The biocatalysis of a crude chlorophyllase extract, obtained from the biomass culture of Phaeodactylum tricornutum, in neat organic solvent media was investigated. The addition of selected excipients, including crown ether (enzyme:crown ether, 135:1--2.7:1, w/w), dextran (enzyme:dextran, 1:2--1:0.25, w/w), Span 40 and Span 60 (enzyme:Span, 1:2, w/w) and sodium bis (2-ethylhexyl) sulfosuccinate (enzyme:AOT, 1:12.6, w/w), to the crude solid enzyme preparation decreased the chlorophyllase activity. The effects of selected parameters, including solvent hydrophobicity (Log P, 2.40--4.45), initial water activity (aw, 0.44--0.97), agitation speed (0--200 rpm), reaction temperature (25--45°C) and enzyme concentration (1.67--5.3 mg solid enzyme/mL) on chlorophyllase activity, were investigated using a crude solid enzyme. The experimental findings showed that the highest chlorophyllase specific activity of 362.4 nmol hydrolyzed chlorophyll/g solid enzyme/min and bioconversion yield of 90.7% were obtained with the hexane/octanone mixture (98.7:1.3, v/v), aw of 0.90, agitation speed of 200 rpm, reaction temperature of 35°C and enzyme concentration of 3.33 mg solid enzyme/mL.

Optimization of the culture conditions for the biomass production of the alga Phaeodactylum tricornutum was investigated in relation to chlorophyllase activity. To obtain the highest total chlorophyllase activity, the biomass of P. tricornutum was harvested after a period of 7 days of incubation during which the incubation temperature was maintained at 18°C for 18 h during the day and 10°C for 6 h during the night. During culture incubation, illumination was provided by fluorescent lamps projecting an incident intensity of 330 mumol/m2.s and the pH of the culture was maintained at 8.4, adjusted by a stream flow of CO 2. The hydrolytic activity of a partially purified chlorophyllase extract, obtained from the fresh biomass of P. tricornutum, was investigated in an aqueous/miscible organic solvent system containing refined bleached deodorized (RBD) canola oil, and chlorophyll or pheophytin as substrate. The effect of a wide range of oil contents, chlorophyll and pheophytin concentrations, acetone concentrations, incubation temperatures and agitation speeds on the enzyme activity was studied. The optimum reaction conditions for chlorophyllase biocatalysis were determined to consist of 20% oil, 10% acetone and a 200 rpm agitation speed with optimum temperatures and substrate concentrations of 35°C and 12.6 muM for chlorophyll, and 30°C and 9.3 muM for pheophytin. The presence of RBD canola oil showed an inhibitory effect on chlorophyllase activity whereas acetone acted as an activator at low concentrations and an inhibitor at higher ones. Moreover, chlorophyllase showed a limited affinity towards pheophytin as substrate compared to that obtained for chlorophyll. Selected samples of crude commercial canola oil were analyzed for their green pigment content using high-performance liquid chromatography and chlorophyllase biocatalysis was investigated on eight varieties of crude commercial canola oil. The chlorophyllase activity was lower in th

A partially purified chlorophyllase, obtained from alga Phaeodactylum tricornutum, was assayed for its hydrolytic activity towards the pheophytin in ternary micellar systems of hexane/Tris-HCl/surfactant. A wide range of surfactants, sorbitans (Span 20, 40, 60, 80 and 85) and polysorbates (Tween 20, 40, 60, 80 and 85), was used. The use of either 50 $ mu$M of Span 85 or 1 $ mu$M of Tween 80 increased the hydrolytic activity of chlorophyllase by 110 and 23%, respectively. The optimum values of pH, enzyme content, incubation time and temperature for the hydrolytic activity of chlorophyllase were determined as 8.25, 8 $ mu$g protein/ml, 60 min and 27.5$ sp circ$C, respectively. The enzyme was assayed for its hydrolytic activity in the most appropriate ternary system containing Span 85 with purified pheophytin, as well as chlorophyll derivatives, as substrates. Moreover, the values of $V sb{ rm max}/K sb{ rm m}$ ratio for chlorophyllase, using the partially purified pheophytin as substrate, in ternary systems with Span 85 and Tween 80 as surfactants, were 0.15 and 0.08, respectively; however, the value of $V sb{ rm max}/K sb{ rm m}$ ratio for the enzyme, in the ternary system with Span 85, using purified pheophytin as substrate was 0.07. The addition of optimized amounts of individual membrane lipids, L-$ alpha$-phosphatidylcholine, L-$ alpha$-phosphatidyl-DL-glycerol and $ beta$-carotene increased the hydrolytic activity of chlorophyllase, using partially purified pheophytin as substrate, by 50, 36 and 10%, respectively, for Span 85, and 30, 48 and 15%, respectively, for Tween 80; in addition, these lipids increased the enzyme activity by 6, 23 and 31%, respectively, in the Span 85 media, using purified pheophytin as substrate. Phytol showed a competitive inhibitory effect on chlorophyllase activity in both Span and Tween systems containing partially purified pheophytin substrate; however, phytol had an uncompetitive inhibitory effect on the enzyme activity in the S

