Faculty of Applied Sciences

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Assessment of microalgal ACCase and rbcl gene expression as a function of nutrient and metal stress
(2017) Singh, Poonam; Bux, Faizal; Kumari, Sheena K.; Guldhe, Abhishek
Microalgae are considered to be a potential feedstock for biodiesel production. However, the main concern with regard to the large scale microalgal biodiesel production process is its competence and economic viability. The commercial realization of microalgal biodiesel production requires substantial impetus towards development of efficient strategies to improve lipid yields upstream. Nitrogen (N) and phosphorus (P) stress during cultivation are the widely used lipid accumulation strategies for microalgae. However, these individual nutrient stress strategies are associated with compromised biomass productivity which hampers overall lipid productivity. Lipid enhancement strategies based on light, temperature and CO2 are associated with technological barriers for scale up and incur additional cost. Thus, the main aim of this study was to develop an integrated, easily applicable and scalable lipid enhancement strategy based on nutrients and metals such as N, P, iron (Fe), magnesium (Mg), calcium (Ca) and EDTA stress for selected indigenous microalgal strains. The effect of metal concentrations individually and in combination on microalgal lipids and biomass production is a scarcely exploited area. In this study, a novel approach involving individual as well as combined metals and EDTA stress under N and P limited conditions for lipid enhancement in microalgae was investigated. Microalgal growth physiology, photosynthetic performance, biochemical composition (lipid, carbohydrate and protein) and expression of selected key genes involved in photosynthesis (rbcL) and fatty acid biosynthesis (accD) were studied both under selected individual and combined stress conditions. Out of seven microalgal isolates obtained during the initial isolation and screening process, two strains were selected for lipid enhancement study based on their growth rates, biomass yields, lipid content and lipid productivities. The strains were later identified as Acutodesmus obliquus and Chlorella sorokiniana based on both morphological characteristics and phylogenetical analysis. The selected strains were thereafter subjected to different cultivation conditions involving varying metal, EDTA and nutrient stress conditions. A significant increase in lipid productivity was observed when the concentrations of Fe, Mg and EDTA were increased and Ca was decreased to degree in the N and P stress BG11 medium. For A. obliquus, a highest lipid productivity of 80.23 mgL-1d-1 was achieved with the developed strategy under limited N (750 mg L-1) condition which was 2.18 fold higher than BG11 medium and 1.89 fold higher than N limited condition alone. Similarly, for C. sorokiniana, highest lipid productivity of 77.03 mgL-1d-1 was achieved with the developed strategy under limited N (500 mgL-1) and P (10 mgL-1) which was 2.67 fold higher than BG11 medium and 2.35 fold higher than N and P limited condition alone. For both the microalgal strains, Fe was the most significant trace metal affecting their lipid productivity. These above observations were further confirmed through photosynthetic performance analysis and gene expression studies. At mid log phase, 6.38 and 5.15 fold increases in the expression levels of rbcL gene were observed under combined stress (OCMS+OE) as compared to the control (BG11) condition in A. obliquus and C. sorokiniana respectively. This also resulted in an increased expression level of accD gene involved in lipid biosynthesis to 10.25 fold and 9.79 fold in A. obliquus and C. sorokiniana respectively at late log phase. The results from expression studies of rbcL and accD genes were in compliance with biomass yields, photosynthetic performance, protein yield and lipid productivities for both the strains under different cultivating conditions. The universal applicability of the above strategy was confirmed by applying it to five other microalgae strains isolated in this study which resulted in considerable increase in their overall lipid productivity under optimized conditions. Attempts were made to scale up the lab scale study to open circular pond (3000L) cultivation for A. obliquus. Results showed a 2.08 fold increase in lipid productivity under optimized conditions compared to the control, which emphasizes the scalability of the developed strategy even under uncontrolled conditions. In conclusion, the developed combined metal and EDTA stress strategy not only assisted in alleviating the biomass productivity but also enhanced the lipid accumulation which resulted in overall increased lipid productivity under N and P limited condition. Furthermore, the improved carbohydrate and protein productivities observed with the developed lipid enhancement strategy make it suitable for biorefinery approach with multiple products. An improvement in lipid profile and high biodiesel conversion were also observed with this universally applicable and scalable lipid enhancement strategy confirming their potential applicability during large scale cultivation for biodiesel production.
