Capacity of fatty acid profiles and substrate utilization patterns to describe differences in soil microbial communities associated with increased salinity or alkalinity at three locations in South Australia

Fatty acid methyl ester (FAME) profiles, together with Biolog substrate utilization patterns, were used in conjunction with measurements of other soil chemical and microbiological properties to describe differences in soil microbial communities induced by increased salinity and alkalinity in grass/legume pastures at three sites in SE South Australia. Total ester-linked FAMEs (EL-FAMEs) and phospholipid-linked FAMEs (PL-FAMEs), were also compared for their ability to detect differences between the soil microbial communities. The level of salinity and alkalinity in affected areas of the pastures showed seasonal variation, being greater in summer than in winter. At the time of sampling for the chemical and microbiological measurements (winter) only the affected soil at site 1 was significantly saline. The affected soils at all three sites had lower organic C and total N concentrations than the corresponding non-affected soils. At site 1 microbial biomass, CO 2-C respiration and the rate of cellulose decomposition was also lower in the affected soil compared to the non-affected soil. Biomarker fatty acids present in both the EL- and PL-FAME profiles indicated a lower ratio of fungal to bacterial fatty acids in the saline affected soil at site 1. Analysis of Biolog substrate utilization patterns indicated that the bacterial community in the affected soil at site 1 utilized fewer carbon substrates and had lower functional diversity than the corresponding community in the non-affected soil. In contrast, increased alkalinity, of major importance at sites 2 and 3, had no effect on microbial biomass, the rate of cellulose decomposition or functional diversity but was associated with significant differences in the relative amounts of several fatty acids in the PL-FAME profiles indicative of a shift towards a bacterial dominated community. Despite differences in the number and relative amounts of fatty acids detected, principal component analysis of the EL- and PL-FAME profiles were equally capable of separating the affected and non-affected soils at all three sites. Redundancy analysis of the FAME data showed that organic C, microbial biomass, electrical conductivity and bicarbonate-extractable P were significantly correlated with variation in the EL-FAME profiles, whereas pH, electrical conductivity, NH 4-N, CO 2-C respiration and the microbial quotient were significantly correlated with variation in the PL-FAME profiles. Redundancy analysis of the Biolog data indicated that cation exchange capacity and bicarbonate-extractable K were significantly correlated with the variation in Biolog substrate utilization patterns.

Patterns of diversity in fatty acid composition in the Australian groundnut germplasm collection

Knowledge of the amounts and types of fatty acids in groundnut oil is beneficial, particularly from a nutritional standpoint. Germplasm evaluation data for fatty acid composition on 819 accessions of groundnut (Arachis hypogaea L.) from the Australian Tropical Field Crops Genetic Resource Centre, Biloela, Queensland were examined. Data for eight quantitative fatty acid descriptors have been documented. Statistical assessment, via methods of pattern analysis, summarised and described the patterns of variation in fatty acid composition of the groundnut accessions in the Australian germplasm collection. Presentation of the results from principal components analysis and hierarchical cluster analysis using a biplot was shown to be a very useful interpretative tool. Such a biplot enables a simultaneous examination of the relationships among all the accessions and the fatty acids. Unlike that information available via database searches, the results from contribution analysis together with the biplot provide a global picture of the diversity available for use in plant breeding programs. The use of standardised data for eight fatty acids, compared to using three specific fatty acids, provided a better description of the total diversity available because it remains relevant with possible changes in the nutritional preferences for fatty acids. Fatty acid composition was found to vary in relation to the branching pattern of the accessions. This pattern is generally indicative of the botanical types of groundnuts; Virginia (alternate) compared to Spanish and Valencia (sequential) botanical types.

Weight-related differences in glucose metabolism and free fatty acid production in two South African population groups

