Crude glycerol is a major byproduct if the biodiesel industry. Biodiesel manufacturers are currently facing the challenges of appropriate disposal of this waste material. Crude glycerol is expensive to purify for use in food, cosmetic, and pharmaceutical industries and therefore, alternative methods for use of this crude glycerol are needed. A promising alternative is to use this crude glycerol as a carbon source for microalgae fermentation. In this project, we investigated the use of crude glycerol as a less expensive substrate for the fermentation of the microalgae <i>Schizochytrium limacinum</i> and <i>Pythium irregulare</i> which are prolific producers of omega-3 polyunsaturated fatty acids. Omega-3 fatty acids have many beneficially effects on treating human diseases such as cardiovascular diseases, cancers, and neurological disorders. In addition, the omega-3 fatty acids docosahexaenoic acid (DHA) has been shown to be an important factor in infant brain and eye development. The first part of this study focused on the continuous fermentation of <i>S. limacinum</i>, a prolific producer of DHA. The objective of this study was to examine the algal cellular physiology and maximize its DHA productivity. Two important parameters used in continuous fermentation were studied: dilution rate (D) and feed glycerol concentration (S₀). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day⁻¹ and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day⁻¹ and S₀ = 90 g/L. The cells had a true growth yield of 0.283 g/g, a maximum specific growth rate of 0.692 day⁻¹, and a maintenance coefficient of 0.2216 day⁻¹. The second part of this study focused on morphology issues with <i>P. irregulare</i>, a prolific producer of eicosapentaenoic acid (EPA). <i>P. irregulare</i> has a filamentous morphology, which can make fermentation difficult. The mycelium can stick to the agitation blades resulting in mechanical problems. In addition, this filamentous morphology prevents adequate amounts of oxygen from reaching some cells resulting in decreased productivities. The focus of this research was to control the fermentation conditions to make the algae grow in small pellets, a morphology more suitable for fermentation. In flask culture studies, pellets were formed at an agitation speed of 110 rpm in both regular and baffled flasks. Baffled flasks resulted in pellet formation at 90 and 130 rpm as well. Fermentation studies resulted in pellet formation at agitation speeds of 150 and 300 rpm. Pellets were better able to form when a baffle was not in place. In addition, agitation speed influenced pellet size, with smaller pellets forming at the higher agitation speed. Overall, this study showed that crude glycerol can be used as a carbon source for the continuous fermentation of <i>S. limacinum</i> with high DHA productivity and the morphology of <i>P. irregulare</i> could be controlled by manipulating culture conditions, mainly agitation speed. These results show the potential for scale-up studies for both algal species. / Master of Science

The ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), in the free fatty acid (FFA) form, has been demonstrated,to reduce adenoma number and size in patients with familial adenomatous polyposis. However, the mechanistic basis of the antineoplastic activity of EPA in the colorectum remains unclear. We tested the hypothesis that EPAFFA negatively modulates synthesis of and signaling by prostaglandin (PG) E2 in human colorectal cancer (CRC) cells.,EPA-FFA induced apoptosis of cyclooxygenase (COX)-2-positive human HCA-7 CRC cells in vitro. EPA-FFA in cell,culture medium was incorporated rapidly into phospholipid membranes of HCA-7 human CRC cells and acted as,a substrate for COX-2, leading to reduced synthesis of PGE2 and generation of PGE3. Alone, PGE3 bound and activated,the PGE2 EP4 receptor but with reduced affinity and efficacy compared with its "natural" ligand PGE2. However,,in the presence of PGE2, PGE3 acted as an antagonist of EP4 receptor-dependent 3',5' cyclic adenosine,monophosphate induction in naturally EP4 receptor-positive LoVo human CRC cells and of resistance to apoptosis,in HT-29-EP4 human CRC cells overexpressing the EP4 receptor. We conclude that EPA-FFA drives a COX-2dependent "PGE2-to-PGE3 switch" in human CRC cells and that PGE3 acts as a partial agonist at the PGE2 EP4 receptor.