Accelerated warming of high latitude systems of the northern hemisphere is expected to cause significant changes to the hydro-ecology of Arctic lakes. To record comprehensive and meaningful baseline hydrological, limnological, and ecological conditions to which future change can be compared, all available environmental information generated on Noell Lake, NWT was compiled and synthesized. Data included: physical and geographical characteristics (bathymetric and drainage basin attributes); general regional climatology; water quality (nutrients, major anions/cations, dissolved oxygen, dissolved organic carbon); biological composition (fish community, macrophyte, phytoplankton, epiphyton and epipelon surveys) and seasonal patterns in primary productivity (as measured by chlorophyll-a (Chl-a)). A field-monitoring study was conducted from September 2010 to July 2013 assessing the application, reliability, and quality control/quality assurance of a newly developed automated buoy-based Arctic Lake Monitoring System (ALMS). The ALMS continuously measured a range of lake limnological and water quality parameters under both open-water and under-ice conditions. Overall, the ALMS provided a usable, uninterrupted record of changes in measured environmental, hydrological, and limnological parameters in both the epilimnion and hypolimnion. Noell Lake was determined to be spatially homogeneous with respect to the limnological measurements taken and, thus, the data recorded by the instrument arrays were determined to be representative of the lake as a whole. In addition to the measurements made by environmental sensors mounted on the buoy and mooring components, an augmentary array of in-situ sampling campaigns and controlled experiments were conducted to produce a continuous and comprehensive description of daily and seasonal changes to the hydrological and limnological conditions of Noell Lake. The continuous data series confirmed that Noell Lake is dimictic, with mixing events occurring in August and June, and hypoxic oxygen conditions occurring in March. Nutrient limitation experiments revealed that autotrophic productivity in Noell Lake was nitrogen-limited. Compiling data from existing literature involved >700 northern, high-latitude lakes; patterns in temporal and latitudinal changes in Arctic lake primary productivity (as measured by open-water, epilimnion Chl-a) and geochemistry were assessed. The key hypothesis tested was whether Arctic lakes are showing increased primary productivity (i.e., “greening”), through time and by latitude, similar to that documented for Arctic terrestrial systems. In general, significant decreases in lake Chl-a was observed in Arctic and sub-Arctic lakes over a ≈50 year time span. Separation of lakes by latitudinal bands revealed that trends in the lower Arctic region (60.00-69.99 Degrees North) showed a significant decreasing time trend, while high Arctic lakes displayed no trends. Corresponding temporal trends of total phosphorous (TP), total nitrogen (TN), and dissolved organic carbon (DOC) differed depending on the latitude of the lakes. Re-evaluation of the original northern-lake productivity models developed by Flanagan et al. (2003) through the use of the new, independent datasets (>700 lakes) as well as the addition of other environmental variables (DOC, dissolved inorganic carbon, lake depth, conductivity, and ice-cover) showed that the original models were valid and the most parsimonious in predicting variation in algal biomass in northern latitude lakes. Only measures of dissolved nutrients (TP, TN) and latitude are required to predict autotrophic water column productivity. / Graduate

