Theses and dissertations (Applied Sciences)
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Item Evaluation of toxicity and biochemical characterisation of a microalgal diatom(2023-05) Beekrum, Lamees; Amonsou, Eric Oscar; Odhav, Bharti; Lalloo, RajOne ofthe critical challengesthat we face in the 21st century isthe need to feed an ever-increasing human population with increasingly limited natural resources. Microalgae have emerged as a potentialsolution for global food security as a sustainable biological food source for humans due to their nutrient-rich composition, particularly rich protein and bioactive compoundsthat provide potential benefits for human health. By establishing microalgae as a new food platform, we can increase the supply of these essential productsto address global demandsin a more efficient and environmentally sustainable way. These under-exploited organisms have been consumed in the human diet for thousands of years. Microalgae cultivation does not compete with land and resources required for traditional crops and has a superior yield compared to terrestrial crops. Diatoms are a major group of microalgae in the phytoplankton community and have the potential to be engineered into cell factoriesforthe sustainable production of bioactive compoundsin food and nutraceutical industries. This study aimed to characterise a rapidly growing marine diatom in terms of its toxicity and biochemical profile. This was done by evaluating the safety profile and biochemical composition, characterising the soluble protein, and investigating the carbohydrate profile with specific emphasis of β-glucan and its effect of cardioprotective properties on ferric-induced oxidative cardiac injury in a rat model. Based on Basic Local Alignment Search Tool (BLAST) analysis, the strain showed the closest similarity to Amphora sp. (JF834543.1) with 99.5% and istherefore represented as Amphora sp., accession number MW721231. The bacterial reverse mutation assay found no evidence of mutagenicity on the methanolic, aqueous, and hexane extracts of Amphora sp. and was found to exert low levels of cytotoxicity against Peripheral Blood Mononuclear Cells (PBMC). A 28- day acute oral toxicity assessment on male Wistar rats showed an absence of adverse effects and mortality in the rats. The biomass exhibited a low lipid profile, modest protein content, notable amino acid content, and excellent carbohydrate and mineral content. Results of this study for antioxidant assays displayed low to moderate activities. Protein extracted using three-phase partitioning (TPP) treatment showed that the protein concentration and total amino acid content were substantially higher in the protein-enriched biomass extract when compared to the dried biomass. The solubility of the protein-enriched biomass extract increased with the increase in pH within the range of pH 2 to pH 12. The biomass consisted of a simple monosaccharide profile comprising glucose, rhamnose, and mannose, and a β-glucan content of approximately 9%. The cardioprotective properties ofthe β-glucan extract on ferric-induced oxidative cardiac injury did not improve the glutathione (GSH) level significantly, it led to increased superoxide dismutase (SOD) and catalase activities, while depleting malondialdehyde (MDA), NO (nitric oxide), low-density lipoprotein cholesterol levels, and simultaneously elevating triglycerides and high-density lipoprotein (LDL) cholesterol levels. GC-MS analysis revealed a complete depletion of the lipid metabolites. Our results advocate the protective capabilities of the β-glucan extract against ferric-induced oxidative cardiac injury as portrayed by its ability to stall oxidative stress and modulate cardiac lipid metabolism while inhibiting the acetylcholinesterase and lipase activities. These results display that the β-glucan extract could be utilized as an alternative for the development of nutraceuticals for maintaining cardiac health. The diversity of food bioactive molecules obtained from microalgae makes these microorganisms a bioresource with full potential of exploitation in the food industry. The richness of compounds in microalgae can contribute to develop an algal-based food industry, focusing on producing and utilizing microalgae for innovative functional food products. Overall, this study demonstrated the potential utilization of the diatom, Amphora sp. as a potential ingredient and nutraceutical in foods.Item An integrated approach for biofuel and fertilizer production using microalgae grown in wastewater(2022-09) Musetsho, Pfano; Bux, Faizal; Renuka, Nirmala; Guldhe, AbhishekMicroalgae