This creative dissertation is a novel titled Into the Attic. The novel tells the story of Sullivan Young, a junior at a small liberal arts college in central Pennsylvania in the mid-2000s, and James Shelley, a young literature professor at the college, with whom Sullivan initiates an affair. The narrative switches between the points of view of these two men, neither of whom is happy with the person he is becoming, and develops around the fears each has about the relationship. The novel is concerned with character, sexuality, and power; in order to explore these issues fully within Sullivan and Shelley’s relationship, the novel alternates between the two characters’ points of view, thus attempting to illustrate the way the negotiation of power in the relationship also alternates between them as the relationship develops. The novel relies heavily on Sullivan’s and Shelley’s experiences as gay men and uses explicit sex scenes to develop character; therefore, my critical introduction examines the implications of writing a political or social “other” and references writers and critics—such as Dorothy Allison, Wayne Booth, Tillie Olsen, Jacqueline Jones Royster, Eve Kosofsky Sedgwick, and Calvin Thomas—who have informed my understanding of writing “the other.”

No description available.

No description available.

Between 1910 and 1926, Hazrat Inayat Khan, a musician and Sufi master from the Chishtiyya order in India, introduced Sufism to the United States of America and Europe. He established a Western Sufi order, the International Sufi Movement, with groups in America, England, and across Europe. In the early 1930s this Western Sufism was introduced into Australia. The aim of this study is to explore and analyse the origins, nature and development of the Sufi Movement in Australia with particular attention to the relationship between the spiritual teacher and the disciple. The study, in its various aspects, and as a whole, makes a contribution to understanding religious change, religion and New Religious Movements in Australia, Sufism in the West and the murshid-mureed relationship. Using what has been termed a “dynamic of religions” framework and Baumann’s model of religious adaptation in cross-cultural circumstances, the study employs a combination of in-depth fieldwork, participant observation, interviews and historiography to identify and analyse both continuity and change in Sufi Movement groups in contemporary Australia. The study found that the early phases of transplantation from India to Europe and America, as well as the struggles within the group after the death of Inayat Khan, substantially shaped the Western Sufism that arrived in Australia. The most significant early adaptations included redefining the relationship between Sufism and exoteric Islam, gender equality, formation of a formal organisation, and the use of music to spread the Sufi Message. Addressing issues identified in the literature on the master-disciple relationship in Eastern traditions in the West, the study describes and analyses the murshid-mureed (master-disciple, spiritual teacher-student) relationship in Inayat Khan’s time, and in contemporary Australia. It shows that Sufism’s development in the West has been interconnected with understandings and practice of this central relationship. The study shows that the continued strength of the murshid-mureed relationship alongside emerging alternatives, the factors affecting change generally in the Sufi Movement, as well as the growing popularity of Sufism in Australia, demonstrate the need to consider not only East-West aspects of transplantation, but also the influences of modernity on spiritual traditions. As well as documenting the Australian history, the study describes the details of the variety of practices and rituals, forms of community and organisation, and forms of the murshid-mureed relationship, found in contemporary Australian groups. This provides insight into the intricacies of Sufi practice in Australia and provides a basis for comparison with other groups. Sufi practice within these groups in Australia includes highly innovative developments alongside preservation of the tradition as taught by Inayat Khan. It also reflects a tension between the spiritual and organisational hierarchy, and concerns for democracy and local autonomy.

Polyhydroxyalkanoates (PHAs) are polyesters produced and stored intracellularly as reserve materials for carbon and energy by a wide range of prokaryotic microorganisms under imbalanced nutritional conditions. PHA production by pure culture has been extensively studied, enabling high production yields and a variety of polymer characteristics. Poly(3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV) (PHBV) has been commercially available under the trade name Biopol. However, the high production costs of these biopolymers are a major barrier for their widespread acceptance as substitute for traditional non-biodegradable polymers. The main costs are associated with maintaining the sterile conditions required by pure cultures, the use of expensive feed materials (eg. sugars) and also the polymer extraction process. Producing PHAs using mixed cultures (such as activated sludge) can drastically simplify the production process and significantly reduce the feed costs due to the use of cheap substrates. It also has the additional benefits in reusing waste materials. To date, substantial efforts have been put into improving PHA productivity in mixed cultures, with the characteristics of these bio-polyesters largely unexplored. The main product is typically PHB, which has several inherent deficiencies in properties. These include brittleness due to its high crystallinity, and thermal instability near its melting point of 175-180&degC. To overcome the drawbacks of PHB, non-3HB monomer units are incorporated in the bio-polymerisation process, but