Dissecting the molecular mechanisms of producing biofuel and value-added products by cadmium tolerant microalgae as sustainable biorefinery approach
Introduction
The frightening statistics of escalating fuel prices, water pollution, and intensified global warming in the past decade have become an inevitable concern worldwide. With the concomitant rise in global population, the energy requirements are envisaged to grow 47 % by 2050, along with the rise in carbon emission and pollutant load in aquifers due to rapid industrialization [1], [2]. Such a scenario demands a sustainable and carbon–neutral process to assure the social and economic well-being of society [3]. In this context, microalgae-derived oil is the best-suited alternative to petroleum that could curb fuel scarcity by linking wastewater treatment, reducing carbon emission, and bioenergy production to attain carbon neutrality in an eco-friendly and economical manner [4], [5]. Microalgae can utilize wastewater as a low cost nutrient source to produce biomass that can be further used as feedstock for biofuel production [6], [7]. Though wastewaters are suitable for the growth of algal cells, the presence of toxic pollutants such as cadmium (Cd) limits its applicability to most of the algal strains [8].
Cd is a carcinogenic pollutant that is widely present in the wastewaters of various industries such as electroplating, battery manufacturing, dyes/paints etc [9]. Cd can readily bind to sulfhydryl, carboxyl, and phosphate functional groups contributing to net negative charge on the microalgal cell's surface. Cd is translocated inside the cells through ions channels or transporter proteins constitutively present on the cell membrane [10], [11], [12], [13], [14], [15], [16]. The enhanced intracellular concentration of Cd alters the physiological activity of microalgal cells resulting in inhibited growth and impaired photosynthesis [17], [18]. Despite the severe toxicity index of Cd, some microalgal strains inherit the ability to tolerate and adapt to the toxic environment by the virtue of their metabolic flexibility [18], [19]. Algal cells adapt and tolerate Cd toxicity by regulating the oxidative stress responses by enhancing the production of antioxidants (enzymes and metabolites) and metal-chelating peptides/proteins [17], [20]. Another general response of algal cells to adapt to such abiotic stress is channelizing the available carbon pools toward the synthesis of energy compounds such as carbohydrates and lipids [21], [22], [23]. Such alteration in the energy molecules not only relieves the oxidative stress in algal cells but also offers the feedstock for sustainable production of microalgae-derived biofuels [22], [24]. Along with biofuels, stress-induced production of other value-added products such as extracellular polysaccharides (EPS), also offer an impressive biorefinery framework for the cost-effective production of algal biodiesel [25], [26].
Bioprospecting Cd-tolerant algal species under a biorefinery approach for producing biofuels and value-added products requires a thorough understanding of the cellular rewiring mechanism for Cd tolerance at the molecular level [27]. Recent studies have provided biochemical evidence on microalgae-based Cd remediation and biodiesel production [12], [15], [16], [28], [29]. Further, transcriptomics/proteomics technologies have been deployed on microalgae Chlamydomonas reinhardtii, Dunaliella sp., and Auxenochlorella sp. to highlight Cd toxicity [30], and redox homeostasis in algal cells under Cd exposure [31], [32], [33]. With the advancements in existing omics techniques, metabolomics is a robust methodology to characterize small metabolites involved in various biological reactions that could facilitate anticipating the cellular and physiological state of cells under certain conditions [34], [35], [36], [37]. Further, integrating the metabolic profiling with biochemical responses and transcriptional expression of candidate genes of interest will enable us to get mechanistic insights into integrated dynamics of Cd toxicity, adaptive responses, and lipid augmentation in the microalgal cell. Interestingly, no such study unraveling the molecular mechanisms of Cd tolerance correlated with lipid/EPS production with comprehensive metabolomics analysis is available till date.
