INVESTIGATING BIOCHEMICAL INDICATORS OF TREATMENT EFFICIENCY IN WASTE STABILISATION PONDS THROUGH THE USE OF FLOW CYTOMETRY

Transcription

1 INVESTIGATING BIOCHEMICAL INDICATORS OF TREATMENT EFFICIENCY IN WASTE STABILISATION PONDS THROUGH THE USE OF FLOW CYTOMETRY Amy Hinchliffe Supervisor: Prof. Anas Ghadouani School of Environmental Systems Engineering Faculty of Engineering, Computing and Mathematics The University of Western Australia

2 This page has intentionally been left blank Page 2

3 Abstract Amy Hinchliffe Waste stabilisation ponds (WSPs) offer an extremely sustainable wastewater treatment option. As such, it is critical that a comprehensive understanding of these systems is developed. Due to a lack of fast and accurate techniques for assessing wastewater, the microbiology of these complex systems is poorly understood. However, recent advances in molecular technologies are providing alternative tools for analysing wastewater ecosystems. One tool that has tremendous potential in the field of wastewater is Flow Cytometry (FCM). This project investigates the potential for FCM to identify microbial community characteristics of WSPs and attemptes to establish links between treatment plant efficiency and microbiology. This study engages the use of FCM to identify communities of auto-fluorescent cells present in wastewater samples from five WSPs located in the Great Southern Region of Western Australia. Samples were analysed on a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA), the instrument was equipped with 3 lasers emitting at 405nm, 488nm and 633nm. Fluoresence characteristics were analysed on dot plots, with logarithmic scales showing different emissions detected by various filters. A Fluoroprobe was used in combination with FCM to give an indication of the types of species that were likely to be present. Additionally, water quality sampling was undertaken to provide information on the different wastewater environments and how well the ponds were functioning. Results from FCM analysis showed that significant differences in microbial community characteristics exist between various WSPs. FCM results also showed that the FluoroProbe can be used to give an indication of species abundance and diversity, however it could not be used to determine specific species. To assist with identification of specific species, FCM should be integrated with other detection methods including plating, imaging, cell sorting and DNA sequencing. Furthermore, relationships between FCM results and treatment plant efficiency were found to be inconclusive. Further monitoring with FCM in conjunction with water quality monitoring is necessary before biochemical indicators of treatment efficiency can be established. Overall, this study has shown that FCM has a promising future as a rapid monitoring tool for wastewater treatment systems. The monitoring of WSPs with FCM could potentially provide a valuable tool for tracking changes in microbial community characteristics. This will not only develop concepts fundamental to wastewater treatment ecology, but will also assist with understanding the potential that FCM has in the field of wastewater. Page 3

4 Acknowledgements Amy Hinchliffe I would like to thank the following people for their support throughout this project; My supervisor, Anas Ghadouani for his guidance and continuous support with the project. I am especially grateful your kind encouragement and reassurance through the more stressful times. The Water Corporation for their support and funding of this project. In particular I would like to thank the Water Corporation group from Great Southern Region for their cooperation, Ken Eade for his enthusiasm and the plant operators for their patience and assistance during on site sampling. Tracey Lee-Pullen at CMCA for her help with flow cytometry. I am appreciative of the enthusiasm she showed for the project and the time that she gave to assist me with the use of the flow cytometer. Elke Reichwaldt for all her help and direction she gave me. The fieldwork would not have been possible without your organisation and assistance. Liah Coggins for the time she devoted to answering my many questions and her willingness to assist me in all stages of this project. Dani Barrington, George Gaylard and Isabelle Laurion for their assistance with fieldwork. And finally, a special thank you to my family and friends, both within and outside of SESE, for their everlasting support and understanding. Page 4

5 Table of Contents Amy Hinchliffe Abstract... 3 Acknowledgements... 4 List of Figures... 8 List of Tables Abbreviations INTRODUCTION LITERATURE REVIEW WASTE STABILISATION PONDS Biological wastewater treatment objectives Treatment processes Treatment process performance Need for further investigation FLOW CYTOMETRY Principles of FCM Flow cytometric analysis Application to wastewater AIMS AND OUTCOMES APPROACH AND METHODOLOGY STUDY SITES PILOT VISIT WATER SAMPLING ENVIRONMENTAL PARAMETERS FLUOROPROBE FLOW CYTOMETRY Sample preparation Calibration Evaluation Page 5

6 4.6.4 Data analysis WATER QUALITY RESULTS FLUOROPROBE FLOW CYTOMETRY Identification Quantification ENVIRONMENTAL PARAMETERS WATER QUALITY DISCUSSION FLUOROPROBE FLOW CYTOMETRY Identification Quantification ENVIRONMENTAL PARAMETRES WATER QUALITY Tambellup water quality Cranbrook water quality Other significant water quality results CONCLUSIONS RECOMMENDATIONS IDENTIFICATION QUANTIFICATION VIABILITY FUTURE OF FCM IN THE FIELD OF WASTEWATER REFERENCES APPENDICES Appendix 1: FluoroProbe Data Page 6