OBJECTIVE The effects of free fatty acids (FFA), leptin, tumour necrosis factor (TNF) alpha and body fat distribution on in vivo oxidation of a glucose load were studied in two South African ethnic groups. DESIGN AND MEASUREMENTS Anthropometric and various metabolic indices were measured at fasting and during a 7h oral glucose tolerance test (OGTT). Body composition was measured using bioelectrical impedance analysis and subcutaneous and visceral fat mass was assessed using a five- and two-level CT-scan respectively. Glucose oxidation was evaluated by measuring the ratio of (13)CO(2) to (12)CO(2) in breath following ingestion of 1-(13)C-labelled glucose. SUBJECTS Ten lean black women (LBW), ten obese black women (OBW), nine lean white women (LWW) and nine obese white women (OWW) were investigated after an overnight fast. RESULTS Visceral fat levels were significantly higher (P < 0.01) in obese white than black women, despite similar body mass indexes (BMIs). There were no ethnic differences in glucose oxidation however; in the lean subjects of both ethnic groups the area under the curve (AUC) was higher than in obese subjects (P < 0.05 for both) and was found to correlate negatively with weight (r = -0.69, P < 0.01) after correcting for age. Basal TNF alpha concentrations were similar in all groups. Percentage suppression of FFAs at 30 min of the OCTT was 24 +/- 12% in OWW and - 38 +/- 23% (P < 0.05) in OBW, ie the 30 min FFA level was higher than the fasting level in the latter group. AUC for FFAs during the late postprandial period (120 - 420 min) was significantly higher in OWW than OBW (P < 0.01) and LWW (P < 0.01) and correlated positively with visceral fat mass independent of age (r = 0.78, P < 0.05) in the OWW only. Leptin levels were higher (P < 0.01) both at fasting and during the course of the OCTT in obese women from both ethnic groups compared to the lean women. CONCLUSIONS Glucose oxidation is reduced in obese subjects of both ethnic groups; inter- and intra-ethnic differences were observed in visceral fat mass and FFA production and it is possible that such differences may play a role in the differing prevalences of obesity-related disorders that have been reported in these two populations.

The fatty acid synthase inhibitor triclosan : repurposing an anti-microbial agent for targeting prostate cancer

Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer.

Molecular markers of obesity and diabetes

Recently it has been shown that the consumption of a diet high in saturated fat is associated with impaired insulin sensitivity and increased incidence of type 2 diabetes. In contrast, diets that are high in monounsaturated fatty acids (MUFAs) or polyunsaturated fatty acids (PUFAs), especially very long chain n-3 fatty acids (FAs), are protective against disease. However, the molecular mechanisms by which saturated FAs induce the insulin resistance and hyperglycaemia associated with metabolic syndrome and type 2 diabetes are not clearly defined. It is possible that saturated FAs may act through alternative mechanisms compared to MUFA and PUFA to regulate of hepatic gene expression and metabolism. It is proposed that, like MUFA and PUFA, saturated FAs regulate the transcription of target genes. To test this hypothesis, hepatic gene expression analysis was undertaken in a human hepatoma cell line, Huh-7, after exposure to the saturated FA, palmitate. These experiments showed that palmitate is an effective regulator of gene expression for a wide variety of genes. A total of 162 genes were differentially expressed in response to palmitate. These changes not only affected the expression of genes related to nutrient transport and metabolism, they also extend to other cellular functions including, cytoskeletal architecture, cell growth, protein synthesis and oxidative stress response. In addition, this thesis has shown that palmitate exposure altered the expression patterns of several genes that have previously been identified in the literature as markers of risk of disease development, including CVD, hypertension, obesity and type 2 diabetes. The altered gene expression patterns associated with an increased risk of disease include apolipoprotein-B100 (apo-B100), apo-CIII, plasminogen activator inhibitor 1, insulin-like growth factor-I and insulin-like growth factor binding protein 3. This thesis reports the first observation that palmitate directly signals in cultured human hepatocytes to regulate expression of genes involved in energy metabolism as well as other important genes. Prolonged exposure to long-chain saturated FAs reduces glucose phosphorylation and glycogen synthesis in the liver. Decreased glucose metabolism leads to elevated rates of lipolysis, resulting in increased release of free FAs. Free FAs have a negative effect on insulin action on the liver, which in turn results in increased gluconeogenesis and systemic dyslipidaemia. It has been postulated that disruption of glucose transport and insulin secretion by prolonged excessive FA availability might be a non-genetic factor that has contributed to the staggering rise in prevalence of type 2 diabetes. As glucokinase (GK) is a key regulatory enzyme of hepatic glucose metabolism, changes in its activity may alter flux through the glycolytic and de novo lipogenic pathways and result in hyperglycaemia and ultimately insulin resistance. This thesis investigated the effects of saturated FA on the promoter activity of the glycolytic enzyme, GK, and various transcription factors that may influence the regulation of GK gene expression. These experiments have shown that the saturated FA, palmitate, is capable of decreasing GK promoter activity. In addition, quantitative real-time PCR has shown that palmitate incubation may also regulate GK gene expression through a known FA sensitive transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), which upregulates GK transcription. To parallel the investigations into the mechanisms of FA molecular signalling, further studies of the effect of FAs on metabolic pathway flux were performed. Although certain FAs reduce SREBP-1c transcription in vitro, it is unclear whether this will result in decreased GK activity in vivo where positive effectors of SREBP-1c such as insulin are also present. Under these conditions, it is uncertain if the inhibitory effects of FAs would be overcome by insulin. The effects of a combination of FAs, insulin and glucose on glucose phosphorylation and metabolism in cultured primary rat hepatocytes at concentrations that mimic those in the portal circulation after a meal was examined. It was found that total GK activity was unaffected by an increased concentration of insulin, but palmitate and eicosapentaenoic acid significantly lowered total GK activity in the presence of insulin. Despite the fact that total GK enzyme activity was reduced in response to FA incubation, GK enzyme translocation from the inactive, nuclear bound, to active, cytoplasmic state was unaffected. Interestingly, none of the FAs tested inhibited glucose phosphorylation or the rate of glycolysis when insulin is present. These results suggest that in the presence of insulin the levels of the active, unbound cytoplasmic GK are sufficient to buffer a slight decrease in GK enzyme activity and decreased promoter activity caused by FA exposure. Although a high fat diet has been associated with impaired hepatic glucose metabolism, there is no evidence from this thesis that FAs themselves directly modulate flux through the glycolytic pathway in isolated primary hepatocytes when insulin is also present. Therefore, although FA affected expression of a wide range of genes, including GK, this did not affect glycolytic flux in the presence of insulin. However, it may be possible that a saturated FA-induced decrease in GK enzyme activity when combined with the onset of insulin resistance may promote the dys-regulation of glucose homeostasis and the subsequent development of hyperglycaemia, metabolic syndrome and type 2 diabetes.