this generally requires the addition of specific and often complex precursor substrates. Glycogen accumulating organisms (GAOs) are emerging as an attractive alternative to other heterotrophic PHA producers due to their special metabolism. GAOs were initially identified as competitors to the polyphosphate accumulating organisms (PAOs) in alternating anaerobic/aerobic wastewater treatment systems. Under anaerobic conditions, GAOs generate energy and reducing power from glycogen hydrolysis, which are used for taking up carbon sources (eg. acetate or propionate) and their synthesis into PHAs as intracellular storage products. Under aerobic conditions, the stored PHAs is partly oxidised for energy generation with the remainder used for biomass growth and glycogen replenishment. The anaerobic hydrolysis of glycogen yields both acetyl-CoA and propionyl-CoA which can be condensed to form 3-hydroxybutyryl-CoA and 3-hydroxyvaleryl-CoA, the precursors of 3HB and 3HV. Therefore GAOs are capable of producing multiple 3- hydroxyalkanoate (3HA) monomers even when only a single carbon source (eg., acetate) is supplied. The potential of producing copolymers without addition of particular monomer-relevant carbon sources provides GAOs an advantage over other mixed culture heterotrophs in terms of polymer quality. So far, two main bacterial species have been identified to display the GAO phenotype, namely, Candidatus Competibacter phosphatis (henceforth referred to as Competibacter) and Defluviicoccus vanus-related GAOs belonging to the Alphaproteobacteria phylum (henceforth referred to as DvGAOs). This thesis focuses on the PHA production by GAOs. The capability of GAOs to produce heterogeneous PHAs from a single carbon source is investigated by characterising the PHA products and optimising the polymer productivity. Moreover, DvGAOs are comprehensively studied for their ability to yield novel four-monomer copolymers of 3HB, 3HV, 3-hydroxy-2-methylvalerate (3HMV) and 3-hydroxy-3- methylbutyrate (3HMB) with controllable composition and favourable physical properties. The main contributions from this thesis are summarised below. The polymers consisting of 3HB, 3HV and minor amounts of 3HMV and 3HMB, produced by Competibacter-dominated GAOs using acetate as a sole carbon source, were identified to be true copolymers based on a detailed characterisation using &sup1H and ¹&sup3C Nuclear Magnetic Resonance (NMR) spectroscopy. The monomer sequence distribution analysis using a known polymer characterisation model suggests that these polyesters are mixtures of random copolymers and thus likely possess desirable properties for practical use, which was confirmed experimentally. This demonstrates that GAOs have a true potential for producing high-quality polymers from a simple and cheap carbon source. The PHAs produced by GAOs under anaerobic and aerobic conditions were characterized using size exclusion chromatography (SEC) and differential scanning calorimetry (DSC). The acetate-derived copolymers possessed characteristics similar to those of commercially available PHBV products. The anaerobically produced PHA contained a relatively constant proportion of non-3HB monomers (30±5 C-mol%), irrespective of the amount of acetate assimilated. In contrast, under aerobic conditions, GAOs only produced 3HB monomers from acetate causing a gradually decreasing 3HV fraction. The 3HV fraction thus obtained ranged from 7 to 35 C-mol%, depending on the amount of acetate supplied under aerobic conditions. The PHAs produced under solely anaerobic conditions featured lower melting points and crystallinity, higher molecular weights, and narrower molecular weight distributions compared to the aerobically produced polymers. However, the anaerobic production yield was limited at 28% of dry cell weight (DCW) due to the shortage of glycogen, while aerobic production obtained a maximum polymer content of 41% based on DCW. To increase the PHA yield from anaerobic production, a novel three-stage strategy was developed. It was demonstrated to be an effective approach to optimise both the quantity and quality of the copolymers produced by GAOs. Using the Competibacterdominated GAO culture, up to 48 wt% poly(3HB-co-3HV-co-3HMV) based on DCW was achieved from acetate as the sole carbon source, close to the highest copolymer yield reported to date produced by mixed cultures but using specific precursor substrates. The optimisation method comprised of an aerobic famine, an aerobic feast, and an anaerobic feast period. The glycogen storage was enhanced through the initial two aerobic periods and hence increased the energy and reducing power available for the final anaerobic polymer synthesis step. The terpolymers/copolymers thus produced displayed high molecular weights (380-460 kg/mol) with a narrow distribution range. A feeding strategy based on pH-stat was demonstrated to achieve the automatic control of feed addition. Using a highly enriched DvGAO mixed culture (95±3%) copolymers of 3HB, 3HV, 3HMV and 3HMB with controllable monomer fractions were obtained from acetate and propionate substrates. Through manipulating the ratio of acetate and propionate in the medium, the 3HB and 3HMV monomer portions could be varied extensively (10 to 66 mol% 3HB and 2 to 41 mol% 3HMV). The microstructure study revealed that the PHAs produced were likely random copolymers or mixtures of random copolymers. These PHA products were demonstrated to possess favourable properties. The weight-average molecular weights were in the range 390-560 kg/mol, while the DSC traces showed melting temperatures in the range of 70 to 161 &degC, glass transition temperatures between -8 and 0 &degC, and melting enthalpies (ΔHm) between 9.1 and 31.5 J/g. The incorporation of considerable amounts of 3HMV and 3HMB constituents greatly lowered the crystallinity, potentially providing the ductile