The activated sludge process underlies the most popular systems used for treating both domestic and industrial sewage. These systems require a rapid separation of the biomass (organized into aggregates called flocs) from the liquid phase in clarifiers, a process that is rarely met in practice (Jenkins et al 2003). Most activated sludge systems suffer from the formation of very stable microbiological foams in their aeration tanks at times which many believe represents the last major unsolved problem encountered in the sewage treatment process (Soddell 1998). These foams cause major environmental, cosmetic, operational and health related problems, as they can contain serious opportunistic bacterial pathogens (Soddell 1998). It is difficult to estimate globally the annual cost to the industry caused by foaming, but it has been suggested to run into several billion dollars, bearing in mind that more than 100,000 activated sludge plants operate around the world (Jenkins, personnel communication).
Of great important to Australia is the environmental impact of foaming on the health and attractiveness of our rivers, streams and oceans. For example, the treatment plants operated by Melbourne Water and South East Water, the industrial partners in this application, frequently suffer from foams that ends up being discharge into the sea at Gunnamatta beach. These foams are more than an environmental nuisance, as they pose a health risk to the users of this area by acting as a potential vector for pathogen contact.
It is now clear that a diverse range of bacteria are responsible for episodes of foaming (Soddell 1998). Because these are so different phylogenetically and where known, in their ecophysiology (Eales et al 2005; Carr et al 2006), it appears unlikely that the foam problem can ever be controlled by a single engineering based strategy. In our view, the unequivocal identification of the foam causing microbes in each individual wastewater treatment plant community needs to be fully detailed first, and only then can an appropriate targeted control strategy be selected. This requirement is of particular importance in the control strategy we propose in this project.
This project seeks to investigate how the microbiological problem of foaming might be controlled by the use of bacteriophages to limit the growth of the problem organisms. This approach offers many advantages over the largely unsuccessful, engineering- based strategies currently available for controlling foaming. Foremost, is the specific targeted removal of the problem causing bacteria, while leaving unharmed the useful strains required for successful plant operation. In addition, it is a more environmentally friendly approach than the currently used strategy of adding non-specific toxic chemicals in an attempt to control the growth of the problematic foaming bacteria.
The aims of this project are:
A. Develop and apply DNA based microarrays for precise identification of the bacteria present in individual foam samples to allow targeted control strategies based on foam- specific communities.
B. Isolate lytic phages (in addition to those already isolated by our group) infective for the major filamentous bacteria identified by our group as being associated with foaming incidents in plants operated by South East Water and Melbourne Water.
C. Characterize these phages for their potential as biocontrol agents. This work will involve determining their host ranges using our unique collection of closely related host organisms, and developing sensitive methods to enable both the hosts and phages to be studied in situ.
D. Undertake laboratory scale reactor experiments using the isolated lytic phages to assess their role in controlling foaming. This work will involve determining the required phage dosages and composition against the targeted bacterial populations under a range of industrially relevant operational conditions.
E. Investigate and develop suitable methodologies for using phages to treat, or prevent, foaming problems in full-scale activated sludge plants.
Australia is a water limited country. A major problem we face is ensuring we will have an adequate supply of high quality water to meet our future needs. This is of particular concern given the increasing evidence of climate change and its likely impact on rainfall patterns. Communities will soon need to consider recycling treated waste water. This is an expensive option in which the final cost is largely dependent on its initial quality and safety of the treated water used. Foaming is a major operational disorder in activated sludge plants around the world (Soddell,1998). It is critical that sound strategies for preventing or curing this problem be found, especially as foaming incidents markedly reduce the quality and safety of treated effluents. While the highly innovative control strategy proposed in this grant is intended to be applied to plants operated by Melbourne Water and South East Water, it should be recognized that the approach has wider utility, in being applicable to other treatment plants around the world suffering from this problem. Furthermore, the strategy, if successful, is likely to be suitable for treating the related problem of bulking by filamentous microbes. Consequently, this research project fits very well into the research priority “Water - a critical resource”. If successful, not only will it improve the environmental health of our rivers and oceans, but it will also substantially reduce the cost of producing a treated wastewater suitable for other uses.
