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ÖgeImproving raceway reactor productivity via vortex induced vibrations for cost effective microalgae production(Graduate School, 2023-09-15)Microalgae research has been becoming more and more common in the last decades due to a number reasons including need for sustainable energy and fuel production, concerns about climate change and orientation of people to biobased products. Microalgae is considered as an excellent feedstock to meet peoples future demands in these fields. Microalgae converts inorganic carbon into sugar using radiative energy in the process so called "photosynthesis". It can grow on non arable land and does not compete with agricultural food products, can assimilate waste products such as flue gas and wastewater and convert them into biomass. Microalgae cultivation is carried out for both remediation of waste streams and commercial purposes. Algal wastewater treatment is a hot topic in environmental engineering and poses several advantages compared to conventional wastewater treatment processes such as reduction of aeration costs. While algal nutrient removal is more common, certain microalgae species can assimilate organic carbon, making it an interesting alternative to activated sludge process. However, algal wastewater treatment is limited to several community scale facilities. Commercial scale algal biomass production is dominated by food and feed industry. While Spirulina and Chlorella are the most commonly cultivated species, cultivation of Haematococcus and Dunaliella is common due to their ability to synthesize high value products such as astaxhanthin and carotenoids. Microalgae based biodiesel is considered as among the main candidates to replace fossil fuels as algae can accumulate lipids up to 70% their dry weight and it can be said that most of the research effort involving microalgae is towards this subject. However, algal biodiesel is not economically feasible yet due to high costs of cultivation, harvesting and other downstream processes. Microalgae cultivation systems are generally classified as open and closed systems. Closed systems offer a more controlled environment with higher light availability; light paths being couple of centimeters to 10 cm. Microalgae growth rate and biomass concentration is higher in this type of systems; however much higher capital and operating costs, as well as upscaling issues strongly limits their utilization. Open systems on the other hand are much easier to build and operate. Raceway ponds is the most common microalgae cultivation system. A raceway can be defined as an oblong channel where culture medium is most commonly circulated with the help of a paddlewheel. A single pond can occupy an area up to 4 hectars. Depth of the pond is kept 20-30 cm to ensure light penetration and flow velocity is typically 0.2-0.3 m/s. While 90% of commercial scale microalgae production is carried out in raceway ponds it has strong disadvantages compared to closed photobioreactors such as limited light availability and vulnerability to environmental and climatic conditions. Among these, light availability is perhaps the most important bottleneck for optimization of low cost microalgae biomass production. Limited light availability results from very limited vertical mixing in long straight channels of raceway ponds. Improving vertical mixing can be achieved by introducing more turbulent to flow by increasing flow velocity, which is energy intensive. Thus, energy efficient systems for improving vertical mixing and creating light dark cycles in raceway ponds is a strong necessity for making algal products more economically attractive. xxii Aim of this thesis to improve vertical mixing in raceway ponds without increasing operational costs. Method to improve vertical mixing is implementation of vortex induced vibrations. Vortex induced vibration is a form of flow induced motion whereby a body becomes excited, with vortices shed from its surface. These vortices, when they shed and leave the surface, exert force on the cylinder. When a vortex separates from the top part, the cylinder feels a downward force. When it separates from the bottom, the direction of the force is then upwards. When the cylinder is allowed to move in the direction perpendicular to the flow, cylinder moves up and down. This is a periodic motion and will last forever as the fluid continues to flow. Vortex induced vibrations make use of the flow energy and convert this power of the fluid to oscillate the cylinder. Within the scope of the thesis vortex induced vibration systems are used to improve vertical mixing in raceway ponds without any additional energy input. Vortex induced cylinder oscillation requires flow uniformity along the width of the channel where the system was implemented. For this, first, flow field of existing raceway pond at the roof of ITU Environmental Engineering Department was numerically investigated using CFD code, to see if it is available for implementation