Browsing by Subject "microalgae"

The trend of energy policy in European Union as well as in international context has lately been to increase the share of renewable biofuels. The causes for this are global warming, shrinking reserves of fossil fuels and governments' aspiration for energy independence. Microalgae have shown to be a potential source of biofuels. Though cultivation of microalgae has a long history, has production for fuel yet been unprofitable. Production has become more effective as cultivation has shifted from open ponds to controlled photobioreactors but to achieve effective cultivation methods substantially more understanding on the ecophysiology of microalgae is needed. The aim of my thesis was to research the optimal light intensity and temperature of photosynthesis for three microalgae (Chlorella pyrenoidosa, Euglena gracilis and Selenastrum sp.), which are the main parameters limiting the level of photosynthesis in nutrient rich environments such as photobioreactor. The research strains were incubated in eight light intensities (0,15-250 µmol m-2 s-2) and in 5-6 temperatures (10-35 °C). Photosynthetic activity was determined with radiocarbon method which is based on the stoichiometry of photosynthesis. The purpose of radiocarbon method is to estimate how much dissolved carbon dioxide do the algae assimilate when photosynthesizing. In the method the algae are incubated in light and dark bottles where certain amount of radiocarbon (14C) has been added as a tracer. The algae fix 14C in the proportion to available 12C. 14C method has become the most common way to measure the photosynthesis of microalgae. All of the algal strains grew in 10-30 °C but C. pyrenoidosa was the only one which grew also in 35 °C. The data was analyzed by fitting them with two photosynthesis-light intensity relationship models and one photosynthesis-temperature relationship model and as a result values of essential parameters, i.e. optimal light intensity (Iopt) and temperature (Topt) for photosynthesis, could be estimated. The model which gave the best fit was chosen to describe the photosynthesis-light intensity relationship. The optimal light intensity for C. pyrenoidosa ranged between 121–242 µmol m-2 s-2 and optimal temperature was 15 °C. Corresponding values for E. gracilis were 117-161 µmol m-2 s-2 and 24,1 °C, and for Selenastrum sp. 126-175 µmol m-2 s-2 and 16,7 °C. Q10-values were also determined. With all research strains, the level of photosynthesis increased as light intensity and temperature grew until optimal values were reached. The strains tolerated higher light intensities in warmer temperatures but after reaching the optimal temperature, the level of photosynthesis did not increase any more with elevating temperature. Robust algal strains, i.e. strains, that are most adaptable in terms of light intensity and temperature, are the most prominent ones for biofuel production. From these research strains the most adaptable strain in terms of light intensity was C. pyrenoidosa and in terms of temperature Selenastrum sp. C. pyrenoidosa had superior carbon fixation rate in relation to cell size. Therefore it can be concluded that C. pyrenoidosa is the most suitable algal strains for biofuel applications of the strains assessed here.

Microalgae are promising raw materials for food- and biotechnology because they contain a lot of proteins, unsaturated fatty acids, pigments, vitamins and minerals. There are few studies on vitamin B in microalgae and some of them are based on partly inaccurate methods. Microalgae in general, analytical methods regarding their analysis and how they use vitamins were discussed in the literature part of this thesis. The structures, chemical properties and occurrence in foods as well as commonly used analytical methods of the vitamins in question were presented. The aim of the experimental part of this thesis was to analyse commercially marketed microalgae supplements (Chlorella sp. and Arthrospira sp. (spirulina)) and laboratory-grown microalga (Euglena gracilis) as potential sources of folate, niacin, vitamin B2 and B12. Contents of vitamin B12, B2 and niacin were analysed using UHPLC method separately validated for each vitamin. The total folate content was analysed microbiologically and folate vitamers by using UHPLC. The vitamin B12 was analysed microbiologically and the active forms of vitamin B12 were confirmed using LC-MS. Acid hydrolysis was used in analysing niacin content. The total folate content in chlorella supplements was of the same order when analysed microbiologically or with UHPLC. Instead, in spirulina supplements the microbiologically analysed total folate content was higher than the total folate content based on the sum of folate vitamers analysed with UHPLC. At most, the total folate content of E. gracilis -sample was 3-fold higher than in commercial microalgae supplements. Especially in spirulina supplements, the vitamin B12 contents were clearly higher when analysed microbiologically than they were when analysed with UHPLC. The difference was most likely due to pseudocobalamin that resembled vitamin B12. On average E. gracilis -samples had higher vitamin B2 content than the commercial supplements. E. gracilis -samples and chlorella supplements contained more niacin than spirulina supplements. According to this thesis, commercially marketed microalgae supplements contained different amounts of vitamin B. Chlorella sp. was proved to be a great source of folate, vitamin B12 and niacin and moderate source of B2. The majority of vitamin B12 in Arthrospira sp. (spirulina) was pseudocobalamin. Despite that, spirulina supplements proved to be a moderate source of vitamin B12. On average, E. gracilis had the highest vitamin B content and it would potentially be an excellent source of vitamin B – if it was accepted for food use.

