). The 50 ^il reaction contained 25 nl of PGR Master Mix (Promega, Madison), 1 ^il of each primer (10 ^M), 40 - 50 ng DNA template and nuclease-free water. The cycling conditions used for the PGR were as follows: 3 minutes of initial denaturation at 95^G and 30 cycles of 20 seconds at 95^G, 20 seconds at annealing temperature 55^G, and 2.5 minutes at 72^G. A final extension was carried out for 10 minutes at 72®G. The PGR products were purified by using the Purelink PGR Purification kit (Invitrogen, San Diego). 3.2.6 Sequencing of 16S rDNA Purified PGR products were amplified for sequencing according to the manufacturers instructions for the ABI 3730 Gapillary Sequencer. Briefly, 20 fil reactions contained 3.5 |il of 5 x buffer, 3.2 |nl of 27F primer (1 ^M), 1 Big Dye Terminator, 20 - 50 ng of 16S rDNA template and molecular grade water. The conditions used for the sequencing PGR were as follows: 25 cycles of 10 seconds at 96®G, 5 seconds at 50^C, 4 minutes at 60^C. The tubes were held at 4®C before products were purified and precipitated using the phenol/butanol method (130). Briefly, the products were resuspended in 80 |il of sterile milli Q water. Samples were transferred to fresh 1.5 ml microfuge tubes containing 100 |il of phenol and vortexed for 5 seconds, centrifuged at 16,060 x g (Hettich, Tuttlingen) for 4 minutes and the aqueous phase transferred to a fresh 1.5 ml microfuge tube containing 900 |LI1 of butan-l-ol. The tube was vortexed for 10 seconds, and centrifuged at 16,060 x g for 10 minutes. The supernatant was discarded, and the pellet was air-dried and sequenced at the Ramaciotti Centre for Gene Function and Analysis (UNSW) using an AB3730 DNA Analyzer (Applied Biosystem, Waltham). 3.2.7 Aggregation assays Inter-generic co-aggregation among bacteria isolated from the same sample type (floes, supernatant or PM) was assessed as previously described with modifications (74). Liquid cultures of isolates were grown to late exponential growth phase, harvested by centrifugation at 16,060 x g for 10 minutes, washed twice in 3 mM NaCl solution containing 0.5 mM CaCl2 and resuspended in the NaCl-CaCl2 solution to the initial optical density of 1 at 600 nm. In pairs, 1 ml of cell suspension of each of the isolates were mixed together in Hungate tubes. The tubes were incubated at room temperature (25^C) with shaking at 100 rpm. Optical density (600 nm) of the mixture was measured every fifteen minutes for a total of 1.5 h. Optical density of individual isolate suspensions were used as negative controls. Blastomonas natatoria (strain 2.8) and Micrococcus luteus (strain 2.13) which have been shown previously to co-aggregate were used as a positive control (109). Isolates were assessed for auto-aggregation by visual observation of cells settling due to gravity. Overnight cultures of isolates were incubated statically to settle on the bench for five minutes. Observations of granulation, clumping and density of media (cloudy or not cloudy) were recorded. 3.3 Results 3.3.1 Isolation and identification of cultivable bacteria Cultivable bacterial isolates from S and F phases of AS as well as from PM surfaces were cultured on solid R2A agar. A total of twenty, thirteen and fifteen isolated colonies were identified for S, F and PM samples respectively. On solid agar, the colonies had simple morphologies of small yellow, small white or white and slimy. The 16S rRNA gene of each bacterial isolate was amplified and sequenced (Table 3.1). Table 3.1: Phylogenetic identity of isolates Isolate origin Strain ID Phylogenetic affiliation (Accession number) Pressurised membrane PM2 Microbacterium lacticum (EF204396.1) PM11,PM9, PM5 Microbacterium pyrexiae (DQ673323.1) PM15,PM13 Microbacterium laevaniformans (EU879962.1) PM16 Microbacterium sp. (AY779522.1) PM7,PM18 Uncultured Actinobacteria (AJ318140.1) PMl Uncultured bacteria (GQ002732.1) PM3 Pseudomonas sp. (EU652471.1) PM8 Staphylococcus cohnii (GQl69065.1) Supernatant S8,S4,S14, S7, S3, S2 Microbacterium lacticum (EF204396.1) S15, S9,S19, Microbacterium pyrexiae (DQ673323.1) S17 Microbacterium laevaniformans (EU879962.1) S1,S13, S20 Microbacterium sp. (FJ006893.1) S5, S6 Uncultured Actinobacteria (AJ318140.1) S16 Sphingomonas sp. (FJ605417.1) S12 Uncultured Alphaproteobacteria (CU918780.1) Sil Xanthomonas sp. (DQ213024.1) Floes F16,F4, F8 Microbacterium lacticum (EF204396.1) F12,F13,F6 Microbacterium pyrexiae (DQ673323.1) Fl Microbacterium laevaniformans (EU879962.1) F14 Microbacterium phyllosphaerae (DQ365561.1) F7 Microbacterium sp. (AY779522.1) Fll Microbacterium flavescens (EU714363.1) Microbacterium sp. was the most abundant lineage in the culture collections of the three sample types. Six other phylotypes were unique for six isolates, hence were not abundant in the culture collections. Microbacterium is a genus