The ability of micro–algae to respond to diverse and often rapidly changing habitats has been attributed to the versatility of their cellular lipids. Amongst these the energy rich triacylglycerols (TAG) have attracted considerable attention due to their potential use as feedstock for renewable biofuel. Although micro-algae have considerable advantages over other biofuel sources there are constraints to their utilisation. Improvements are required in certain areas including efficiencies in production and enhanced lipid yields if micro-algal biofuels are to become commercially feasible. To achieve this, genetic and metabolic manipulation will be essential and therefore a greater understanding of the lipid biosynthetic pathways is required. In this study the expression of genes putatively involved in TAG biosynthesis in the diatom Phaeodactylum tricornutum was investigated, for the first time, with CO2 supplementation and low pH stress over the entire growth cycle. This molecular analysis was combined with physiochemical examination of the lipid accumulation in the micro-algal model. The results indicated that TAG accumulation was enhanced by CO2 supplementation and occurred predominantly during the stationary growth phase. The molecular analysis revealed increased expression for three genes of interest, encoding enzymes involved in the acyl dependent pathway: Glycerol-3-phosphate acyl transferase (GPAT) -7198728 (Phatrdraft_50031), lysophosphatidic acyl transferase (LPAAT) -7196550 (Phatrdraft_42446) and phosphotidic acid phosphatase (PAP) -7195747 (Phatrdraft_40261) in cultures supplemented with CO2. Under the same conditions up-regulation of a gene involved in the first committed step of fatty acid biosynthesis, Acetyl CoA carboxylase (ACC) -7194806 (Phatrdraft_54926) was also observed. Overall, this study provides further insight on the specific genes linked with increased TAG production in P. tricornutum and the identification of references genes suitable for normalisation of qPCR data across the growth cycle and under CO2 supplementation, thus providing the tools needed for future molecular studies of P. tricornutum lipid production.

Microalgae synthesize and store large volumes of lipids (potentially over 25% of dry weight) which could provide a renewable source of biodiesel. Traditional extraction techniques often produce poor lipid yields particularly from microalgae with robust cell walls. This project investigated the role of power ultrasound as a cell disruption step in lipid extraction from four microalgal species. Nile Red staining was used to assess the time when ultrasound induced increased membrane permeability in each species and lipids were extracted using an ultrasound assisted Bligh and Dyer extraction method. A 20 kHz probe system (40% amplitude, 0.086 W/cm3) caused increased lipid recovery from dry biomass in all cases; D. salina (no cell wall) from 15 to 22.5% of dry biomass after 1 minute (26% when stressed with 35 g/L NaCl). C. concordia (thin cell wall) from 7.5 to 10.5% of dry biomass after 2 minutes (27% with 25% nitrogen reduction in growth media). N. oculata (thick cell wall) from 6.5 to 10% of dry biomass after 16 minutes (31.5% when stressed with 30 g/L NaCl). The stressed cultures yield could be improved to 35% when ultrasound was combined with S070 beating beads. Chlorella sp. (thick cell wall) from 6.3 to 8.7% of dry biomass, after 16 minutes (44% was achieved when harvested at day 9 instead of 15). A Dual Frequency Reactor (16 and 20 kHz, 0.01 W/cm3) flow system with S070 beads demonstrated that high lipid extraction yields could be achieved on a larger level with N. oculata. After 4:48 minutes sonication 24% lipid recovery was achieved. This system could theoretically increase daily microalgal oil production from 3.96 to 5.76 L per day when compared to conventional techniques, at an extra production cost of only 2.9 p/litre (1.5% increase). D. salina, N. oculata and C. concordia resumed normal growth following sonication at 20 kHz after 1-20 days (8 minutes treatment for D. salina, 60 minutes treatment for N. oculata and 16 minutes treatment for C. concordia). It was found that the supernatant of sonicated D. salina and C. concordia when added to established cultures were able to boost their growth.