7 Appendix 2: Environmental Parameters Appendix 3: Water Quality Data Page 7

8 List of Figures Amy Hinchliffe Figure 1 Symbiosis between algae and bacteria in WSP environment (The Water Treatments n.d.) Figure 2 Schematic representation of WSP processes (Metcalf and Eddy 1991) Figure 3 A schemcatic rendition of Flow Cytometry displaying hydrodynamic focussing of cells and excitation of cells with a laser (MacManus 2010) Figure 4 Map showing locations of the sample sites at the Tambellup WSP. Where green arrows represent the direction of water flow between the ponds and red dots indicate the sample location Figure 5 Photograph illustrating algae chains. Wag-P2-S2 (Right) showing visible algae flocculation, Wag-P1-S2 showing some flocculation and Wag-P1-S1 (Left) showing little flocculation Figure 6 Line graphs illustrating decreasing total chlorophyll-a concentrations with increasing treatment level at different WSPs; Mount Barker (top left), Cranbrook (top right), Boddington (bottom left), Wagin (bottom right). Note that FluoroProbe did not record data correctly for Bdt-P2-S1 and Bdt-P2-S2, therefore results are missing Figure 7 Graphs illustrating increasing total chlorophyll-a concentrations with increasing treatment level at Tambellup WSP Figure 8 Flow cytometric dot plot of Bdt-P1-S1 (sonicated) after gating strategies were applied. Figure displays the fluorescence characteristics of the sample on three logarithmic scales (units: fluorescence/cell). The axes show excitation with different lasers and emissions detected by various filters. Two distinct populations are highlighted in blue (population 1) and orange (population 2) Figure 9 Flow cytometric dot plot of Wag-P1-S1 (sonicated) after gating strategies were applied. Figure displays the fluorescence characteristics of the sample on three logarithmic scales (units: fluorescence/cell). The axes show excitation with different lasers and emissions detected by various filters. Two distinct populations are highlighted in blue (population 1) and orange (population 2) Figure 10 Flow cytometric dot plot of Tam-P2-S1 (sonicated) after gating strategies were applied. Figure displays the fluorescence characteristics of the sample on three logarithmic scales (units: fluorescence/cell). The axes show excitation with different lasers and emissions detected by various filters. A distinct population is highlighted in blue (population 1) Page 8

9 Figure 11 Flow cytometric dot plot of Tam-P2-S2 (sonicated) after gating strategies were applied. Figure displays the fluorescence characteristics of the sample on three logarithmic scales (units: fluorescence/cell). The axes show excitation with different lasers and emissions detected by various filters. A distinct population is highlighted in blue (population 1) Figure 12 Flow cytometric dot plot of Wag-P4-S1 (sonicated) after gating strategies were applied. Figure displays the fluorescence characteristics of the sample on three logarithmic scales (units: fluorescence/cell). The axes show excitation with different lasers and emissions detected by various filters. Three distinct populations are highlighted in blue (population 1), organge (population 2), green (population 3) Figure 13 Flow cytometric pseudo-colour density plot of Bdt-P1-S1, Forward Scatter-Area Vs Forward Scatter-Height i) Sonicated sample ii) Unsonicated sample Figure 14 Flow cytometric dot plot of Bdt-P5-S2 after gating strategies were applied i) Sonicated ii) Unsonicated. Figure displays the fluorescence characteristics of the sample on two logarithmic scales (units: fluorescence/cell). The axes show excitation with different lasers and emissions detected by various filters. Two distinct populations are highlighted in blue (population 1) and orange (population 2) Figure 15 Flow cytometric dot plot of MtB-P3-S1 after gating strategies were applied i) Sonicated ii) Unsonicated. Figure displays the fluorescence characteristics of the sample on two logarithmic scales (units: fluorescence/cell). The axes show excitation with different lasers and emissions detected by various filters. Two distinct populations are highlighted in blue (population 1) and orange (population 2) Figure 16 Flow cytometric dot plot of Tam-P1-S2 after gating strategies were applied i) Sonicated ii) Unsonicated. Figure displays the fluorescence characteristics of the sample on two logarithmic scales (units: fluorescence/cell). The axes show excitation with different lasers and emissions detected by various filters. Two distinct populations are highlighted in blue (population 1) and orange (population 2) Figure 17 Flow cytometric pseudo-colour density plot of Wag-P1-S2. Figure displays the fluorescence characteristics of the sample on two logarithmic scales (units: fluorescence/cell). The axes show excitation with different lasers and emissions detected by various filters Figure 18 Flow cytometric pseudo-colour density plot of Bdt-P6-S2. Figure displays the fluorescence characteristics of the sample on two logarithmic scales (units: Page 9

10 fluorescence/cell). The axes show excitation with different lasers and emissions detected by various filters Figure 19 Flow cytometric pseudo-colour density plot of i) pure Microcystis aeruginosa culture ii) Tam-P3-S1 iii) Tam-P3-S2. Figure displays the fluorescence characteristics of the samples on logarithmic scales (units: fluorescence/cell). The x-axis: excitation with 405nm laser, blue fluorescence emission, y-axis: excitation with 488nm laser, far-red fluorescence emission Figure 20 Flow cytometric pseudo-colour density plot of of i) pure Microcystis aeruginosa culture ii) Tam-P3-S1 iii) Tam-P3-S2. Figure displays the fluorescence characteristics of the samples on logarithmic scales (units: fluorescence/cell). The x-axis: excitation with 488nm laser, far-red fluorescence emission, y-axis: excitation with 633nm laser, far-red fluorescence emission List of Tables Table 1 Summary of design details for Boddington, Wagin, Tambellup, Cranbrook and Mount Barker Table 2 Details for sample codes Table 3 Details of the lasers detectors and filters that the BD FACSCanto II flow cytometer is equipped with. PMT (photomultiplier tube) is the type of detector (CMCA 2012) Table 4 Guidelines for effluent quality following various levels of treatment (NWQMS 1997) Table 5 Table detailing significant FluoroProbe data as concentration (µg chlorophyll-a/l) (top) and percentage of total concentration (bottom). Includes samples that displayed the highest and lowest total concentration, the highest and lowest percentage of green algae, Table 6 Water quality data for Tambellup Table 7 Table detailing the variation in water quality data between new primary ponds and old primary ponds Page 10