are recognized as potential candidates for resource recovery from wastewater and are projected for biorefinery models. Therefore, this study was undertaken to evaluate the potential of poultry litter and municipal wastewater as nutrient and water sources, for the cultivation of Acutodesmus obliquus for lipids production for biodiesel application. The efficacy of lipid extracted biomass (LEA) as fertilizer for mung bean crops was also assessed in microcosm. A. obliquus cultivation in acid pre-treated poultry litter extract (PPLE) showed maximum biomass production of 1.90 g L-1 , which was 74.67% and 12.61% higher than the raw poultry litter extract (RPPE) and BG11 respectively. Higher NO3-N, NH3-N, and PO4-P removal of 79.51%, 81.82%, and 80.52% respectively were observed in PPLE as compared to RPLE treatment. The highest biomass (140.36 mg L-1 d -1 ), lipids (38.49 mg L-1 d -1 ), and carbohydrates (49.55 mg L-1 d -1 ) productivities were observed in the PPLE medium. The application of LEA as a fertilizer for mung bean crops showed improvement in plant growth and soil microbial activity. A maximum increase in organic carbon (59.5%) and dehydrogenase activity (130.8%) was observed in LEA amended soil which was significantly higher than chemical fertilizer (CF) control in 30 days. Whilst plant fresh weight and leaf chlorophyll in the LEA amended soil was comparable to whole algal biomass (WA) and CF control. The findings of the present study could be a basis for sustainable biorefinery for the valorization of wastewater for the production of microalgae-derived biofuel and byproducts for agricultural applications.Item Optimization of biomass and lipids production from microalgae using wastewater in a pilot scale raceway pond(2021) Rawat, Ismail; Bux, FaizalMicroalgae provide a sustainable renewable solution for the production of commodity products such as liquid biofuels. There are numerous benefits to using algae for the production of biofuels, however, the cost of production is a major hurdle to commercial-scale development. Major factors influencing the production of algae are the cost of nutrients, availability of water, contamination, and grazers. Research into algal biomass for biofuels production at laboratory scale does not translate directly to cultivation at large scale due to the change in cultivation conditions and the constant flux of environmental factors. This study focuses on the upstream processes of cultivation of biomass in a ~ 1146 m2 raceway pond. It demonstrates biomass productivity under different climatic conditions and utilisation of post-chlorinated wastewater as a water and nutrient source. The study further elucidates the population dynamics of the system and provides insight into the challenges faced during the cultivation of algae at large scale. An indigenous Scenedesmus sp. gave biomass productivity of 31.23 g/m2 /d with lipid production of 29.6 % lipid/g DCW in a 10 m2 raceway pond in a greenhouse using BG11. Biomass productivity was reduced to 13.09 g/m2 /d with a lipid content of 22.9 % lipid/g DCW under 3-fold higher irradiance. Biomass productivity of circular 3000L ponds at the large scale site resulted in the highest biomass and acceptable lipid content using 250mg/L NaNO3 although significantly lower than the 10 m2 raceway ponds. Wastewater has shown potential to replace conventional media. Post-chlorinated wastewater was found to have low levels of nitrogen and phosphorus but contained metals that act as micronutrients for algae. Supplemented wastewater proved to be an effective growth. Six individual runs of a covered 1146 m2 raceway pond driven by paddlewheel were conducted over 15 months. The average water temperature ranged from 20.61±0.68°C during mid-winter to 31.03±2.22°C in late summer. Daylight ranges from 10.25 to 14 hours in winter and summer respectively. The highest average light intensity was 359.00±212.71 µmol/m2 /s from Mid-winter to early spring and 645.44±330.58 µmol/m2 /s in late summer. Biomass productivities were low ranging from 2.7 to 7.34 g/m2 /d for most runs of the raceway pond, mainly due to the long periods of cultivation. Average productivity at day 7 for all raceway runs was 7.25 g/m2 /d. Adaptive Neuro-Fuzzy Inference System (ANFIS) modelling of the system elicited that the major factors affecting biomass productivity in the raceway pond were light intensity, pH, and depth for the raceway pond. The model showed that maximum biomass productivity is possible at a depth between 20 and 22 cm at light intensities between 200 and 400 µmol/m2 /s. pH