and tough materials required for many practical applications. The anaerobic metabolism of DvGAOs with acetate and propionate was found to be well described by the metabolic models previously proposed for GAOs and verified with experimental data obtained with other types of GAO cultures. The results suggested DvGAOs likely use metabolic pathways similar to those used by other GAOs for anaerobic acetate and propionate uptake. However, when both acetate and propionate were present simultaneously, DvGAOs took up these two carbon sources sequentially, with propionate uptake preceding acetate uptake. As a result, mixtures of 3HV&3HMV-rich copolymers and 3HB&3HV-rich copolymers were formed. Through model-based analysis, it was hypothesised that DvGAOs prefer propionate in order to maximise their production of PHAs with the same glycogen consumption, which would enhance their growth potential in the following aerobic period. This feature may explain the more efficient PHA production by DvGAOs with propionate as the carbon substrate compared to acetate. Despite very low acetate consumption when propionate was available in excess, the presence of acetate considerably stimulated the uptake of propionate in comparison to the case where only propionate was present. This was likely due to the difference of the intracellular adenosine triphosphate (ATP) level in the two cases. A lower intracellular ATP level detected in the simultaneous presence of acetate and propionate might stimulate the glycolysis process resulting in a higher propionate uptake rate. This thesis shows that GAOs have indeed a good potential as cost-effective PHA producers. They are able to efficiently generate true copolymers of up to four 3HA monomers with desirable properties from simple carbon sources. Through manipulating the feed composition, comonomer fractions can vary in a wide range resulting in a variety of polymer properties. The contribution from this work could be very useful for the current drive to cost-effectively produce good quality PHAs to replace conventional petrol-derived polymers.

Nitrogen removal in biological wastewater treatment plants by nitrification and denitrification can be short-cut via nitrite instead of the traditional nitrate and has the potential for up to a 25% reduction in aeration and 40% reduction in COD requirements. While the potential monetary savings for nitrogen removal via nitrite are significant, the difficulty lies in achieving specific inhibition or removal of the nitrite oxidising bacteria (NOB; those that oxidise nitrite to nitrate) whilst retaining ammonia oxidising bacteria (AOB; those that oxidise ammonia to nitrite). In approaching this general problem, characterisation of NOBs (both Nitrobacter and Nitrospira) were used to determine, in conjunction with AOB data, reactor operating factors which may lead to NOB removal. From literature, the AOBs have been hypothesised to have a higher oxygen affinity (lower oxygen half saturation constant, K[subscript]o) than NOBs. The K[subscript]o values of both Nitrospira and Nitrobacter enrichments (NOBs) and a Nitrosomonas enrichment (AOB) were determined with floc size distributions that indicated oxygen mass transfer was negligible as 0.54 ± 0.14 mg.L¯¹, 0.43 ± 0.08 mg.L¯¹ and 0.033 ± 0.003mg.L¯¹, respectively. The relative AOB and NOB K[subscript]o values confirm the hypothesised difference in oxygen affinity. The growth rate values of the same Nitrosomonas and Nitrobacter enrichments were determined using a novel nitrifier growth rate method (developed in this thesis) as 0.47 ± 0.09 day¯¹ and 0.60 ± 0.03 day¯¹, respectively. Therefore, whilst the NOB maximum growth rate was determined as greater than that of the AOB maximum growth rate at saturated dissolved oxygen (DO) concentration, at low DO concentration the maximum growth rate of AOBs was determined as greater than the maximum growth rate of the NOBs. Hence, washout of NOBs was achieved by operating a continuous reactor at a DO concentration (0.4 mg.L¯¹) and a sludge retention time of 2.4 days which allowed for AOB retention but not NOBs. The results from the continuous reactor experiments suggested that the growth rate of NOBs may not be exclusively greater than the AOB growth rate, contrary to previous thought. Therefore, if aeration is terminated at the end of ammonium oxidation in a batch process (such as a sequencing batch reactor; SBR) with accumulated nitrite remaining, then the relative population of AOBs would eventually dominate. Indeed this concept was investigated using an SBR treating domestic wastewater (TKN concentration of about 43 mgN.L¯¹) with a pre-denitrification configuration and only the termination of aeration at the end of ammonium oxidation (aerobic duration control) as the selection factor for AOBs. The process proved effective in achieving a steady state whereby 80% nitrification to nitrite (20% to nitrate) was observed. Investigation of the cause of nitrification to nitrite by a calibrated ammonium and nitrite oxidation model showed the aerobic duration control as the key selection factor for AOBs. The results of numerous simulations with the same computer model also verified the importance of a greater AOB growth rate than a NOB growth rate in achieving sustained nitrite as the product of nitrification. The processes developed in this thesis to remove NOBs, i.e. low DO concentration and aerobic duration control could theoretically be implemented in current operational wastewater treatment plants. The low DO concentration selection factor against NOBs would probably be more effective in combination with other selection factors against NOBs (e.g. high temperature) as this would enhance the effectiveness of such processes. However, the aerobic duration control could theoretically be implemented immediately in similar SBR processes treating similar domestic wastewater.

