Theses and dissertations (Applied Sciences)
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Item 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 The use of PAM fluorometry to assess microalgal physiological stress for the production of biodiesel(2011) White, Sarah Anne; Bux, FaizalUnder environmental stress, most microalgae produce intracellular neutral lipids as a storage mechanism. In the biotechnology industry, these lipids are extracted and converted to microalgal biodiesel; however the extent of the stress is not measured for optimum lipid accumulation. In the series of studies undertaken, Pulse Amplitude Modulated (PAM) fluorometry was used to measure three types of physiological stress on Chlorella sp., a freshwater microalgal species. Biomass and lipid yields were also used as indicators of the induced stress conditions. Firstly, nutrient induced physiological stress and the subsequent synthesis of cellular neutral lipids was investigated.Item Characterisation of biodiesel from Litsea glutinosa(2014-08-08) Perumal, Alicia Ann; Odhav, BhartiGlobal warming is a major concern to the world’s population. It is caused by greenhouse gases that result from the burning of fossil fuel. The fossil fuel reserves are rapidly depleting as the needs and wants of man in the world increases. Biodiesel is one of the solutions proposed to remedy this environmental crisis facing the world today. The aim of this study was to characterise the biodiesel that can be produced from the oil of Litsea glutinosa by transesterification. Biodiesel can be used in a diesel engine without modification and be produced from many different natural renewable oil sources such as algae, plants and kitchen waste material. Jatropha curcas has been identified as a potential producer of oil for biodiesel. The biodiesel properties of Jatropha curcas meet the required American Society for Testing and Materials (ASTM) standards. The fruit of Jatropha curcas contains 40.0% lipids. The oil has a saponification number of 202.6 and an iodine value of 93.0. However Jatropha curcas cannot be grown in South Africa because it is a highly invasive plant. Cetane number is the most important parameter of biodiesel. The higher the cetane value, the better the quality of the biodiesel. Oil from Jatropha curcas has a cetane number of 57.1. An alternative is the oil from Litsea glutinosa, which is found as a naturalised free forest along the South African coastline, and is also found in many Asian countries. It has many medicinal properties, however, it is not edible and hence its use for biodiesel does not add to the debate of fuel versus food production. The cetane number of oil from Litsea glutinosa is 64.79, which is ideal for ignition, and the fruit with 61.29% lipids can yield valuable quantities of biodiesel. Thus, the aim of the research was to determine the potential of Litsea glutinosa as a source of biodiesel. Furthermore, to maintain a sustainable source, Litsea glutinosa was micropropagated, and transformation of Litsea glutinosa was attempted for hairy root cultures. The Clevenger apparatus was used to extract fatty acids from dried crushed fruit of Litsea glutinosa. Fatty acids were converted to fatty acid methyl esters by transesterification. Transesterification was conducted in the presence of nitrogen and the reaction was catalysed with a mixture of methanol and sodium hydroxide (NaOH). The ratio 1 : 3 of oil to catalyst mixture was used for optimum transesterification to ensure a forward reaction and it was transferred to a separating funnel to allow the glycerol and fatty acid methyl esters to separate. GC-MS was used to determine the fatty acids. The iodine number, saponification number, acid value, viscosity, kinematic viscosity, density, specific gravity, thermostability, distillation point and sulphur content were determined. The seeds of Litsea glutinosa were germinated and tissue culture callus was produced from the seeds and leaves. The leaves and stems were used to produce hairy root cultures by inoculating them with Agrobacterium rhizogenes. Litsea glutinosa yielded 61% oil, which included 47 fatty acids in the fruit and 24 fatty acids in the seeds. The fatty acid profile of the oils indicated that the predominant fatty acids present were those that are essential for good quality biodiesel. The dominant fatty acids found in the fruit were 65.4% 9-octadecenoic acid and 13.6% hexadecanoic acid. The dominating fatty acids found in the seeds contained 36.3% 9-octadecenoic acid, 13.9%, hexadecanoic acid and 39.1%, dodecanoic acid. The iodine value was 6.3. The saponification value was 274. The acid value was 0.45 mg KOH. g-1. The viscosity was 22.48 mm2. s-1 and the kinematic viscosity was 23.84 mm2. s-1. The density was 942.69 kg. m-3 and the specific gravity was found to be 0.9 g. cm-3. The distillation temperature ranged between 52.2°C to 610.2°C. The sulphur content was found to be 383 µg. ml-1. These characteristics indicate that Litsea glutinosa can be used as a source of biodiesel, because the properties meet the required ASTM standards. However, the production of biodiesel from Litsea glutinosa has not been commercialised because the production of fuel is dependent on the fruit of the plant, which is seasonal. To overcome this, a part of this study investigated micropragation of Litsea glutinosa and transformation of Litsea glutinosa by Agrobacterium rhizogenes into hairy roots and attempts where made to determine whether fatty acid could be produced by these techniques. Callus cultures were grown on MS media and McCowns woody plant media supplemented with 1 ml BAP and 1 ml 2,4-D per 1 L of media. Callus cultures were obtained in the light. However, Litsea glutinosa resisted transformation by Agrobacterium rhizogenes.Item Bioconversion of biodiesel-derived crude glycerol waste to 1,3 propanediol and gellan using adapted bacterial isolates(2013-09-19) Raghunandan, Kerisha; Permaul, KugenThe continual growth of the global biodiesel industry has resulted in a proportional increase in crude glycerol production. The by-production of glycerol waste during the manufacture of biodiesel has, with recent research, proven to hold use as a feedstock for the production of several commodity chemicals. The conversion of glycerol may be carried out by both chemical as well as biological means. The biological conversion of glycerol surpasses chemical conversion with respect to higher yield and selectivity, normal reaction conditions and the use of cheaper biological catalysts. Many microorganisms are known to convert glycerol to different value added products. This study involved the isolation of bacteria from soil and crude glycerol from a local biodiesel plant. Isolates were then used to convert crude glycerol supplemented with salts and a nitrogen source into commercially viable products. Isolates which successfully degraded glycerol were then identified via 16S PCR. A strain of Klebsiella pneumoniae, which is a known producer of 1,3-propanediol (1,3-PDO), was isolated from soil and two strains of Sphingomonas sp., which is a known gellan producer, was isolated from biodiesel waste. Gellan is an exopolysaccharide used in the food, cosmetic and pharmaceutical industries sold commercially as a product known as Gelrite or Gelzan while 1,3-PDO is an important component of fuels and polyesters (used widely in the petroleum industry) and is currently chemically produced. Using crude glycerol for producing 1,3-PDO is a good solution from an economic as well as ecological point of view. K. pneumoniae, Sphingomonas psueudosanguinis and Sphingomonas yabuuchiae were subjected to a series of shake flask fermentations in order to determine optimal growth conditions. This microoganism was able to successfully produce significant amounts of 1,3-PDO and lactic acid using crude glycerol (80 g/l), without pre-treatment (37 and 6.8 g/l respectively). S psueudosanguinis and S. yabuuchiae were both able to produce two of the highest amounts of gellan gum than that reported by other studies using crude glycerol (80 g/l) as a sole carbon source in a minimal medium (50.9 and 52.6 g/l respectively).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.