in the range of 9 to 9.5 correlated positively with light intensity ranging from 200 to 1000 µmol/m2 /s with maximum biomass expected in the region of 400 to 500 µmol/m2 /s. The main algal constituents for the raceway ponds were Scenedesmus obliquus, Scenedesmus dimorphus, Chlorella, Keratococcus, and species of unidentified cyanobacteria. Either Scenedesmus or Chlorella was dominant for extended periods. Bacteria in open systems can have a positive or negative effect on the growth of microalgae but is dependent on the strains of microalgae and bacteria as well as prevailing conditions making these systems highly complex. Rhodobacteraceae, Plactomycetaceae, Xanthomonadaceae, Flavobacteriaceae, Phycisphaeraceae, Comamonadaceae, and Cyclobacteriaceae were found to be the major families of bacteria that proliferate at different levels during the cultivation period in the circular ponds and the raceway pond. These families of bacteria have several beneficial traits to algae cultivation however further investigation is required. Modelling the system revealed that pH, depth, and light intensity were factors having a substantial effect on biomass productivity. As the system was carbon limited addition of CO2 (preferably a waste stream) could significantly enhance the overall biomass productivity. A major factor negatively affecting biomass productivity was the size of the pond. Inadequate mixing impacts biomass productivity in terms of access to nutrients and gaseous exchange. Shorter periods of cultivation resulted in higher productivities. For the scale of the system, semi-continuous harvesting would be required to achieve shorter residence time. This must be balanced against the energy utilization and cost of harvesting potentially lower culture densitiesItem Application of lipid extracted algae in feed and energy production(2019) Ansari, Faiz Ahmad; Bux, Faizal; Gupta, Sanjay Kumar; Guldhe, AbhishekMicroalgae are well considered to be promising feedstocks for biodiesel production. Microalgae can be grown under different types of cultivation conditions and their biomass has tremendous potential to be used as biofuel feedstock and for other applications such as feed, food, cosmetics, pharmaceutical etc. Despite the many benefits and the significant development in the field of microalgal biodiesel production, there are several challenges including high cultivation cost and developing efficient downstream processing methods. The biomass production cost is high, which significantly hinders the use of microalgae as a feedstock. Most of the available literature is focused on upstream, single strain and single product strategy, where mainly algal lipids are used for biofuel production. Hence, for improving the sustainability of the algal biofuel production processes and related process economics, a multiple applications approach using integrated biorefinery and exploiting microalgae for environmental benefits is required. To explore the microalgal biorefinery concept it is vital to understand the various cultivation conditions and applications of biomass in different sectors. There are various strategies, which have potential to make algal biofuel technologies more economically feasible and environmentally sustainable. Use of alternative culture media, improving the biomass production and the efficiency of downstream processing (drying, cell disruption, lipid extraction etc.) algal biofuel technology economical. Utilizing lipid-extracted algae (LEA) for energy and aqua feed application will maximize overall economic return and will leave minimal residues as by-product. The major focus of this thesis was to utilize LEA as substrate for biomethane production and protein source in aquaculture feed. However, effect of preceding steps such as microalgae cultivation, biomass drying and cell disruption on major metabolites extraction was also studied. Microalgae were cultivated in different medium (domestic wastewater and BG11) and their biomass yields and biochemical composition (lipid, protein and carbohydrate) were compared. Different drying and cell disruption techniques were employed for lipid extraction and their effect on lipid, protein and carbohydrate yields were evaluated. The yield of major metabolites on whole cell and LEA were also compared. Suitable solvent systems were selected for optimum lipid extraction from wet and dry biomass with minimal toxic effect on LEA metabolites so that LEA can be further used for biomethane and aquaculture feed production. The choice of microalgae at large scale depends upon the number of factors such as