Yes / We investigated red blood cell (RBC) PUFA profiles, and the predictive value of RBC EPA content for tumour EPA exposure and clinical outcomes, in the EMT study, a randomised trial of EPA in patients awaiting colorectal cancer (CRC) liver metastasis surgery (A.J. Cockbain et al., 2014). There was a significant increase in RBC EPA in the EPA group (n=43; median intervention 30 days; mean absolute 1.26 [±0.14]% increase; P<0.001), but not in the placebo arm (n=45). EPA incorporation varied widely in EPA users and was not explained by treatment duration or compliance. There was little evidence of ‘contamination’ in the placebo group. The EPA level predicted tumour EPA content (r=0.36; P=0.03). Participants with post-treatment EPA ≥1.22% (n=49) had improved OS compared with EPA <1.22% (n=29; HR 0.42[95%CI 0.16–0.95]). RBC EPA content should be evaluated as a biomarker of tumour exposure and clinical outcomes in future EPA trials in CRC patients.

Yes / As pre-clinical and clinical research interest in ω-3 polyunsaturated fatty acids (PUFA) increases, so does the need for a fast, accurate and reproducible analytical method to measure fatty acids (FA) in biological samples in order to validate potential prognostic and predictive biomarkers, as well as establishing compliance in ω-3 PUFA intervention trials. We developed a LC-ESI-MS/MS method suitable for high throughput development to measure FAs and validated it in the context of treatment with the ω-3 PUFA eicosapentaenoic acid (EPA). Uniquely we directly compared the LC-ESI-MS/MS method to a GC-MS protocol. We demonstrated the LC-ESI-MS/MS method is accurate and reproducible, with coefficients of variation consistently below 15% for each PUFA analysed. The relative FA content values correlated well with those obtained by GC-MS (r2 = 0.94, p<0.001 for EPA) in vitro. The data obtained following analysis of FA content of liver tissues from mice fed an eicosapentaenoic acid enriched diet showed similar results to that of published studies in which GC-MS was used. The LC-ESI-MS/MS method allows concomitant analysis of unesterified (free, unbound) and esterified (bound) FAs in biological samples, allowing investigation of different PUFA pools in cells and tissues. / Yorkshire Cancer Research, MRC/NIHR

Eicosapentaenoic acid (EPA, C20:5, n-3) is a long chain crucial unsaturated fatty acid, essential for the regulation of critical biological functions in humans. Its benefits include the therapeutic treatment of cardiovascular disease, schizophrenia and Alzheimer's disease. The fungus Pythium irregulare (ATCC 10951) has great potential as a natural EPA producer. In this study, the electroporation conditions for P. irregulare were determined. The auxotrophic selectable genes ura, trp and his were respectively cloned into the plasmid pESC to construct shuttle vectors. Electroporation with 2.0kV and a 0.2cm cuvette was applied as the most effective condition for heterogeneous genes transformation. The yield and content of EPA and other components of total fatty acids (TFA) were further determined by the FAME approach with GC, as well as the analysis of biomass. The EPA content in P. irregulare with heterologous pESC-TRP vector reached 16.68 mg/g if cultured in auxotrophic medium, which showed a 52.33% increase compared to the wild-type P. irregulare. The maximum of EPA yield was 98.52 mg/L from P. irregulare containing the pESC-URA plasmid, a 32.28% increase over the wild-type. However, the maximum cell dried weight of these two organisms were respectively 6.13g/L and 5.3g/L, significantly less than the 6.80g/L of the wild-type. Not only was a feasible approach detected to electro-transform and increase the EPA yield of P. irregulare, this study also inferred that Ï -6 route was mainly involved in the EPA biosynthesis in this organism. An antisense RNA (asRNA) thermodynamic model was developed to design new asRNA constructs capable of fine-tuning gene expression knockdown. The asRNA technology is now identified as an effective and specific method for regulating microbial gene expression at the posttranscriptional level. This is done by targeting mRNA molecules. Although the study of regulation by small RNAs is advanced in eukaryotes, the regulation of expression through artificially introducing antisense oligodeoxynucleotides into host is still being developed in prokaryotes. To study the thermodynamics of asRNA and mRNA binding, (i) the fluorescence protein genes GFP and mCherry were separately cloned into the common pUC19 vector and (ii) antisense GFP and antisense mCherry DNA fragments were randomly amplified and inserted into the constructed plasmid under the control of an additional plac promoter and terminator. The expression level of fluorescence reporter proteins was determined by plate reader in this combinatorial study. A thermodynamic model to describe the relationship between asRNA binding and observed expression level was created. The study indicates two factors that minimum binding energy of the asRNA-mRNA complex and the percentage of asRNA binding mRNA were crucial for regulating the expression level. The correlation relationship between gene expression level and binding percentage multiplied by the minimum binding energy was found to show a good correlation between the thermodynamic parameters and the observed level of gene expression. The model has the potential to predict the sequence of asRNA and the approach will ultimately be applied to cyanobacteria to increase lipids production. Here, the long-term approach is to build metabolic switches from asRNA that can turn "on/off" various cellular programs and metabolic pathways at will in a fine-tuned manner. This will allow engineers to control metabolic activity in response to reactor conditions. / Master of Science