11 Abbreviations BOD Biochemical Oxygen Demand DO Dissolved Oxygen E.coli Escherichia Coli FCM Flow Cytometry HPC Heterotrophic Plate Counts NWQMS National Water Quality Management Strategy ORP Oxidation Reduction Potential SS Suspended Solids TN Total Nitrogen TP Total Phosphorous WSP Waste Stabilisation Pond WWTP Waste Water Treatment Plant Page 11

12 1. INTRODUCTION Amy Hinchliffe Australia s rapidly expanding population and increasing urbanisation is placing more pressure on water resources than ever before. One of the greatest challenges we face as a nation is sustainability of urban water management (Kershaw 2012). As a result there is an increasing importance for recycled water supplies, and a greater need to develop more environmentally responsible and efficient wastewater treatment technologies. Waste stabilisation ponds (WSPs) provide an entirely natural, highly adaptable and low maintenance method for wastewater treatment that is economically viable for both developed and developing countries (Mara 2004). It is believed that almost all wastewater containing biodegradable constituents can be treated successfully using only biological treatment methods if proper analysis and environmental controls are employed (Metcalf and Eddy 2003). WSPs offer an extremely sustainable alternative to wastewater treatment, and thus there is a critical need to better understand these complex systems. Through studying the internal system of WSPs, concepts fundamental to biological treatment processes can be advanced, ultimately leading to the development of more efficient WSPs. Moreover, by progressing the efficiency, reliability and safety of recycled water, the options for it s reuse will multiply. The microbiology of WSPs is of great interest, particularly in terms of microbiological community structure and the technical significance in treatment efficiency (Loy and Wagner 2002). Despite the enormous technical significance of microbial communities in WSPs, they are poorly understood (Daims et al. 2006). The high complexity of WSPs has meant that finding methods and techniques to explore their microbiology has been challenging. A conventional technique most commonly used to assess the microbiology of aquatic systems is heterotrophic plate counts (HPC). However, this technique is slow and does not accurately represent the total bacterial cells in aquatic environments (Berney et al. 2007). Recent developments of advanced molecular tools and technologies are now providing the opportunity for faster, more extensive and accurate analysis of wastewater. One of these techniques is Flow Cytometry (FCM). FCM provides a fast, accurate and quantitative method for analysing the microbiology of aquatic environments (Berney et al. 2007). There are a number of papers that discuss the application of FCM to wastewater however few have actually carried out field trials. Egli and Hammes (2010) suggest that there is tremendous potential for FCM to replace conventional HPC and provide an alternative tool for the analysis of wastewater. Page 12

13 This project investigates the potential for FCM to be used in the field of wastewater. More specifically, FCM will be employed to explore differences in microbiological community characteristics between five different WSPs. It is also intended that biochemical indicators, which are prevalent in efficient WSPs, will be identified through the use of FCM. Page 13

14 2. LITERATURE REVIEW 2.1 WASTE STABILISATION PONDS The term waste stabilisation pond (WSP) is used to describe any pond or pond system that is designed to treat wastewater using natural methods. Like all wastewater treatment plants (WWTPs), the aim of WSPs is to reduce the environmental impacts and health risks that are associated with untreated wastewater. However, unlike advanced WWTPs, WSPs provide an entirely natural, highly adaptable and low maintenance method of wastewater treatment (Mara 2004). They provide an economical solution for wastewater treatment that is suitable for locations where there is a shortage of trained operators or funding. WSPs are now regarded as the preferred wastewater treatment method in many parts of the world. In some areas of Latin America, Africa and Asia, WSPs are being used for wastewater treatment of large populations, while in Australia, WSPs are being used for smaller rural communities (Ramadan and Ponce n.d.) Biological wastewater treatment objectives WSPs are widely used to treat a range of domestic and industrial wastewater containing a range of characteristics, including, containing nutrients (mainly nitrogen and phosphorous), faecal microbes (which includes viruses, bacteria and protozoans) and both organic and inorganic chemicals. Treatment requirements differ depending on how the effluent is being reused or discharged. The overall objectives of biological treatment of wastewater as defined by Metcalf and Eddy (2004) are to; 1) Transform, dissolve and particulate biodegradable constituents into acceptable end products; 2) Capture suspended and unsettled colloidal soils into a biological floc or sludge; 3) Transform or remove nutrients and in some cases remove specific recalcitrant or toxic organic compounds Treatment processes WSPs use a combination of biological, chemical and physical processes to remove unwanted wastewater constituents. WSPs typically consist of shallow man-made ponds operating singly or with several ponds either in series or parallel, depending on the treatment objectives. A series design is typically used when a higher level of treatment is required, while a parallel system is used when operational flexibility is desirable (Gloyna 1971). Generally, treatment Page 14

15 begins in a primary facultative pond which then feeds into one or more polishing ponds. Microbiology plays a central role in wastewater treatment with WSPs Facultative pond A facultative lagoon is the term commonly given to the first pond that treats raw wastewater. Facultative WSPs are typically 1m-2.5m deep and vary in size depending on the loading rates and required hydraulic retention times (Gloyna 1971). The facultative pond functions to remove solids and organic content contained within the raw effluent. The effect of gravity settles heavy solids to the bottom of the ponds, forming a layer of sludge on the pond floor, while lighter solids remain suspended in the water column. Aerobic conditions of surface waters are maintained by wind action and photosynthetic processes of algae. A symbiotic relationship between algae and bacteria exists in WSPs, as shown in Figure 1. Figure 1 Symbiosis between algae and bacteria in WSP environment (The Water Treatments n.d.). Oxygen produced by photosynthetic algae is utilised by bacteria to breakdown organic matter. By-products of bacterial metabolism, including carbon dioxide, provide essential vitamins for the growth of algae. In addition to driving bacterial oxidation of organic materials, algae have also been found to assist in the removal of heavy metals, nutrients and pathogens (Gloyna 1971). Algal concentrations are typically between µg chl-a/litre depending on nutrient loading, sunlight and temperature (Mara 1987). Dissolved oxygen (DO) Page 15