their adaptability to large-scale cultivation, biomass production, major metabolites content, robustness towards the open system cultivation and contamination. In this study, S. obliquus and C. sorokiniana were cultivated in wastewater and BG11 medium at laboratory scale. Both strains are indigenous to KwaZulu-Natal. C. sorokiniana showed lower biomass and major metabolites (lipid, protein and carbohydrate) production at large scale compared to S. obliquus. Considering better adaptability to open cultivation, high biomass and metabolites yields, S. obliquus strain was selected for the LEA application study. Microalgae species, C. sorokiniana and S. obliquus were cultivated on BG11 and using different ratios of raw domestic wastewater and post-chlorinated wastewater as nutrient media. The cultivation of S. obliquus and C. sorokiniana showed biomass yield of 1.2-3.5 and 0.78-1.8 g L-1 in BG11 medium, respectively. While biomass yield observed in wastewater was 0.59-1.59 g L-1 for S. obliquus and 0.67-1.45 g L-1 for C. sorokiniana. The higher biomass yield in BG11 medium attributed to the higher nutrient contents in this medium compared to wastewater. The lipid contents for S. obliquus and C. sorokiniana were 20 and 16.5% dry cell weight (DCW), respectively when grown using BG11 medium. While increases in lipid contents of 26.25 and 29.4% DCW were found for S. obliquus and C. sorokiniana, respectively when cultivated using wastewater. Similarly, carbohydrate contents for S. obliquus and C. sorokiniana were 18 and 17% DCW, respectively for BG11 medium. Increased in carbohydrate contents of 25% for S. obliquus, 28.4% DCW for C. sorokiniana were observed for wastewater. Microalgae tend to accumulate more lipids and/or carbohydrates under nutrient stress condition. The nitrogen and phosphorus contents in wastewater are lower than BG11 medium, which were responsible for stressed condition for microalgae. With limited nutrients in wastewater compared to BG11 medium, growth of microalgae is also lower which resulted in lower protein content. Protein content for S. obliquus and C. sorokiniana in BG11 medium were 37.83-48.8 and 25-35.3% DCW, respectively. The protein contents for S. obliquus and C. sorokiniana in wastewater medium were 16.4-27.29 and 15.8-27.3% DCW, respectively. The biochemical composition depends upon the nutrient composition of the medium and cultivation conditions. The two selected microalgae have shown potential for nutrient removal while cultivated in wastewater. The removal efficiency by S. obliquus was found to be 76.13% for COD, 98.54% for nitrogen and 97.99% for phosphate. Microalgae C. sorokiniana cultivation in wastewater removed 69.38% COD, 86.93% nitrogen and 68.24% phosphates. Increased lipid accumulation in the cells was also recorded in stressed conditions due to low nutrient availability from wastewater. After harvesting of microalgae from culture media, the water content in thick algal slurry (>85% DCW) lowers the products recovery. To overcome this challenge drying and cell disruption are required to enhance the efficiency of lipid extraction. Where drying and cell disruption increase the viability of biomass for lipid extraction process. Three biomass-drying techniques viz. sun, oven and freeze-drying and four-cell disruption techniques viz. microwave, sonication, osmotic shock and autoclave disruption were studied for their effect on recovery of major metabolites from S. obliquus. Microalgae metabolites recovery from whole cell and LEA were analysed and compared. The results showed that after lipid extraction, LEA still contained comparable protein to whole algae biomass however, the carbohydrate concentration was reduced. Oven drying exhibited the highest recovery of all the major metabolites followed by freeze-drying; sun drying however, showed lower yields. Despite lower metabolites recovery sun-drying technique is preferable at large scale due to its easy application and cost-effective nature. The main drawback of sun drying technique is weather dependence and required longer period to dry. The microwave and autoclave microalgal cell disruption improved the lipid yield but loss of other compounds was observed. In osmotic shock treatment, due to poor cell disruption efficiency low lipid were obtained and comparably lower protein loss was noticed during lipid extraction. Lipid extraction is crucial step for microalgae biodiesel production. Solvent-assisted lipid extraction is widely used technique for lipid recovery from dry or wet algae biomass. In a biorefinery approach, it is vital to choose appropriate