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Lobosphaera Incisa בקנה מידה DGLA : אופטימיזציה של ייצור דסאטוראז

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Lobosphaera Incisa בקנה מידה DGLA : אופטימיזציה של ייצור דסאטוראז מוטנט חסר דלתא5- דסאטוראז של Lobosphaera incisa: אופטימיזציה של ייצור DGLA בקנה מידה גדול ובחינת השימוש במיקרואצה כתוסף מזון בדגים מחקר לשם מילוי חלקי של הדרישות לקבלת תואר "דוקטור לפילוסופיה" מאת איזבל קאמפוס פורטוגל הוגש לסינאט אוניברסיטת בן גוריון בנגב כ"ח אדר תשע"ז 26.3.2017 באר שבע מוטנט חסר דלתא5- דסאטוראז של Lobosphaera incisa: אופטימיזציה של ייצור DGLA בקנה מידה גדול ובחינת השימוש במיקרואצה כתוסף מזון בדגים מחקר לשם מילוי חלקי של הדרישות לקבלת תואר "דוקטור לפילוסופיה" מאת איזבל קאמפוס פורטוגל הוגש לסינאט אוניברסיטת בן גוריון בנגב אישור המנחה: פרופ' דינה זילברג פרופ' אינה חוזין-גולדברג פרופ' סמי בוסיבא אישור דיקן בית הספר ללימודי מחקר מתקדמים ע"ש קרייטמן ____________________ כ"ח אדר תשע"ז 26.3.2017 באר שבע Delta-5 desaturase-deficient mutant of Lobosphaera incisa: optimizing up-scaled production DGLA by the microalga and testing its application as a dietary supplement in fish Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY by Isabel Campos Portugal Submitted to the Senate of Ben-Gurion University of the Negev March 2017 Beer-Sheva Delta-5 desaturase-deficient mutant of Lobosphaera incisa: optimizing up-scaled production DGLA by the microalga and testing its application as a dietary supplement in fish Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY by Isabel Campos Portugal Submitted to the Senate of Ben-Gurion University of the Negev 26 th of March, 2017 Beer-Sheva This work was carried out under the supervision of Prof. Sammy Boussiba Prof. Inna Khozin-Goldberg Prof. Dina Zilberg In the Department: Albert Katz International School for Desert Studies Faculty: The Jacob Blaustein Institutes for Desert Research Research-Student's Affidavit when Submitting the Doctoral Thesis for Judgment I Isabel Campos Portugal, whose signature appears below, hereby declare that (Please mark the appropriate statements): I have written this Thesis by myself, except for the help and guidance offered by my Thesis Advisors. The scientific materials included in this Thesis are products of my own research, culled from the period during which I was a research student. ___ This Thesis incorporates research materials produced in cooperation with others, excluding the technical help commonly received during experimental work. Therefore, I am attaching another affidavit stating the contributions made by myself and the other participants in this research, which has been approved by them and submitted with their approval. Date: 26.03.2017 Student's name: Isabel Campos Portugal Signature: ABSTRACT: Microalgae possess the potential for numerous commercial applications, among them the production of long-chain polyunsaturated fatty acids (LC-PUFA), as the most efficient primary producers of LC-PUFA. Losses associated with diseases and stress pose a significant burden in the fish aquaculture industry and use of appropriate immune-modulatory dietary supplements can serve as a partial solution, for example in the form of fatty acid supplementation to the feed. The Δ5 desaturase deficient mutant of Lobosphaera incisa (code name P127) is the richest green source of the omega-6 LC-PUFA, dihomo-γ-linolenic acid (DGLA). DGLA is known to possess anti-inflammatory effects that are important in preventing/treating diseases that include excessive activation of the immune system. Being the precursor for anti-inflammatory eicosanoids, DGLA is assumed to induce an anti-inflammatory effect. Its benefit has been demonstrated in several studies with mammals, but it has never been tested in fish. The commercial use of this microalga as a source of this valuable fatty acid requires optimization of production conditions in order to maximize its yield. In this research, I optimized the growth of L. incisa Δ-5 desaturase mutant and DGLA production by this microalga, aiming to achieve up scaled production of DGLA. The effect of different light regimes and the initial microalgal inoculum (expressed as chlorophyll concentration, ICC) on DGLA productivity in different cultivation facilities was investigated. The effects of two different light regimes in culture under nitrogen (N) starvation in 100 L panels, including the application of continuous light regime (CLR) of 240 µmol photons m-2 s-1 and biphasic light regime (BLR) which include a change in light intensity after day 4 from 200 µmol photons m-2 s-1 to 280 µmol photons m-2 s-1, were investigated. Further, the productivity of the cultures under N starvation for total fatty acids and DGLA in indoor and outdoor culture facilities was compared. The highest DGLA productivity was obtained in N-starved cultures exposed to biphasic light regime. Under CLR, it was possible to determine that the accumulation of oleic acid (OA) occurred faster than the accumulation of DGLA and thus impaired DGLA productivity, while under relatively low light we did not obtain resonable productivity of DGLA. Thus, we