Environmental stresses applied to microalgae for high lipid production

Abstract

Natural resources are a major part of global economy, and a number of regulations have been implemented to increase the demand for renewable energy resources. There is a great deal of potential in biomass energy, since it is a low-cost and sustainable form of energy. In recent years, there has been significant research on the use of vegetable oil in the production of biodiesel as a competitive fuel to oil-based diesel, which results in significantly less environmental damage. The use of renewable energy sources such as biomass, water, and solar is becoming increasingly prevalent in an attempt to minimize environmental problems and emissions from fossil fuels. A very attractive solution would be to use microalgae as a fuel source since they can store high levels of lipids and can be fueled by solar energy. Due to their low space requirements and high lipid content, microalgae are preferred by researchers over plants and other types of energy sources. The majority of current studies on microalgae focus on macroalgae that produce high levels of lipids. Accordingly, stress conditions, adaptations, or genetic manipulations are being investigated. Autotrophs and heterotrophs produce a large amount of lipids, making them ideal candidates for biofuel production. In spite of this, fossil fuel production still has a relatively low cost in comparison to the production of fuel from algae. Due to these reasons, using microalgae as biodiesel is not economically feasible. Furthermore, in order for microalgae to be economically competitive, it is necessary to increase lipid production efficiency. It is likely that the increased efficiency of lipid production has been attributed to both an increase in the amount of lipid produced and a concurrently high rate of biomass production. According to previous studies, microalgae under stress induce biomass production, biomass inhibition, or significant changes in the structure of biochemical substances. They produce higher levels of lipids, proteins, carbohydrates, and fatty acids, as well as higher quality methyl ester fatty acids. It has been determined that microalgae that are intended for use as biofuels are required to produce a high level of biomass, a high level of lipid, and a high level of fatty acid methyl ester quality. A major objective of this study is to reduce the cost of producing lipids that can be used in the production of biodiesel, which can be used in place of fossil fuels. Thus, an extremely significant step will be taken to obtain microalgae efficient enough to compete economically with fossil fuels. In this study, the microalgae, mainly Auxenochlorella protothecoides, which have both heterotrophic and phototrophic growth properties, were investigated to obtain microalgae with suitable biomass, lipid, and lipid composition. The thesis is organized into ten chapters. In order to evaluate the sustainability of microalgae biodiesel production under stressful environmental conditions, various nutrient stress factors will be examined in relation to growth parameters, such as growth kinetics, biomass, lipid and fatty acid methyl ester composition, chlorophyll content, and carotene content. Based on the results obtained, it was determined whether or not biodiesel-quality lipids could be obtained. In the next step, multiple effects of different stress factors were examined, and optimum parameter values were derived using the surface response approach. The final section of the study examined the effect of the two-stage growth process on microalgae lipid production. Studying the addition or deprivation of ferrous sulfate at different concentrations revealed that only a concentration of 0.2 mM and 14.4 mM ferrous sulfate maintained the lipid in high-quality biodiesel. Additionally, Auxenochlorella protothecoides displayed growth properties even at a concentration of 21.6 mM iron sulfate. At 1.08 mM ferrous sulfate concentration, the highest biomass was obtained (1520 mg / L), while the highest saturated fatty acids were obtained at 1.44 mM ferrous sulfate concentration. Despite no significant variation in lipid production, iron deprivation led to the greatest amount of lipid (59.6%). When nitrogen starvation, deprivation, and excess addition were applied, only biomass grown under 0.8 mM NH4Cl met the biodiesel standard. In this case, the amount of lipid measured is 53.8%. However, the biomass obtained in this case is 1.7 times lower than that obtained in a nitrogen-containing medium. This study illustrates that biomass decreases under stress in response to an increase in lipid content. Through participation in various cell metabolic pathways via changing some enzyme activities, plant hormones are able to change the metabolism of plants, including acceleration or deceleration of cell growth and stimulation of cell biochemical products. Abscisic acid hormone is an inhibitory growth hormone used in plants. Adding this hormone to the growth environment of Auxenochlorella protothecoides may lead to more lipid production. According to this theory, abscisic acid was added in a different medium than in other studies, which served as a source of carbon for glycerol. Under 2.5 µM to 40 µM abscisic acid concentrations, microalgal lipids met high-quality biodiesel standards. It was also concluded that a concentration of 2.5 µM abscisic acid promotes growth. Additionally, high levels of lipids were found at concentrations of 2.5 µM and 10 µM abscisic acid. This thesis also examines the effects of multiple stress factors on microalgae lipids and biomass by using the surface response method and experimental results were correlated with quadratic equations, and optimum conditions for maximum biomass and maximum lipid were determined. For this purpose, in the first study, the acetate parameters known as the carbon source and pH as an indicator of hydrogen ion concentration were selected as variables. The growth experiments of batch microalgal growth have been conducted in different pH and acetate-containing media, which were modeled in three dimensions using surface response methodology, resulting in situations where high biomass and high lipid conditions were achieved and obtained as a result of this model. A second study concerning multiple stresses examined the effects of magnesium deprivation and sodium chloride salt on chlorophyll, carotene, biomass, lipid, protein, and carbohydrate parameters in growth media. Biomarkers of stress such as reactive oxygen species and malondialdehyde, a product of lipid peroxidation, confirmed these changes. Surface response methodologies were used to obtain three-dimensional graphs of the results, and magnesium and sodium chloride concentrations, which were likely to maximize biomass and lipid production, were calculated. An increase in lipid content while a decrease in biomass is generally insufficient to develop a strain with improved biodiesel properties. A robust strain with high lipid and biomass content may be obtained through chemical mutagenesis such as ethyl methane sulfonate. In this study, suitable mutants have been selected considering a selective environment which is a kind of ACCase inhibitor herbicide. In the final stages of this thesis, this study examined the effect of different forms of nitrogen (nitrate and ammonia) on the growth of mixed microalgal cultures in anaerobic digestate. The Biodiesel properties of lipids obtained from microalgae cultured under salt and iron stress were evaluated for their suitability as biodiesel feedstock. This thesis revealed that it is necessary to develop systems that make biomass available as a source of energy so as to reduce the use of fossil fuels as an alternative energy source. Furthermore, results were obtained to support microalgae-based biodiesel production, which would contribute to the lack of literature on multiple stress and singular stress.