Combustion analysis of microalgae methyl ester in a common rail direct injection diesel engine

In this study, the biodiesel properties and effects of blends of oil methyl ester petroleum diesel on a CI direct injection diesel engine is investigated. Blends were obtained from the marine dinoflagellate Crypthecodinium cohnii and waste cooking oil. The experiment was conducted using a four-cylinder, turbo-charged common rail direct injection diesel engine at four loads (25%, 50%, 75% and 100%). Three blends (10%, 20% and 50%) of microalgae oil methyl ester and a 20% blend of waste cooking oil methyl ester were compared to petroleum diesel. To establish suitability of the fuels for a CI engine, the effects of the three microalgae fuel blends at different engine loads were assessed by measuring engine performance, i.e. mean effective pressure (IMEP), brake mean effective pressure (BMEP), in cylinder pressure, maximum pressure rise rate, brake-specific fuel consumption (BSFC), brake thermal efficiency (BTE), heat release rate and gaseous emissions (NO, NOx,and unburned hydrocarbons (UHC)). Results were then compared to engine performance characteristics for operation with a 20% waste cooking oil/petroleum diesel blend and petroleum diesel. In addition, physical and chemical properties of the fuels were measured. Use of microalgae methyl ester reduced the instantaneous cylinder pressure and engine output torque, when compared to that of petroleum diesel, by a maximum of 4.5% at 50% blend at full throttle. The lower calorific value of the microalgae oil methyl ester blends increased the BSFC, which ultimately reduced the BTE by up to 4% at higher loads. Minor reductions of IMEP and BMEP were recorded for both the microalgae and the waste cooking oil methyl ester blends at low loads, with a maximum of 7% reduction at 75% load compared to petroleum diesel. Furthermore, compared to petroleum diesel, gaseous emissions of NO and NOx, increased for operations with biodiesel blends. At full load, NO and NOx emissions increased by 22% when 50% microalgae blends were used. Petroleum diesel and a 20% blend of waste cooking oil methyl ester had emissions of UHC that were similar, but those of microalgae oil methyl ester/petroleum diesel blends were reduced by at least 50% for all blends and engine conditions. The tested microalgae methyl esters contain some long-chain, polyunsaturated fatty acid methyl esters (FAMEs) (C22:5 and C22:6) not commonly found in terrestrial-crop-derived biodiesels yet all fuel properties were satisfied or were very close to the ASTM 6751-12 and EN14214 standards. Therefore, Crypthecodinium cohnii- derived microalgae biodiesel/petroleum blends of up to 50% are projected to meet all fuel property standards and, engine performance and emission results from this study clearly show its suitability for regular use in diesel engines.

Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

Interaction of erythrocyte eicosapentaenoic acid and physical activity predicts reduced risk of mild cognitive impairment

Objectives To evaluate relationships between self-reported physical activity, proportions of long-chain omega-3 polyunsaturated fatty acids (LCn3) in erythrocyte content (percentage of total fatty acids) and risk of mild cognitive impairment (MCI) in older adults. Method A cross-sectional study was conducted. Community-dwelling male and female (n = 84) participants over the age of 65 years with and without MCI were tested for erythrocyte proportions of the LCn3s eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Physical activity was measured using a validated questionnaire. Results The interaction between erythrocyte EPA, but not DHA, and increased physical activity was associated with increased odds of a non-MCI classification. Conclusion An interaction between physical activity and erythrocyte EPA content (percentage of fatty acids) significantly predicted MCI status in older adults. Randomised control trials are needed to examine the potential for supplementation with EPA in combination with increased physical activity to mitigate the risk of MCI in ageing adults.

Ethyl-eicosapentaenoic acid in first-episode psychosis : a 1H-MRS study

Ethyl-eicosapentaenoic acid (E-EPA) is an omega-3 fatty acid that has been used in a range of neuropsychiatric conditions with some benefits. However, its mechanism of action is unknown. Here, we investigate its effects on in vivo brain metabolism in first-episode psychosis (FEP). Proton magnetic resonance spectroscopy at 3 T was performed in the temporal lobes of 24 FEP patients before and after 12 weeks of treatment in the context of a larger double-blind, placebo-controlled E-EPA augmentation study. Treatment group effects for glutathione (F1,12=6.1, p=0.03), and a hemisphere-by-group interaction for glutamine/glutamate (F1,20=4.4, p=0.049) were found. Glutathione increased bilaterally and glutamate/glutamine increased in the left hemisphere following E-EPA administration. Improvement in negative symptoms correlated with metabolic brain changes, particularly glutathione (r=-0.57). These results suggest that E-EPA augmentation alters glutathione availability and modulates the glutamine/glutamate cycle in early psychosis, with some of the metabolic brain changes being correlated with negative symptom improvement. Larger confirmatory studies of these postulated metabolic brain effects of E-EPA are warranted.

Mass spectrometric study of the dissociation of Group XI metal complexes with fatty acids and glycerolipids : Ag2H+ and Cu2H+ ion formation in the presence of a double bond

Results of mass spectrometric studies are reported for the collisional dissociation of Group XI (Cu, Ag, Au) metal ion complexes with fatty acids (palmitic, oleic, linoleic and a-linolenic) and glycerolipids. Remarkably, the formation of M2H+ ions (M = Cu, Ag) is observed as a dissociation product of the ion complexes containing more than one metal cation and only if the lipid in the complex contains a double bond. Ag2H+ is formed as the main dissociation channel for all three of the fatty acids containing double bonds that were investigated while Cu2H+ is formed with one of the fatty acids and, although abundant, is not the dominant dissociation channel. Also. Cu(I) and Ag(I) ion complexes were observed with glycerolipids (including triacylglycerols and glycerophospholipids) containing either saturated or unsaturated fatty acid substituents. Interestingly. Ag2H+ ion is formed in a major fragmentation channel with the lipids that are able to form the complex with two metal cations (triacylglycerols and glycerophosphoglycerols), while lipids containing a fixed positive charge (glycerophospocholines) complex only with a single metal cation. The formation of Ag2H+ ion is a significant dissociation channel from the complex ion Ag-2(L-H)(+) where L = Glycerophospholipid (GP) (18:1/18:1). Cu(I) also forms complexes of two metal cations with glycerophospholipids but these do not produce Cu2H+ upon dissociation. Rather organic fragments, not containing Cu(I), are formed, perhaps due to different interactions of these metal cations with lipids resulting from the much smaller ionic radius of Cu(I) compared to Ag(I) (C).

Evaluation of a pilot-scale oil extraction from microalgae for biodiesel production