of vortex induced vibration systems. Flow velocity is kept as 0.3 m/s. Paddlewheel was removed from the domain to decrease computational effort and k- ɛ was chosen as turbulence model. In the CFD analyses, the raceway pond was modified with one, two and three flow deflectors and width of the central divider was increased to 5 and 10 cm. It has been seen that by installing 3 semi-circular flow deflectors in the bends of the pond, uniform flow along channel width could be achieved. Existing raceway pond was modified in this way and vortex induced vibration system, which consists of a cylinder with 6 cm diameter and two springs was installed to pond. Continuous cylinder oscillation was achieved with 6.5 cm vertical amplitude and 1.24 s-1 oscillation frequency while water level was 0.3 m. Impact of this cylinder motion on vertical mixing was numerically analyzed using CFD code. To simulate the cylinder oscillation, governing equations of vortex induced vibration was implemented to model as user defined function. Flow velocity was kept as 0.3 m/s as in the experiments and k- Ω SST was chosen as turbulence model. Model was run under steady conditions until the dynamic equilibrium was reached. After this, model was run for 10 seconds to investigate VIV motion. Model output revealed that vertical motion of flow covered 2/3 of pond depth. Cylinder oscillation directs flow upwards with a magnitude of 0.3 m/s and creates high frequency light dark cycles to effectively utilize so called flashing light effect. Light to dark cut off point was assumed as 3 cm below culture surface and average frequency of L/D cycles in the first 60 cm downstream of the cylinder for uppermost, neutral and lowermost cylinder positions were calculated as 21.17 s-1, 5.28 s-1 and 2.33 s-1, respectively. Pure culture of Chlorella vulgaris was grown comparatively to assess the effect of VIV on biomass production capacity. Culture was first grown under laboratory conditions in 10 L plastic bottles. Temperature was kept constant at 28 oC and Bald's Basal Medium was used as growth medium. Culture was grown for 1 week in laboratory and after that transferred to open ponds at the roof of ITU Environmental Engineering department. Culture was further grown for 1 week to acclimate outdoor conditions and diurnal cycle. VIV system was removed from the pond and culture was grown in two identical ponds for additional one week two make sure identical ponds demonstrated the same performance in terms of biomass production capacity. VIV system was implemented to one of the ponds at the end of this week and comparative cultivation xxiii with and without VIV system was carried out for one week. Biomass growth was monitored by optical density measurement under wavelength of 540, 690 and 750 nm. Experiments revealed that VIV increased biomass production capacity in the pilot scale raceway pond with 3 m channel length and 1 m total width by over 20%. Amplitude response of cylinder achieved in the pilot scale raceway pond for 0.3 m/s flow velocity was lower compared to literature. To investigate the reason of this and to investigate effect of VIV on vertical mixing and light dark cycles in the raceway pond in detail, flow visualization technique was applied. Particle image velocimetry using LED illumination was applied under experimental conditions mentioned above. Frame rate of PIV camera was 165 FPS and focal length was 35 mm. Flow visualization experiments without the VIV system revealed that flow velocity decreases through pond depth in the paddlewheel driven system with a 4.5 cm bottom clearance. Distribution of horizontal flow velocity could be modeled with 2nd order polynomial. This uneven distribution of flow velocity through the depth suppresses cylinder motion which resulted in lower amplitude response compared to literature. Several other equipment such as archimedes pumps, centrifugal pumps, airlift pumps and propellers are proposed to replace the paddlewheel for further exploitation of effect of VIV motion on vertical mixing and thus light availability and biomass production capacity. Indeed, it has been reported that propellers and airlift pumps are more energy efficient than paddlewheels. Furthermore, paddle induced circulation would become more disadvantageous when culture depth was increased due to mechanical reasons. Flow field in the raceway pond when vortex induced vibration system was implemented was analyzed using particle imaginary technique. Vertical component of flow was in accordance with CFD analyzes in general. 75 cells were selected at the downstream of VIV cylinder and were tracked for 20 cm in horizontal direction, until they disappeared from the other side of cameras projection area. Initial cell positions were set as three equidistant planes through the depth of the raceway channel to represent the average situation. Flow visualization experiments revealed that 33% of selected cells entered high frequency light dark cycles with the help of VIV. Average frequency of light dark cycle was found to be 35.69 s-1 with a light fraction of 0.49. 