designed a BLR which starts with a relatively low light provision until day 4 in order to assure photoacclimation of the N-staved culture and achieve the balanced production of DGLA and OA, and then switching to higher light intensity which allowed a higher production rate of DGLA, due to the sustained DGLA deposition into storage triacylglycerol (TAG). As the goal of the experiments was to achieve higher total fatty acid (TFA) and DGLA production and we observed that BLR in the 100L panel indoor produced III more TFA and DGLA, 723.1 and 216.2 mg L-1, respectively, as compared with the CLR which produced 641.5 and 185.3 mg L-1 TFA and DGLA, respectively. The lower production rate was obtained in the tubular photobioreactor (PBR) outdoor, where the TFA and DGLA production were 568.5 and 148.4 mg L-1, respectively. Principal component analysis (PCA) was used as an exploratory analysis of the DGLA productivity as a function of Ratio Chlorophyll/Dry Weight – Chl (%DW), DGLA index, Ratio Carotenoid/Chlorophyll, Dry Weight, Oleic acid, Dihomo-γ- linolenic acid, Total Fatty Acid, Chlorophyll and Carotenoid for all 16 experiments in 100 L flat panel nitrogen starvation under different light regimes ( BLR and CLR). The PCA indicated that the Chl (%DW) parameter induced the strongest biological responses in terms of algal lipid production along with the DGLA index. This can be explained by maintaining optimal Chl content and its significant effect on the DGLA productivity. Maximal culture content of DGLA in outdoor production was lower compared with the maximum obtained in the laboratory. Next, the effects of dietary administration of the algal biomass enriched in DGLA on immune parameters and survival of guppy fish upon parasite challenge were studied. The complement system comprises of a large number of different plasma proteins that work together to opsonize pathogens and thereby initiate a cascade of inflammatory responses which help fight an infection. This study presents a method to analyze complement activity in tissue homogenates of fish and compares the use of sheep and rabbit red blood cells for the analysis. Analysis in serum samples of different fish species is also presented. Results revealed large variabilities in complement activity between different fish species and within species of fish from different origin (different farms). Analyzing the effect of freeze-thaw cycles reveled substantial reduction in complement activity due to freezing and thawing, especially in body homogenate samples. To evaluate the effects of a DGLA-rich triacylglycerol-enriched diet, guppies (Poecilia reticulate) were fed a diet supplemented with P127 at different levels. Fish were examined for growth, survival and immune competence, as well as resistance to parasitic infection by Tetrahymena sp. Two trials were performed in which fish were fed a diet supplemented with different levels of P127, containing 5%, 10% and 20% of broken dry algae and non-supplemented control. Fish were fed with the different diets for a period of 4 or 6 weeks (in trials 1 and 2 respectively) at 2% of their body weight per day. Mortality was monitored throughout the trial. After 2 or 3 (in trials 1 and 2 respectively) weeks of feeding, fish were challenged with Tetrahymena sp., a parasite that is considered to be a significant disease problem in guppies. Fish were sampled for immune IV analyses prior to the challenge and for fatty acid analyses prior to the challenge and 2 or 5 days after challenge (in trials 1 and 2 respectively). In the first trial a mixed population of males and females was used. Results revealed a trend in reduced mortality in males, but not in females supplemented with P127, thus in the second trial only males were used. In the second trial ongoing mortality was monitored prior to the Tetrahymena sp. challenge, and accumulated to 25.5% in the control, as compared to the significantly lower level of 14.5% in the 20% P127 supplemented treatment group. Similarly, cumulative mortality following challenge with Tetrahymena sp. was significantly lower in the 20% supplemented group compared with control, reaching levels of 32.8% and 52.1% respectively. Dietary supplementation with DGLA significantly affected body and liver lipid content, significantly increasing DGLA content, more in liver than in the body as well as altering ARA/DGLA ratio, as compared with the non- supplemented control. Interestingly, ARA content, which was similar among treatment groups prior to challenge, increased following challenge, thus also affecting the ARA/DGLA ratio, which increased in all treatment groups after challenge. This suggests that some of the DGLA was converted to ARA, presumably to increase the proportion of ARA-originating pro- inflammatory prostaglandins (as opposed to the anti-inflammatory prostaglandins originating from DGLA). Dietary supplement with P127 also resulted in elevated lysozyme, but not anti- protease levels. Overall, dietary supplementation with 20% P127 positively affected fish survival and resistance to infection.
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