Green microalgae are an important source of natural products such as β-carotene, and have recently become objects of intense study for producing biodiesel and valuable recombinant proteins. Application of chloroplast engineering in microalgae is limited by the availability of tools for genetically engineering the chloroplast of commercially important species. The phytoene desaturase gene of a previously isolated norflurazon tolerant mutant of Chlamydomonas reinhardtii was isolated and sequenced. A thymine to guanine transversion in exon 2 changes codon 131 resulting in a F131V mutation that is located in the NADP binding site domain on the primary structure. This mutation clusters with three conserved amino acids, whose substitution confers norflurazon tolerance in other species, in a pocket on a 3-D structure of the protein. The pocket identifies the target site of norflurazon. The side pocket is at the opening of a tunnel leading to the enzyme's NADP binding site. The mutant gene was cloned and used as marker for glass-bead mediated nuclear transformation of C. reinhardtii using direct selection with 5 μM norflurazon. Integration was by illegitimate recombination and transformants were able to grow in media containing 150 μM norflurazon. Transformants exhibited cross tolerance to fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wildtype. This allows mutant pds gene to act as a dual negative/positive selectable marker that is conditional on the herbicide used. The F131V mutation was introduced into a synthetic gene encoding a Dunaliella salina phytoene desaturase that contained codons used frequently in C. reinhardtii chloroplast genes. The 1.8 kbp CpPDS1 gene was assembled from 74 oligonucleotides by overlap PCR. The coding sequence was inserted into a Dunaliella tertiolecta chloroplast targeting vector that integrated the CpPDS1 sequence into the ycf3-trnL-rbcL region of the plastome. The resulting vector was transformed into D. salina and D. tertiolecta chloroplasts using particle bombardment with plasmid coated gold microprojectiles. Norflurazon tolerant colonies were isolated and the D. salina and D. tertiolecta clones were shown to contain a pds gene integrated in the plastome using PCR analyses. Transformation of the CpPDS1 gene into C. reinhardtii chloroplasts by rescue of an atpB mutation only gave rise to herbicide tolerant colonies if the presequence was removed. Industrial production of algae in large volumes is limited by the availability of light to drive algal growth. This problem was addressed by expressing fluorescent protein Katushka in the chloroplast of C. reinhardtii which converts yellow light to red light. The Katushka gene was transformed into chloroplasts using vector pB10, which was constructed to rescue a deletion in the chloroplast atpB gene in the mutant CC373 strain. The Katushka coding sequence was codon-optimised for expression in chloroplasts and expressed from three different promoter and 5' UTRs (atpA, atpB and psbD). C. reinhardtii wild type cells were able to grow under either blue or red LED lights but grew best when both were present. Wild type cell grew poorly in yellow LED lighting. Cells expressing Katushka in the chloroplast exhibited enhanced autotrophic growth in yellow light and under conditions where yellow light was present and red light was limiting. The improvement in growth was related to the levels of Katushka fluorescence detected in chloroplast transformants, which was highest for the atpA promoter and UTR.

No description available.