solvents for the optimum lipid extraction whilst having minimal effect on the remaining metabolites (protein and carbohydrates) in LEA. LEA could be used for energy generation or aquaculture feed applications. Six commonly used organic solvents/ solvent systems were used for lipid extraction from wet and dry biomass. The results showed that the lipid extraction efficiency depends strongly on types of biomass as well as solvent systems selected. Lipid extraction from wet algal biomass could reduce the processing steps and save energy incurred in drying. However, the water present in wet algal slurry acts as a barrier, which results in lower lipid yield compared to the dry biomass. The results revealed that among all six-selected solvents, chloroform: ethanol (1:1 v/v) was most effective if wet biomass used specifically for lipid purpose only. To explore the biorefinery concept, isopropanol/hexane composition is the most suitable solvent system because it is less toxic and resulted in high protein (20.07% DCW) and carbohydrate (22.87%) yields in LEA. For dry algal biomass, chloroform: methanol (2:1 v/v) is an appropriate solvent system if biomass used especially for lipid (19.25%) extraction. If LEA to be used for energy and/or aquaculture feed application, DCM: methanol was found to be a suitable solvent system, which gave 32.79% protein and 26.92% carbohydrate yield. Comparatively hexane has lower lipid recovery but shown higher protein and carbohydrate yield in LEA. Due to less toxic, easy to scale up and inexpensive, hexane is preferable as a solvent for lipid extraction if LEA is to be further utilized at large scale for energy or feed application. Anaerobic digestion (AD) of organic residues is well-researched technology for biomethane production. Whole microalgae and LEA has promising potential for biomethane production. The anaerobic sludge used as inoculum for microalgal biomass digestion. Biomethane production from whole algae and products extracted algae highly depends on sludge to algae biomass ratio for higher methane production. The extraction of metabolites also changes the biochemical composition of residual biomass, which can affect the biomethane production. It is vital to understand the effect of various product-extracted algae and as well as pre-treated algae on the biochemical methane potential. In order to compare biomethane potential, four types of biomass were selected namely sun dried powder algae (SDPA), mild heat-treated algae (MHTA), LEA (using hexane as lipid extracting solvent) and protein-extracted algae (PEA). The average methane (CH4) production rate was ~ 2.5 times higher for protein and lipid extracted algae than for whole algae SDPA and MHTA whilst the cumulative CH4 production was higher for pre-treated algae. Highest cumulative CH4 production (318.7mL CH4 g-1 VS) was found for MHTA followed by SDPA (307.4mL CH4 g-1 VS). The CH4/CO2 ratios of 1.5 and 0.7 were observed for MHTA and LEA, respectively. Outcome of this objective revealed that pre-treatment process disrupts the microalgae cell walls, exposing intracellular material and increasing the surface area. The product-extracted algae changes the elemental composition, which decreases the cumulative gas yield CH4/CO2 ratio. Presence of high nitrogen in the form of protein produces ammonia (NH3) which inhibits the methane production. Therefore, it is imperative to use PEA biomass to improve the methane production yield than the whole cell biomass. Due to escalating price and unstable supply of fish meal (FM), alternative protein sources are used in aqua feed, however these sources do not meet to the requirement. The use of less expensive protein source in aquaculture feed as alternative to FM is required. Microalgae are primary producers in the food chain as well as a natural food for fish. Microalgal biomass is comprised of proteins, lipids, carbohydrates, pigments and many other bioactive compounds. The microalgal proteins have an appropriate balance of all essential amino acids, while lipids are rich in polyunsaturated fatty acids (omega-3 fatty acids, EPA, DHA). Whole algae contain all required ingredients while LEA also contain protein, carbohydrates, vitamins, bioactive compounds even though most of the lipid soluble nutrients have been removed. Thus, microalgae have promising potential to be used in aquaculture feed. Aquaculture production continues to increase globally, to meet the aquaculture feed demand algae supplemented aquaculture feed will play an important role in providing good quality fish. In this study, approximately 200 kg of