16 concentration in the ponds varies diurnally but generally, concentrations are lowest during the night when light is unavailable for photosynthesis. DO concentrations gradually rise after sunrise and reach a maximum level in the afternoon (Katima 2005). Furthermore, production of oxygen occurs to the depth at which light can penetrate and provide necessary sunlight for photosynthesis (Ramadan and Ponce n.d.). Beyond this depth, light deficiencies create anaerobic environments near the pond floor. This supports the existence of reducing organisms that assist in the removal of sludge through digestion. Intermediate depths of the pond support facultative micro-organisms that are capable of oxidising both the dissolved and suspended organics from the raw wastewater and the products of anaerobic reactions at the bottom of the pond (Gloyna 1971). These processes are depicted in Figure 2 below. Figure 1.1. Schematic representation of daytime WSP operation (Metcalf and Eddy, 1991). Figure 2 Schematic representation of WSP processes (Metcalf and Eddy 1991) Polishing ponds Polishing ponds, also known as maturation or aerobic ponds, sit in series with facultative ponds and are used for final polishing of wastewater. They are usually slightly shallower Page 16

17 than primary ponds (1m-1.5m) and facilitate higher populations of aerobic organisms. Polishing ponds provide a higher level of treatment of wastewater than facultative ponds alone and are primarily concerned with removing nutrients and pathogens. The reduced eutrophic state of polishing ponds supports highly diverse populations of plankton. Algae and zooplankton can often develop at high densities in the final stages of wastewater treatment (Pearson et al. 1987, Pearson 1990). In some cases, a final pond maybe used as a storage pond. In water that still contains high nutrient levels, the stagnant water conditions of storage ponds can provide ideal conditions for cyanobacteria blooms. At small concentrations cyanobacteria are important components of WSPs (Martins et al. 2011). However, in large concentrations, the occurrence of cyanobacteria presents a risk to the health of the water bodies receiving effluent. Martin et al (2011) discusses cyanobacteria ecology in wastewater treatment plants and reveals that, while the complex interactions between cyanobacteria and other microbial communities are not fully understood, it has been found that cyanobacteria can disrupt microorganisms essential to biological treatment processes, consequently affecting treatment efficiency Treatment process performance Biological, physical and chemical processes are essentially controlled by the retention time and the environmental conditions of the stabilisation pond, including temperature, ph and the degree of mixing influenced by wind (Schultz 2005) Retention time The retention time is the time it takes for wastewater to pass through the treatment system. Any pond system requires a steady inflow and outflow rate to maintain the intended retention time and conserve treatment processes. If the influent load is too large, important microorganisms may be flushed out of the system (Ramadan and Ponce n.d.). Facultative ponds can have a wide range of hydraulic retention times ranging from a few days to a few weeks. Generally, longer retention times lead to a higher quality of treatment, although if the water is held for too long it can have detrimental effects Environmental conditions Environmental factors such as temperature, ph and wind can limit survival and growth of organisms and therefore influence treatment efficiency. Optimal growth generally occurs within a narrow range of temperature and ph values unique to different types of bacteria and other microorganisms (Metcalf and Eddy 2003). Page 17

18 Due to the shallow design of the waste stabilisation ponds the temperature can vary significantly annually and diurnally, easily affecting the biological processes within the ponds. Optimum temperature ranges depend on the classification bacteria, with some bacteria and algae strains able to survive temperatures as high as while other species can survive in cooler conditions below (Metcalf and Eddy 2003). Warmer temperatures are typically associated with higher reaction rates and thus more efficient treatment. Typically water temperatures close to, or around provide the most ideal conditions for treatment. ph is also a key factor in the growth rate of microorganisms. Katima et al. (2005) found that optimal ph for heterotrophic bacteria and algae growth occurs between 6 and 8. However, peak algae activity and oxygen production is also often associated with ph values as high as 9. These environments can kill bacteria that are less tolerant of basic conditions, including faecal coliforms. Wind is an important factor in maintaining environmental conditions. The degree of mixing caused by wind action is important for distributing oxygen around the pond system. Additionally, wind conditions are important for transporting algae into the zone of effective light penetration (Gloyna 1971). Changes in the degree of mixing caused by annual and diurnal wind changes may lead to a reduction in the quantity of algae and a shift from one dominant species of algae to another Need for further investigation Biological wastewater treatment has been applied for more than a century. However, there is still little known about the microbiology that inhabits these ecosystems (Daims et al. 2002; Correia et al. 2007). The high complexity of WSPs has meant that finding methods and techniques to explore the microbiology has been challenging. A conventional technique commonly used to assess the microbiology of aquatic systems is heterotrophic plate counts (HPCs), an approach that involves isolating pure cultures of microorganisms. The cultures are then characterised using microscopy and by observing their interactions with other substances (Metcalf and Eddy 2003). This technique is slow and since not all species can be isolated, does not accurately represent the total bacterial cells in aquatic environments (Berney et al. 2007). Recent developments of advanced molecular tools and technologies are now providing the opportunity for faster, more extensive and accurate analysis of wastewater (Daims et al. Page 18

