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.

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.

The following research has focused on the utility of Ulva as an indicator of seawater nitrogen loading. Evaluation was made in three ways: 1) Observation of large-scale geographic variation in nitrogen status in natural populations around New Zealand in summer and winter, 2) Laboratory-based experimental assessment of the biochemical responses of N-indices in Ulva to nitrogen enrichment, and 3) Culturing standardized test-Ulva under low nutrient conditions which could be deployed into a variety of field situations. Seawater inorganic nutrient (nitrate, nitrite, ammonium and phosphate) concentrations and nitrogen (N)-indices (free amino acids, chlorophyll and total tissue nitrogen) in natural Ulva populations from 32 sites around New Zealand were compared. Sites were divided into 6 environmental categories: sheltered rural, exposed rural, rock pools, sheltered urban, exposed urban, and nitrogen-enriched urban sites. Seawater nutrient concentrations were highly variable between all sites in summer and winter. However, in the summer enriched urban sites had the highest mean total inorganic nitrogen concentrations and Ulva with the highest mean levels of all N-indices compared with any other environmental category. In the winter, Ulva contained more nitrogen (reflected in all N-indices) compared with Ulva in the summer, particularly in populations growing in colder southern seawater on more exposed coasts. The increase in Ulva N-status was not explained by increased seawater inorganic nitrogen concentrations. With univariate and multivariate statistical approaches it was shown that there was a significant effect of seawater temperature and site exposure on N-status in Ulva. Compared with other N-indices, stable nitrogen isotopes (δ15N) from Ulva growing in enriched urban sites had the widest range (4.77 ± 0.04 ‰ to 15.16 ± 0.03 ‰) of values compared with all other categories in both summer and winter. Conversely, Ulva from exposed rural sites had the lowest range of δ15N values compared with any other category (6.7 ± 0.1 to 8.8 ± 0.1 ‰) and showed no seasonal change in mean values (7.8 ‰ and 7.6 ‰ for summer and winter, respectively). In addition, δ15N values in Ulva were the only N-index that showed a significant difference between urban and rural categories. To test the relationship between inorganic nitrogen concentration in seawater and the responses of biochemical nitrogen indices in Ulva pertusa, several experiments were conducted in an outdoor, flow-through culture apparatus, in summer and winter. In this apparatus effects of ammonium concentration, nitrogen source (nitrate and ammonium), light and seawater motion were investigated. Of the same three N-indices examined in natural Ulva populations (free amino acids, chlorophyll and total tissue nitrogen), increases in free amino acids, particularly asparagine, provided the strongest indicator of increases in nitrogen availability. In addition, while tissue nitrogen and chlorophyll also increased with seawater nitrogen concentration, it was apparent that these indices were also strongly influenced by light, and probably season. Rates of ammonium assimilation provided no overall measure of the availability of nitrogen in seawater and were clearly affected by season. Similarly, growth rates in Ulva only showed a response to nitrogen addition in summer months. Stable isotopes of nitrogen (δ15N) in Ulva provided a clear distinction between natural and synthetic nitrogen sources, but more importantly, showed only minor fractionation (ranging from 1.3 ‰ to -1.9 ‰) of 15N supplied from synthetic nitrate and ammonium under both light-saturating and light-limiting conditions. To further develop Ulva as a standardized test-organism it was cultured in low-nutrient (non-polluted) seawater to deplete internal storage pools of nitrogen. Each month the resulting test-Ulva was then placed in surface-moored growth enclosures at a range of coastal sites around Auckland and then monitored for one year. In winter there were increases in seawater inorganic nitrogen concentrations and concomitant increases in free amino acid content. However, tissue nitrogen and chlorophyll content in test-Ulva showed similar increases (possibly saturating) across all sites suggesting that seasonal increases in these N-indices were also due to other seasonal factors (e.g., surface irradiance and / or seawater temperature). On the other hand, the total free amino acid pool showed strong differences between a low-nitrogen reference site and the other study sites all year round. It was probable that test-Ulva was integrating differences in tidally-averaged nitrogen loading that were not reliably detected in instantaneous seawater samples. In addition to N-indices in test-Ulva, levels of tissue heavy metals and stable isotopes of nitrogen showed strong differences with higher values of both typically found in urban environments compared with values found in non-polluted reference sites. It is concluded that several abiotic and biotic factors affect nitrogen status in Ulva, but the average nitrogen concentration in seawater, and the physical factors of temperature, light and water motion, appear to be the overarching determinants. It is further suggested that in combination with Ulva tissue δ15N values, tissue nitrogen and the free amino acid pool, as quantitative biochemical measures of nitrogen availability, are likely to provide useful information on both the amount and composition of nitrogen entering coastal environments. / Foundation for Research, Science and Technology. Auckland Regional Council.

Cirsium arvense (L.) Scop. (Californian, Canada, or creeping thistle) is an exotic perennial herb indigenous to Eurasia that successfully established in New Zealand (NZ) approximately 130 years ago. Presently, C. arvense is considered one of the worst invasive weeds in NZ arable and pastoral productions systems. The mechanism most commonly invoked to explain the apparent increased vigour of introduced weeds is release from natural enemies. The enemy release hypothesis (ERH) predicts that plants in an introduced range should experience reduced herbivory, particularly from specialists, and that release from this natural enemy pressure facilitates increased plant performance in the introduced range. In 2007 broad surveys were carried out in NZ and central Europe in order to quantify and compare growth characteristics of C. arvense in its native vs. introduced range. Additionally, permanent field plots were established in NZ and Europe where natural enemies were excluded with the use of insecticide and fungicide applications, and compared with controls (ambient natural enemy pressure). The impact of the specialist leaf-feeding beetle, Cassida rubiginosa Müller, which was recently released in NZ as a biological control agent against thistles, was also assessed. From the field surveys, significantly more endophagous herbivory was present in the native range compared to the introduced range, as predicted by the ERH. Endophagous herbivory in NZ was solely from the capitulum-feeding weevil, Rhinocyllus conicus (Frölich), and was only found in the North Island surveys. No stem mining attack was found anywhere in NZ. The proportion of shoots attacked by the specialised rust pathogen, Puccinia punctiformis (Str.) Röhl., was similar in both the native and introduced ranges. Interestingly, this has casted doubt on the idea that stem-mining vectors, such as Ceratapion onopordi Kirby, are important for transmission of the rust pathogen. Contrary to the ERH, there were no significant difference in plant performance between the native and introduced ranges, or differences could be explained by simple climatic factors. Climate tended to be more favourable for growth of C. arvense in NZ. In the permanent field plots in the native range, population growth of C. arvense was significantly greater where natural enemies were excluded, suggesting that insect herbivores and pathogens might have a regulating influence on the population growth of this plant. Furthermore, the probability of shoots transitioning to the reproductive growth stage was enhanced when insect herbivores were excluded, indicating that natural enemies might influence plant development. The biological control agent C. rubiginosa reduced the growth of C. arvense, although the impact of this herbivore was minimal in comparison to interspecific plant competition. Thus, although there is reduced specialist natural enemy pressure in NZ, the growth of C. arvense is not significantly different from in its native range. Nevertheless, there is some evidence that natural enemies in the native range might have a regulating influence on the population dynamics of the plant, and that the specialist herbivore, C. rubiginosa, can impact the plant in certain conditions.