The study introduces and demonstrates the viability of the continuous flow reactor (CFR) system for the production of bio-crude oil (BCO) from wet microalgae. Preliminary experiments conducted in the CFR system in hot compressed water (HCW) were successful in converting wet microalgae into liquid BCO. However, the synthesis and aggregation of high boiling point (HBP) components of BCO and the accumulation of char in the tubular piping of CFR system were identified as the limiting factor to the viability of the system. The aggregation of HBP components and the accumulation of char result to system blockage which prevents the continuous flow of the liquefaction product mixture in the CFR system. Inhibiting the reactions leading to the formation of HBP components and char will improve the performance of the CFR system. Therefore, the study seeks to incorporate co-solvents in the liquefaction reaction media in an attempt to inhibit or minimize the prevalence of HBP components of BCO. As such, different co-solvents were screened for their influence on improving the quality of BCO with respect to its boiling point profile (BPP), initial and final boiling point, as well as the amount of char recovered from each experiment. Only one co-solvent was chosen for further exploration in the CFR system. Batch liquefaction reactor’s (BLR) made up of stainless steel were used to carry out the co-solvent screening experiments. These experiments were carried out at a constant temperature (280 °C), pressure (75 bar), and co-solvent concentration (10 wt.%), at varying residence times. Solvent extraction with dichloromethane (DCM) was performed on the liquefaction product mixture to separate the products, viz. BCO, char and water soluble components. The extracted BCO was analysed through simulated distillation (SimDis) to obtain the BPP. The BPP properties of the BCO samples, from different liquefaction media, and the amount of char recovered were highly influenced by the addition of a co-solvent. The final boiling point (FBP) of tetralin, heptane, and n-octanol BCO products were significantly reduced to below 500 °C for all tested residence times except at 20 minutes. The residence time also proved to be influential in the processing of wet microalgae. n-Octanol was selected as the optimal performing co-solvent and was used for the continuous liquefaction of wet microalgae in the CFR system. The CFR system was modified by adding a co-solvent feed line into the continuous system since n-octanol was insoluble in water. The n-octanol pump was set at different flow rates, 0.2, 0.3, and 0.4 g/min, which resulted in a concentration of about 10 wt.% in the reactor feed. The concentration of n-octanol had a significant influence on the BPP of BCO components. The FBP’s were reduced with an increase in n-octanol concentration. The initial boiling point (IBP) of n-octanol BCO was increased to just above 100 °C which was required for the stability of the BCO product. The components of BCO were identified by GCMS. n-Octanol also proved to affect the composition of the BCO with respect to its components. HCW BCO components were significantly different from those identified from n-octanol BCO. A second co-solvent (tetralin) was used to prove whether the difference on the components of BCO was affected by n-octanol. The results proved that indeed the addition of different solvents in liquefaction reaction media favours the formation of different components. The amount of char formed was also reduced when using a co-solvent. A decrease in the oxygen/nitrogen compounds was also observed in the presence of a co-solvent, thus improving BCO properties.

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011. / The 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. / National Research Foundation.