No description available.

Polyhydroxyalkanoates (PHAs) are polyesters produced and stored intracellularly as reserve materials for carbon and energy by a wide range of prokaryotic microorganisms under imbalanced nutritional conditions. PHA production by pure culture has been extensively studied, enabling high production yields and a variety of polymer characteristics. Poly(3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV) (PHBV) has been commercially available under the trade name Biopol. However, the high production costs of these biopolymers are a major barrier for their widespread acceptance as substitute for traditional non-biodegradable polymers. The main costs are associated with maintaining the sterile conditions required by pure cultures, the use of expensive feed materials (eg. sugars) and also the polymer extraction process. Producing PHAs using mixed cultures (such as activated sludge) can drastically simplify the production process and significantly reduce the feed costs due to the use of cheap substrates. It also has the additional benefits in reusing waste materials. To date, substantial efforts have been put into improving PHA productivity in mixed cultures, with the characteristics of these bio-polyesters largely unexplored. The main product is typically PHB, which has several inherent deficiencies in properties. These include brittleness due to its high crystallinity, and thermal instability near its melting point of 175-180&degC. To overcome the drawbacks of PHB, non-3HB monomer units are incorporated in the bio-polymerisation process, but this generally requires the addition of specific and often complex precursor substrates. Glycogen accumulating organisms (GAOs) are emerging as an attractive alternative to other heterotrophic PHA producers due to their special metabolism. GAOs were initially identified as competitors to the polyphosphate accumulating organisms (PAOs) in alternating anaerobic/aerobic wastewater treatment systems. Under anaerobic conditions, GAOs generate energy and reducing power from glycogen hydrolysis, which are used for taking up carbon sources (eg. acetate or propionate) and their synthesis into PHAs as intracellular storage products. Under aerobic conditions, the stored PHAs is partly oxidised for energy generation with the remainder used for biomass growth and glycogen replenishment. The anaerobic hydrolysis of glycogen yields both acetyl-CoA and propionyl-CoA which can be condensed to form 3-hydroxybutyryl-CoA and 3-hydroxyvaleryl-CoA, the precursors of 3HB and 3HV. Therefore GAOs are capable of producing multiple 3- hydroxyalkanoate (3HA) monomers even when only a single carbon source (eg., acetate) is supplied. The potential of producing copolymers without addition of particular monomer-relevant carbon sources provides GAOs an advantage over other mixed culture heterotrophs in terms of polymer quality. So far, two main bacterial species have been identified to display the GAO phenotype, namely, Candidatus Competibacter phosphatis (henceforth referred to as Competibacter) and Defluviicoccus vanus-related GAOs belonging to the Alphaproteobacteria phylum (henceforth referred to as DvGAOs). This thesis focuses on the PHA production by GAOs. The capability of GAOs to produce heterogeneous PHAs from a single carbon source is investigated by characterising the PHA products and optimising the polymer productivity. Moreover, DvGAOs are comprehensively studied for their ability to yield novel four-monomer copolymers of 3HB, 3HV, 3-hydroxy-2-methylvalerate (3HMV) and 3-hydroxy-3- methylbutyrate (3HMB) with controllable composition and favourable physical properties. The main contributions from this thesis are summarised below. The polymers consisting of 3HB, 3HV and minor amounts of 3HMV and 3HMB, produced by Competibacter-dominated GAOs using acetate as a sole carbon source, were identified to be true copolymers based on a detailed characterisation using &sup1H and ¹&sup3C Nuclear Magnetic Resonance (NMR) spectroscopy. The monomer sequence distribution analysis using a known polymer characterisation model suggests that these polyesters are mixtures of random copolymers and thus likely possess desirable properties for practical use, which was confirmed experimentally. This demonstrates that GAOs have a true potential for producing high-quality polymers from a simple and cheap carbon source. The PHAs produced by GAOs under anaerobic and aerobic conditions were characterized using size exclusion chromatography (SEC) and differential scanning calorimetry (DSC). The acetate-derived copolymers possessed characteristics similar to those of commercially available PHBV products. The anaerobically produced PHA contained a relatively constant proportion of non-3HB monomers (30±5 C-mol%), irrespective of the amount of acetate assimilated. In contrast, under aerobic conditions, GAOs only produced 3HB monomers from acetate causing a gradually decreasing 3HV fraction. The 3HV fraction thus obtained ranged from 7 to 35 C-mol%, depending on the amount of acetate supplied under aerobic conditions. The PHAs produced under solely anaerobic conditions featured lower melting points and crystallinity, higher molecular weights, and narrower molecular weight distributions compared to the aerobically produced polymers. However, the anaerobic production yield was limited at 28% of dry cell weight (DCW) due to the shortage of glycogen, while aerobic production obtained a maximum polymer content of 41% based on DCW. To increase the PHA yield from anaerobic production, a novel three-stage strategy was developed. It was demonstrated to be an effective approach to optimise both the quantity and quality of the copolymers