Faculty of Applied Sciences
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Item 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, AbhishekMicroalgae 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.Item 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, FaizalIn 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.Item Cytotoxicity and gene expression of selected apoptotic markers in the human laryngeal carcinoma cell line (HEp-2) by Bulbine spp. fractions(2013-07-30) Singh, Rishan; Reddy, Lalini; Permaul, KugenApoptosis, or programmed cell death, is a process which is pivotal in eliminating damaged, infected, or unwanted cells from the body. It has been studied in numerous types of cell lines ranging from normal to infected cell lines, and there have been a wide range of studies on apoptosis in laryngeal cancer because this type of cancer has become one of the most common types of head or neck cancer due to the high incidence of alcohol consumption, tobacco smoking and chewing of betel quid amongst populations. Laryngeal cancer is usually treated with radiotherapy or is surgically removed, but due to the loss of the function of the larynx after surgery, it has been suggested that alternative strategies or ways of treating laryngeal cancer are required. This has prompted the use of, and research in the field of, plant medicine to combat laryngeal cancer. Plant medicine has been used for centuries by the Chinese, Indian and Arabian population in Uhani, Ayurveda and Siddha as a form of replacing conventional medicine with complementary and alternative medicine, these include many plants from the family Asphodelaceae, which have become marketable commodities owing to their medicinal values and traditional uses. Amongst these plants, the genus Bulbine has been used as a form of natural medicine in rural Africa and they are also exploited for their aloe vera properties as well as their possession of phytochemical compounds such as isoflavanoids, nor-lignans, naphthalene derivatives, anthracene and poly prenylated flavonoids. There has been a compelling amount of literature on the traditional uses of the Bulbine spp. because these are linked to the Bulbine spp. having secondary metabolites such as pyroles, chromones, coumarins, bianthraceane, benzene as well as alkaloids. However, for Bulbine natalensis and B. frutescens, the plants of interest in this study, the location of anticancer compounds in them are the only amounts of information available. It has been reported, traditionally, that B. natalensis possesses the anticancer potential in the roots, while the anticancer potential for B. frutescens is in the leaves. However, this requires scientific clarification. Therefore, this study was conducted to assess programmed cell death or apoptosis by analysing the responses of the human laryngeal carcinoma cell line (HEp-2) to crude aqueous and organic (50% and 100% ethanol) fractions of B. natalensis and B. frutescens. In order to have achieved this, the HEp-2 cell line was exposed to the above mentioned fractions at three different final concentrations (20, 2 and 1μg/ml) and assessed for cytotoxicity using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay as an indicator of cell death after fraction utilisation (3 days) for 5 and 8 days. The differences in the potency of the Bubline spp. fractions were confirmed using the non-parametric ANOVA test. Thereafter, selected fractions were screened for apoptotic potential using reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the expression of bax and caspase-3 biomarkers, which are the biomarkers that participate in mitochondrial, endoplasmic reticulum and receptor-ligand mechanism of apoptosis. The fractions of B. frutescens were selected relative to those of B. natalensis for the RT-PCR procedure (read section 3.4.1. for the selection procedure) and links between the cytotoxicity and gene expression results were analysed. It was found that the B. natalensis fractions had a much greater cytotoxic effect on the HEp-2 cell line compared to fractions of B. frutescens by the fifth day of the MTT assay. On the eight day of incubation, there was an increase in HEp-2 cell line proliferation by the fractions of both plant species administered. The fractions selected for bax and caspase-3 gene expression analysis for B. natalensis were the: 20 μg/ml root and corm aqueous fractions, 20 μg/ml leaf and corm 100% ethanol fractions, 20 μ g/ml corm 50% ethanol fraction, 2 μg/ml root aqueous fraction, 2 μg/ml leaf 100% ethanol fraction and the corm 1 μg/ml aqueous and 50% ethanol fractions. The fractions that were compared to B. natalensis were the 20 μg/ml root and leaf aqueous and 100% ethanol fractions respectively, the 2 μg/ml root aqueous fraction and the 2 μg/ml leaf 100% ethanol fraction. It was found from RT-PCR analysis that all of the B. natalensis fractions tested induced expression of caspase-3, which indicated that those fractions were capable of inducing apoptosis in laryngeal carcinoma in vitro, since caspase-3 is the molecular indicator of apoptosis. The aqueous B. frutescens root fraction, did not induce expression of caspase-3 gene, although it caused expression of bax. This implied that the root aqueous B. frutescens fraction, may be involved in some other form of cell death, other than apoptosis. It was also found that there was variability in the response of the HEp-2 cell line to the Bulbine spp. fractions because of the variation in bax expression among fractions of different concentration. It