A number of different strategies for the control of sludge foaming have been attempted around the world (Jenkins et al 2003; Tandoi et al 2006). These strategies were developed empirically, with little or no understanding of the identity of the causative bacteria or their physiological properties. Consequently, they have proven unpredictable and difficult to apply in any general manner. In many cases gram-positive bacteria (the Mycolata) with cell walls containing complex long chain fatty acids called mycolic acids are responsible for the foaming problems. Control strategies tried have ranged from engineering approaches, such as redesigning the treatment plants to include an extra reactor or selector (hoping that the conditions established there will discourage growth of the Mycolata, but allow the rest to flourish), to the application of non-specific chemicals like chlorine (Jenkins et al 2003; Soddell, 1998; Wanner 2002). Other disinfectants, including ozone, have been used with some success on foaming problems caused by Gordonia amarae in Japan, but this technology is prohibitively expensive and its general applicability to other bacteria is not known (T. Mino, personal communication). The real risk with applying non-selective chemicals is that all the activated sludge bacterial populations are harmed, not just those responsible for the problem, and hence can cause disruption of the sludge treatment process from which plants 36 only recover slowly. Alternatives, such as identifying the dominant bacteria and then applying a set of empirical rules of thumb to try to eliminate a particular filament type (Jenkins et al 2003) are theoretically more attractive. Unfortunately, with the exceptions of the foaming organisms Microthrix parvicella and possibly Skermania piniformis (Soddell 1998), most strains are not identified adequately to allow such an approach. Furthermore, the factors that determine why a particular strain dominates a community are not understood to a level that it is possible to be confident that a particular strategy will work, making the outcome of plant operational manipulations almost impossible to predict. We believe that a completely novel approach to the foaming problem is required, one that can be targeted specifically at the problematic strains alone via the use of strain-specific phages. There is no reason to believe that the same approach could not be applied to the other major problem of bulking caused by filamentous bacteria in activated sludge. These microbes prevent adequate separation of the solids and liquids phase by forming flocs that then fail to settle in the clarifier, resulting again in biosolids leaving the plant in the effluent (Wanner 2002).
Bacteriophages are the most abundant and genetically diverse group on earth (Weinbauer 2004). They are typically found in very large numbers in all environments where their host bacteria exist, and play important roles in natural bacterial community dynamics and are responsible for 10% to 50% of the total bacterial mortality (Fuhrman & Schwalbach 2003; Weinbauer 2004). Furthermore, as phage infection rate is dependent on host cell population density, it has been hypothesized that phages play a strong role in maintaining microbial community diversity by preferentially killing the dominant strains – the “kill the winners” hypothesis (Thingstad & Lingnell 1997). Experiments involving the addition of phages specific to the dominant members of microbial communities have supported this hypothesis and strongly suggest that phages play an important role in nature in suppressing strain dominance and maintaining community diversity (Fuhrman & Schwalbach 2003; Suttle 1994).
This ability of phages to control microbial communities has been exploited as a means to treat bacterial infections in humans (Chanishvili et al 2001). Work in various regions of the world, including France, Russia, India, Egypt, United States and Canada in the pre-antibiotic days of the early 20th century demonstrated that phages could be used to successfully treat bacterial infections in humans (reviewed in Sulakvelidze and Kutter 2005). Increasing concern over antibiotic resistance in bacterial strains has renewed interest around the world in developing new phage therapies for the treatment of human and animal disease (Alisky et al 1998; Chanishvili et al 2001). Phage therapy has several advantages over chemotherapy, including specificity of attack and effectiveness in dealing with multi-antibiotic resistant strains (Barrow 2001). Others have proposed using phages to control food borne pathogenic bacteria, bacterial infections in plants and aquaculture, and even cyanobacterial blooms from eutrophied rivers (Modi et al 2001; Lawrence et al 2002; Thiel 2004). In the wastewater treatment process, Withey et al (2005) have enthusiastically supported exploring phages as a means for controlling pathogens and improving sludge digestibility.
Activated sludge systems are rich reservoirs for phages with densities exceeding 1010 per mL (Withey et al 2005). Prior work in our laboratory by Dr J. Thomas using a host enrichment approach led to the successful isolation and subsequent characterization of a range of phages infective for a number of the Mycolata associated with sludge foaming problems (Thomas et 37 al 2002; Thomas, 2005). Some of these phages were monovalent in attacking a single Mycolata single strain, while others were polyvalent and able to lyse strains from several different genera. This work confirms that lytic phages are present in activated sludge systems and can be isolated relatively easily. To date, lytic phages have been obtained for species of the foam-associated genera of Gordonia, Nocardia, Rhodococcus, Mycobacterium and Tsukamurella. The wide range of hosts for which phages have been isolated suggests there is no reason to believe that additional phages capable of infecting and lysing other Mycolata strains, as well as strains responsible for bulking problems, are not also present in these communities.