within the Actinobacteria phylum of which many filamentous genera have been known for causing foaming and bulking problems in activated sludge. A) • Microbacterium lacticum • Microbacterium pyrexiae • Microbacterium iaevaniformans a Microbacterium sp. • non-sequenced isolates • Microbacterium fiavescens Microbacterium phyiiosphaerae B) • Microbacterium lacticum • Microbacterium pyrexiae • Microbacterium Iaevaniformans Microbacterium sp. M non-sequenced isolates • Uncultured Actinobacteria • Uncultured bacteria M Pseudomonas sp. • Staphylococcus cohnii C) • Microbacterium lacticum • Microbacterium pyrexiae • Microbacterium Iaevaniformans Microbacterium sp. • non-sequenced isolates • Uncultured Actinobacteria MSphingomonas sp. • Uncultured Alphaproteobacteria • Xanthomonas sp. Figure 3.1: Pie charts showing the proportions of distinct phylotypes among bacterial isolates from A) FIocs, B) Pressurised membrane, C) Supernatant. Microbacterium phylotypes present in all sample types (floes, supernatant and membrane surfaces) are labelled in grey. The proportion of each distinct sequence in the culture collections of floes (Figure 3.1 A), pressurised membrane (Figure 3.1 B) and supernatant (Figure 3.1 C) is presented in Figure 3.1. DNA extractions for eight isolates were unsuccessful (two S, three F and three PM isolates). For statistical purposes, these isolates were included and labelled as non-sequenced isolates in the figures. A number of phylotypes were abundant among isolates from all three sample types, including Microbacterium lacticum, Microbacterium pyrexiae, Microbacterium laevaniformans and unidentified Microbacterium sp., making up 61% of F isolates (Figure 3.1 A), 46% of PM isolates (Figure 3.1 B) and 65% of S isolates (Figure 3.1 C). While M. lacticum was abundant among isolates from S (30%) and F (22%), only one isolate out of fifteen PM isolates belonged to the M. lacticum species (7%). A number of isolate phylotypes were unique to specific samples. For example, a Pseudomonas sp. and Staphylococcus cohnii were only present on pressurised membrane surfaces, whilst Sphingomonas sp., Xanthomonas sp. and an Alphaproteobacteria with no close cultured relatives were isolated only from the planktonic phase of the reactor. Culture collections of membrane and supernatant samples were more phylogenetically diverse than flocs, as floe isolates belong to the Microbacterium genus (77%) (Figure 3.1 A) with two species M. flavescens and M phyllosphaerae being floc-specific. There was no significant distinction between the culture collections of isolates from three sample types. Rather, the culture collections were dominated by the genus Microbacterium with a small number of unique phylotypes in each collection (with the exception of flocs whose isolates all belonged to the Microbacterium genus). 3.3.2 Detection of A^-acyl-L-homoserine lactone activity Due to their significance in the regulation of biofilm formation, the production of AHLs by isolates was investigated. Out of 48 bacterial isolates tested, only the Alphaproteohacteria sp. from the supernatant culture collection (SI2) gave a positive response in the A. tumefaciens A136 assay. A blue halo around the inoculum-well with a radius of 0.6 cm could be seen after 24 hours of incubation (Figure 3.2). a) b) Figure 3.2: AHL-like activity of a) the positive control P. aeruginosa 18A and b) the S12 isolate im the A. tumefaciens A136 assay. This finding suggested AHL-mediated gene expression did not play a major role in biofilm or floe formation in this system. Since this signalling pathway is more specific for Gram-negative bacteria, the result was in agreement with the relatively low frequency of Proteobacteria isolated. 3.3.3 Motility assays All bacterial isolates were tested for three types of motility: swimming, swarming and twitching. There was no correlation between the origin of isolates and motility observed (Table 3.2). Only two isolates exhibited swimming motility, five isolates possessed swarming motility and none of the isolates showed twitching motility. With the exception of isolate PM6, the swimming isolates belonged to Proteobacteria sp. whilst all of swarming isolates belonged to Microbacterium sp. Table 3.2: Motility of bacterial isolates from floes (F), supernatant (S), and pressurised membrane (PM). Isolates Phylogeny Swimming Swarming Twitching PM3 Pseudomonas sp. + - PM5 M pyrexiae - + PM6 non-sequenced isolate - + PM15 M. laevaniformans - + S15 M. pyrexiae - + S12 Uncultured a-Proteobacteria + - F8 M. lacticum _ + 3.3.4 Cell surface hydrophobicity Surface hydrophobicity of bacterial isolates was determined by an assay quantifying the adhesion of cells to hydrocarbons. Differences in absorbance of the aqueous phase of cell suspensions reflect the amount of cells adhering to the hexadecane