STUDIES ON STRESS INDUCED TAG ACCUMULATION IN MICROALGA SCENEDESMUS sp. IITRIND2

Abstract

Energy crisis, water shortage and pollution are among the major challenges confronting the sustainable environmental existence. Currently, fossil fuels fulfil 80 % of the world‟s primary energy requirements of which 58 % is consumed by the transportation sector. Renewable energy derived from sustainable feedstocks can reduce the load on the fossil fuels and curb the greenhouse gasses (GHG) emissions. Biofuels specifically biodiesel production has increased over the years (2.8 billion gallons in 2016) due to its renewability, reduced carbon emissions, unburned hydrocarbons and particulate emissions as compared to petro diesel engines. Biodiesel is a mixture of fatty acid methyl esters (FAMEs) derived from plant oils, animal fats, and waste cooking oils. However, plant oil derived biodiesel cannot be sustainable as edible oils compete with the food consumption. Further, non-edible oils and waste cooking oils have high amount of free fatty acids (FFA) which are undesirable for biodiesel production. Non edible oils also demand large areas of land reserves and water resources, thus competing with food crops. Additional to the fuel crisis, heavy metals disembogue into water bodies have also led to loss of aquatic life, bioaccumulation and biomagnification of toxins in the food chain. Removal of heavy metals from the water sources is critical as they are non-biodegradable, recalcitrant and can abolish the self-purification ability of aquatic bodies leading to toxic water supply. Among the heavy metals, arsenic (As) is one of the major toxin contaminating the groundwater in many countries including India, China, Vietnam, Bangladesh and Pakistan. United States Environmental Protection Agency (USEPA) has classified arsenic as Group 1 carcinogen based on human epidemiological data. European Union has fixed a maximum limit up to 10 μg ml-1 for As contamination. However, exposure to As even in low quantities (0.1 μg ml-1) can increase the risk of skin, kidney, lung and bladder cancers. Currently various physico-chemical treatment methods are employed including ion-exchange, precipitation, solar distillation for removal of As from water bodies. However, methods listed above produce As laden discards which needs to be treated further before being finally discharged into water bodies. In view of the current scenario, utilization of microorganisms specifically photosynthetic green microalgae has been considered as potential cell factories for the production of biodiesel and as prospective bioremediators. They have faster doubling time, require less land, high photosynthetic ability and biomass production as compared to energy crops such as rapeseed and soybean. Furthermore, they have a unique ability to adapt to various ix environmental conditions ranging from fresh water to marine and even waste water along with mitigation of of CO2. Under adverse conditions such as nutrient limitation, temperature, pH, light intensity, heavy metal etc. microalgae can accumulate up to 50-60 % of lipids (dry cell weight) making them as desirable feedstocks for biodiesel production. These stored lipids or triacylglycerol (TAGs) contain fatty acids ranging from C12-C24 that are identical to plant oils (Jatropha, Palm, and Soya). Moreover, microalgae have high metal binding capacity and they can remove arsenic by adsorbing the heavy metal onto its surface as its cell wall is composed of different functional groups which act as binding sites for heavy metals followed by intracellular metabolism. Despite the great potential of microalgae for biodiesel and mitigation of arsenic, its utilization on large scale is still a long road ahead. To bridge this gap, reduction in the cost of algal production encompassing bioprospecting of potential high lipid accumulating strains, utilization of sea water for cultivating microalgae and integrating As removal with biodiesel production are quintessential. Further, understanding the molecular adaption mechanisms in response salt and metal stress in prospective oleaginous microalgal strains could lead to successful modifications in the expression of key lipid/salt/metal associated enzymes resulting in overall improved productivity. This could be achieved by through knowledge of algal-omics which can then be manipulated to achieve desirable outcomes. Keeping the above view in mind, the specific details of thesis chapters (1-5) are as follows: Chapter 1 gives an overview on energy crisis and arsenic pollution with the role of microalgae as a potential feedstock for biodiesel production and as a budding tool for mitigating arsenic from the contaminated waters. A detailed literature survey of algal omics techniques (proteomics, metabolomics and lipidomics) and their role in identification of potential genetic engineering targets for boosting TAG accumulation have also been discussed. Chapter 2 deals with the bioprospecting of indigenous high biomass and lipid accumulating microalgal strains capable of growing sea water. A total of four strains (Scenedesmus sp. IITRIND2, Chlamydomonas debarayna IITRIND3; Chlorella sp., and Tetradesmus obliquus IITRIND1) were isolated from a fresh water lake and three procured strains (Chlorella minutissima, Scenedesmus abundans