從微藻提煉的生物燃料，是化石燃料和其他生物燃料的優良替代品。藻類生物燃料屬碳中性，因為微藻為光自養生物，能經光合作用吸收二氧化碳，並將之轉化成碳氫化合物和脂肪。碳氫化合物和脂肪可用以提煉生物燃料。此外，微藻可以吸收廢水中的污染物作生長的營養，同時作污水處理。 / 本研究項目的目的為透過下述方法，降低藻類生物燃料的生產成本，並提高藻株的脂肪含量： (1) 篩選可以在污水自養培育，並有高產油量的微藻菌株，(2) 以兩階段培養方法，用處理過的污水作培養，從而提高油脂產，(3) 透過微藻毒理測試，和水質化學分析，研究處理後的污水中影響微藻生長的污染物和有毒物質。 / 這個研究中使用從沙田污水處理廠收集的二級處理污水，其水質亦被研究。幾種微藻菌株分別為小球藻 (Chlorella pyrenoidosa)，叢粒藻 (Botryococcus braunii) 和微綠球藻 (Nannochloropsis oculata)，從鰂魚池水分離出的小球藻 (Chlorella sp.1)，及兩種從處理污水中分離出的小球藻(Chlorella sp. 2, Chlorella sp. 3)。微藻菌株分別在培養基和處理污水中培養，並比較在兩種情況下的脂肪，脂肪酸，碳水化合物，蛋白質含量，生物質量和總有機碳。結果發現，雖然經處理的污水中營養成分非常低 (<0.11 mg / L活性磷，<9.68 mg / L硝酸根，<0.5 mg / L鉀離子)，所有研究的微藻菌株都能存活。在兩階段培養法下，首先以「氮含量充足階段」(培養基)提高生物質量，然後以「氮含量不足階段」(經處理污水) 培養，培養成本可以降低，同時提高脂肪生產率。在兩階段培養法下，叢粒藻的脂肪生產率比在人工培養基和經處理污水高2.6倍和7.13倍。 / 沙田污水處理廠處理的污水水質良好，並無驗出有害重金屬，雙酚A(BPA)，四溴雙酚A(TBBPA)和2,3,7,8-四氯二苯並二噁英(TCDD)。從藻類產生的生物燃料將不含有重金屬。 / 在這個研究中的叢粒藻 (Botryococcus braunii)，微綠球藻 (Nannochloropsis oculata)和小球藻 (Chlorella sp.1)都可以容忍雙酚A(BPA)，四溴雙酚A(TBBPA)，二氯苯氧氯酚 (TCS)和2,3,7,8-四氯二苯並二噁英(TCDD)。他們可以培育在其他來源的經處理污水。 / 利用經處理污水於兩階段培養法，是一種新的、更經濟的增加微藻油脂產量方法，亦可以配合任何其他方法，以減低藻類生物燃料的製造成本。 / Biofuel from microalgae can be an excellent substitute of fossil fuel and other biofuels. Algal biofuel is carbon neutral as microalgae are photoautotrophic. Through photosynthesis, microalgae can capture and convert carbon dioxide to hydrocarbons or lipids which can be used for biofuel production. Besides, microalgae can use pollutants from wastewater as nutrients for growth, which can serve as a wastewater treatment process. / The aims of the project are to lower the cost of algal biofuel production and boost up lipid content of algal strains by (1) screen a microalgal strain that can be cultivated in treated sewage autotrophically and give high oil yield, (2) use two phase cultivation, with treated sewage as medium, to boost up lipid productivity, (3) investigate heavy metals and some organic pollutants that may exist in treated sewage and can affect algal growth by performing algal toxicity test and chemical analysis of treated sewage. / The secondarily treated sewage used in this project was collected from the Sha Tin Sewage Treatment Works. The quality of the secondarily treated sewage was monitored. Chlorella pyrenoidosa, Botryococcus braunii and Nannochloropsis oculata from commercial source, and Chlorella sp. 1 isolated from tilapia fish pond water, and two species of algae, Chlorella sp. 2 and Chlorella sp. 3, isolated from treated sewage were investigated. Microalgal strains are compared by investigating the content of lipid, fatty acid, carbohydrate, protein, biomass and total organic carbon when cultivated in culture medium and treated sewage. Results found that although nutrients in treated sewage were very low (<0.11 mg/L reactive phosphorus, <9.68 mg/L nitrate and <0.5 mg/L potassium ion), all the microalgae investigated could grow reasonably well. Using two phase cultivation, with an initial nitrogen sufficient phase (artificial media) for biomass production, followed by nitrogen limitation phase (treated sewage), cost of cultivation could be reduced and the overall lipid productivity could be increased. Under the two phase cultivation, the lipid productivity of Botryococcus braunii was 2.6 and 7.13 fold higher than cultivated in artificial medium and treated sewage respectively. / Treated sewage from the Sha Tin Sewage Treatment Works was in good quality without harmful concentrations of heavy metal and BPA, TBBPA and TCDD. The microalgae could not absorb or adsorb significant amount of the harmful substances and the algal biofuel produced would not contain heavy metals. All the microalgae investigated in this project could tolerate BPA, TBBPA, TCS and TCDD. They could be cultivated in treated sewage from other sources. / Two phase cultivation using treated sewage is a new way for increasing lipid productivity from microalgae economically and can be combined with any other means for producing algal biofuel with lowest cost. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Kwan, Ka Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 104-113). / Abstracts also in Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.Vi / Table of Contents --- p.viii / List of Figures --- p.Xii / List of Plates --- p.Xvi / List of Tables --- p.xviii / Abbreviations --- p.xx / Chapter 1. --- General introduction / Chapter 1.1 --- Fossil fuel, the major energy source nowadays --- p.1 / Chapter 1.2 --- Disadvantages of using fossil fuel --- p.3 / Chapter 1.3 --- Biofuel --- p.5 / Chapter 1.4 --- Disadvantages of traditional biofuel production --- p.8 / Chapter 1.5 --- Characteristics of microalgae --- p.9 / Chapter 1.6 --- Biofuel from microalgae --- p.14 / Chapter 1.7 --- Nutrients for microalgae related to lipid production --- p.18 / Chapter 1.8 --- Current research on algal biofuel --- p.19 / Chapter 1.9 --- Two phase cultivation as a new way for lipid production --- p.24 / Chapter 1.10 --- Objectives --- p.24 / Chapter 2. --- Biofuel production under two phase cultivation with artificial medium and treated sewage / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Algal strains collection and isolation --- p.28 / Chapter 2.2.2 --- Artificial culture media --- p.29 / Chapter 2.2.2.1 --- Bristol’s Medium (BM) --- p.29 / Chapter 2.2.2.2 --- Modified Bold 3N medium (MBM) --- p.31 / Chapter 2.2.3.3 --- F/2 medium (F/2) --- p.33 / Chapter 2.2.3 --- Water quality of treated sewage --- p.33 / Chapter 2.2.3.1 --- Chemical and biological condition --- p.34 / Chapter 2.2.3.2 --- Total organic carbon and total nitrogen (TOC/TN) --- p.35 / Chapter 2.2.3.3 --- Reactive phosphate --- p.35 / Chapter 2.2.3.4 --- Nitrate --- p.37 / Chapter 2.2.3.5 --- Ammonia --- p.39 / Chapter 2.2.3.6 --- Metal elements --- p.40 / Chapter 2.2.4 --- Cultivation conditions --- p.40 / Chapter 2.2.5 --- Growth monitor of microalgae in artificial medium and treated sewage --- p.41 / Chapter 2.2.6 --- Comparison of microalgae cultivated in artificial media and treated sewage --- p.42 / Chapter 2.2.6.1 --- Large scale cultivation --- p.42 / Chapter 2.2.6.2 --- Cell morphology --- p.43 / Chapter 2.2.6.3 --- Cell harvesting --- p.44 / Chapter 2.2.6.4 --- Dried biomass --- p.44 / Chapter 2.2.6.5 --- Lipid content --- p.45 / Chapter 2.2.6.6 --- Fatty acid profile --- p.46 / Chapter 2.2.6.7 --- Extraction of carbohydrates and protein --- p.48 / Chapter 2.2.6.8 --- Carbohydrate content --- p.48 / Chapter 2.2.6.9 --- Protein content --- p.49 / Chapter 2.2.7 --- Two phase cultivation --- p.50 / Chapter 2.2.8 --- Statistical analysis --- p.50 / Chapter 2.3 --- Results --- p.51 / Chapter 2.3.1 --- Water quality of treated sewage --- p.51 / Chapter 2.3.2 --- Nutrient contents in artificial medium --- p.54 / Chapter 2.3.3 --- Growth of microalgae in artificial medium and treated sewage --- p.54 / Chapter 2.3.3.1 --- Cell morphology and cell size --- p.57 / Chapter 2.3.3.2 --- Biomass --- p.59 / Chapter 2.3.3.3 --- Lipid content --- p.61 / Chapter 2.3.3.4 --- Fatty acid profile --- p.63 / Chapter 2.3.3.5 --- Carbohydrates content --- p.66 / Chapter 2.3.3.6 --- Protein content --- p.67 / Chapter 2.3.4 --- Two phase cultivation --- p.69 / Chapter 2.4 --- Discussion --- p.74 / Chapter 2.4.1 --- Water quality of treated sewage and nutrients in artificial medium --- p.74 / Chapter 2.4.2 --- Growth of microalgae in artificial medium and filtered treated sewage --- p.75 / Chapter 2.4.3 --- Microalgae cultivated in artificial media and treated sewage --- p.76 / Chapter 2.4.4 --- Two phase cultivation --- p.81 / Chapter 3. --- Possible toxic effect on algal growth from chemicals in sewage / Chapter 3.1 --- Introduction --- p.84 / Chapter 3.2 --- Materials and methods --- p.85 / Chapter 3.2.1 --- Analysis of dissolved metals by ICP --- p.85 / Chapter 3.2.2 --- Organic compounds --- p.86 / Chapter 3.2.3 --- Algal bioassay --- p.87 / Chapter 3.3 --- Results --- p.88 / Chapter 3.3.1 --- Dissolved metals and metalloids --- p.88 / Chapter 3.3.2 --- Organic compounds --- p.88 / Chapter 3.3.3 --- Algal bioassay --- p.91 / Chapter 3.4 --- Discussion --- p.97 / Chapter 4. --- Conclusion and future prospectives --- p.99 / Chapter 4.1 --- Summary --- p.99 / Chapter 4.2 --- Genetic engineering --- p.100 / Chapter 4.3 --- Further study --- p.102 / Chapter 4.4 --- Conclusion --- p.102 / Chapter 5. --- References --- p.104

Submitted in fulfillment of the requirements for the degree of Doctor of Philosophy: Biotechnology, Durban University of Technology, Durban, South Africa, 2017. / Fossil 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. / D

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011. / Due 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.