This project aims to investigate a revolutionary approach to tackling the problems of activated sludge foaming. Instead of trying to solve microbiological problems using poorly understood engineering approaches, or by applying toxic bactericidal chemicals, we intend to adopt a completely fresh strategy by exploiting a targeted biocontrol method using naturally occurring bacteriophages to specifically and safely eliminate the causative bacteria in an environmentally harmless manner. We believe our approach may provide, for the first time in the history of foaming problems, a practicable solution based on sound scientific principles. If successful, the impact on water treatment technology in Australia will be profound.
Conventional methods for ‘identifying’ problem bacteria in foams are based entirely on microscopic characteristics, especially their morphology and response to a few staining reactions (Jenkins et al 2003; Tandoi et al 2006). With the possible exception of the unbranched M. parvicella (Rosetti et al 2005), microscopic identification is inadequate, and only allows recognition of filament morphotypes (Seviour & Blackall 1998). For example, bacteria recognized microscopically by their right-angled branched filaments as belonging to the GALO (Gordonia amarae-like organisms) morphotype, are now known to contain members of several different bacterial genera. More recently, molecular methods have been employed to allow the in situ identification of strains and overcome these problems (Kampfer & Wagner, 2002; Martins et al 2004; Carr et al 2005). Our group at La Trobe University has been among the pioneers in this field and has been working on activated sludge foaming for about 15 years. We have designed targeted FISH probes for many of the causative filamentous bacteria (Seviour & Blackall 1998) which are now widely used around the world (Martins et al 2004). We have also isolated into pure culture many of bacteria associated with sludge foams, including several previously undescribed genera and species (Soddell 1998; Soddell et al 2006). This work has provided us with an invaluable and unrivalled collection of Mycolata pure cultures with corresponding FISH probes for application to this project (Eales & Seviour, unpublished).
In 2001, the Biotechnology Research Centre at La Trobe University was awarded an ARC Linkage Grant (LP0211598) in collaboration with South East Water to investigate the causes and control of foams in their treatment plants. This study involved surveying the microbial community compositions of these foams using both culture dependent and culture independent methods, including applying a range of FISH probes. The data obtained indicated that the major filaments present were strains of M. parvicella, Gordonia amarae and Dietzia maris, although not all plants had the same communities. However, one limitation with FISH is that only single populations can be probed for each analysis, and so the organisms identified earlier may only represent only the dominant populations there. Certainly the 16S rRNA profiles we obtained using denaturing gradient gel electrophoresis (DGGE) suggested a higher level of bacterial biodiversity in these communities, and filaments that were also seen during this study included a range of other non-G. amarae GALO (Eales et al unpublished). Work in a laboratory scale treatment system in this project found that daily dosing of the reactor with polyaluminium chloride (PAX) inhibited the growth of M. parvicella and prevented its foams. DGGE profiling of the treated and non- treated activated sludge communities suggested that PAX specifically targets M. parvicella, but had no effect on G. amarae (Eales et al, unpublished). This work suggests that while PAX dosing may prove an appropriate treatment for foams caused by M. parvicella, new methods are required for other members of the Mycolata.
For more than a decade, we have performed routine monthly microscopic examinations of both the foams and mixed liquor samples from the Easter Treatment Plant in Carrum, Victoria, run by Melbourne Water. Many of the bacteria observed in these plants have been cultured and their 16S rRNA genes sequenced. We now believe that the foams at Carrum are dominated by G. amarae and G. defluvii, with the majority of the bulking problems caused by the morphotype type 021N (Seviour, unpublished). However, for the same reasons given above, whether we have comprehensively characterized these communities is unlikely. Consequently, for the control strategy proposed in this application to succeed, a precise and unequivocal identification of the foaming populations in each plant is essential. This information, together with that acquired from our earlier work with both our industrial partners, provide us with a strong foundation for investigating alternative strategies for controlling the Mycolata foams seen in their plants.