molecules. Hydrophobicity is an index assessing the tendency of a particle to mix with water. The higher this value is, the more hydrophobic the isolate is on its surface and the less likely the isolate exists in the water phase. Isolates from the planktonic phase of the reactor (supernatant isolates) were expected to have a low hydrophobicity index whilst higher hydrophobicity indeces were expected for both floe and pressurised membrane isolates. The results obtained reflected this trend (Table 3.3). Table 3.3: Cell surface hydrophobicity of bacterial isolates (average of duplicates). Isolates Cell surface hydrophobicity Supernatant S17 0% S12 SI WSBBSBSKm S16 S2 S13 84% S7 89% S20 100% Floes F1 F6 F16 F8 60% F4 61% F13 79% F12 80% F7 83% F14 87% Pressurised membrane PMll PM8 50% PM14 68% PM2 74% PMl 91% A higher proportion of supernatant isolates tested possessed lower hydrophobicity (5 out of 8 had a hydrophobicity index of 50% or less) than floes (3 out of 9) and pressurised membrane (1 out of 5) isolates. This indicated that surface hydrophobicity could be an important factor that defines the microbial community structures in niches such as floes, membrane surface and planktonic phase. 3.3.5 Auto-aggregation Auto-aggregation of isolates was examined by visual observation of overnight cultures, left statically for five minutes (Table 3.4). Table 3.4: Auto-aggregation of isolates Isolates Phylogeny % of total Supernatant S9 M pyrexiae SIO Non-sequenced isolate 20% Sll Xanthomonas sp. S19 M pyrexiae Floes F1 M. laevaniformans FIO Non-sequenced isolate Fll M.ßavescens 46% F12 M. pyrexiae F13 M. pyrexiae F15 Non-sequenced isolate Pressurised membrane PM3 Pseudomonas sp. PM5 M pyrexiae PM6 Non-sequenced isolate PM9 M pyrexiae 46% PM13 M. laevaniformans PM14 Non-sequenced isolate PM15 M laevaniformans A higher proportion of F and PM isolates auto-aggregated (46%) when compared with supernatant isolates (20%). Most of these isolates are Microbacterium sp. 3.3.6 Biofílm formation on polystyrene plates The ability of isolates to form biofilms was assessed in 24-well polystyrene plates. Biomass of 1, 2 and 3 day-old biofilms was quantified by staining with 0.3% crystal violet (Figure 3.3). The bacterial strain P. aeruginosa PAOl was included as positive control. The majority of the isolates obtained from the planktonic phase did not form extensive biofilms when compared with P. aeruginosa PAOl (Figure 3.3 A), with the exceptions of SI 1, SI2, SI 3 and S20 (Figure 3.3 B). Sequencing of these isolates revealed two of them (SI 1 and SI 2) VJQXQ Xanthomonas sp. and Alphaproteobacteria sp. respectively, and two of them (SI3 and S20) were Microbacterium sp. Remarkably, all of these isolates possess either swimming motility (SI2), high surface hydrophobicity (SI3 - 84% and S20 - 100%) or were auto-aggregating (SI 1). There were 4 bacterial isolates out of 11 tested from F samples that produced more biofílm than the positive control organism, P. aeruginosa PAOl (Figure 3.3 D). Isolates F12, F14, F4 and FIO were all identifíed as Microbacterium sp. and had either high surface hydrophobicity (80%, 87%, 61% respectively) or were auto- aggregating (FIO), suggesting that hydrophobicity and auto-aggregation are important in biofílm formation. Similarly for PM isolates, out of ten isolates, only 2, PM3 (Pseudomonas sp.) and PM14 (non-sequenced isolate) formed thick biofílms on the surface of polystyrene plates (Figure 3.3 F). While PM3 possessed swimming motility, PM14 was relatively hydrophobic (68%). A) B) «» P «e- S16 S3 U 1,40- $2 * S8 m 2 Day C) D) u- m HSH Fta •• m F10 i1 1-0- m: 'S- F4 8 0.5- 0.0- E) F) mi mil p. PM2 Hfr. f>M18 J > PM3 mr ^ PM1? •*- pMte T"" Dsy Figure 3.3: Biomass of 1, 2 and 3 day-old biofilm on polystyrene plates of supernatant (A, B), floes (C, D) and pressurised membrane (E, F) isolates. P. aeruginosa PAOl was included as positive control. Data presented are averages of triplicates. Error bars represent standard deviation. Within the same niche, some isolates were better at forming biofilms (B, D, and F) than others (A, C, and E) when compared with P. aeruginosa PAOl (broken line). There was no link between the origin of isolates (S, F or PM) and their ability to form biofilms. However, isolates that formed extensive biofilms also exhibited traits known to be associated with biofilm formation in physiological assays such as swimming motility, hydrophobicity and auto-aggregation. Co-aggregation between isolates was also examined in this study. However, the assay did not reveal any significant co-aggregation between isolates. This could be due to low species diversity in the culture collection. 