microalgal biomass was harvested for the feed application. Due to lower toxicity, ease of availability and ease of recovery from mixture, hexane was used as a lipid extracting solvent at pilot scale to generate LEA. The 44 weeks (from juvenile to finisher stage) feeding trials were conducted to evaluate the effect of whole and LEA supplementation of S. obliquus strain on growth performance, disease tolerance, feed utilization, physiological activity, and fillet biochemical composition of Nile tilapia (Oreochromis niloticus). In the first trial, fish were fed with an algae free diet (control) and four experimental diets (2.5, 5, 7.5 and 10 wt%) as protein source of dried S. obliquus. The study showed that microalgae could be used as a protein supplement in the Tilapia feed for enhancement of morphological characteristics and nutritional value. The 7.5% and 10% supplementation of whole algal biomass in tilapia feed showed significant improvement in weight and length of the fish compared to the control. The daily body weight gain was 0.25 g higher in experimental groups than the control. The hepatosomatic index percentage was also higher in fish feed when 7.5% whole algae was used in fish feed as a protein source. The results also showed that 7.5% and 10% have better specific growth rate (1.57 and 1.5%), daily body weight gain (1.1 and 0.86 g), overall body weight gain (427.16 and 331.48 g), protein assimilation (43.96 and 40.46%) higher than the control diet fed fish. The survival rate of fish were 100% at every inclusion level. In second trial (44 weeks), two supplementations (7.5 and 10 wt%) of LEA as protein source were used in Nile tilapia diets. Results showed 7.5% and 10% LEA supplemented feed shown better growth performance than control. The protein content were 42.2%, 41.3% and 36.1% in tilapia fed with 7.5%, 10% LEA and control feed, respectively. The body weight gain, tilapia fed with 7.5% LEA shown 357 g while 10% LEA and control have 331.78 g, and 330.08 gm, respectively. The application of whole and LEA of S. obliquus in tilapia feed, shown appropriate supplementation level for tilapia feed at demonstration scale. This thesis presents advances in knowledge in the field of microalgae biorefinery research for pilot scale operations. This research work has covered various aspects such as effect of drying, cell disruption and lipid extraction on whole and LEA metabolites yield. The extraction of lipid from wet and dry microalgal biomass using various solvent systems provides a new insight for the selection of appropriate solvent systems, which can be used for the large-scale lipid extraction. The study on LEA for biomethane production enhances the understanding about the effects of different pre-treatments and product extractions on biomethane production. The results revealed that the supplementation of whole cell and LEA using S. obliquus for tilapia feed is safe therefore, can be used as an alternative protein source. The findings of this study have both academic and industrial value.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 Mitigation of carbon dioxide from synthetic flue gas using indigenous microalgae(2017) Bhola, Virthie Kemraj; Bux, Faizal; Swalaha, Feroz MahomedFossil carbon dioxide emissions can be biologically fixed which could lead to the development of technologies that are both economically and environmentally friendly. Carbon dioxide, which is the basis for the formation of complex sugars by green plants and microalgae through photosynthesis, has been shown to significantly increase the growth rates of certain microalgal species. Microalgae possess a greater capacity to fix CO2 compared to terrestrial plants. Selection of appropriate microalgal strains is based on the CO2 fixation and tolerance capability, both of which are a function of biomass productivity. Microalgal biomass could thus represent a natural sink for carbon. Furthermore, such systems could minimise capital and operating costs, complexity, and energy required to transport CO2 to other places. Prior to the development of an effective CO2 mitigation process, an essential step should be to identify the most CO2-tolerant indigenous strains. The first phase of this study therefore focused on the isolation, identification and screening of carboxyphilic microalgal strains (indigenous to the KwaZulu-Natal province in South Africa). In order to identify a high carbon-sequestering microalgal strain, the physiological effect of different concentrations of carbon sources on microalgae growth was investigated. Five indigenous strains (I-1, I-2, I-3, I-4 