19 2006). New insights include a greater understanding of the presence and role of specific bacteria species (Metcalf and Eddy 2003). Some new, alternative methods for the identification and classification of microorganisms take advantage of unique nucleic acid sequences that exist in the DNA of cells. Restrictive fragment length polymorphism is a technique that extracts DNA from cells in mixed microbial communities and determines a genetic fingerprint of the community (Metcalf and Eddy 2003). Other emerging analysis techniques involve using fluorescence and fluorescence staining of cells to acquire information about microbial communities, including fluorescence in situ hybridisation (FISH) and flow cytometry (FCM). A number of papers have demonstrated the use of FISH with application to wastewater to identify bacterial species (Bjornsson et al. 2002; Liu et al. 2001; Wagner et al. 1993). With regard to FCM, the application to aquatic microbiology has been discussed by a number of papers, including Caron et al. (2000), Boon et al. (2010) and Egli and Hammes (2010); however there are few reports that actually concern the study of algal and bacterial communities in wastewater using FCM. The technical significance of microbiology in biological wastewater treatment is enormous and despite recent developments of more advanced molecular tools, there is still much to learn about in terms of the ecology of WSPs. In order to understand the effects of microbiology in treatment efficiency it is necessary to unravel complex microbial community behaviours by applying new molecular methods to wastewater. This study deals specifically with FCM and the application of this technique to the field of wastewater 2.2 FLOW CYTOMETRY FCM has been used in the medical field since the 1960s when it was introduced for the analysis of mammalian cells (Boon et al. 2010). Since then, the capabilities of FCM has greatly improved, opening the door for FCM to be used in a wide range of applications, including aquatic microbiology. FCM has only recently been introduced to study aquatic ecology but according to Boon et al. (2010), it has significant potential and scientists are only beginning to discover these possibilities. FCM provides fast, accurate and quantitative microbial analysis (Berney et al. 2007). It is a robust technique which is adaptable to microbial cells from different types of environments irrespective of their cultivability. Page 19

20 2.2.1 Principles of FCM Particles are suspended in a stream of fluid that passes through an excitation light source, usually a laser. A key feature of FCM is that cells are presented to the laser individually, allowing analysis to be made on a single-cell level (Egli and Hammes 2010). The interaction between the light beam and the particle causes excitation. As the cells leave their excited state, energy is released as photons of light. A number of detectors are aligned with the light source to collect the scattered light and emitted photons, or fluorescent light (Figure 3). The photons are then directed into specific channels using filters. There are three types of filters; long pass filters, which transmit all wavelengths greater than the specified wavelength; short pass filters, which transmit all wavelengths less than the specified wavelength; and band pass filters, which transmit a specific band of wavelength. Based on specific fluorescence and scatter characteristics information about the cells can be derived. Figure 3 A schemcatic rendition of Flow Cytometry displaying hydrodynamic focussing of cells and excitation of cells with a laser (MacManus 2010). A number of microbes in aquatic environments exhibit auto-fluorescence characteristics due to the presence of photo-pigments. One of the most common photo-pigments found in aquatic samples is chlorophyll. This pigment is critical for photosynthesis and can be found in algae and cyanobacteria species. A number of marine biology studies have applied FCM to explore auto-fluorescent properties of plankton and have successfully derived information about community structure and abundance (Gasol and Giorgio 2000). For detection of non-auto Page 20

21 fluorescent species, FCM is usually combined with fluorescent cell staining methods. A number of fluorescent labels, or fluorophores, are available, some of which bind exclusively to certain cell features, thereby allowing specific cellular communities to be detected (Egli and Hammes 2010) Flow cytometric analysis Flow cytometric analysis can be performed at different flow rates and settings. Typical flow rates are between μl/min and a detection of up to 10,000 events/sec is possible (Egli and Hammes 2010). A number of software programs are available to assist with FCM data analysis. Amongst them are FlowJo, FCS Express, CytoPaint, CellQuest Pro, and Cytospec. These programs allow the operator to focus on specific areas by drawing shapes around regions of interest; this is commonly referred to as gating (Egli and Hammes 2010) Application to wastewater One specific application within the field of aquatic microbiology that is using FCM is microbial monitoring in drinking water and wastewater systems. Berney et al. (2007) has used FCM to assess microbial parameters in drinking water and suggests that there is tremendous potential for FCM to replace conventional analysis techniques, like HPCs, and provide an alternative tool for wastewater analysis. A great deal of information can be gained about microbial cells and microbial communities present in wastewater from FCM analysis, including total cell counts, cell size, viability analysis and detection of specific species Total cell counts Total cell counts are one of the most straightforward and useful applications of FCM. Upon sampling a total cell count can be immediately attained. A number of papers have investigated the application of FCM total cell counting in drinking water including Berney et al. (2007) and Andrews et al. (2003). The study performed by Berney et al. (2007) investigated FCM total bacterial cell counts as a descriptive microbiological parameter for drinking water, while the research conducted by Andrews et al. (2003) looked at enumeration of water-borne bacteria using viability assays and FCM. The studies have demonstrated that the total cell number can provide an indication of the changes in water quality during treatment, with total cell concentration tending to be greatest in raw wastewater and smallest in treated effluent. These papers also discuss the need for further research. Page 21