The chlorophyll degradation pathway is central to a number of plant processes including senescence and fruit ripening. However, the regulation of the chlorophyll degradation pathway enzymes is not well understood. The aim of this thesis was to elucidate the genetic mechanisms that control changes in pigment composition leading to fruit flesh yellowing in kiwifruit. Actinidia deliciosa and A. chinensis fruit, which are green and yellow, respectively, provide an opportunity to study the regulation of chlorophyll levels. The expression of genes that code for enzymes of the chlorophyll and cytokinin metabolic pathways was measured using qRT-PCR. Candidates for chlorophyll degradation regulatory points were then characterised for functionality by transient transformation in N. benthamiana. The endogenous cytokinin levels were measured in kiwifruit and transient activation assays were carried out with the promoters of key candidate genes. Overall, expression of the chlorophyll degradation genes was elevated in yellow fruit and expression of biosynthetic genes was higher in green fruit. The chlorophyll degradation-associated protein, STAY-GREEN2 (SGR2), was more highly expressed in yellow fruit, and transient over-expression of SGR was sufficient to drive chlorophyll degradation. Expression of isopentenyl transferase (IPT), the rate-limiting step for cytokinin biosynthesis, showed an increase towards maturity in green fruit, but not in yellow fruit. However, both fruit had similar high levels of cytokinin nucleotides and free bases. A gene coding for O-glucosylation was also highly expressed in green fruit. Green fruit contained higher levels of cytokinin O-glucosides and ribosides towards maturity, suggesting differences in cytokinin signalling, which could lead to regulation of chlorophyll levels via activation of the SGR promoter by transcription factors. It is likely that the chlorophyll degradation pathway and cytokinin metabolism are linked. The differential expression of cytokinin response regulators could lead to differential regulation of cytokinin levels in the fruit of the two species, and possibly differential regulation of the chlorophyll degradation pathway. Progress towards elucidation of the control of chlorophyll levels provides knowledge of this key process in kiwifruit and potentially gene-based markers for breeding new kiwifruit cultivars.

Information regarding the structure and function of living tissues and cells is instrumental to the advancement of cell biology and biophysics. Nonlinear optical microscopy can provide such information, but only certain biological structures generate nonlinear optical signals. Therefore, structural specificity can be achieved by introducing labels for nonlinear optical microscopy. Few studies exist in the literature about labels that facilitate harmonic generation, coined "harmonophores". This thesis consists of the first major investigation of harmonophores for third harmonic generation (THG) microscopy. Carotenoids and chlorophylls were investigated as potential harmonophores. Their nonlinear optical properties were studied by the THG ratio technique. In addition, a tunable refractometer was built in order to determine their second hyperpolarizability (γ). At 830 nm excitation wavelength, carotenoids and chlorophylls were found to have large negative γ values however, at 1028 nm, the sign of γ reversed for carotenoids and remained negative for chlorophylls. Consequently, at 1028 nm wavelength, THG signal is canceled with mixtures of carotenoids and chlorophylls. Furthermore, when such molecules are covalently bonded as dyads or interact within photosynthetic pigment-protein complexes, it is found that additive effects with the γ values still play a role, however, the overall γ value is also influenced by the intra-pigment and inter-pigment interaction. The nonlinear optical properties of aggregates containing chlorophylls and carotenoids were the target of subsequent investigations. Carotenoid aggregates were imaged with polarization-dependent second harmonic generation and THG microscopy. Both techniques revealed crystallographic information pertaining to H and J aggregates and β-carotene crystalline aggregates found in orange carrot. In order to demonstrate THG enhancement due to labeling, cultured cells were labeled with carotenoid incorporated liposomes. In addition, Drosophila melanogaster larvae muscle as well as keratin structures in the hair cortex were labeled with β-carotene. Polarization-dependent THG studies may be particularly useful in understanding the structural organization that occurs within biological structures containing carotenoids and chlorophylls such as photosynthetic pigment-protein complexes and carotenoid aggregates in plants and alga. Further, artificial labeling with carotenoids and chlorophylls may be useful in clinical applications since they are nontoxic, nutritionally valuable, and they can aid in visualizing structural changes in cellular components.

The on-going calibration and validation of visible satellite imagery remains a core activity of the scientific community in pursuit of high quality data characterising the oceanic chlorophyll field, providing input to models assessing primary productivity and the potential role of the oceans in climatic regulation. This work serves to examine the operational characteristics of semi-analytical algorithms that are designed to derive key optical properties from space born observations of ocean colour. The collection of water samples contemporaneously with precision profiled radiometry conforming to similar spectral bands to those of the NASA Sea Viewing Wide Field of view Sensor (SeaWiFS) was planned and executed, with appropriate field sampling techniques developed in accordance with the SeaWiFS Ocean Optics protocols (Mueller and Austin, 1995). Data were collected during extensive fieldwork sampling at a near coastal survey site and during two deep Atlantic research programmes. Historical and recently developed algorithms designed to retrieve the diffuse attenuation coefficient at 490nm and chlorophyll-a pigment concentrations from upwelling radiances were applied to the optical data, to compare the mathematically retrieved in-water properties with the values measured in-situ. The radiometric data were then used to generate general and local algorithm modifications to assess possible differences in the mathematically retrieved values. Statistical analyses of the errors in mathematical retrieval of in-water properties identified stmctured variability resulting from the empirical approach to algorithm generation, supporting the point of view that locally constrained algorithms provide a method of achieving significantly improved results. The problems associated with the derivation of semi-analytical algorithms are then discussed and errors analysed. The new algorithms generated here are found to compare well with their source data and with work by other investigators. Systematic variability was found within the data sets and the affect this has on the determinations is discussed. It is suggested that data users be afforded details of the equations employed in the production of readily available remote sensing products, placing them in a position where they are better able to assess the data in the context of their work.