3.4 Discussion Whilst use of cultivation-independent methods based on 16S rRNA gene sequences provides usefiil insights into community structure of bacterial biofilms, the actual mechanisms of interactions of individual bacterial species with the membrane surfaces cannot be identified. Previously, cultivation and characterisation of RO membrane-bound isolates revealed the physiological traits that allow for the attachment and growth of these isolates on membrane surfaces (55, 98). In this study, bacterial cells were isolated from three different micro-environments: hollow fibre membrane surfaces, floes and supernatant phases of AS, and characterised for physiological traits known to be important for the attachment and proliferation on membrane surfaces. The sequencing results revealed the majority of isolates belong to the Microbacterium genus of the Actinobacteria phylum. The Microbacterium isolates included M. lacticum, M. pyrexiae, M. laevaniformans, M. phyllosphaerae and M. flavescens. This is in contrast to previous results on the sequencing of DNA isolated from sludge and membranes which revealed that Proteobacteria dominated the community with only one representative of the Actinobacteria phylum identified (Chapter 2, Figure 2.8). However, it should be noted that these studies were performed 2 months apart, the early colonisation after 7 months and the isolation of strains after 9 months of stable operation. Whether this shift in microbial diversity was due to the different sampling methods used (culture-independent vs culture- dependent) or to shifts in the community itself is unknown. However, all of the operating parameters remained the same. Fluctuations in bacterial community structure in MBR AS is a well documented phenomenon (139), and results presented in the previous chapter indicated that the bacterial community structure in the bioreactor was highly dynamic (Chapter 2, Figure 2.2). Microbacterium species are characterized by the presence of N-glycolyl residues in the cell walls, having isoprenoid quinones MK-11 and MK-12 (126). The genus belongs to the Microbacteriaceae family and is in the same Micrococcineae suborder as Tetrasphaera (Figure 3.4). The Microbacterium genus comprises a large group of bacteria with diverse properties. Like Tetrasphaera species, a number of Microbacterium species are known for their structural role in sludge flocculation. In fact, extracellular polymeric substances (EPS) produced by Microbacterium genus have been proposed to be good bioflocculants for treating settling problems in sludge (124). It is therefore, not surprising to find that all isolates derived fi-om floes belong to this genus. Phylum: Actinobacteria \ r A ctinobacteriaceae A ctinomycetales y Suborder: Micrococcineae Family: Microbacteriaceae ¥simi\y:Intrasporangiaceae Genus Microbacterium Genus Tetrasphaera Figure 3.4: A schematic diagram showing the phylogenetic relationship between the Microbacterium genus and Tetrasphaera genus within the Actinobacteria phylum. In contrast to previous findings suggesting that Proteobacteria are the dominant colonisers of membrane surfaces, bacterial species isolated fi-om the pressurised membrane in this study was predominantly Microbacterium species. Considering how dominant the Microbacterium genus was in the fiocs and supernatant phases of this MBR (making up 77% and 65% of isolates respectively), the presence of these species on the membrane surface could have been simply their physical deposition on the membrane due to extraction of liquid fi-om the reactor. Nevertheless, their eariy presence on membrane surfaces may have an effect on physico-chemical properties of the membrane surface and thereby influence subsequent colonisation. In addition to Microbacterium, Pseudomonas sp. (PM3) and S. cohnii (PM8) were also identified as part of the sessile community on the membrane surface, both of which have previously been isolated fi-om membrane surfaces (148). Physiological traits relevant to biofilm initiation and formation were characterised for all isolates. Studies of biofilm formation in pure culture have revealed it is a complex phenomenon, involving many factors and regulatory pathways. The process is even more complex on membrane surfaces due to hydrodynamics and complex bacterial community interactions. Bacteria are likely to have more than one strategy for the initiation and formation of biofilms on membrane surfaces. Pang et al. (2005) showed that different membrane-bound isolates possess different characteristics, which promote attachment and proliferation on RO membranes. Some facilitate bacterial adhesion by possessing high cell surface hydrophobicity (as Dermacoccus sp. strain R012 and Microbacterium sp. strain R018) and others by low surface charge (as Rhodopseudomonas sp. strain R03). Our results on cell surface hydrophobicity supported previous observations that membrane-bound isolates were more hydrophobic than suspended solid isolates (55). Conversely, the ability of isolates to form biofilms on polystyrene surfaces was not dependent on the source of isolation, rather, isolates that possessed swimming motility (SI2 or PM3) or high surface hydrophobicity (S20, F14, F12, S13, PM14) formed better biofilms than the P. aeruginosa PAOl positive control. In contrast to previous studies which suggested membrane isolates grew slowly when compared to strains isolated from the suspended solids (55), there was no significant difference in the growth rates of isolates investigated (data not shown). Similarly, results from the co-aggregation assay did not yield any conclusive data as had been previously observed in aquatic bacteria by Rickard et al. (2000). In this chapter, we present data on the physiological traits that have been shown to be important for surface colonisation of bacterial strains isolated from the: membrane surface, supernatant and floe phases of activated sludge from a nitrifying MBR. The three culture collections were dominated by Microbacterium species, which have been isolated frequently from activated sludge. Our physiological studies showed these Microbacterium sp. isolates exhibited various properties that may assist them in colonising the membrane surface, including hydrophobic surface, swimming motility and auto-aggregation. Further investigation of membrane colonisation by Microbacterium sp. may provide information that can lead to prevention of colonisation initiated by these organisms. Chapter 4 General discussion Membrane bioreactors (MBRs) represent an attractive alternative to conventional activated sludge processes (ASP) because of their ability to produce effluent of high clarity and their high adaptability to current ASP plants. MBRs combine membrane filtration with biological treatment where a membrane unit can be placed outside or submerged directly into the activated sludge (AS) reactor. Physical separation of AS biomass and the treated water through membrane filtration produces effluent of such high clarity that the need for subsequent treatment steps can be eliminated. The biggest challenge for MBR technology is the fouling of membrane surfaces that reduces productivity by lowering flux rates and increasing energy requirements. Membrane fouling also leads to the need for membrane cleaning and ultimately shortens membrane lifetime. The study of membrane fouling has attracted more than one third of the research studies investigating MBR operation (142). Most of these studies focused on the membrane-fouling phenomenon as an increase in transmembrane pressure (TMP) in response to different engineering parameters (85, 140, 143). Membrane fouling, however, is a result of not only biomass accumulation due to liquid extraction but also of the formation and development of a microbial biofilm on the membrane surface. Considering many microbial factors (e.g., EPS, soluble microbial products) related to membrane fouling, it is reasonable to presume that a biofilm approach to the study of the fouling process may provide the key to novel anti-fouling strategies. Microbiological studies in MBRs have predominantly characterised micro- organisms responsible for substrate degradation or assessed the dynamics of microbial community structure within the AS in response to changing operating parameters (69, 85). Studies into biofilm formation of pure bacterial cultures on static surfaces show it is a sequential process that starts with the formation of a conditioning film as soon as the substratum is submerged into the liquid medium (77). Biofilm formation in MBRs is complicated by the fact that not only the membrane is exposed to a complex microbial community within the AS but also by the presence of complicated hydrodynamics. The constant suction pressure creates a micro- environment on the membrane surface where the nutrient concentration is high, providing an ideal environment for attachment and growth of bacteria. This study examined the effects of the hydrodynamics on the identity of early bacterial colonisers on hollow fibre membrane surfaces. A bench-scale, nitrifying MBR treating artificial waste was employed. The hollow fibre ultrafiltration (UF) membrane was made of polypropylene with an average pore diameter of 0.04 ^im. The bacterial community composition of early biofilms on the membrane surface under different hydrodynamic conditions (pressurised and non-pressurised) and of the AS were examined by culture-independent, molecular-based methods of PCR- denaturing gradient gel electrophoresis (PCR-DGGE) and PGR cloning of 16S rRNA genes. Using amplified ribosomal DNA restriction analysis (ARDRA) and 16S ribosomal DNA gene sequencing, Zhang et al. (2006) showed that bacterial community structure in the suspended biomass could be very different from that observed on the