and I-5) and a reference strain (I-0: Coccolithus pelagicus 913/3) were subjected to CO2 concentrations of 0.03 - 15% and NaHCO3 of 0.05 - 2 g/1. The logistic model was applied for data fitting, as well as for estimation of the maximum growth rate (µmax) and the biomass carrying capacity (Bmax). Amongst the five indigenous strains, I-3 was similar to the reference strain with regards to biomass production values. The Bmax of I-3 significantly increased from 0.214 to 0.828 g/l when the CO2 concentration was increased from 0.03 to 15% (r = 0.955, p = 0.012). Additionally, the Bmax of I-3 increased with increasing NaHCO3 concentrations (r = 0.885, p = 0.046) and was recorded at 0.153 g/l (at 0.05 g/l) and 0.774 g/l (at 2 g/l). Relative electron transport rate (rETR) and maximum quantum yield (Fv/Fm) were also applied to assess the impact of elevated carbon sources on the microalgal cells at the physiological level. Isolate I-3 displayed the highest rETR confirming its tolerance to higher quantities of carbon. Additionally, the decline in Fv/Fm with increasing carbon was similar for strains I-3 and the reference strain (I-0). Based on partial 28S ribosomal DNA gene sequencing, strain I-3 was found to be homologous to the ribosomal genes of Chlorella sp. The influence of abiotic parameters (light intensity and light:dark cycles) and varying nutrient concentrations on the growth of the highly CO2 tolerant Chlorella sp. was thereafter investigated. It was found that an increase in light intensity from 40 to 175 umol m2 s-1 resulted in an enhancement of Bmax from 0.594 to 1.762 g/l, respectively (r = 0.9921, p = 0.0079). Furthermore, the highest Bmax of 2.514 g/l was detected at a light:dark cycle of 16:8. Media components were optimised using fractional factorial experiments which eventually culminated in a central composite optimisation experiment. An eight-factor resolution IV fractional factorial had a biomass production of 2.99 g/l. The largest positive responses (favourable effects on biomass production) were observed for individual factors X2 (NaNO3), X3 (NaH2PO4) and X6 (Fe-EDTA). Thereafter, a three-factor (NaNO3, NaH2PO4 and Fe-EDTA) central composite experimental design predicted a maximum biomass production of 3.051 g/l, which was 134.65% higher when compared to cultivation using the original ASW medium (1.290 g/l). A pilot scale flat panel photobioreactor was designed and constructed to demonstrate the process viability of utilising a synthetic flue gas mixture for the growth of microalgae. The novelty of this aspect of the study lies in the fact that a very high CO2 concentration (30%) formed part of the synthetic flue gas mixture. Overall, results demonstrated that the Chlorella sp. was able to grow well in a closed flat panel reactor under conditions of flue gas aeration. Biomass yield, however, was greatly dependent on culture conditions and the mode of flue gas supply. In comparison to the other batch runs, run B yielded the highest biomass value (3.415 g/l) and CO2 uptake rate (0.7971 g/day). During this run, not only was the Chlorella strain grown under optimised nutrient and environmental conditions, but the culture was also intermittently exposed to the flue gas mixture. Results from this study demonstrate that flue gas from industrial sources could be directly introduced to the indigenous Chlorella strain to potentially produce algal biomass while efficiently capturing and utilising CO2 from the flue gas.Item Design and operation of a laboratory scale photobioreactor for the cultivation of microalgae(2011) Bhola, Virthie; Bux, FaizalDue to greenhouse gas emissions from fossil fuel usage, the impending threat of global climate change has increased. The need for an alternative energy feedstock that is not in direct competition to food production has drawn the focus to microalgae. Research suggests that future advances in microalgal mass culture will require closed systems as most microalgal species of interest thrive in highly selective environments. A high lipid producing microalga, identified as Chlorella vulgaris was isolated from a freshwater pond. To appraise the biofuel potential of the isolated strain, the growth kinetics, pyroletic characteristics and photosynthetic efficiency of the Chlorella sp was evaluated in vitro. The optimised preliminary conditions for higher biomass yield of the selected strain were at 4% CO2, 0.5 g l-1 NaNO3 and 0.04 g l-1 PO4, respectively. Pulse amplitude modulation results indicated that C. vulgaris could withstand a light intensity ranging from 150-350 μmol photons m-2s-1. The