produced by GAOs. Using the Competibacterdominated GAO culture, up to 48 wt% poly(3HB-co-3HV-co-3HMV) based on DCW was achieved from acetate as the sole carbon source, close to the highest copolymer yield reported to date produced by mixed cultures but using specific precursor substrates. The optimisation method comprised of an aerobic famine, an aerobic feast, and an anaerobic feast period. The glycogen storage was enhanced through the initial two aerobic periods and hence increased the energy and reducing power available for the final anaerobic polymer synthesis step. The terpolymers/copolymers thus produced displayed high molecular weights (380-460 kg/mol) with a narrow distribution range. A feeding strategy based on pH-stat was demonstrated to achieve the automatic control of feed addition. Using a highly enriched DvGAO mixed culture (95±3%) copolymers of 3HB, 3HV, 3HMV and 3HMB with controllable monomer fractions were obtained from acetate and propionate substrates. Through manipulating the ratio of acetate and propionate in the medium, the 3HB and 3HMV monomer portions could be varied extensively (10 to 66 mol% 3HB and 2 to 41 mol% 3HMV). The microstructure study revealed that the PHAs produced were likely random copolymers or mixtures of random copolymers. These PHA products were demonstrated to possess favourable properties. The weight-average molecular weights were in the range 390-560 kg/mol, while the DSC traces showed melting temperatures in the range of 70 to 161 &degC, glass transition temperatures between -8 and 0 &degC, and melting enthalpies (ΔHm) between 9.1 and 31.5 J/g. The incorporation of considerable amounts of 3HMV and 3HMB constituents greatly lowered the crystallinity, potentially providing the ductile and tough materials required for many practical applications. The anaerobic metabolism of DvGAOs with acetate and propionate was found to be well described by the metabolic models previously proposed for GAOs and verified with experimental data obtained with other types of GAO cultures. The results suggested DvGAOs likely use metabolic pathways similar to those used by other GAOs for anaerobic acetate and propionate uptake. However, when both acetate and propionate were present simultaneously, DvGAOs took up these two carbon sources sequentially, with propionate uptake preceding acetate uptake. As a result, mixtures of 3HV&3HMV-rich copolymers and 3HB&3HV-rich copolymers were formed. Through model-based analysis, it was hypothesised that DvGAOs prefer propionate in order to maximise their production of PHAs with the same glycogen consumption, which would enhance their growth potential in the following aerobic period. This feature may explain the more efficient PHA production by DvGAOs with propionate as the carbon substrate compared to acetate. Despite very low acetate consumption when propionate was available in excess, the presence of acetate considerably stimulated the uptake of propionate in comparison to the case where only propionate was present. This was likely due to the difference of the intracellular adenosine triphosphate (ATP) level in the two cases. A lower intracellular ATP level detected in the simultaneous presence of acetate and propionate might stimulate the glycolysis process resulting in a higher propionate uptake rate. This thesis shows that GAOs have indeed a good potential as cost-effective PHA producers. They are able to efficiently generate true copolymers of up to four 3HA monomers with desirable properties from simple carbon sources. Through manipulating the feed composition, comonomer fractions can vary in a wide range resulting in a variety of polymer properties. The contribution from this work could be very useful for the current drive to cost-effectively produce good quality PHAs to replace conventional petrol-derived polymers.

Polyhydroxyalkanoates (PHAs) are polyesters produced and stored intracellularly as reserve materials for carbon and energy by a wide range of prokaryotic microorganisms under imbalanced nutritional conditions. PHA production by pure culture has been extensively studied, enabling high production yields and a variety of polymer characteristics. Poly(3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV) (PHBV) has been commercially available under the trade name Biopol. However, the high production costs of these biopolymers are a major barrier for their widespread acceptance as substitute for traditional non-biodegradable polymers. The main costs are associated with maintaining the sterile conditions required by pure cultures, the use of expensive feed materials (eg. sugars) and also the polymer extraction process. Producing PHAs using mixed cultures (such as activated sludge) can drastically simplify the production process and significantly reduce the feed costs due to the use of cheap substrates. It also has the additional benefits in reusing waste materials. To date, substantial efforts have been put into improving PHA productivity in mixed cultures, with the characteristics of these bio-polyesters largely unexplored. The main product is typically PHB, which has several inherent deficiencies in properties. These include brittleness due to its high crystallinity, and thermal instability near its melting point of 175-180&degC. To overcome the drawbacks of PHB, non-3HB monomer units are incorporated in the bio-polymerisation process, but this generally requires the addition of specific and often complex precursor substrates. Glycogen accumulating organisms (GAOs) are emerging as an attractive alternative to other heterotrophic PHA producers due to their special metabolism. GAOs were initially identified as competitors to the polyphosphate accumulating organisms (PAOs) in alternating anaerobic/aerobic wastewater treatment systems. Under anaerobic conditions, GAOs generate energy and reducing power from glycogen hydrolysis, which are used for taking up carbon sources (eg. acetate or propionate) and their synthesis into PHAs as intracellular storage products. Under aerobic conditions, the stored PHAs is partly oxidised for energy generation with the remainder used for biomass growth and glycogen replenishment. The anaerobic hydrolysis of glycogen yields both acetyl-CoA and propionyl-CoA which can be condensed to form 3-hydroxybutyryl-CoA and 3-hydroxyvaleryl-CoA, the precursors of 3HB and 3HV. Therefore GAOs are capable of producing multiple 3- hydroxyalkanoate (3HA) monomers even when only a single carbon source (eg., acetate) is supplied. The potential of producing copolymers without addition of particular monomer-relevant carbon sources provides GAOs an advantage over other mixed culture heterotrophs in terms of polymer quality. So far, two main bacterial species have been identified to display the GAO phenotype, namely, Candidatus Competibacter phosphatis (henceforth referred to as Competibacter) and Defluviicoccus vanus-related GAOs belonging to the Alphaproteobacteria phylum (henceforth referred to as DvGAOs). This thesis focuses on the PHA production by GAOs. The capability of GAOs to produce heterogeneous PHAs from a single carbon source is investigated by characterising the PHA products and optimising the polymer productivity. Moreover, DvGAOs are comprehensively studied for their ability to yield novel four-monomer copolymers of 3HB, 3HV, 3-hydroxy-2-methylvalerate (3HMV) and 3-hydroxy-3- methylbutyrate (3HMB) with controllable composition and favourable physical properties. The main contributions from this thesis are summarised below. The polymers consisting of 3HB, 3HV and minor amounts of 3HMV and 3HMB, produced by Competibacter-dominated GAOs using acetate as a sole carbon source, were identified to be true copolymers based on a detailed characterisation using &sup1H and ¹&sup3C Nuclear Magnetic Resonance (NMR) spectroscopy. The monomer sequence distribution analysis using a known polymer characterisation model suggests that these polyesters are mixtures of random copolymers and thus likely possess desirable properties for practical use, which was confirmed experimentally. This demonstrates that GAOs have a true potential for producing high-quality polymers from a simple and cheap carbon source. The PHAs produced by GAOs under anaerobic and aerobic conditions were characterized using size exclusion chromatography (SEC) and differential scanning calorimetry (DSC). The acetate-derived copolymers possessed characteristics similar to those of commercially available PHBV products. The anaerobically produced PHA contained a relatively constant proportion of non-3HB monomers (30±5 C-mol%), irrespective of the amount of acetate assimilated. In contrast, under aerobic conditions, GAOs only produced 3HB monomers from acetate causing a gradually decreasing 3HV fraction. The 3HV fraction thus obtained ranged from 7 to 35 C-mol%, depending on the amount of acetate supplied under aerobic conditions. The PHAs produced under solely anaerobic conditions featured lower melting points and crystallinity, higher molecular weights, and narrower molecular weight distributions compared to the aerobically produced polymers. However, the anaerobic production yield was limited at 28% of dry cell weight (DCW) due to the shortage of glycogen, while aerobic production obtained a maximum polymer content of 41% based on DCW. To increase the PHA yield from anaerobic production, a novel three-stage strategy was developed. It was demonstrated to be an effective approach to optimise both the quantity and quality of the copolymers produced by GAOs. Using the Competibacterdominated GAO culture, up to 48 wt% poly(3HB-co-3HV-co-3HMV) based on DCW was achieved from acetate as the sole carbon source, close to the highest copolymer yield reported to date produced by mixed cultures but using specific precursor substrates. The optimisation method comprised of an aerobic famine, an aerobic feast, and an anaerobic feast period. The glycogen storage was enhanced through the initial two aerobic periods and hence increased the energy and reducing power available for the final anaerobic polymer synthesis step. The terpolymers/copolymers thus produced displayed high molecular weights (380-460 kg/mol) with a narrow distribution range. A feeding strategy based on pH-stat was demonstrated to achieve the automatic control of feed addition. Using a highly enriched DvGAO mixed culture (95±3%) copolymers of 3HB, 3HV, 3HMV and 3HMB with controllable monomer fractions were obtained from acetate and propionate substrates. Through manipulating the ratio of acetate and propionate in the medium, the 3HB and 3HMV monomer portions could be varied extensively (10 to 66 mol% 3HB and 2 to 41 mol% 3HMV). The microstructure study revealed that the PHAs produced were likely random copolymers or mixtures of random copolymers. These PHA products were demonstrated to possess favourable properties. The weight-average molecular weights were in the range 390-560 kg/mol, while the DSC traces showed melting temperatures in the range of 70 to 161 &degC, glass transition temperatures between -8 and 0 &degC, and melting enthalpies (ΔHm) between 9.1 and 31.5 J/g. The incorporation of considerable amounts of 3HMV and 3HMB constituents greatly lowered the crystallinity, potentially providing the ductile and tough materials required for many practical applications. The anaerobic metabolism of DvGAOs with acetate and propionate was found to be well described by the metabolic