This project seeks to isolate and characterize additional bacteriophages specific for each individual bacterial community associated with foaming and bulking problems in plants operated by Melbourne Water and South East Water. The intention is to develop methodologies, applicable to these and other treatment plants, which will control the growth of the foam-causing filamentous bacterial in a manner that is environmentally friendly, cost effective and non-disruptive to the other microorganisms in the systems. We see this project progressing in the following chronological sequence.
We have recently developed 16S rRNA based DNA microarrays for community fingerprinting of the foaming communities in treatment plants, allowing the simultaneous identification of more than 120 of the filamentous bacteria and their close relatives known to be associated with foaming and bulking in activated sludge systems. (Eales & Seviour unpublished). These microarrays have been manufactured and validated, and recently applied to several foaming communities from S.E Water plants, where earlier work had already identified their major populations. Early data suggest that these microarrays could confirm their presence, but also detected other populations not previously recognized there. Consequently these systems appear suitable for our stated purpose of community fingerprinting, and will thus be used routinely together initially with other microscopic methods.
Although we already possess several Mycolata specific phages from earlier work carried by our group (Thomas et al 2002; Thomas 2005), we will attempt to isolate additional bacteriophages capable of lysing other Mycolata strains. This work will concentrate on those bacterial strains that we know occur in the biomass and foams of the plants operated by Melbourne Water and South East Water, as indicated from the microarray studies. Water samples from theses plants, as well as other wastewater plants, will be screen for Mycolata phages using a modification of the standard phage plaque assay developed in our laboratory (Thomas 2005).
The isolation, propagation and study of bacteriophages in the laboratory ideally requires pure cultures of the host bacterial cells. We probably have had more success in culturing the activated sludge filamentous bacteria (especially members of the Mycolata) than any other group and hold a wide selection of taxonomically diverse strains in our culture collection, including many of the GALO (Soddell 1998). However, it is also the aim of this project to expand our search and propagate other filamentous bacteria commonly associated with foaming and bulking incidents, including Nostocoida limicola II and several of the Eikelboom filament morphotypes, and hopefully M. parvicella (a notoriously difficult microorganism to cultivate). We then aim to isolate phages that lyse these organisms. In particular, we wish to isolate phages specific for pathogenic strains previously recovered from sludge foams (eg Nocardia asteroides, Rhodococcus equi and Mycobacterium spp) (Soddell 1998), as these bacteria present significant potential hazards to human health.
The host range of the phages isolated in Stage 2 will be determined using both culture dependent and culture independent methods with the aim of identifying phages, or collections of phages, that can lyses the majority of the foam causing Mycolata strains. Individual isolated phages will be screened using our modified standard plaque assays against our extensive collection of cultured Mycolata strains to determine their host specificity. Further in situ studies will be undertaken using a combination of FISH and fluorescently labeled phage particles (Hennes et al 1995). This culture-independent approach will allow phage specificity to be studied in both cultured and uncultured strains, as well as in the microbiologically complex foam environment. Such an approach will ensure that the phages isolated will be able to lyse the majority of Mycolata strains contributing to the foaming problem.
We propose to characterize the phages isolated in Stage 2 using electron microscopy and modern molecular techniques. Techniques to be used include Restriction Fragment Length Polymorphic DNA fingerprinting (RFLP) genome typing, DNA-DNA or RNA-RNA cross hybridisation, and partial and complete phage genome sequencing (Jardillier et al 2005; Paul & Sullivan 2005). This work is critical to ensure that we isolate a genetically diverse range of phages so as to minimizing the likelihood of phage resistance occurring.
Identification of Mycolata strains in activated sludge systems is currently far from straightforward and requires highly skilled personnel and expensive equipment (Soddell 1998). We propose to develop a diagnostic system using fluorescent Mycolata phages that will allow the unambiguous identification of problematic microbes in the complex activated sludge environments. This project will involve identifying a set of Mycolata phages from those isolated in Stages 2 and 3 that can be labeled with different fluorophores and used to 40 “fingerprint” the Mycolata bacterial community in situ. This approach promises a technically simple method for strain identification that will be suitable for use in industry with relatively simple equipment and semi-skilled staff.