Zooplankton play a vital role in the world's oceans in terms of transport of carbon out of the surface layer and providing food for fish. Zooplankton are patchily distributed on all scales, and this has important consequences for both sampling and understanding their role in the ocean. The distribution of zooplankton on different scales forms the focus of this study. Three Atlantic Meridional Transect (AMT) cruises were carried out and data made available from three previous cruises. Zoo plankton data were collected using a combination of vertical nets and using an optical plankton counter (OPC) sampling from the pumped seawater supply. Validation of methods showed that the OPC data could reliably be converted to carbon and numerical abundance estimates for open ocean conditions. Spectral analysis suggested that surface zooplankton heterogeneity followed a power law relationship over several scales. Over the 30 to 1000 km range this was approximately -1, and for smaller and larger scales the slope was reduced. Chlorophyll was less patchy, following temperature and salinity over the same range with a slope of -1.8. Analysis of large scale heterogeneity showed clear latitudinal trends in diversity, particularly evident in the copepod genera, with low diversity at high latitudes. The size structure appeared to be more closely related to the productivity of the area, with high zooplankton biomass associated with larger zooplankton. Regions with similar copepod communities were identified. These were found to be similar to other pelagic regions, but less closely related to watermasses or production regimes. Multiple linear regression of surface zooplankton biomass showed a strong relationship with the physics (temperature and salinity), chlorophyll and the time of day, accounting for 55% of the variability. Use of the regression equations to predict new transects gave R²=0.34. Improvement could be made by dividing the transect into smaller regions. Neural networks gave enhanced predictability (R² = 0.77 for the training set, and R²= 0.47 for the novel set) with a simpler model, although similar variables were important. This study has shown that copepods show latitudinal gradient in diversity, associated with seasonality, and form regions of similarity that do not conform to biogeochemical provinces or the watermasses. Neural networks may be used to predict zooplankton abundance from a few readily available parameters.

No description available.

Marine phytoplankton ecophysiological state and primary production measurements have typically been controversial due to potential impacts of measurement techniques. Advances in chl-a fluorescence techniques have provided a means for rapid, non-invasive measurement of electron transport through photosystem 2 (PSII) in dilute phytoplankton suspensions. While studies on higher plants have outlined a close relationship between PSII electron transport and carbon fixation, results from studies on microalgae reveal significant variations in the relationship. Three species of phytoplankton representing three major taxonomic groups of the marine phytoplankton were used in this study: (1) Chaetoceros muelleri CS176 Lemmermann (Bacillariophyta), (2) Isochrysis galbana CS177 Parke (Haptophyta) and, (3) Nannochloropsis oculata CS179 (Droop) Hibberd (Ochrophyta, eustigmatophyte). Each species was cultured in semicontinuous culture and primary production was estimated using oxygen evolution and carbon fixation techniques and compared against predictions based on chl-a fluorescence measurements. It was found that predicted values of primary production both under-estimated and overestimated actual carbon fixation measured via radioisotope (14C) techniques. This variation was primarily explained by probable errors in the assumed values for PSII density. The relationship between oxygen evolution or carbon fixation with chl-a fluorescence-derived measures was commonly linear below the light saturation parameter, with a departure from linearity occurring at higher irradiances. This departure from linearity was greatest in cultures adapted to low light conditions. At higher light intensities alternative electron pathways such as the Mehler reaction and/or chlororespiration are likely to be more active in low light-adapted cultures, leading to this greater non-linearity. Chl-a fluorescence measurements were also found to be a useful in characterising ecophysiology using photosynthesis-versus irradiance curves. However, an important caveat on this is the measurement of PSII density (çPSII) rather than use of an assumed value as changes in çPSII can have a profound impact on light curve parameters. A field study in Fremantle Harbour found a healthy (negligible nutrient starvation), diatom dominated, phytoplankton community. Results suggest that phytoplankton are able to begin boosting photosynthetic capability just prior to morning twilight. Waters in the harbour were well mixed via tidal motion and substantial midday photoinhibition was not observed. Data suggest levels of primary production at the mouth of the harbour are similar to those of coastal waters in the plume of the Ocean Reef wastewater outfall.