In this context, the current study comprehends the integration of molecular mechanism of Cd tolerance, Cd induced carbon flux channelization towards lipid and value-added by-products in a Cd tolerant green microalga with detailed physiological changes. The current study elucidates the bioprospecting of natural robust microalgae for biorefinery approach by integrating green energy and bioremediation of toxic heavy metals in a sustainable manner. Cd induced changes in the lipid profiles, extracellular polysaccharide (EPS) content, and responsive metabolome of Scenedesmus sp. IITRIND2 was analyzed in detail to uncover the decisive pathways for regulating Cd toxicity in cells. Further, ultrastructural changes were visualized by cross-sectional transmission electron microscopy (TEM), and molecular evidences were evaluated by quantifying the gene expression levels for carbohydrate/lipid pathways along with Cd detoxification using RT-PCR analysis. The outcomes of the study provided crucial insights into the shifting and allocation of carbon flux towards lipid biosynthesis, and strengthened antioxidant machinery upregulating the synthesis of glutathione, sugars and osmolytes to prevent Cd toxicity. Moreover, the enhanced fraction of EPS under Cd stress also indicated an integrated network regulating the tradeoff among lipid and polysaccharides to mitigate Cd stress. Overall, the current investigation elucidated the key interactions among the molecular pathways of Cd detoxification and lipid/polysaccharide production to validate the sustainable and viable framework of microalgae derived biorefinery.
Section snippets
Reagents
All the media component for microalgae cultivation and cadmium chloride (CdCl2) was procured from SRL and Hi Media, India respectively. Reagents for NMR experiments such as deuterium oxide (D2O) and trimethylsilylpropanoic acid (TSP) was purchased from Sigma, India. All the solvents used in this study were of HPLC grade with 99 % purity. Chemicals for biochemical assays like oxidized glutathione (GSSG) and reduced glutathione (GSH), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB),
Results
A robust microalgal strain shows tolerance towards toxic heavy metals like Cd by the virtue of their metabolic flexibility. Scenedesmus sp. IITRIND2 is one such potent algal strain that sustains in 25 ppm of Cd, mitigates 80 % of it, while augmenting ∼33 % of lipid content [12]. Deciphering the molecular mechanism behind the Cd induced lipid augmentation in algal cells could unveil potential target pathways for tailoring microalgae as a sustainable feedstock for biorefinery approach. Such
Mechanistic insights into the molecular mechanisms involved in Cd tolerance and lipid accumulation in Scenedesmus sp. IITRIND2
The adaptive flexibility of microalgal cells to accumulate energy compounds under stress is one of the most attractive features proffering a sustainable solution coupling bioremediation, biofuel, and value-added compounds production in a biorefinery framework. Needless to mention, the total product yield largely depends on the nature of toxicant/wastewater, and the choice of algal strain. Over the last decade, extensive efforts have been made to integrate the remediation of Cd and biofuel
Conclusions
The current investigation elucidated the mechanism of Cd tolerance in Scenedesmus sp. IITRIND2 that enabled augmentation of lipids and EPS. The microalga rewired its cellular machinery by regulating multiple pathways comprising of amino acid metabolism, citric acid cycle, starch/sugar metabolism, glycerolipid metabolism, and glutathione metabolism to tolerate Cd stress. The intricate defense response of cells established the crucial role of ascorbate–glutathione and phytochelatin based
CRediT authorship contribution statement
Shweta Tripathi: Investigation, Formal analysis, Visualization, Writing – original draft. Manikyaprabhu Kairamkonda: Investigation, Formal analysis. Payal Gupta: Investigation, Formal analysis. Krishna Mohan Poluri: Conceptualization, Project administration, Supervision, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
ST acknowledge the MHRD fellowship from IIT-Roorkee for pursuing Ph.D. KMP acknowledge the support of Grants GKC-01/2016-17/212/NMCG-Research from NMCG-MoWR, Govt. of India, and BEST-18-KMP/IITR/109 from Bharat Energy Storage Pvt. Ltd (BEST), India. The authors sincerely thank the NMR, other analytical facilities provided by Institute Instrumentation Centre (IIC) at IIT-Roorkee, and SAIF, AIIMS-New Delhi for TEM facility.
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