<p>Rapid growth of energy consumption and greenhouse gas emissions from fossil fuels have promoted extensive research on biofuels. <a>Algal biofuels have been considered as a promising and environmentally friendly renewable energy source</a>. However, several limitations have inhibited the development of cost-effective biofuel production, which includes unstable cultivation caused by invading organisms and high cost of lipid extraction. This dissertation aims to investigate photochemical approaches to prevent culture collapse caused by invading organisms and biological approaches for the development of cost-effective lipid extraction methods.</p><p> </p><p>As a chemical-free water treatment technology, ultraviolet (UV) irradiation has been widely applied to inactivate pathogens but has not been used in algal cultivation to control invading organisms. To evaluate the potential of using UV irradiation to control invading algal species and minimize virus predation, <i>Tetraselmis sp. </i>and <i>Paramecium bursaria Chlorella virus 1</i> (PBCV-1) were examined as challenge organisms to evaluate effectiveness of UV disinfection. The concentration of viable (reproductively/infectively active) cells and viruses were quantified by a most probable number (MPN) assay and a plaque assay. A low-pressure collimated-beam reactor was used to investigate UV₂₅₄ dose-response behavior of both challenge organisms. A medium-pressure collimated-beam reactor equipped with a series of narrow bandpass optical filters was used to investigate the action spectra of both challenge organisms. Both challenge organisms showed roughly five log₁₀ units of inactivation for UV₂₅₄ doses over 120 mJ/cm². the most effective wavelengths for inactivation of <i>Tetraselmis</i> were from 254 nm to 280 nm, in which the inactivation was mainly attributed to UV-induced DNA damage. On the contrary, the most effective wavelength for inactivation of PBCV-1 was observed at 214 nm, where the loss of infectivity was mainly attributed to protein damage. These results provide important information for design of UV reactors to minimize the impact of invading organisms in algal cultivation systems.</p><p> </p><p>Additionally, a virus-assisted cell disruption method was developed for cost-effective lipid extraction from algal biomass. Detailed mechanistic studies were conducted to evaluate infection behavior of <i>Chlorovirus </i>PBCV-1 on <i>Chlorella sp.</i>, impact of infection on mechanical strength of algal cell wall, lipid yield, and lipid distribution. Viral disruption with multiplicity of infection (MOI) of 10^-8 completely disrupted concentrated algal biomass in six days. Viral disruption significantly reduced the mechanical strength of algal cells for lipid extraction. Lipid yield with viral disruption increased more than three times compared with no disruption control and was similar to that of ultrasonic disruption. Moreover, lipid composition analysis showed that the quality of extracted lipids was not affected by viral infection. The results showed that viral infection is a cost-effective process for lipid extraction from algal cells as extensive energy input and chemicals required by existing disruption methods are no longer needed.</p><p> </p><p>Overall, this dissertation provides innovative approaches for the development of cost-efficient algal biofuels. Application of UV disinfection and viral disruption significantly reduces chemical consumption and improves sustainability of algal biofuel production.<br></p>