44% of cells entered light limited zone from dark zone as a result of VIV motion. Pilot scale RWP has 3 m channel length, which means, compared to full scale facilities, cells pass through paddlewheel, where vertical mixing happens, more frequently. In other words, pilot scale RWP is more effectively mixed compared to full scale systems. By installing one VIV cylinder, it can be said that a 2nd vertical mixing point was created in the pond. On the other hand, real scale RWPs have much higher channel lengths, thus effect of paddle induced vertical mixing in these systems would be less pronounced. In these long channel sections, cells near the surface will become "over- charged" after a certain period of time. On the other hand, cells at lower parts of pond depth will reside in photobiologically inactive parts of the pond for a prolonged period. As indicated above, VIVs can cycle cells between photobiologically active and inactive parts of channel and increase number of cells that perform photosynthesis in one circulation around pond. Thus, it is believed that the effect of VIV could be more pronounced in larger ponds.
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ÖgeLipid production by Yarrowia lipolytica growing on food waste(Graduate School, 2023-05-17)Biodiesel production from plants and vegetable oils or different organic wastes as feedstock for microorganisms, can helps to decrease the consumption of fossil fuels and generation of greenhouse gases along with improve the economy. Applying organic wastes as feedstock for oleaginous yeasts is an economic technique in order to replace fossil-derived diesel with biodiesel as a clean and green fuel. Using food waste (FW) as a rich organic carbon source for cultivation of oleaginous yeast is considered as a promising environmentally friendly approach to achieve microbial lipid as the source of biodiesel. In this thesis, FW was collected from refectory of Istanbul Technical University. It contained cooked and uncooked food which dried and filtered after collection. Dark fermentation process was carried out with the collected FW and rumen microorganisms as inoculum. The rumen was taken from sheep stomach. The output of fermentation process was collected and applied as substrate for cultivation of oleaginous yeast Yarrowia lipolytica. The Soluble COD (SCOD) and TKN concentrations of fermented food waste (FFW) were 48.400 ± 0.49 and 0.907 ± 0.01 g/L, respectively and pH of the medium was 5.44 ± 0.05. Different concentrations of FFW (diluted with distilled water) were applied as growth medium of Y. lipolytica. The medium which was applied with no dilution was identified as the optimum one. In all applied mediums the growth was monitored as optical density (OD600) and the highest OD of 36.11 was observed in medium with FFW with no dilution. Nitrogen is considered as an essential nutrient for synthesis of cell materials and metabolites by microorganism. In terms of nitrogen depletion along with excess amount of carbon in the culture, carbon uptake rate is limited that causes metabolic activities shift towards lipid storage instead of cell proliferation. This approach was used in this thesis to increase lipid content of Y. lipolytica cultivated on FFW. Different carbon sources of glucose, glycerol, and potassium acetate along with five different COD/TKN ratios of 75, 100, 125, 150 and 175 were selected. Carbon sources were added to the medium in early stationary phase to increase carbon concentration of FFW and obtain favorable COD/TKN ratios. In order to identify the appropriate fermentation time to collect the biomass to evaluate its lipid content, biomass samples were collected at both early and late stationary phase in YPD (the optimum growth medium for yeast growth) and FFW medium. The results indicated lipid content of 21.54 ± 1.4 and 14.97 ± 0.51% lipid along with biomass concentration of 9.63 ± 0.24 and 8.34 ± 0.82 g/L in early and late stationary phase respectively, for YPD medium. These values were 19.5 ± 0.5 and 15.52 ± 0.31% lipid content along with 8.31 ± 0.51 and 7.10 ± 0.34 g/L biomass generation for Y. lipolytica cultivated on FFW medium. Results illustrated the early stationary phase as the optimum fermentation point to obtain highest lipid content and microbial cell because the biodegradation of yeast cell and intracellular lipid is occurred by time during stationary phase. This biodegradation caused drop of intracellular lipid content and yeast cell quantity at the end of stationary phase. In COD/TKN 75, lipid content of biomass was 26.7 ± 0.5, 34.2 ± 1.12 and 33.2 ± 1.59% with biomass concentration of 9.50 ± 0.37, 8.40 ± 0.72 and 9.32 ± 0.12 g/L for mediums contain glucose, glycerol, and potassium acetate, respectively. By increasing the ratio to 100, lipid content of medium with glucose increased to 29.2 ± 0.28, in medium contains glycerol lipid content was 34.3 ± 2.16 and in medium with potassium acetate the