microfiltration flatsheet membrane. These differences were observed after 4 hours of cross-flow filtration. In the current study, the examination of bacterial community profiles on hollow fibre membrane surfaces submerged for different durations showed profiles from both pressurised and non-pressurised membranes resembled the AS profile after 4 hours of exposure. Hence, to investigate the influence of hydrodynamics on bacterial community structure on membrane surfaces, we thus focused on the early colonisation events in the first three hours of inoculation. DGGE profiling of the bacterial community structures on both pressurised and non- pressurised hollow fibre membranes revealed the presence of Tetraspaera elongata indicating that this organism binds onto membrane surfaces instantly and indiscriminately of the hydrodynamic context, suggesting it could play an important role in the fouling of MBR membranes. The Tetrasphaera genus belongs to the Actinobacteria phylum of which many genera have been isolated from AS systems and shown to play dominant roles in foaming and bulking (119). T. elongata was only recently isolated and proposed as a new member of the Tetrasphaera genus based on its phenotypic and phylogenetic distinctiveness (43). The Tetrasphaera genus is characterised by the presence of phosphate granules in the cells, suggesting they are important micro-organisms for the biological phosphorous removal. Whilst the ability to accumulate poly-phosphate might contribute to their dominance in activated sludge under alternating aerobic-anaerobic conditions that support phosphorus removal, proliferation of Tetrasphaera within the bioreactor used for this study was not expected due to the total aerobic conditions applied. This was evident in the sequencing results of the AS in the reactor at the time, which showed that T elongata was the only representative not only of the Tetrasphaera genus but also of the Actinobacteria phylum. The presence of T. elongata so early on pressurised membranes could have been the result of mass transfer by suction pressure during liquid extraction. However, the finding that T. elongata was also present on the membrane surface free of hydrodynamics (non-pressurised membrane) and after an extremely short exposure time to activated sludge (0 h) suggests this was not the case. With no surface structures, it is not clear how T. elongata was able to colonise the membrane surfaces. The only characteristic that is likely to contribute to their surface colonisation is the distinct ability of T elongata to form linear clumps of up to 4 \im long, which could increase exposure to the membrane surface. Other surface properties that can influence the cell-membrane interactions need to be further investigated in order to elucidate the colonisation mechanism of T elongata to the membrane surface. Studies of surface colonisation by pure bacterial cultures have revealed valuable knowledge on the mechanisms by which cells attach. Most of these studies, however, were investigating biofilm formation on a static substratum of flow cell systems, which are different from surfaces used in engineering processes (such as the permeable substratum of membrane surface). Bacterial mechanisms used form biofilms on these engineering surfaces, therefore, could be very different. Moreover, characterisation of bacterial species from different niches within the bioreactor also revealed differences in physiological traits that gave the bacteria advantages to proliferate within different micro-environments. For example, Jinhua et al. (2006) demonstrated that membrane-bound isolates exhibit higher surface hydrophobicity than isolates from the suspended sludge. Culture collections of isolates from three different micro-environments (planktonic and floe phases of activated sludge and membrane surface) from the same bioreactor were collected and their physiological characteristics important for biofilm initiation and proliferation such as motility, hydrophobicity, aggregation and quorum sensing (QS) were compared. The collections were dominated by the Microbacterium genus of the Actinobacteria phylum (slightly more on membrane surface and floe phase than planktonic phase). This was in contrast to the results of the early colonisation study where T. elongata was the only representative of Actinobacteria in the activated sludge. With the bioreactor under stable operation, these dramatic changes in community structure may have occurred during the two-month period between the sampling events. Fluctuations in bacterial community composition had been demonstrated in the early colonisation study when bacterial community of the sludge floes were monitored for more than two months. Due to the complex interactions inside the bioreactor, fluctuations in bacterial profiles are