Submitted in fulfillment for the requirements for the degree of Doctor of Technology: Biotechnology, Durban University of Technology, Durban, South Africa, 2015. / Main focus of this study is to investigate the enzymatic-conversion of microalgal lipids to biodiesel. However, preceding steps before conversion such as drying of microalgal biomass and extraction of lipids were also studied. Downstream processing of microalgae has several challenges and there is very little literature available in this area. S. obliquus was grown in the pilot scale open pond cultivation system for biomass production. Different techniques were studied for biomass drying and extraction of lipids from harvested microalgal biomass. Effect of these drying and extraction techniques on lipid yield and quality was assessed. Energy consumption and economic evaluation was also studied. Enzymatic conversion of microalgal lipids by extracellular and whole cell lipase application was investigated. For both applications, free and immobilized lipases from different sources were screened and selected based on biodiesel conversion. Process parameters were optimized using chosen extracellular and whole cell lipases; also step-wise methanol addition was studied to improve the biodiesel conversion. Immobilized lipase was studied for its reuse. Final biodiesel was characterized for its fuel properties and compared with the specifications given by international standards. Enzymatic conversion of microalgal lipids was compared with the conventional homogeneous acid-catalyzed conversion. Enzymatic conversion and chemical conversion were techno-economically investigated based on process cost, energy consumption and processing steps. Freeze drying was the most efficient technique, however at large scale economical sun drying could also be selected as possible drying step. Microwave assisted lipid extraction performed better compared to sonication technique. Immobilized P. fluorescens lipase in extracellular application and A. niger lipase in whole cell application showed superior biodiesel conversion. The extracellular immobilized P. fluorescens lipase showed better biodiesel conversion and yields than the immobilized A. niger whole cell lipase. Both the enzyme catalysts showed lower biodiesel conversion compared to conventional chemical catalyst and higher processing cost. However, techno-economic analysis showed that, the reuse potential of immobilized lipases can significantly improve the economics. Fewer purification steps, less wastewater generation and minimal energy input are the benefits of enzymatic route of biodiesel conversion. Microalgae as a feedstock and lipase as a catalyst for conversion makes overall biodiesel production process environmentally-friendly. Data from this study has academic as well as industrial significance. Conclusions from this study form the basis for greener and sustainable scaling-up of microalgal biodiesel production process. / D

High rate algae oxidation ponds (HRAOP) for domestic wastewater treatment generate biosolids that are predominantly microalgae. Consequently, HRAOP biosolids are enriched with minerals, amino acids, nutrients and possibly contain plant growth regulator (PGR)-like substances, which makes HRAOP biosolids attractive as fertiliser or PGR. This study investigated HRAOP biosolids as a starting material for a natural, cost-effective and readily-available eco-friendly organic fertiliser and/or PGRs. Various HRAOP extract formulations were prepared and their effect on plant growth and development was evaluated using selected bioassays. Initial screening included assessing the effect on change in specific leaf area, radish cotyledon expansion as an indicator of PGR-like activity, and seed germination index (GI). More detailed studies on fertiliser efficacy and PGR-like activity utilised bean (Phaseolus vulgaris) and tomato (Solanum lycopersicum) plants. Combined effects of sonicated (S) and 40% v/v methanol (M) extract (5:1 SM) had impressive plant responses, comparable to Hoagland solution (HS). Other potentially fertiliser formulations included 0.5% M, 1% M, 2.5% S and 5% S formulations. The 5:1 SM and 5% S showed greater PGR-like activity, promoting cotyledon expansion by 459 ± 0.02% and 362 ± 0.01%, respectively. GI data showed that none of the formulations negatively impacted germination. Further investigation showed that the 5% S formulation increased leaf length, width and area by 6.69 ± 0.24, 6.21 ± 0.2 