Thesis (MSc (Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Long chain omega-3 fatty acids such as eicosapentaenoic acid (EPA) are essential for the regulation of critical biological functions in humans and other mammals. Fish oil as the main dietary source of EPA holds several disadvantages and alternative sources and production processes such as microbial fermentation are increasingly being investigated. Therefore the aim of the first part of this study was to evaluate brewers’ spent grain (BSG) as substrate for the production of EPA by solid state fermentation with 29 fungal strains representing different Mortierella species. The effect of a 10% (w/w) linseed oil (LSO) supplement on EPA production was also studied. Consequently, fungal inoculated BSG was incubated at 22oC for three days to obtain optimal fungal growth, before the incubation temperature was lowered to 16oC for the following eight days. Cultures were then harvested and dried, followed by lipid extraction and analyses using gas chromatography. All the strains were found to produce EPA on BSG, while addition of the LSO improved the EPA yield of most strains. The strains which produced the highest levels of EPA on BSG supplemented with LSO were Mortierella antarctica Mo 67 and Mortierella epicladia Mo 101, which respectively produced 2.8 mg and 2.5 mg EPA per g of BSG. During the second part of the study eight Mortierella strains were used to study EPA production via solid state fermentation of sunflower press cake (SPC). Similar culture conditions and analytical methods were used as in the first part of the study. The effect of supplementing the SPC substrate with 10% (w/w) LSO was studied with regard to the supplement’s impact on EPA production and on the highly unsaturated fatty acid (HUFA) profile of the fermented substrate. Addition of LSO improved EPA yield of most strains on SPC, leading to a reduction in the average arachidonic acid (ARA):EPA ratio from 50.68 to 3.66. The ratio of HUFA to saturated and monoenoic fatty acids, was increased significantly (t=5.75, p=0.05) by the addition of LSO, with higher desaturation levels among the 20-carbon fatty acids. Addition of LSO also had a positive effect (r = 0.9291, p = 0.001) on the relative amount of long chain fatty acids (C≥20) produced. The strains which produced the highest levels of EPA on SPC supplemented with LSO were Mortierella alpina Mo 46 and Mortierella basiparvispora Mo 88, which produced 6.4 mg and 5.8 mg EPA per g of sunflower press cake, respectively. Fungi belonging to the genus Mortierella successfully converted LSO supplemented agro-processing wastes, such as BSG and SPC, to materials containing EPA, thereby adding value to these substrates. These EPA-enriched waste substrates could eventually find applications as animal or fish feed or as a source of EPA and other HUFA for the growing omega-3 market in the neutraceutical and therapeutics industry. / AFRIKAANSE OPSOMMING: Langketting omega-3 vetsure soos eikosapentaenoë suur (EPS) is noodsaaklik vir die regulasie van kritiese biologiese funksies in mense en ander soogdiere. Visolie, die mees belangrike EPS-bron in die dieet, hou verskeie nadele in en alternatiewe bronne sowel as produksie-prosesse, soos mikrobiologiese fermentasie, word dus toenemend ondersoek. Die doel van die eerste gedeelte van hierdie studie was dus om gebruikte brouersgraan (GBG) te ëvalueer as ‘n substraat vir die produksie van EPS deur soliede staat fermentasie met 29 fungus isolate wat verskillende Mortierella spesies verteenwoordig. Die uitwerking van byvoeging van 10% (m/m) lynsaadolie (LSO) op EPS-produksie is ook bepaal. Gevolglik is fungus-geïnokuleerde GBG vir drie dae by 22oC geïnkubeer om optimale fungusgroei te verkry, waarna die inkubasie temperatuur verlaag is na 16oC vir die volgende agt dae. Kulture is hierna ge-oes en gedroog, gevolg deur lipied ekstraksie en analise met behulp van gaschromatografie. Al die isolate het EPS geproduseer op die GBG substraat, terwyl byvoeging van LSO die EPS-opbrengs van die meeste isolate verbeter het. Die isolate wat die hoogste vlakke van EPS op GBG wat met LSO verryk is, geproduseer het, was Mortierella antarctica Mo 67 en Mortierella epicladia Mo 101, wat onderskeidelik 2.8 mg en 2.5 mg EPS per g GBG geproduseer het. Tydens die tweede gedeelte van die studie is agt Mortierella isolate gebruik om die produksie van EPS deur soliede staat fermentasie van sonneblom perskoek (SPK) te ondersoek. Kultuurtoestande en analitiese metodes soortgelyk aan die eerste gedeelte van die studie is gebruik. Die uitwerking van byvoeging van 10% LSO tot die SPK substraat is ondersoek met betrekking tot die impak van die byvoeging op EPS produksie asook op die profiel van hoogs onversadigde vetsure (HOVS) van die gefermenteerde substraat. Die byvoeging van LSO tot SPK het die EPS opbrengs van meeste isolate verbeter en het tot ‘n verlaging in die gemiddelde arachidoonsuur (ARS):EPS verhouding vanaf 50.69 tot 3.66 gelei. Die verhouding van HOVS tot versadigde en mono-onversadigde vetsure, is betekenisvol (t=5.75, p=0.05) verhoog deur die byvoeging van LSO, met hoër vlakke van onversadigheid onder die 20-koolstof vetsure. Byvoeging van LSO het ook ‘n positiewe uitwerking (r = 0.9291, p = 0.001) op die relatiewe aantal langketting vetsure (C≥20) gehad. Die isolate wat die hoogste vlakke van EPS geproduseer het op LSO-verrykte SPK, was Mortierella alpina Mo 46 en Mortierella basiparvispora Mo 88, wat onderskeidelik 6.4 mg en 5.8 mg EPS per g SPK geproduseer het. Fungi wat aan die genus Mortierella behoort, het LSO-verrykte agroprosesserings afvalprodukte, soos GBG en SPK, suksesvol omgeskakel na materiale wat EPS bevat, en sodoende waarde toegevoeg aan hierdie substrate. Die EPS-verrykte afvalsubstrate kan uiteindelik toepassings vind as diere- of visvoer of as bron van EPS of ander HOVS vir die groeiende omega-3 mark in die neutraseutiese en terapeutiese industrie.