frequently observed AS. Membrane surfaces used in water purification processes such as MBRs offer a unique environment for biofilm development. The permeate extraction generates a convective transport that not only transports salt and nutrients to the membrane surface but also assists the cells through the hydrodynamic layers, bringing them indiscriminately to the surface (51). While motility has been shown to play an essential role for bacterial attachment and proliferation on static surfaces, motility may not play a major role in biofilm formation on membrane surfaces. This was cleariy demonstrated by Pang et al. in a study where three out of four isolates from reverse osmosis (RO) membrane surfaces {Dermacoccus R012, Microbacterium R018 and Rhodopseudomonas R02) did not exhibit swimming motility. We observed similar results in our culture collections. Only 2 isolates out of 48 exhibited swimming motility, both belonging to the Proteobacteria group. Quorum sensing is a type of communication system that regulates expression of phenotypes that are important in high-density bacterial communities, such as biofilms. Bacteria produce signalling molecules that diffuse freely across cell membranes, allowing the cells to detect their surrounding neighbours. When the autoinducer reaches a threshold concentration, it triggers expression of specific phenotypic characteristics fi*om the whole population. Quorum sensing has been shown to affect different aspects of biofilm dynamics such as architecture, stress resistance, and sloughing (60). While specific QS systems have been found to be exclusive for specific bacteria, including acylated homoserine lactones (AHLs) in Gram-negative bacteria and peptide-mediated signalling system in Gram-positive, other systems such as the autoinducer 2 (AI2) is prevalent in both Gram-positive and Gram-negative bacteria. AHLs have been shown to influence bacterial community composition and function in AS (132). These biofilm regulatory systems are attractive targets for biofilm control strategies. Indeed, recent work by Yeon et al. (2009) showed strong correlation between concentration of AHL signalling molecules in the bioreactor and an increase in transmembrane pressure (TMP). They showed that degradation of the signalling molecules could significantly retard the TMP increase. In contrast, only 1 isolate (an a-Proteobacteria) was shown to produce AHLs in this study. AHL quorum sensing systems are specific for Gram- negative bacteria, while most isolates in the culture collections in this study were Gram-positive. The role of QS in biofilm formation on membrane surfaces requires further investigation. Due to hydrophobic-hydrophilic interactions, bacteria with increased hydrophobic properties orient themselves in away that limits contact with the water. Hydrophobic bonding, hence increases bacterial adhesive characteristics to membrane surfaces. There was indeed a higher proportion of isolates from floes and pressurised membrane that possess a high hydrophobicity compared with isolates from the planktonic culture collections. Bacterial aggregation may include co-aggregation, which is aggregation among genetically distinct bacteria, or auto-aggregation among the same bacterial species. Bacterial aggregation can contribute to biofilm development via recognition and binding of single cells in suspension to biofilm cells, or aggregation in suspension followed by the subsequent adhesion of this aggregate to the developing biofilm. Bacterial aggregation is frequently observed for isolates from AS studies. Efficient aggregation amongst flocculating bacteria helps them to remain in the rich nutrient environment of the AS, which is reported to be essential for proper settling of floes. Alternatively, aggregation has also been proposed to play structural roles in aerobic granulation, a suspended biofilm growth in which floes are converted into compact granules of micro-organisms. Interestingly, bacterial strains with low substrate degradation rate but high aggregation activity were previously found to be dominant in compact granules (54). None of the previous studies, however, investigated bacterial aggregation of isolates from membrane surfaces. Our study showed that there was no significant co- aggregation among isolates, but auto-aggregation was prevalent among membrane- bound and floe isolates. Low co-aggregation activity could be attributed to low phylogenetic diversity within the collections. A high number of isolates from the Microbacterium genus in the collections could mean that these bacteria were less likely to come into contact with other genetically distinct bacteria. This was supported by the observation that most of the auto-aggregating isolates belong to the Microbacterium genus. 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