mm and 41.55 ± 0.2 mm². All formulated fertiliser extracts had no adverse effect on chlorophyll content and plant nutrient balance as indicated by C:N (8-10:1) ratio. In addition, plants appeared to actively mobilise nutrients to regions where needed as evidenced by a shift in shoot: root ratio depending on C, N and water availability. Furthermore, 5% S caused a 75% increase in tomato productivity and had no effect on bean productivity. Whereas, 5:1 SM and 1% M formulation improved bean pod production by 33.3% and 11%, respectively but did not affect tomato production. Harvest index (HI) however indicated a 3% reduction in tomato productivity with 5:1 SM and little or no enhancement in bean productivity with both 5:1 SM and 5% S treatments. Bean plants treated with 5:1 SM and 5% S produced larger fruits, which could be an indication of the presence of a PGR effect. Overall, HRAOP biosolids extracts prepared and investigated in this study demonstrated both fertiliser characteristics and PGR-like activity with performances comparable and in some cases exceeding that of commercial products. However additional research is needed to confirm presence of PGR-like activities and fertiliser efficacy.

Submitted in fulfillment of the requirements of the degree of Master of Applied Science in Biotechnology, Durban University of Technology, 2015. / In light of the world’s declining fossil fuel reserves, the use of microalgal biodiesel has come to the forefront as a potentially viable alternative liquid fuel. The depleting freshwater reserves make the feasibility of this concept questionable. The use of wastewater reduces the requirement for depleting freshwater supplies. This project aimed to determine the viability of municipal domestic wastewater effluent as a substrate for microalgal growth, in order to generate an economical and environmentally friendly source of biofuel. Wastewater effluents from three domestic wastewater treatment plants were characterized in terms of known microalgal nutrients viz., ammonia, phosphate and nitrates. Phosphate concentrations varied throughout the year and were found to be low (< 3 mgL-1) whilst ammonia and nitrate concentrations ranged from 0 to 10 mgL-1 throughout the experimental period. These wastewaters were found to be suitable for cultivating microalgae. The study explored the cultivation of Chlorella sorokiniana on pre- and post-chlorinated domestic wastewater effluent to assess their potential as a medium for high microalgal culture density and lipid production. Post-chlorinated wastewater effluent was found to be superior to pre-chlorinated wastewater effluent, as evident by the higher biomass concentration. This wastewater stream did not contain high concentrations of bacteria when compared to pre-chlorinated wastewater effluent. Nitrogen is an essential nutrient required for regulating the growth and lipid accumulation in microalgae. Cultures growing in post-chlorinated effluent had a lifespan of 18 d. Residual nitrogen in wastewater effluent supported microalgal growth for limited periods. Supplementation using cheap, readily available nitrogen sources was required for optimal biomass and lipid production. Urea, potassium nitrate, sodium nitrate and ammonium nitrate were evaluated in terms of biomass and lipid production of C. sorokiniana. Urea showed the highest biomass yield of 0.216 gL-1 and was selected for further experimentation. Urea concentrations (0–10 gL-1) were assessed for their effect on growth and microalgal physiology using pulse amplitude modulated fluorometry. A concentration of 1.5 gL-1 urea produced 0.218 gL-1 biomass and 61.52 % lipid by relative fluorescence. Physiological stress was evident by the decrease in relative Electron Transport Rate from 10.45 to 6.77 and quantum efficiency of photosystem II charge separation from 0.665 to 0.131. Gas chromatography analysis revealed that C16:0, C18:0, C18:1, C18:2 and C18:3 were the major fatty acids produced by C. sorokiniana. Wastewater effluent has been considered an important resource for economical and sustainable microalgal biomass/lipid production. The study showed that C. sorokiniana was sufficiently robust to be cultivated on wastewater effluent supplemented with urea. The results indicate that supplemented wastewater effluent was an acceptable alternative to conventional media. Using a relatively cheap nitrogen source like urea can certainly improve the techno-economics of large scale biodiesel production.