Doctor of Philosophy / Department of Human Nutrition / Mark D. Haub / Mark D. Haub / Psychological problems and human error are leading causes of death and disability among military service members. Strategies to improve the psychological health and cognitive performance of those in the military are much needed. Recent advances in neuroscience suggest that omega-3 fatty acids may play an important role in the psychological well-being of those in the military. The purpose of this research was to explore the relationship between omega-3 status and psychological outcome variables among soldiers deploying to combat. Data collection was preceded by the development and reliability testing of a novel food frequency questionnaire (FFQ) designed to capture intake from contemporary sources of omega-3 fatty acids including functional foods and supplements. Based on the instrument assessment study (Chapter 2) conducted among university students (n = 165), this FFQ appears to be a comprehensive and reliable (n = 54, ρ = 0.86, p < 0.001) instrument for measuring docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) intakes in young adults. As described in Chapter 3, intake of EPA + DHA as estimated by the FFQ was positively correlated (r = 0.39, p < 0.001) with biomarker measurements of omega-3 status. Primary data were obtained from a volunteer sample of soldiers (n = 272) scheduled for deployment to Iraq. Preliminarily analyses revealed relationships between attention deficit hyperactivity disorder (ADHD) screening scores and psychological outcome variables (Chapter 4). Primary analyses (Chapter 5) indicated intake of EPA + DHA was not significantly correlated with mood, nor were omega-3 exposure variables correlated with cognitive performance based on the required p value (< 0.001) calculated using the Bonferroni correction for multiple tests. Among participants with EPA + DHA intakes at or below the median, omega-3 HUFA was related (p < 0.002) to happiness (β = -0.46), depression (β = 0.44), and fatigue (β = 0.43). Although exploratory in nature, the results of this study suggest a relationship between omega-3 fatty acids and mood. Given the current concerns regarding the psychological health of those in the military, additional research is warranted.