The literature review introduced the chemistry of sterols and presented the sterols found in microalgae, and placed emphasis on the analytical methods used for studying sterols in microalgae. A brief discussion about application of microalgae-derived sterols was also included. The aim of this work was to learn about the sterol compositions in microalgae: Euglena gracilis and Selenastrum sp.. The common analytical methods of sterols are not suitable when applied to microalgae. Traditional alkaline hydrolysis may lead to an underestimation of total sterol content, because it cannot break acetal bond in steryl glycoside (SG). Additional acid hydrolysis for determining SG may lead to isomerization or decomposition of Δ7-sterols, which are the main sterols in green algae. A combination of alkaline hydrolysis and enzymatic hydrolysis was performed in this study. Firstly, sterol contents were determined using two methods: direct saponification and accelerated solvent extraction followed by saponification. Secondly, sterol classes: free sterol (FS), steryl ester (SE), and SG were determined by fractionation using solid phase extraction, followed by alkaline hydrolysis (FS and SE) and enzymatic hydrolysis (SG). Sterols were quantified using an internal standard and determined by GC-FID as their trimethylsilyl ether derivatives and identified by GC-MS. Euglena gracilis contained three major sterols: ergosterol and corbisterol, and Selenastrum sp. contained Δ7-ergosterol, chondrillasterol, and Δ7-chondrillasterol. Sterol contents ranged from 0.68-3.24 mg/g dry matter in Euglena gracilis, of which ergosterol constituted 68-93%. Sterol content in Selenastrum sp. was > 9 mg/g dry matter, with 36% Δ7-ergosterol, 12% chondrillasterol, and 52% Δ7-chondrillasterol. Comparison between the two extraction methods showed that ASE had a lower sterol yield than direct saponification. In E. gracilis, SE compromised 20-24%, FS 60-65%, and SG 11-12%. In Selenastrum, SE compromised only 1%, FS 74%, and SG 25%. The findings suggested that data on sterol composition ought to be viewed with caution. Underestimation of total sterol content may result from missing remarkable amounts of SG in certain microalgae species.

The literature review deals with the basics of microalgae, microalgal cultivation and harvest, and the organism Euglena gracilis. The carbohydrates found in E. gracilis are discussed, with the focus on the storage carbohydrate paramylon. The review also deals with effects of cultivation conditions on composition of microalgae. The aim of the experimental work was to investigate carbohydrate composition in E. gracilis, and in this way increase the knowledge of the microalgae. E. gracilis cultivated in five different environments was studied for content of the beta-glucan paramylon, as well as free sugars and oligosaccharides. As the method used for determination of paramylon content was a gravimetric method, a glucose measurement, protein determination and size-exclusion chromatography were performed on the paramylon isolated. In addition, the effect of supercritical fluid extraction (SFE) of the biomass to extract high value compounds on the overall carbohydrate composition and content was also investigated. In addition, the SFE samples were also analysed according to the AOAC method for dietary fibre The paramylon content in the E. gracilis biomass was between 22 and 40 % of the dry biomass. SEC analysis of this paramylon isolated showed that it was of molecular weight around 150 kDa, but that it was not only paramylon that had been isolated, but the isolates also contained impurities. This was also confirmed by the analysis of glucose and protein in the isolates. Possible compounds that can have been isolated with the paramylon are leftover peptides bound to the tight paramylon structure, chlorophyll, or glycoproteins. The most abundant sugars found in E. gracilis biomass were mannitol, trehalose and glucose, with a total content of and the total content of the samples were from between 2.4 and 14.9 % of the E. gracilis of the total dry mass. There were also some other unquantified free sugars, such as lactose seen in the E. gracilis biomass. The oligosaccharide content was considered low and not further quantified.