pyrolitic studies under inert atmosphere at different heating rates of 15, 30, 40 and 50 ºC min-1 from ambient temperature to 800 oC showed that the overall final weight loss recorded for the four different heating rates was in the range of 78.9 to 81%. A tubular photobioreactor was then designed and utilised for biomass and lipid optimisation. The suspension of microalgae was circulated by a pump and propelled to give a sufficiently turbulent flow periodically through the illuminated part and the dark part of the photobioreactor. Microalgal density was determined daily using a Spectrophotometer. Spectrophotometric determinations of biomass were periodically verified by dry cell weight measurements. Results suggest that the optimal NaNO3 concentration for cell growth in the reactor was around 7.5 g l-1, yielding maximum biomass of 2.09 g l-1 on day 16. This was a significant 2.2 fold increase in biomass (p < 0.005) when compared to results achieved at the lowest NaNO3 cycle (of 3.8 g l-1), which yielded a biomass value of 0.95 g l-1 at an OD of 1.178. Lipid accumulation experiments revealed that the microalga did not accumulate significant amounts of lipids when NaNO3 concentrations in the reactor were beyond 1.5 g l-1 (p > 0.005). The largest lipid fraction occurred when the NaNO3 concentration in the medium was 0.5 g l-1. Results suggest that the optimal trade-off between maximising biomass and lipid content occurs at 0.9 g l-1 NaNO3 among the tested conditions within the photobioreactor. Gas chromatograms showed that even though a greater number of known lipids were produced in Run 8, the total lipid percentage was much lower when compared to Runs 9-13. For maximal biomass and lipid from C. vulgaris, it is therefore crucial to optimise nutritional parameters such as NaNO3. However, suitable growth conditions for C. vulgaris in a tubular photobioreactor calls for innovative technological breakthroughs and therefore work is ongoing globally to address this.Item Screening for indigenous algae and optimisation of algal lipid yields for biodiesel production(2011) Rawat, Ismail; Bux, FaizalThe depletion of global energy supplies coupled with an ever increasing need for energy and the effects of global warming have warranted the search for alternate renewable sources of fuel such as biodiesel. First generation biofuels are not sustainable enough to meet long term global energy requirements and more recently there has been concern expressed as to the potential negative implication of crop based biofuels in the form of negative energy balances and potentially no greenhouse gas benefit due to land utilisation not being taken into account. Microalgae have shown great promise as a sustainable alternative to first generation biofuels. They have faster growth rates, have greater photosynthetic efficiencies, require minimal nutrients and are capable of growth in saline waters which are unsuitable for agriculture. Microalgae utilise a large fraction of solar energy and have the potential to produce 45 to 220 times higher amounts of triglycerides than terrestrial plants. The use of microalgae for biodiesel production requires strain selection, optimisation and viability testing to ascertain the most appropriate organism for large scale cultivation. This study focuses on bioprospecting for indigenous lipid producing microalgae, screening, selection and optimisation of growth and lipid yields with respect to nutrient limitation. Further we have ascertained the sustainability of a selected species of microalgae in open pond system. Chlorella sp. and Scenedesmus sp. were found to be dominant amongst the isolates. Strains we selected and underwent media selection and growth and lipid optimisation trials. BG11 media was selected as the most appropriate media for the growth of the selected Chlorella and Scenedesmus strains. Little variation in growth was observed for both cultures ten days into cultivation under varying nitrate concentrations. Phosphate optimum was shown to be 0.032g/l for Scenedesmus sp and 0.04g/l for Chlorella sp. Best lipid yield determined during exponential growth was achieved in cultures with 0.3g/L to 0.6g/L nitrate and phosphate as per BG11 medium. pH optimisation showed that cultures may be adapted to growth at higher pH over time. The optimum pH range for growth was determined to be narrow and was found to be between pH 10 and pH 11. Chlorella sp. was shown to be sustainable as a dominant culture in open pond system. Open pond systems however are prone to contamination by other species of microalgae within weeks of inoculation.