22 Challenges of total cell counts include separating cells of interest from background signals (of the machine and samples) and ensuring cells are being analysed in single cell suspension. The latter is particularly a concern for aquatic samples that contain algal communities that may have a tendency to agglomerate into chains, or flocs Cell size Forward scatter is assumed to be proportional to cell size and can therefore be used to estimate size differences amongst bacterial populations. It should be emphasized however that it only provides a rough indication of particle size. Other factors that affect the scattering of light include cell structure, chemical composition, refractive index and angle of detection (Caron 2000) Viability FCM also allows for viability analysis of cells when combined with the correct viability staining methods. There exist a number of dyes that can be used to test whether cells are dead, alive or in some in-between state (e.g damaged but still alive). Propium iodide is commonly used to test membrane integrity, while ethidium bromide is used to test cells efflux pump functioning. Other dyes test for membrane potential, esterase activity and bacterial respiration (Egli 2010). Stains work very differently for different organisms and environments, therefore it is important to base stain selection on the intended purpose of the viability test. In some studies, fixation is a pre-analysis requirement. Fixation of samples may be needed to preserve samples that cannot be processed immediately after sampling, or to ensure that samples do not pose a health risk to the laboratory environment they are analysed in. Fixation protocols should effectively preserve nucleic acids and protect the auto-fluorescence properties of cells (Gasol and Giorgio 2000). Three common protocols that are used in microbial flow cytometry are glutaraldehyde, formaldehyde and formalin. There substances are typically used at 1-2% final concentration. There are a number of disadvantages to fixation. The main disadvantage of sample fixing is that viability based stains cannot be applied to fixed cells. Other problems include alteration of scatter and fluorescence properties. To minimise the drawbacks of fixation, cells should be rapidly frozen, preferably in liquid nitrogen (Caron 2000; Gasol and Giorgio 2000) Detection of specific species Another use of FCM is for the detection of specific microbial groups or microbial species. FCM can be used to detect specific groups of bacteria using a number of methods. These Page 22

23 methods can be used independently of one another or combined for more comprehensive analysis. 1) Cell sorting Many flow cytometers are equipped with fluorescence-activated cell sorting (FACS) equipment. FACS provides a method for sorting of cells based on specific light scattering and fluorescent characteristics of each cell. The collected cells may then be analysed further to determine the specific species groups. 2) Fluorophores Fluorescently labelled probes or marker molecule that bind to certain cell features can be used as a means of detecting specific species (Egli and Hammes 2010). One common practice involves using fluorescent dyes that bind stoichiometrically to DNA. Species can be identified based on fluorescence which is proportional to DNA content (CMCA 2012). Nucleic acid dyes that are commonly used for aquatic environment samples include the SYTO series and SYBR series, with the most popular being SYBR Green (Boon et al. 2010). However a challenge for aquatic samples is finding appropriate fluorophores that will bind with specific target cells. 3) Pure Cultures Another practice that can assist with the detection of specific species is to run pure cultures of microorganisms that are likely to be present in the sample. This can help to create fluorescent fingerprints. If a sample exhibits the same characteristics as the known cultures, it is likely that that species is present in the sample. 4) Microscopy Microscopy is a technique that is commonly integrated with FCM analysis and cell sorting. Microscopy involves using microscope to view cells that cannot be seen with the unaided eye. A visual representation of the sample on a microscopic level can help to develop information about specific cellular groups and identify specific species. Page 23

24 3. AIMS AND OUTCOMES The specific aims of the study are; 1) Investigate the potential for FCM to be used in the field of wastewater. Literature indicates that the potential for FCM to be used in the field of wastewater is enormous, however there have been few papers published that have actually applied FCM to analyse the complex microbial populations that are found in wastewater. This project will investigate the potential for FCM to be used in the field of wastewater by analysing samples taken from several WSPs in the South West region of Western Australia. 2) Through the innovative use of FCM, investigate microbial community characteristics of several WSPs. Despite the growing application of biological wastewater treatment there is still little known about the microbiology that inhabit these ecosystems. Through the use of FCM, this study will investigate microbial community characteristics of several WSPs. In doing so, a deeper understanding of WSPs ecology will be developed. 3) Identify biochemical indicators that are prevalent in efficient WSPs. It is also intended that biochemical indicators, which are prevalent in efficient WSPs, can be identified through the use of FCM. This will be achieved by exploring differences in microbial community characteristics between several WSPs. Results will be used to establish links between treatment plant performance and microbiology. This type of research will ultimately lead to the development of more efficient wastewater treatment designs and processes. Page 24

25 4. APPROACH AND METHODOLOGY 4.1 STUDY SITES Five WSPs from the Great Southern region were selected for this study; Boddington, Wagin, Tambellup, Cranbrook, and Mt Barker. These WSPs have been established by the Water Corporation to treat domestic wastewater to a secondary effluent standard using only a series of biological processes. Treated effluent is either released to the environment, where it is reused to irrigate local agricultural pastures and tree lots, or allowed to infiltrate into the ground. At the Boddington WSP, a water recycling scheme is in place in which up to 0.5 mega litres/day of effluent is treated with chlorine and then pumped to Boddington Gold Mine for industrial process water (Water Corporation n.d.). Site selection was influenced by plant design and location. To ensure comparability of data, only plants that treat water to a secondary effluent standard were selected, and plants that use additional chemical or physical treatment such as alum or aerators were ruled out. Additionally, it was important that sites were accessible to Perth in a day trip so that samples could be collected and transported to appropriate storage facilities at The University of Western Australia. Most of these plants do not have any issues complying with the National Water Quality Management Strategy Guidelines for sewerage systems, with the exception of the Cranbrook plant. Cranbrook s WSP has a history of producing poor quality effluent that does not meet the standards for reuse. Table 1 below provides a summary of the design and details for each of the WSPs. Page 25