Once a range of suitable Mycolata phages has been identified, we propose to develop methods for pilot-scale production. This work will involve identifying the idea host strain for each phage and the most suitable conditions for growing the host in 10 L+ volumes. The ideal parameters for phage production, such a host growth conditions and density, and the timing of phage addition, will be investigated, along with methods for efficient harvesting of the phage particles. Identification of suitable long-term storage conditions will also be undertaken. The methods developed will be performed with later larger production scale needs in mind.
Our group has extensive experience running laboratory scale activate sludge systems that will enable us to examine the effects of adding Mycolata phages on foaming under conditions that simulate full-scale waste treatment plants. Such systems will allow us to determine if phages are able to control foaming and what application processes and doses are required. This technology will then be applied at selected full-scale plants of each partner.
This project forms an ideal project for the training of two PhD students in an area of critical importance to Australia’s future. It will provide considerable intellectual challenges involving a combination of basic and applied research directed towards solving an important industrial and environmental problem. Furthermore, it will provide an opportunity for young scientists to work in an active research environment under the supervision of international recognized experts in the field who have a substantial track record of successful research training. Upon graduation, the PhD students will be knowledgeable in a wide range of innovative molecular biology techniques directed at tackling practical problems. These skills will be in great demand in a water-limited country like Australia. Water reuse and recycling is of increasing importance and both PhD students will benefit from having practical experience in this area and will be well placed to contribute significantly to improving how we use our scarce water resources.
The Biotechnology Research Centre (BRC) at La Trobe University has long standing working relationships with both industrial partners. Our laboratory has been carrying out consultancy work and has undertaken several small research projects to identify the filamentous bacteria associated with foaming in Melbourne Water treatment plant over the past decade. S.E. Water was an industrial partner on an earlier ARC Linkage grant with the BRC attempting to understand better the microbiology of foaming and exploring possible new methods for foaming control. Regular scientific meetings and seminar presentations have taken place between us, particularly over the past 3 years, to collectively consider how best to tackle this foaming problem, because it is of increasing concern to both. Because their foaming communities appear to differ in their bacterial composition, we decided jointly that working in collaboration on the approach outlined here was the best way forward. This application is the outcome of these prolonged discussions. Curing foaming would improve the performance of their treatment plants in a profound way and help enable them to produce Class A water from their treatment plants. We believe that grounds for optimism for our continued collaboration on this and other projects are well founded, when the history of our prolonged professional interaction over more than a decade is considered.
Their staff have regularly attended many of the short professional courses we run for wastewater treatment professionals dealing with these operational problems. In a sense, the project represents a logical extension and expansion of our earlier productive interactive work. Both partners and their wastewater treatment staff have long record of accomplishment and commitment to collaborative ARC Linkage grants and in undertaking scientific research into activated sludge systems. This has helped develop in both partner a culture where the value of research is recognized. The increasingly stringent discharge license requirements demanded by government bodies for producing Class A water, has given solving problems like foaming (which markedly reduces the quality of the treated effluent) a very high priority. Their strong commitment to support this work is indicated by their joint cash and in kind contribution of $198,000 over the three years of the project.
Most wastewater treatment plants in Australia are activated sludge processes and most suffer from episodes of foaming. The consequences of this on water quality and human health is severe. Not only are the aesthetics of the receiving bodies of water affected, but also the discharged solids represent a major pollution problem, including the possible spread of pathogenic bacteria to areas of public use like rivers and beaches. If foaming (and bulking) could be prevented or controlled, with the potential pathogenic bacteria removed, then our environment would be cleaner and safer. The contamination at Boag’s Rocks by outfalls from the plants operated by Melbourne Water and South East Water is of particular topical relevance, but it is not the only location in Australia where foaming is of concern. This project will provide direct benefits to regional Australia. Many wastewater treatment plants and associated receiving bodies of water including ocean outfalls are often located in regional zones adjacent to metropolitan centres. Melbourne is an excellent example as both industrial partners operate plants that discharge their treated sewage into regional areas, which then suffer the environmental and health consequences of foaming incidents. It should be noted that the majority of the experimental work will be carried out at La Trobe University’s, Bendigo campus. This project will contribute directly to the conduct of high quality research at a key regional university campus and support an ongoing research culture. Such projects are essential if regional research capabilities and infrastructure are to be maintained and developed. Equally, this project offers high quality students from the regional areas excellent opportunities for first class postgraduate research training rather than having to leave (usually permanently) for the capital cities.
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