models previously proposed for GAOs and verified with experimental data obtained with other types of GAO cultures. The results suggested DvGAOs likely use metabolic pathways similar to those used by other GAOs for anaerobic acetate and propionate uptake. However, when both acetate and propionate were present simultaneously, DvGAOs took up these two carbon sources sequentially, with propionate uptake preceding acetate uptake. As a result, mixtures of 3HV&3HMV-rich copolymers and 3HB&3HV-rich copolymers were formed. Through model-based analysis, it was hypothesised that DvGAOs prefer propionate in order to maximise their production of PHAs with the same glycogen consumption, which would enhance their growth potential in the following aerobic period. This feature may explain the more efficient PHA production by DvGAOs with propionate as the carbon substrate compared to acetate. Despite very low acetate consumption when propionate was available in excess, the presence of acetate considerably stimulated the uptake of propionate in comparison to the case where only propionate was present. This was likely due to the difference of the intracellular adenosine triphosphate (ATP) level in the two cases. A lower intracellular ATP level detected in the simultaneous presence of acetate and propionate might stimulate the glycolysis process resulting in a higher propionate uptake rate. This thesis shows that GAOs have indeed a good potential as cost-effective PHA producers. They are able to efficiently generate true copolymers of up to four 3HA monomers with desirable properties from simple carbon sources. Through manipulating the feed composition, comonomer fractions can vary in a wide range resulting in a variety of polymer properties. The contribution from this work could be very useful for the current drive to cost-effectively produce good quality PHAs to replace conventional petrol-derived polymers.

Polyhydroxyalkanoates (PHAs) are polyesters produced and stored intracellularly as reserve materials for carbon and energy by a wide range of prokaryotic microorganisms under imbalanced nutritional conditions. PHA production by pure culture has been extensively studied, enabling high production yields and a variety of polymer characteristics. Poly(3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV) (PHBV) has been commercially available under the trade name Biopol. However, the high production costs of these biopolymers are a major barrier for their widespread acceptance as substitute for traditional non-biodegradable polymers. The main costs are associated with maintaining the sterile conditions required by pure cultures, the use of expensive feed materials (eg. sugars) and also the polymer extraction process. Producing PHAs using mixed cultures (such as activated sludge) can drastically simplify the production process and significantly reduce the feed costs due to the use of cheap substrates. It also has the additional benefits in reusing waste materials. To date, substantial efforts have been put into improving PHA productivity in mixed cultures, with the characteristics of these bio-polyesters largely unexplored. The main product is typically PHB, which has several inherent deficiencies in properties. These include brittleness due to its high crystallinity, and thermal instability near its melting point of 175-180&degC. To overcome the drawbacks of PHB, non-3HB monomer units are incorporated in the bio-polymerisation process, but this generally requires the addition of specific and often complex precursor substrates. Glycogen accumulating organisms (GAOs) are emerging as an attractive alternative to other heterotrophic PHA producers due to their special metabolism. GAOs were initially identified as competitors to the polyphosphate accumulating organisms (PAOs) in alternating anaerobic/aerobic wastewater treatment systems. Under anaerobic conditions, GAOs generate energy and reducing power from glycogen hydrolysis, which are used for taking up carbon sources (eg. acetate or propionate) and their synthesis into PHAs as intracellular storage products. Under aerobic conditions, the stored PHAs is partly oxidised for energy generation with the remainder used for biomass growth and glycogen replenishment. The anaerobic hydrolysis of glycogen yields both acetyl-CoA and propionyl-CoA which can be condensed to form 3-hydroxybutyryl-CoA and 3-hydroxyvaleryl-CoA, the precursors of 3HB and 3HV. Therefore GAOs are capable of producing multiple 3- hydroxyalkanoate (3HA) monomers even when only a single carbon source (eg., acetate) is supplied. The potential of producing copolymers without addition of particular monomer-relevant carbon sources provides GAOs an advantage over other mixed culture heterotrophs in terms of polymer quality. So far, two main bacterial species have been identified to display the GAO phenotype, namely, Candidatus Competibacter phosphatis (henceforth referred to as Competibacter) and Defluviicoccus vanus-related GAOs belonging to the Alphaproteobacteria phylum (henceforth referred to as DvGAOs). This thesis focuses on the PHA production by GAOs. The capability of GAOs to produce heterogeneous PHAs from a single carbon source is investigated by characterising the PHA products and optimising the polymer productivity. Moreover, DvGAOs are comprehensively studied for their ability to yield novel four-monomer copolymers of 3HB, 3HV, 3-hydroxy-2-methylvalerate (3HMV) and 3-hydroxy-3- methylbutyrate (3HMB) with controllable composition and favourable physical properties. The main contributions from this thesis are summarised below. The polymers consisting of 3HB, 3HV and minor amounts of 3HMV and 3HMB, produced by Competibacter-dominated