abstract: Carbon dioxide (CO2) levels in the atmosphere have reached unprecedented levels due to increasing anthropogenic emissions and increasing energy demand. CO2 capture and utilization can aid in stabilizing atmospheric CO2 levels and producing carbon-neutral fuels. Utilizing hollow fiber membranes (HFMs) for microalgal cultivation accomplishes that via bubbleless gas-transfer, preventing CO2 loss to the atmosphere. Various lengths and geometries of HFMs were used to deliver CO2 to a sodium carbonate solution. A model was developed to calculate CO2 flux, mass-transfer coefficient (KL), and volumetric mass-transfer coefficient (KLa) based on carbonate equilibrium and the alkalinity of the solution. The model was also applied to a sparging system, whose performance was compared with that of the HFMs. Typically, HFMs are operated in closed-end mode or open-end mode. The former is characterized by a high transfer efficiency, while the latter provides the advantage of a high transfer rate. HFMs were evaluated for both modes of operation and a varying inlet CO2 concentration to determine the effect of inert gas and water vapor accumulation on transfer rates. For pure CO2, a closed-end module operated as efficiently as an open-end module. Closed-end modules perform significantly worse when CO2-enriched air was supplied. This was shown by the KLa values calculated using the model. Finally, a mass-balance model was constructed for the lumen of the membranes in order to provide insight into the gas-concentration profiles inside the fiber lumen. For dilute CO2 inlet streams, accumulation of inert gases -- nitrogen (N2), oxygen (O2), and water vapor (H2O) -- significantly affected module performance by reducing the average CO2 partial pressure in the membrane and diminishing the amount of interfacial mass-transfer area available for CO2 transfer. / Dissertation/Thesis / Masters Thesis Chemical Engineering 2018

Global warming is becoming more concerned by the public. The escalating atmospheric CO₂ level has introduced the intensification of greenhouse effect which brought enormous impact to the environment and climate. Among different methods for CO₂ mitigation, biological treatment on CO₂ emission using microalgae is regarded to be more economical and beneficial. On the other hand, countries around the world are projected to face water scarcity in the coming decades. Therefore alternatives to the finite fresh water resources for consumption have to be explored. Seawater has been introduced for toilet flushing in Hong Kong since 1950s. The flushing water is mixed with the consumed fresh water in the sewage treatment process to give a sewage effluent with high salinity, which still contains nutrients to support the growth of algal cells. Using sewage effluent as an algal culture medium not only can have a lower operation cost while saving fresh water for food crops, but also the effluent can be purified before discharging. Besides Hong Kong, there is an increase in numbers of cities incorporating seawater in toilet flushing systems as an economical and sustainable solution to fresh water scarcity. / Taking sustainable development into consideration, the use of algal cell for the fixation of CO₂ in saline sewage effluent is proposed, which should be an effective mitigation for CO₂ emissions, removal of nutrient in sewage effluent as well as production of useful products such as biofuel feedstock. In order to find out a suitable algal species for CO₂ the bioremediation, commercially available algal strains were compared against the locally isolated species on the growth and CO₂ removal efficiency in saline sewage effluent. Chlorogonium capillatum, the algal strain isolated from a local fish pond, was found to be the best candidate for CO₂ sequestration and nutrient removal in the non-sterile saline sewage effluent since it could grow much better in the presence of other microorganisms, comparing with the majority of other algal species could not grow under this situation. / The effect of CO₂ enhancement on algal photosynthetic rate and growth was studied in terms of the change in cell number, biomass and lipid production, and the fatty acid profile. The optimisation of CO₂ mitigation was achieved by deploying the response surface methodology (RSM) approach with a model describing the change in CO₂ consumption rate being developed. In the minimal run resolution V (MR5) screening