INTEGRATED OMICS-BASED MOLECULAR MAPPING OF CADMIUM TOLERANT MICROALGA TO ADDRESS ENVIRONMENT-ENERGY NEXUS

Abstract

Industrialization and technological advancements in the last decade have transformed the globe to provide an easier and more efficient social outlook. Nonetheless, the undesirable consequences of these expansions accompany environmental pollution through the release of industrial effluents, and unmanaged greenhouse gas (GHG) emissions in the ecosystem. The disposal of such toxic pollutants to local water bodies not only imposes a high risk of ecological vulnerability to aquatic habitats but also to the well-being of the humans. Heavy metal pollution is one such category of hazardous substances having an acclaimed history of toxicity in various life forms. Amongst different heavy metals, the teratogenic and mutagenic nature of cadmium (Cd) makes it a group I human carcinogen, and has been ranked 7th in Agency for Toxic Substances and Disease Registry (ATSDR's) Priority List of Hazardous Substances. Cd is primarily released into the environment through anthropogenic activities such as iron and steel production, the manufacture/disposal of rechargeable cadmium-nickel batteries, electroplating, etc. Numerous physicochemical techniques such as precipitation, ion exchange, membrane filtration, etc., have been used for Cd removal but they suffer with limitations of lower efficiency, generation of metal-laden discards and operation/maintenance costs. Overcoming such demerits, microalgal-based remediation has emerged as an eco-friendly and cost-effective solution for efficient heavy metal removal from wastewater. The ease of cultivation, short generation time, unicellular morphology, and rapid uptake capacity of metals are a few imperative properties of microalgae that make its utilization privileged over existing techniques. Microalgal cells bind to Cd by the virtue of functional groups on their cell surface resulting in biosorption. The adsorbed Cd ions are eventually accumulated inside the intracellular organelles such as chloroplast, vacuoles, or mitochondria leading to bioaccumulation. Cd ions inside the cells can inflict severe damage to essential metabolic pathways like photosynthesis, carbohydrate, protein, and lipid biosynthesis by displacing the crucial metal ions that act as co-factor to regulate the activity of the involved enzymes. Indeed, for adaptation, microalgal cells needs to rewire their metabolic responses as per the demand of the stressor. Interestingly, some robust microalgal strains apart from tolerating stress, they generate biomass feedstock for alternative applications. Indeed, an integrated approach combining both heavy metal/Cd removal and biofuel production establishes a sustainable biorefinery approach. Other than a sustainable biorefinery, molecular mapping of such tolerant algal strain with advanced omics-based approaches would help to uncover new genetic engineering targets to develop super algal strains with desired traits.In this context, the present thesis comprehends the utility of an oleaginous microalgal strain, Scenedesmus sp. IITRIND2 for efficient Cd removal, coupled with the production of value-added products like biodiesel and extracellular polysaccharide (EPS). Along with detailed morphological and biochemical characterization under Cd stress, the adaptive response of Scenedesmus sp. IITRIND2 was mapped across its transcriptome and metabolome to gain mechanistic insights into the key molecular pathways. Further, the strategy to improve the inherent capability of Cd tolerance in Scenedesmus sp. IITRIND2 was also explored. For such systematic investigation, the aim of the project was categorized into five separate chapters (Chapter 1-5) and has been described as follows: Chapter 1 provides a detailed overview of the global scenario of energy crisis and water pollution. With the facts and figures related to escalating energy demands and heavy metal toxicity in ecological concern, the chapter entails the benefits of deploying microalgal strains to address the energy-environment paradigm under one roof. The role of microalgal cells in producing a wide range of bioenergy products, and the critical pathways involved for the same have been discussed. Further, different types of organic and inorganic pollutants with a major focus on heavy metal pollutants have been outlined. The chapter enlists the existing conventional techniques for heavy metal remediation and its comparative assessment with microalgal-based remediation technology. The general strategies for heavy metal detoxification by microalgal cells such as extracellular precipitation, ion exchange, peptide/organic ligand chelation for biosorption and internalization through surface carrier proteins, sequestration in organelles, entrapment by thiol peptide, and efflux through pumps/organic ligands have been discussed. Establishing the intracellular response of cells, advanced omics techniques such as transcriptomics, proteomics, metabolomics, lipidomics, etc., have been reviewed. The literature studies listing the progress in the integration of heavy metal remediation and biofuel production by microalgal cells, the research gaps, and specific aims to address the same have been thoroughly presented. Chapter 2 presents the screening and selection of a Cd tolerant microalgal strain, followed with the detailed biochemical characterization of the same. Amongst the six microalgal strain, Scenedesmus sp. IITIND2 showed excellent tolerance to Cd with IC50 value of ~32 ppm and phenomenal removal efficiency (~80%) when exposed to 25 ppm of Cd. Such a high uptake of Cd by the cells was accompanied with increased total lipid content (~33% of dry cell weight). The microalgae cells accustomed their carbon pools towards lipid biosynthesis on the expenditure of carbohydrate and protein pools to reinstate cellular redox balance aiding their survival under Cd stress. Additionally, the elevated level of ROS, lipid peroxidation, glycine-betaine, and antioxidant enzymes evidenced the activation of efficient antioxidant machinery for alleviating the Cd stress. Further, analysis of the fatty acid methyl ester (FAME) presented a steady increase in saturated and polyunsaturated fatty acids with biodiesel properties complying with the American and European fuel standards. In a nutshell, the current study highlights an integrative approach combining the phycoremediation of Cd ions along with lipid production as a sustainable concept of biorefinery. Chapter 3 dissects the molecular mechanisms behind the adaptive response of a novel Cd-tolerant microalgae, Scenedesmus sp. IITRIND2 by de novo transcriptomics profiling, biochemical analysis, and TEM-based ultrastructural changes. Among the several omics-based approaches, de novo transcriptomics offers powerful platforms to map the differential gene expression of a non-model, but commercially relevant microorganism under certain stress. The comprehensive transcriptomics profiling of Scenedesmus sp. IITRIND2 under Cd stress revealed the significant role of membrane transporters in the uptake and sequestration of Cd inside intracellular organelles. Further, significantly upregulated gene expression of malic enzyme, acetyl CoA carboxylase, starch synthase, pyruvate kinase etc., confirmed the rewired carbon flux towards lipid accumulation and extracellular polysaccharide synthesis. Additionally, Cd-responsive upregulation of glutathione synthesis genes along with key transcriptional factors (MYB, bZIP) provided mechanistic insights into the role of GSH in antioxidant defense and lipid synthesis. The outcomes of the study highlighted the crucial molecular networks and crosstalk between the antioxidant and carbon assimilation pathways that aided Scenedesmus sp. IITRIND2 to survive under Cd stress with enhanced lipid content. Chapter 4 provides a detailed molecular metabolic profiling of the Cd spiked Scenedesmus sp. IITRIND2 through NMR-based metabolomics, gene expression, and biochemical analysis to validate the pathways involved in Cd tolerance and the carbon flux channelization resulting in enhanced lipid/extracellular polysaccharide (EPS) production. The metabolic rewiring in the tricarboxylic acid (TCA) intermediates, amino acid metabolism, and starch metabolism presented the robust machinery of the cells to channelize the carbon pools for producing neutral lipids of vehicular quality under Cd stress. Further, microalgal cells instigated a defense network comprising organic osmolytes (betaine, ethylene glycol, sugars), antioxidants (glutathione, ascorbate, etc.,) and amino acids (glycine, glutamate, alanine, valine, threonine) suggesting the interplay of glutathione-ascorbate and probable phytochelatin based detoxification pathways as double edge sword to impart tolerance and detoxification of Cd. Moreover, stress-induced response constituted significant changes in the membrane fluidity and production of biodiesel quality lipids along with soluble EPS having paramount biotechnological applications. Overall, key findings of the investigation validated the perspective of a viable and green solution to address the socio-economic concerns of microalgal biorefinery. Chapter 5 provides a strategic development of a microalgal biosorbent system for improved Cd removal efficiency and reusability. Microalgal biomass is considered an effective biosorbent of heavy metals, but encompasses demerits including the small size for their reuse. In this context, a green biosorbent, developed by immobilizing biomass of Scenedesmus sp. IITRIND2 in calcium alginate beads was evaluated for its Cd adsorption capacity. The algal alginate beads exhibited a Cd removal efficiency of ~ 84% from an aqueous medium at 25 ppm. The algal beads followed Langmuir isotherm suggesting primary action of physisorption rather than chemisorption in Cd removal. The adsorption of Cd on the beads was confirmed by surface charge and energy dispersive X-ray analysis. Further, FTIR analysis suggested the role of hydroxyl and carbonyl functional groups in Cd adsorption. Amongst the eight different desorption agents tested, ethylene diamine tetra-acetic acid (EDTA) and nitric acid (HNO3) showed comparable desorption efficiency of ~95-99%. The reusability of microalgal beads was tested for five consecutive cycles with a regeneration efficiency of ~90%. The findings of the present study suggested that green biosorbent made with biomass of a robust microalga holds great opportunity in the removal of cadmium ions from wastewater. In conclusion, the thesis explored the Cd tolerance, lipid augmentation, and EPS production in an oleaginous microalgal strain, Scenedesmus sp. IITRIND2 to propose a sustainable biorefinery framework. The proposed concept was validated comprehensively by biochemical and microscopic evidences. Further, molecular mapping of the entire networks involved in eliciting the adaptive response was done by integrating the information across the transcriptome and metabolome of Cd spiked cells. The outcomes of the study provided strong evidences for robust characteristics of this microalgal strain, and its implication for dual purpose of heavy metal remediation and biodiesel production. In addition, the application and reusability of the algal biomass in the form of algal-alginate beads was established as an effective strategy to remove Cd ions from the water.