GAOs using acetate as a sole carbon source, were identified to be true copolymers based on a detailed characterisation using &sup1H and ¹&sup3C Nuclear Magnetic Resonance (NMR) spectroscopy. The monomer sequence distribution analysis using a known polymer characterisation model suggests that these polyesters are mixtures of random copolymers and thus likely possess desirable properties for practical use, which was confirmed experimentally. This demonstrates that GAOs have a true potential for producing high-quality polymers from a simple and cheap carbon source. The PHAs produced by GAOs under anaerobic and aerobic conditions were characterized using size exclusion chromatography (SEC) and differential scanning calorimetry (DSC). The acetate-derived copolymers possessed characteristics similar to those of commercially available PHBV products. The anaerobically produced PHA contained a relatively constant proportion of non-3HB monomers (30±5 C-mol%), irrespective of the amount of acetate assimilated. In contrast, under aerobic conditions, GAOs only produced 3HB monomers from acetate causing a gradually decreasing 3HV fraction. The 3HV fraction thus obtained ranged from 7 to 35 C-mol%, depending on the amount of acetate supplied under aerobic conditions. The PHAs produced under solely anaerobic conditions featured lower melting points and crystallinity, higher molecular weights, and narrower molecular weight distributions compared to the aerobically produced polymers. However, the anaerobic production yield was limited at 28% of dry cell weight (DCW) due to the shortage of glycogen, while aerobic production obtained a maximum polymer content of 41% based on DCW. To increase the PHA yield from anaerobic production, a novel three-stage strategy was developed. It was demonstrated to be an effective approach to optimise both the quantity and quality of the copolymers produced by GAOs. Using the Competibacterdominated GAO culture, up to 48 wt% poly(3HB-co-3HV-co-3HMV) based on DCW was achieved from acetate as the sole carbon source, close to the highest copolymer yield reported to date produced by mixed cultures but using specific precursor substrates. The optimisation method comprised of an aerobic famine, an aerobic feast, and an anaerobic feast period. The glycogen storage was enhanced through the initial two aerobic periods and hence increased the energy and reducing power available for the final anaerobic polymer synthesis step. The terpolymers/copolymers thus produced displayed high molecular weights (380-460 kg/mol) with a narrow distribution range. A feeding strategy based on pH-stat was demonstrated to achieve the automatic control of feed addition. Using a highly enriched DvGAO mixed culture (95±3%) copolymers of 3HB, 3HV, 3HMV and 3HMB with controllable monomer fractions were obtained from acetate and propionate substrates. Through manipulating the ratio of acetate and propionate in the medium, the 3HB and 3HMV monomer portions could be varied extensively (10 to 66 mol% 3HB and 2 to 41 mol% 3HMV). The microstructure study revealed that the PHAs produced were likely random copolymers or mixtures of random copolymers. These PHA products were demonstrated to possess favourable properties. The weight-average molecular weights were in the range 390-560 kg/mol, while the DSC traces showed melting temperatures in the range of 70 to 161 &degC, glass transition temperatures between -8 and 0 &degC, and melting enthalpies (ΔHm) between 9.1 and 31.5 J/g. The incorporation of considerable amounts of 3HMV and 3HMB constituents greatly lowered the crystallinity, potentially providing the ductile and tough materials required for many practical applications. The anaerobic metabolism of DvGAOs with acetate and propionate was found to be well described by the metabolic models previously proposed for GAOs and verified with experimental data obtained with other types of GAO cultures. The results suggested DvGAOs likely use metabolic pathways similar to those used by other GAOs for anaerobic acetate and propionate uptake. However, when both acetate and propionate were present simultaneously, DvGAOs took up these two carbon sources sequentially, with propionate uptake preceding acetate uptake. As a result, mixtures of 3HV&3HMV-rich copolymers and 3HB&3HV-rich copolymers were formed. Through model-based analysis, it was hypothesised that DvGAOs prefer propionate in order to maximise their production of PHAs with the same glycogen consumption, which would enhance their growth potential in the following aerobic period. This feature may explain the more efficient PHA production by DvGAOs with propionate as the carbon substrate compared to acetate. Despite very low acetate consumption when propionate was available in excess, the presence of acetate considerably stimulated the uptake of propionate in comparison to the case where only propionate was present. This was likely due to the difference of the intracellular adenosine triphosphate (ATP) level in the two cases. A lower intracellular ATP level detected in the simultaneous presence of acetate and propionate might stimulate the glycolysis process resulting in a higher propionate uptake rate. This thesis shows that GAOs have indeed a good potential as cost-effective PHA producers. They are able to efficiently generate true copolymers of up to four 3HA monomers with desirable properties from simple carbon sources. Through manipulating the feed composition, comonomer fractions can vary in a wide range resulting in a variety of polymer properties. The contribution from this work could be very useful for the current drive to cost-effectively produce good quality PHAs to replace conventional petrol-derived polymers.