26 Table 1 Summary of design details for Boddington, Wagin, Tambellup, Cranbrook and Mount Barker. WSP No. Details Load Previous Effluent Ponds Cyanobacteria Quality Problems Boddington 6 - Two primary and secondary ponds Low Yes OK - P1( primary) and P2 (secondary) are more new than P3(primary) and P4 (secondary - Final pond (P6) is a storage pond - Water is pumped from storage pond, chlorinated and reused at local gold mine site Wagin 4 - Two primary ponds (P1 and P2) Med-high Yes OK - Treated effluent is discharged into creek Tambellup 3 - Final pond (P3) is a storage pond Low-med Yes OK - P3 is not currently discharging to the environment as storage volume has not reached maximum Cranbrook 2 - Treated effluent is returned directly back to the environment via outlet pipe High Yes Poor - Has a history of producing poor quality effluent Mt Barker 5 - Two primary ponds - P1 (primary) newer than P2 (primary) Low No OK - Treated effluent is used for irrigation of tree lot 4.2 PILOT VISIT Initial pilot visits to the WSPs at Wagin and Waroona took place to finalise project methodology. 4.3 WATER SAMPLING Following the success of the pilot visits, final sampling was conducted during the second week of September Samples were taken from the inlet and outlet of each pond, with approximately 10 minutes being spent at each sample site. An example of the sampling locations is shown below in Figure 4. Page 26

27 Figure 4 Map showing locations of the sample sites at the Tambellup WSP. Where green arrows represent the direction of water flow between the ponds and red dots indicate the sample location. Water samples were collected from the respective ponds using an open grab sampler from approximately 10 cm below the water surface. Samples were stored in 10 ml screw cap tubes and were labelled according to the plant, pond and sample site they were taken from. Table 2 below details the sample coding. Samples have been referred to by their sample code throughout this paper. Table 2 Details for sample codes. WSP Code Pond Code Sample Site Code Example Code Cranbrook Cran 1 P1 1st sample 1 Tambellup, Pond 3, Sample 2 Tam-P3-S2 Boddington Bdt 2 P2 2nd sample 2 Tambellup Tam 3 P3 Mount Barker MtB 4 P4 Wagin Wag 5 P5 6 P6 A number of precautions were taken during the sampling procedure to prevent any contamination or interference of samples that could deter the results. The precautions were as follows; 1) All water was filtered through 500 μm filter mesh before being funnelled into the storage tubes to remove large plankton that could graze on smaller species; 2) Storage tubes were rinsed three times with sample water prior to being filled; Page 27

28 3) Samples were stored in refrigerated devices while they were transported back to Perth; 4) Upon the arrival at the Laboratory, the 10 ml wastewater samples were fixed with approximately 1 % (final concentration) of glutaraldehyde and immediately frozen until they were analysed. Fixing ensures that samples are preserved and are also safe to use in the FCM laboratory. 4.4 ENVIRONMENTAL PARAMETERS Environmental variables were measured at each of the sample sites using a calibrated TPS WP-81 probe (TPS, Brisbane, Queensland, Australia). These parameters included temperature, ph, dissolved oxygen (DO), conductivity and oxidation reduction potential (ORP). These parameters provide information on the conditions of the different wastewater environments that influence selection, survival and growth of microorganisms. As previously mentioned, optimal growth generally occurs between a ph of 6-8 and at temperatures around 20 o C. DO provides a good indication of the biological activity, high levels of DO indicate an abundance of photosynthesising organisms, while low levels may be associated with low algal concentrations or a high concentration of aerobic microorganisms. ORP measures the ability of the water to permit essential aerobic and anaerobic treatment processes. A positive measurement indicates the degree to which the system is oxidative, while a negative value indicates the water reducing potential (Myers et al. 2006). Positive ORP values are to be expected at the subsurface of WSPs, where anaerobic reactions take place. Conductivity in wastewater is affected by temperature and the presence of inorganic dissolved solids (EPA 2012). Generally, efficient treatment is more effective at removing solids and would therefore show lower conductivity values. Hence, conductivity provides a useful indicator of water quality. 4.5 FLUOROPROBE FluoroProbe analysis was carried out on site using a BBE-Moldaenke FluoroProbe (FluoroProbe, bbe-moldaenke, Kiel, Germany) with at least 10 FluroProbe measurements being recorded at each sample site. The probe contains 5 light emitting diodes that excite auto-fluorescing cells containing chlorophyll-a. These cells are mostly algae. The FluoroProbe quantifies different algae classes based on the unique fluorescence patterns, or fingerprints, of different species. The fingerprints of 4 algae classes are already stored in the FluoroProbe, including green algae, blue-green algae (cyanobacteria), diatoms and Page 28

29 cryptophytes. The concentration of each algae division is given in μg chlorophyll-a/l. An additional fingerprint is also stored within the probe for the detection of yellow substances, which includes coloured dissolved organic matter (Bbe Moldaenke 2007). Due to high concentrations of chlorophyll-a in the ponds, samples were diluted with potable water to ensure concentrations were within the working range of the FluoroProbe. Ratios of sample water to potable water were usually between 1:5 and 1:50. For more specific details of the dilutions refer to the FluoroProbe data in Appendix 1. FluoroProbe data can be correlated with FCM data to give an indication of the types of species that are likely to be detected. 4.6 FLOW CYTOMETRY As discussed FCM was used to analyse water samples taken from the aforementioned sample sites. FCM was applied to these samples to explore the community characteristics of autofluorescent microorganisms Sample preparation Cell staining As only auto-fluorescent characteristics of microorganisms were investigated, cell staining with fluorescent dyes was not necessary Sonication Most samples exhibited some floc formation. This was particularly noticeable with algae, which had a tendancy to form chains (Figure 5). To dissociate the agglomerated cells into single cell suspension, replicates of all samples were made and then sonicated for 2 minutes in an ultra-sonic bath at a frequency of 50 Hz. The optimum sonication time is unknown but based on visual observation 2 minutes was enough time to separate the chains. Page 29