was difficult, from this study, to classify fractions into categories for their mechanism of action, because not all of the fractions that caused the expression of capase-3, induced bax gene expression. Hence, proper conclusions were unable to be made, more so, because all the mechanisms of apoptosis mentioned, involve bax gene activation in order to proceed to completion. Therefore for those Bulbine spp. fractions to which the HEp-2 cell line exhibited a variable response to, it was postulated that cell death or apoptosis occurred through some other unknown mechanism. Overall, the cytotoxicity result didn’t correlate to the gene expression results because fractions that promoted HEp-2 cell line growth by day five, expressed apoptotic markers, which highlighted the sensitivity and accuracy of the cells-to-cDNATM II kit for detecting a few possibly apoptosed cells. This was confirmed by the fact that the HEp-2 cell line used in the MTT cytotoxicity assay and gene expression study had the same passage number and were viable, the latter being achieved because the MTT assay only measures the cytotoxicity of compounds once they have been taken up by viable cells – measuring mitochondrial activities expressed as absorbances. Therefore, the deduction that HEp-2 cell death may be due to bax/caspase-3 expression was valid because the mRNA was isolated from viable HEp-2 cells that had been killed by Bulbine spp. fractions of different polarity. Furthermore, the lack of correlation between the cytotoxicity and gene expression results indicated the amount of HEp-2 cell line proliferation by the fraction out-competes those that died, thereby producing a negative cytotoxicity result. There was a relationship between the traditional information about the anticancer potential for B. natalensis and B. frutescens. For example, the aqueous root fractions of B. natalensis were found to be non-toxic to the HEp-2 cell line, but did express caspase-3, which indicated the possibility of apoptosis. Similarly, the 100% ethanol leaf B. frutescens fractions were non-toxic to the HEp-2 cell line, but were able to induce apoptosis as well. This emphasised that the MTT cytotoxicity assay should be compared with other methods of measuring cytotoxicity when performing studies like this, because although literature has emphasised many advantages of using the MTT cytotoxicity assay in apoptotic studies, this study proved otherwise. When identical HEp-2 cells were treated with the same extract, only some cells were killed (apoptosis) whereas others proliferated. This was because although the cells were identical phenotypically, they were all probably at different phases of the cell cycle resulting in the HEp-2 cells responding variably to the same fraction at different concentrations. It was also found that the responses were concentration independent. For example, the 1 μg/ml B. natalensis corm fraction exhibited the highest toxicity of the three concentrations administered. The lowest cytotoxicity was achieved for the 20 μg/ml fraction – showing a proliferative effect on the HEp-2 cell line. Similarly, the 2 μg/ml aqueous B. natalensis leaf fraction induced the highest cytotoxicity level in the HEp-2 cell line followed by the 1 μg/ml and then the 20 μg/ml fractions. Apart from the genetic variation in identical HEp-2 cells; this indicated that the HEp-2 cell line was selective to particular fractions of the Bulbine spp. for utilisation. Concentration independence and HEp-2 cell preferential selection has been reported in many other studies involving plant fractions/extracts and natural products. This study demonstrated that although all the tested B. natalensis fractions were capable of inducing HEp-2 cell death possibly via. apoptosis (caspase-3 induction), a lack of any link between apoptosis and the cytotoxicity results (hence the 20 μg/ml corm fraction had a negative cytotoxicity but expressed both apoptotic markers), indicated the need for phytochemical screening of both Bulbine spp. in future, to determine the compounds that are responsible for the cytotoxicity and gene expression result outcomes of both Bulbine spp. fractions. Furthermore, procaspase genes also have to be analysed since genes are expressed to form procaspases, which then form active caspases. Although normal cells also express caspase-3 genes during apoptosis, this study focused exclusively on the effect of Bulbine natalensis and B. frutescens fractions (selected relative to the cytotoxicity results of B. natalensis) on the HEp-2 cell line (read cell culture and cytotoxicity discussion for selection of HEp-2 cell line). The validity of this study is confirmed by similar experimental designs that assayed the cytotoxicity of plant-derived or natural compounds on cancer cell lines only, and the detection of apoptosis through caspase- 3 induction and other unrelated methods. This is the first study to report the induction of apoptosis in cancer cell lines by Bulbine spp. fractions using cytotoxicity and the expression of bax and caspase-3 apoptotic markers. It provides insight into the interaction between the HEp-2 cell line and the aqueous and organic fractions of B. natalensis and B. frutescens by analyzing links between cytotoxicity and bax and caspase-3 gene expression; which could probably contribute to drug design with selected Bulbine spp. fractions. Further investigations are required in future, to confirm the possible drug targets of the studied Bulbine spp. fractions in an attempt of assaying their therapeutic importance.