test, it was found that salinity, pH, CO₂ and PO₄³⁻-P levels were influential to CO₂ removal by C. capillatum in non-sterile sewage effluent. Further optimisation of the CO₂ consumption rate was performed using the Box-Behnken design. The results of study showed that C. capillatum was able to deliver its maximum CO₂ consumption rate at 58.96 mg L⁻¹ d⁻¹ at the optimal condition, which was very close to the ordinary condition in the average sewage. In addition, the lipid content of C. capillatum could reach 24.26±2.67% with fatty acid profile conforming to typical biodiesel composition, delivering a high potential for biofuel feedstock production. Together with a high nutrient removal rate, C. capillatum could be used to produce a promising waste-recycling oriented simultaneous treatment system. / Since the CO₂ consumption rate was not dependent to light intensity, the spectral effects on the light-enhanced algal growth and carbon sequestration were investigated to find the best culture condition and how the carbon sequestration process was being influenced. Five LED light spectra were chosen for the analysis and it was found that growth parameters and cell compositions were influenced by the colour of the light very differently. The results indicated that under the irradiation of white LED light, C. capillatum had the highest CO₂ consumption rate and lipid content. Red LED light induced the highest amount of cellular protein as well as the chlorophyll a content. However, the performance of the light dependent reaction of the red LED light culture did not show apparent improvement. Regarding the CO₂ fixation enzyme, the spectral effect on RuBisCO content was marginal and there was no obvious relationship between the light induced CO₂ consumption and solely the light induced RuBisCO content change. Chemical analysis on the algal biomass indicated the C. capillatum culture would be a suitable microbial system to mitigate CO₂ emission, remove nutrients from saline sewage effluent and produce biomass suitable for biofuel production. / This study delivers a bioremediation system which is capable of simultaneous CO₂ mitigation, nutrient removal and biofuel feedstock production with a newly isolated algal species in a waste recycling manner. The findings of this study are not limited to the application locally in Hong Kong, but hopefully all these can also be useful in similar works in other places to help with the sustainable development. / 全球暖化正越來越受到公眾關注，大氣中不斷上升的二氧化碳水平已經加劇了溫室效應，並對環境和氣候帶來了巨大的影響。在不同的二氧化碳緩減方法中，利用生物方法以微藻處理二氧化碳排放被認為是更具效率和回報價值。另一方面，世界各地已經預計會在未來幾十年面臨水荒，因此是有必須要為有限的淡水資源尋找代替品。自五十年代起，香港便引入了海水作沖廁用途。沖廁用水會在污水處理過程中混合經使用過的淡水而產生高鹽度的污水，而污水當中仍含有營養物質，以支持藻類細胞生長。使用污水作為藻類培養介質不但可以降低營運成本，並有助於節省淡水用以耕種糧食作物，而且可以在污水排出前進行純化。除香港以外，一些引入海水用於沖廁系統的城市數目正在增加。 / 考慮到可持續發展，我們提出利用微藻細胞在帶鹽污水中進行二氧化碳固定。這應該是一個有效的緩解二氧化碳排放，清除污水中養份，以及產生有價值產品的綜合方案。為了找出一種合適的微藻進行生物整治，我們比較了市面上買到的品種和於本地環境分離出來的藻種於帶鹽污水中生長和去除二氧化碳的表現。經過實驗後我們確定一種從魚塘中分離出來的綠梭藻(Chlorogonium capillatum)是能夠在未經消毒的帶鹽污水中進行碳封存和去除養份的最佳選擇，因為綠梭藻能夠在其他微生物存在下仍可以良好地生長，反觀其他大多數的藻種就不能在這情況生長。 / 我們從細胞數、生物量、脂質和脂肪酸譜的變化方面研究了二氧化碳量增強對微藻光合速率和生長的影響。我們利用了反應曲面法(Response Surface Methodology)對微藻的二氧化碳緩減進行最佳化，並將二氧化碳緩減率的變化製成模型。在條件篩選實驗，我們找出鹽度、酸鹼、二氧化碳和磷質水平是會影響二氧化碳緩減率。在隨後的二氧化碳緩減率的最佳化後，綠梭藻的最大二氧化碳緩減率為58.69微克每公升每天。研究發現綠梭藻能夠於接近平常環境條件下達成最高效的二氧化碳緩減。除此之外，綠梭藻的脂質含量可達24.26±2.67百份比。加上脂肪酸分佈符合典型的生物柴油成份和具有高度養份去除率，綠梭藻可以用來創造以廢物回收作主導的多功能的生物修復系統。 / 由於二氧化碳緩減率並不依賴於光的強度，我們進行了光譜對微藻生長和碳封存的影響的研究，以找出最佳的培養條件和了解碳吸收的過程如何被影響。我們分析了五種發光二極管光譜，發現燈光顏色對微藻的生長參數和細胞組合物有明顯的影響。結果顯示，在白色燈的照射下，綠梭藻有最高的二氧化碳緩減率和脂質含量。紅色燈引發了最高的細胞蛋白質，以及葉綠素a含量。然而，紅光並沒有明顯提升光依賴反應。關於固定二氧化碳的酶，光譜對羧化/加氧酶的數量變化效果細微。另外，羧化/加氧酶跟二氧化碳緩減率之間沒有明顯關係。從微藻生物質中的化學分析，我們認為綠梭藻是一個合適的微生物系統以達成二氧化碳的排放緩減，去除帶鹽污水中的養份和生產適用於生物燃料製造的生物質。 / 這項研究提供了一個生物修復系統，它能夠以廢品回收方式同時減少二氧化碳排放、去除營養和生產製作生物燃料的原料。這項研究的結果並不只限於香港使用，我們希望這些東西也可以應在其地方類似的工程上，為可持續發展出力。 / Lee, Kwan Yin. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 115-134). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, January, 2017). / 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.