30 Figure 5 Photograph illustrating algae chains. Wag-P2-S2 (Right) showing visible algae flocculation, Wag- P1-S2 showing some flocculation and Wag-P1-S1 (Left) showing little flocculation Filtration Approximately ml of each sample was filtered through 35 μm filter mesh into 5 ml round bottom test tubes. This was to remove large particles or any debris that could cause equipment to clog. It should be noted that for the unsonicated samples, the mesh noticeably filtered out more algae than for the sonicated samples Calibration Prior to analysis, a number of calibrations were conducted to ensure the validity data. Initially a default experiment template was established. The template were created from running a culture of Desmodesmus spp., a genus of algae, and a culture of Microcystis aeruginosa, a genus of freshwater cyanobacteria which has been known to exist in WSPs (Martins et al. 2011). These species gave an indication of the size and fluorescence of cells that was likely to be detected in the samples and provided the basis for developing parameters at which the wastewater samples would be analysed. As an internal standard for fluorescence and size, 1μm and 3μm calibration beads were also used to establish the template setting. Although one setting may not be accurate to represent all the cells in the sample, it provides a starting point Page 30

31 for analysing wastewater using FCM. Ideally a number of settings would be established for analysing different species of interest. This has been done for studies that have applied FCM to marine biology. The fluorescence measured by FCM is entirely dependent on the instrument settings and even when using the same flow cytometer the variability within the instrument performance itself makes comparison between data acquired on different days uncertain (Gasol and Giorgio 2000). To allow for accurate comparison of multiple sets of data acquired on different days, the flow cytometer was calibrated with SPHERO 8 Peak Rainbow Calibration beads at the beginning of each FCM experiment. It was ensured that the median fluorescence values for the beads were similar for each experiment Evaluation Samples were analysed on a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). The instrument is a bench top analyser that is equipped with 3 lasers emitting at fixed wavelengths of 405nm (violet laser), 488nm (blue laser) and 633nm (red laser). Up to eight colours can be detected through various band pass (BP) and long pass (LP) filters. TABLE X below details the emitted light from the three lasers and the fluorescence collected by the various filters. Page 31

32 Table 3 Details of the lasers detectors and filters that the BD FACSCanto II flow cytometer is equipped with. PMT (photomultiplier tube) is the type of detector (CMCA 2012). Each sample was run for two minutes, or until 10,000 events were recorded, on a low flow rate setting Data analysis Following the assessment of all the samples, FCM data was analysed with the software package FlowJo. This software was used to establish boundaries, or gates, thereby highlighting populations of interest. A gate was established in a forward scatter/side scatter plot (FSC-A vs. SSC-A) to discriminate between the cells of interest and background noise. The cells of interest were then analysed across different excitation and emission ranges, and sub-gates were drawn based on the prevalence of clusters of cells across different fluorescent signals. By comparing the gated communities with results from the FluoroProbe, speculations can be made over identification of these species. Page 32

33 4.7 WATER QUALITY The Water Corporation undertakes water sampling once a month at the inlets and outlets of each plant as part of a water quality monitoring routine. It was requested that the Water Corporation organise additional samples to also be taken at the outlet of each pond during the month of September to coincide with sampling for this project. Water quality data provides an indication of how well the ponds function and can be used to identify relationships between treatment efficiency and FCM results. Furthermore, this data is important for confirming whether Cranbrook is currently producing poor quality effluent like previously believed. Indicators of water quality that are tested for include biochemical oxygen demand (BOD), total phosphorous (TP), total nitrogen (TN), suspended solids (SS) and faecal microbes. Definitions for the water quality indicators are as follows (Water Corporation n.d.); 1) BOD: is the measure of unstable organic matter present in the wastewater. It is a measure of the oxygen required by the available microorganisms to break down organic matter. When there are larger quantities of organic waste, BOD is greater due to the fact that there will be more organisms working to decompose the waste. As the waste is consumed, BOD levels will decline. Generally, lower BOD levels are indicative of water of a higher quality. 2) TN and TP: is the sum of all forms of nitrogen and phosphorous present in the wastewater. Both are essential nutrients for plant growth. Excess concentration of TP and TN in freshwater ways can cause blooms of algae of waterweeds to occur. 3) SS: is a measure of the mass of fine inorganic particles suspended in the water column. Treatment process within WSPs aim to remove most of the SS. High concentrations of SS can cause a decrease in the amount of light that can penetrate through the water column. 4) Faecal Microbes include viruses, bacteria and protozoans. Faecal coliforms and faecal streptococci are bacteria that are found in the digestive tracts of humans and animals. Their presence in a water body indicates faecal contamination. If faecal microbes are not treated sufficiently in the treatment process they may pose a serious health risk for humans, potentially causing disease. Faecal microbes are commonly measured as Escherichia Coli (E. Coli,) as this is the main indicator microorganism of faecal contamination, in units of organisms/100ml or cfu/100ml (colony-forming unit/ml). Page 33

34 The Water Corporation manages effluent in accordance with the National Water Quality Management Strategy (NWQMS) Guidelines for Sewerage Systems. Acceptable effluent quality suitable for discharge, as outlined by the NWQMS, is provided in Table 4 below. Table 4 Guidelines for effluent quality following various levels of treatment (NWQMS 1997) Page 34