Intakes of the highly unsaturated fatty acids (HUFA, ≥20 Carbons, ≥3 double bonds) eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3, DHA) greater than 0.25 g/d are currently recommended for health benefits. Targets for omega-3 blood biomarkers have also been proposed based on associations with protection against coronary heart disease mortality. The relationship between diet intakes and blood biomarkers is not well defined, particularly differences between men and women. North American intakes and blood biomarkers of EPA and DHA are typically below recommendations and targets. To address this disparity, adherence to dietary advice strategies to increase EPA + DHA intake was investigated over one year. Adherence was sustained up to 12 weeks and long-term adherence was well characterized by the % of DHA in erythrocytes. For women, n-3 HUFA blood biomarkers increased following nutraceutical or combined strategy dietary advice but not seafood or functional food advice. To assist in the assessment of EPA + DHA intakes, food sources of EPA and DHA in Canada were incorporated into a semi-quantitative, nutrient-specific food frequency questionnaire (FFQ) and validated. The FFQ is an adequate tool for estimating habitual EPA and DHA intake and ranking Canadian adults by their intakes. The blood biomarker response to recommended intakes of 0.25, 0.5 and 1 g/d EPA + DHA was also characterized in adult men and women. Blood n-3 HUFA biomarkers increased in a dose-dependent manner and aligned with blood targets associated with primary cardiac arrest risk reduction. Sex differences in the DHA:EPA ratio in blood observed with low intakes at baseline disappeared following 0.25 g/d EPA + DHA. These findings are applicable towards informing achievable dietary guidelines for EPA + DHA intake and improving measurement of EPA + DHA intake in relation to blood n-3 HUFA biomarkers.

This thesis investigated the effects of omega-3 supplementation (2.0 g/day EPA + 1.0 g/day DHA) for 12 weeks on human skeletal muscle sarcolemmal and mitochondrial membrane fatty acid (FA) composition and whole body energy expenditure in young healthy males. Supplementation resulted in significant incorporation of EPA and DHA into sarcolemmal and mitochondrial membranes, with an increase in total unsaturation of mitochondrial membranes. The incorporation profile of the sarcolemma and mitochondria differed, with the mitochondria mimicking changes in whole muscle. There were no changes in the protein content of mitochondrial and selected proteins involved in energy metabolism, except for a significant increase in the long form of UCP3. Despite changes in membrane FA compositions, there were no changes in whole body substrate oxidation at rest or during exercise. These data demonstrate that omega-3 supplementation for 12 weeks altered the FA composition of sarcolemmal and mitochondrial membranes in human skeletal muscle.

The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), in the free fatty acid (FFA) form, has been demonstrated to reduce adenoma number and size in patients with familial adenomatous polyposis. However, the mechanistic basis of the antineoplastic activity of EPA in the colorectum remains unclear. We tested the hypothesis that EPA-FFA negatively modulates synthesis of and signaling by prostaglandin (PG) E(2) in human colorectal cancer (CRC) cells. EPA-FFA induced apoptosis of cyclooxygenase (COX)-2-positive human HCA-7 CRC cells in vitro. EPA-FFA in cell culture medium was incorporated rapidly into phospholipid membranes of HCA-7 human CRC cells and acted as a substrate for COX-2, leading to reduced synthesis of PGE(2) and generation of PGE(3). Alone, PGE(3) bound and activated the PGE(2) EP4 receptor but with reduced affinity and efficacy compared with its "natural" ligand PGE(2). However, in the presence of PGE(2), PGE(3) acted as an antagonist of EP4 receptor-dependent 3',5' cyclic adenosine monophosphate induction in naturally EP4 receptor-positive LoVo human CRC cells and of resistance to apoptosis in HT-29-EP4 human CRC cells overexpressing the EP4 receptor. We conclude that EPA-FFA drives a COX-2-dependent "PGE(2)-to-PGE(3) switch" in human CRC cells and that PGE(3) acts as a partial agonist at the PGE(2) EP4 receptor.