and Chlorella pyrendosia) were evaluated for salt tolerance by cultivating in artificial sea water (ASW; 35 g/L sea salts). Among the isolated strains, Scenedesmus sp. IITRIND2 was able to grow in ASW and thus was chosen for further experiments. The microalga attained maximum lipid productivity of 83 ± 2 mg/L/d in ASW as compared to the Bold‟s basal media (BBM) (25 ± 1.2 mg/L/d). The increase x in the lipid content was balanced by a sharp decrease in its protein and carbohydrate content. Further, biochemical analysis evidenced that salinity induced oxidative stress resulted in reduced levels of photosynthetic pigments, elevated H2O2, thiobarbituric acid reactive substances, proline, glycine betaine, catalase and ascorbate peroxidase activity attributing to microalga‟s halotolerance. The FAME analysis revealed the dominance of C14:0, C16:0, C18:0, C18:1 and C18:2 fatty acids under halotolerant conditions. Further, the properties of biodiesel resulted under saline conditions were in compliance with ASTM D6751 and EN 14214 fuel standards indicating that the lipid augmented halotolerant algal strains capable of growing in sea water can be formulated as environmental sustainable and economic viable sources for biodiesel production. Chapter 3 characterizes the halotolerance and TAG accumulating mechanism of Scenedesmus sp. IITRIND2 in response to ASW (35 g/L sea salts) as compared to control (0 g/L sea salts) by studying both physiological and molecular responses. On exposure to salinity, the microalga rewired its cellular reserves and ultrastructure, restricted the ions channels, modulated its surface potential along with secretion of extrapolysaccharide to maintain homeostasis and resolve the cellular damage. To gain further insights into the molecular responses under halotolerance conditions, an “integrated omics approach” comprising of metabolomics, proteomics and lipidomics studies was utilized. The obtained results were complemented with real time polymerase chain reaction (RT-PCR). The algal-omics studies suggested a well organised salinity driven metabolic adjustment by the microalga starting from increasing the negatively charged lipids, up regulation of proline and sugars accumulation followed by direction of carbon and energy flux towards TAG synthesis. Further, the algalomics studies indicated both de-novo and lipid cycling pathways at work for increasing the overall TAG accumulation inside the microalgal cells. Such a salt response is unique and different from the well-known halotolerant microalga; Dunaliella salina, implying diversity in algal response with species. Based on the integrated algal-omics studies, four potential genetic targets belonging to two different metabolic pathways (salt tolerance and lipid production) were identified. Chapter 4 illustrates the potential of Scenedesmus sp. IITRIND2 for carbohydrate and lipid accumulation by cultivating in natural sea water in a small-scale custom built photobioreactor. The microalga showed remarkable ability to cope up with different salinity environments under given temperature and light conditions. Such an adaptation was attributed xi to the increase in neutral sugars, such as glucose, mannose, galactose, fucose and ribose, associated with both structural and storage (and potentially osmoprotectant) polymeric carbohydrates. The carbohydrate rearrangements may aid with rewiring the cellular components and membrane permeability in circumventing the detrimental effects of high salinity. The microalga showed high carbohydrate and lipid accumulation, signifying its potential for integrated bioethanol and biodiesel production. (This work was a part of Bioenergy-Awards for Cutting Edge Research (B-ACER), 2016 carried out under the supervision of Dr. Philip T. Pienkos, Strategic Project Lead, NREL, CO, USA). Chapter 5 aims to integrate toxic and carcinogenic heavy metal arsenic (both III and V forms) removal coupled to biodiesel production by Scenedesmus sp. IITRIND2. The microalga was able to tolerate half a gram of As (III) and As (V) forms in synthetic soft water (SSW) triggering lipid production along with efficient removal of the toxic heavy metal from SSW. The bioremediation mechanism was studied by analysing the changes in its biochemical, biophysical and metabolic characteristics. The results suggested that by using a complex interplay of biomolecular, photosynthetic agents and varied metabolites, the microalga tolerated the arsenic stress. The study also revealed that the metabolic changes in the presence of As (III) and As (V) are differential in nature, and were more extensive in the presence of As (V) due to its enhanced toxicity, thus evidencing for variable perturbations in major cellular metabolic pathways. In summary, the thesis highlighted the unique characteristics of a novel fresh water microalga; Scenedesmus sp. IITRIND2 which efficiently adapted to sea water and arsenic contaminated waters along with high biomass and TAG accumulation. Utilization of various biophysical and algal-omics techniques unravelled the physiological and molecular mechanism involved for salt and arsenic tolerance. This comprehensive study will help identifying biomarkers for TAG increment along with salt and metal tolerance that could be potential genetic engineering targets in microalgal strains.