Biodiesel derived from microalgae is one of a suite of potential solutions to meet the increasing demand for a renewable, carbon-neutral energy source. However, there are numerous challenges that must be addressed before algae biodiesel can become commercially viable. These challenges include the economic feasibility of harvesting and dewatering the biomass and the extraction of lipids and their conversion into biodiesel. Therefore, it is essential to find a suitable extraction process given these processes presently contribute significantly to the total production costs which, at this stage, inhibit the ability of biodiesel to compete financially with petroleum diesel. This study focuses on pilot-scale (100 kg dried microalgae) solvent extraction of lipids from microalgae and subsequent transesterification to biodiesel. Three different solvents (hexane, isopropanol (IPA) and hexane + IPA (1:1)) were used with two different extraction methods (static and Soxhlet) at bench-scale to find the most suitable solvent extraction process for the pilot-scale. The Soxhlet method extracted only 4.2% more lipid compared to the static method. However, the fatty acid profiles of different extraction methods with different solvents are similar, suggesting that none of the solvents or extraction processes were biased for extraction of particular fatty acids. Considering the cost and availability of the solvents, hexane was chosen for pilot-scale extraction using static extraction. At pilot-scale the lipid yield was found to be 20.3% of total biomass which is 2.5% less than from bench scale. Extracted fatty acids were dominated by polyunsaturated fatty acids (PUFAs) (68.94±0.17%) including 47.7±0.43 and 17.86±0.42% being docosahexaenoic acid (DHA) (C22:6) and docosapentaenoic acid (DPA) (C22:5, ω-3), respectively. These high amounts of long chain poly unsaturated fatty acids are unique to some marine microalgae and protists and vary with environmental conditions, culture age and nutrient status, as well as with cultivation process. Calculated physical and chemical properties of density, viscosity of transesterified fatty acid methyl esters (FAMEs) were within the limits of the biodiesel standard specifications as per ASTM D6751-2012 and EN 14214. The calculated cetane number was, however, significantly lower (17.8~18.6) compared to ASTM D6751-2012 or EN 14214-specified minimal requirements. We conclude that the obtained microalgal biodiesel would likely only be suitable for blending with petroleum diesel to a maximum of 5 to 20%.

The effect of exercise on the skeletal muscle phospholipidome of rats fed a high-fat diet

The aim of this study was to examine the effect of endurance training on skeletal muscle phospholipid molecular species from high-fat fed rats. Twelve female Sprague-Dawley rats were fed a high-fat diet (78.1% energy). The rats were randomly divided into two groups, a sedentary control group and a trained group (125 min of treadmill running at 8 m/min, 4 days/wk for 4 weeks). Forty-eight hours after their last training bout phospholipids were extracted from the red and white vastus lateralis and analyzed by electrospray-ionization mass spectrometry. Exercise training was associated with significant alterations in the relative abundance of a number of phospholipid molecular species. These changes were more prominent in red vastus lateralis than white vastus lateralis. The largest observed change was an increase of similar to 30% in the abundance of 1-palmitoyl-2-linoleoyl phosphatidylcholine ions in oxidative fibers. Reductions in the relative abundance of a number of phospholipids containing long-chain n-3 polyunsaturated fatty acids were also observed. These data suggest a possible reduction in phospholipid remodeling in the trained animals. This results in a decrease in the phospholipid n-3 to n-6 ratio that may in turn influence endurance capacity.

Effect of temperature and moisture on high pressure lipid/oil extraction from microalgae

Commercially viable carbon–neutral biodiesel production from microalgae has potential for replacing depleting petroleum diesel. The process of biodiesel production from microalgae involves harvesting, drying and extraction of lipids which are energy- and cost-intensive processes. The development of effective large-scale lipid extraction processes which overcome the complexity of microalgae cell structure is considered one of the most vital requirements for commercial production. Thus the aim of this work was to investigate suitable extraction methods with optimised conditions to progress opportunities for sustainable microalgal biodiesel production. In this study, the green microalgal species consortium, Tarong polyculture was used to investigate lipid extraction with hexane (solvent) under high pressure and variable temperature and biomass moisture conditions using an Accelerated Solvent Extraction (ASE) method. The performance of high pressure solvent extraction was examined over a range of different process and sample conditions (dry biomass to water ratios (DBWRs): 100%, 75%, 50% and 25% and temperatures from 70 to 120 ºC, process time 5–15 min). Maximum total lipid yields were achieved at 50% and 75% sample dryness at temperatures of 90–120 ºC. We show that individual fatty acids (Palmitic acid C16:0; Stearic acid C18:0; Oleic acid C18:1; Linolenic acid C18:3) extraction optima are influenced by temperature and sample dryness, consequently affecting microalgal biodiesel quality parameters. Higher heating values and kinematic viscosity were compliant with biodiesel quality standards under all extraction conditions used. Our results indicate that biodiesel quality can be positively manipulated by selecting process extraction conditions that favour extraction of saturated and mono-unsaturated fatty acids over optimal extraction conditions for polyunsaturated fatty acids, yielding positive effects on cetane number and iodine values. Exceeding biodiesel standards for these two parameters opens blending opportunities with biodiesels that fall outside the minimal cetane and maximal iodine values.

Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

Comparative analysis and distribution of Omega-3 lcPUFA Biosynthesis genes in marine molluscs

Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the required enzymes for the synthesis of lcPUFAs.