lipid content was slightly decreased to 31.2 ± 1.60%. In COD/TKN ratio of 125, the significant lipid content of 42.2 ± 1.72% in the medium supplemented with glycerol was measured. The other mediums contain glucose and potassium acetate have intracellular lipid content of 38.7 ± 0.35 and 34.7 ± 3.1%, respectively. Biomass concentration was measured as 18.52 ± 1.97, 12.95 ± 1.95 and 17.60 ± 0.75 g/L in culture sets supplemented with glucose, glycerol, and potassium acetate, respectively. In COD/TKN ratios 150 and 175, the amount of accumulated lipid and generated biomass were decreased that demonstrated the adverse effect of high concentration of carbon source on intracellular lipid accumulation of the cell. In these two ratios the amount of lipid content and cell concentration was 36.7 ± 1.3 and 9.77 ± 0.97; and 38.1 ± 3.0% and 11.57 ± 0.77 g/L, respectively. Highest lipid concentration of 7.61 ± 0.17 g/L was observed in medium with potassium acetate in COD/TKN 100 that is related to high biomass concentration in this experimental set. Lipid concentration started to decrease in higher COD/TKN ratios and dropped to 3.59 ± 0.13 g/L in COD/TKN ratio 150 with glycerol. Although the final pH value of the mediums with COD/TKN 125 and lower was always over 8, in ratios over 125 the pH was dropped to 4.3. This pH indicated formation of secondary metabolites such as organic acids in higher ratios that was due to high glycerol concentration in the mediums. This metabolic shift from lipid accumulation to organic acid generation led to the pH drop of the batch cultures. In next step of the thesis, Yeast Extract (YE), Iron Sulphate (IS) and Trace elements Solution (TS) was supplied to FFW along with glycerol as second carbon source to boost the lipid content further. Firstly, various amount of mentioned components were added to FFW to investigate the optimum concentration of each one to enhance lipid content of the cell. The concentrations of 1000 mg/L YE, 150 mg/L IS, and 5 ml/L TS were identified as the optimum. Different dual and triple combinations of these components along with glycerol were added to the mediums. In COD/TKN 150, accumulated lipid was reached to 44.72 ± 0.31% in the medium contains YE+IS+TS. Accumulated lipid reached to its maximum in ratio 175 i.e., 45.94 ± 0.21% in experimental set supplemented with YE+IS+TS. This value was the highest lipid content obtained in this thesis and by increasing the COD/TKN ratio over 175, the lipid accumulation dropped significantly. The highest concentration of lipid was 7.12 ± 0.12 g/L obtained in ratio 175 in medium supplied with YE+TS. The highest cell production was measured in the same culture with biomass concentration of 16.67 ± 0.27 g/L. Investigation of metabolic behavior of Y. lipolytica in this thesis revealed that in general, by increasing concentration of organic carbon in the medium, the carbon consumption is enhanced as well. In ratio 75, the highest COD consumption was measured in culture contains glycerol as 30.11 g/L COD. In ratio 100, the COD consumption of medium contain glycerol was increased slightly to 32.54 mg/L COD. In ratio 125, the COD consumption of 38.84 g/L was observed that caused the lipid storage of 42.2 ± 1.72% w/w in the cell. By increasing the COD/TKN ratios to 150 and 175, the microbial lipid content decreased although COD consumption was increased. This demonstrated the metabolic shift of Y. lipolytica from lipid generation to secondary metabolite production which caused drop of lipid biosynthesis. By adding YE, IS and TS to the medium, in ratio 150 the lipid content enhanced to 44.72 ± 0.31% with COD consumption of 48.37 g/L and in ratio 175, highest lipid content of 45.94 ± 0.21% was obtained by COD consumption of 49.63 g/L. However, decreasing pH value in experimental sets with COD/TKN over 125 indicated the generation of secondary metabolites in the medium. Additionally, in ratio 200 and 225 despite increase in COD consumption in the culture, the amount of stored lipid was reduced. Gas chromatography analyses of accumulated lipid revealed the fatty acid (FA) composition of stearic acid (C18:0), linoleic acid (C18:2), pentadecanoic acid (C15:1), palmitic acid (C16:0) and heptadecenoic acid (C17:1), similar to the FA profile of plant oil and appropriate for biodiesel generation. This thesis presented the efficient conversion of FFW and glycerol to considerable amount of microbial lipid. It indicated that FW and glycerol as two available and economic carbon sources can be evaluated as feedstock for intracellular lipid accumulation of Y. lipolytica. Additionally, this study demonstrated that the two-stage batch cultivation method by using FFW as initial carbon source and glycerol as the second one, can improve the lipid content of the microbial cell in significant amount. This thesis provides not only an economic waste treatment strategy, but also a sustainable and profitable method of microbial oil production by using carbon rich wastes as substrate for oleaginous yeast.