Investigation of combined effect of nitrogen, phosphorus and iron on lipid productivity of microalgae Ankistrodesmus falcatus KJ671624 using response surface methodology
(Elsevier, 2015-02-15) Singh, Poonam; Guldhe, Abhishek; Kumari, Sheena K.; Rawat, Ismail; Bux, Faizal
Enhancement of lipid accumulation is essential to improve the commercial feasibility of microalgal biodiesel production. An oleaginous microalgal strain, Ankistrodesmus falcatus KJ671624 was evaluated for its potential as a biodiesel feedstock in this study. The collective effect of nutrient (nitrogen, phospho-rous and iron) stresses on the lipid productivity of the selected strain was studied by response surface methodology. The highest lipid content of 59.6% and lipid productivity of 74.07 mg L−1 d−1 was obtained under nutrient stress with nitrogen 750 mg L−1, phosphorus 0 mg L−1 and iron 9 mg L−1. The photosyn-thetic behaviour validates the high lipid productivity under combined nutrient stress condition. Saturated fatty acid composition was increased by 38.49% under selected nutrient stress condition compared to BG11 medium. The enhanced lipid accumulation with suitable lipid proﬁle (C16:0, C18:1, C18:2, C18:3) and biodiesel conversion of 91.54 ± 1.43% achieved in A. falcatus KJ671624 further conﬁrm its potential as a promising feedstock for biodiesel production.
Trends and novel strategies for enhancing lipid accumulation and quality in microalgae
(Elsevier, 2016) Singh, Poonam; Kumari, Sheena K.; Guldhe, Abhishek; Rawat, Ismail; Misra, Rohit; Bux, Faizal
In order to realize the potential of microalgal biodiesel there is a need for substantial impetus involving interventions to radically improve lipid yields upstream. Nutrient stress and alteration to cultivation conditions are commonly used lipid enhancement strategies in microalgae. The main bottleneck of applying conventional strategies is their scalability as some of these strategies incur additional cost and energy. Novel lipid enhancement strategies have emerged to research forefront to overcome these challenges. In this review, the latest trends in microalgal lipid enhancement strategies, possible solutions and future directions are critically discussed. Advanced strategies such as combined nutrient and culti-vation condition stress, microalgae–bacteria interactions, use of phytohormones EDTA and chemical additives, improving light conditions using LED, dyes and paints, and gene expression analysis are described. Molecular approaches such as metabolic and genetic engineering are emerging as the potential lipid enhancing strategies. Recent advancements in gene expression studies, genetic and metabolic engineering have shown promising results in enhancing lipid productivity in microalgae; however environmental risk and long term viability are still major challenges.
Biodiesel synthesis from microalgae using immobilized Aspergillus niger whole cell lipase biocatalyst
(Elsevier, 2016) Guldhe, Abhishek; Singh, Poonam; Kumari, Sheena K.; Rawat, Ismail; Permaul, Kugen; Bux, Faizal
Whole cell lipase catalysis and microalgal feedstocks make overall biodiesel synthesis greener and sustainable. In this study, a novel approach of whole cell lipase-catalyzed conversion of Scenedesmus obliquus lipids was investigated for biodiesel synthesis. Microalgal biodiesel was characterized for its fuel properties. Optimization of process parameters for immobilized Aspergillus niger whole cell lipase-catalyzed biodiesel synthesis was carried out. Highest biodiesel conversion of 53.76% was achieved from S. obliquus lipids at 35 °C, methanol to oil ratio of 5:1 and 2.5% water content based on oil weight with 6 BSPs (Biomass support particles). Step-wise methanol addition was applied to account for methanol tolerance, which improved biodiesel conversion upto 80.97% and gave 90.82 ± 1.43% yield. Immobilized A. niger lipase can be used for 2 batches without signiﬁcant loss in conversion efﬁciency. Most of the fuel properties of biodiesel met the speciﬁcations set by international standards.

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