Rozprawy doktorskie na temat „Microalgal bioreactor”

Microalgae are recognised as a source of lipids for bioenergy, nutrients and pharmaceuticals. Photobioreactors, closed vessels for microalgal cultivation, are known to have high energy consumption due to mixing and aeration. Sparging is commonly used for mixing and gas-liquid mass transfer in photobioreactors, but is energy intensive. The aim of this work was to reduce these energy requirements by optimising conventional sparging and considering surface aeration coupled with mechanical agitation as an alternative. An airlift photobioreactor was used as a base for comparison with two novel, surface aerated reactors: oscillatory baffled and wave photobioreactors. The three bioreactors were compared in terms of power input, mixing, CO2 mass transfer, algal growth and lipid production. Prior to comparison, each photobioreactor was optimised based on these parameters. To calculate power input, isothermal gas expansion equations were used for sparged systems and calorimetry was used for mechanically agitation systems. Mixing was investigated using a salt tracer and phenolphthalein indicator and mass transfer was measured using the gassing-in method. Scenedesmus sp., a high lipid-producer, was cultivated in low nitrate media across a range of mixing rates in each photobioreactor.In the airlift photobioreactor a critical minimum CO2 supply rate (of 2.7×10-5 m s-1) was found, below which carbon was limiting and above which energy was spent on sparging without increased productivity (0.20 g L-1 d-1 biomass; 0.03 g L-1 d-1 lipid). In the oscillatory baffled reactor, insufficient mass transfer limited algal productivity (0.11 g L-1 d-1 biomass; 0.02 g L-1 d-1 lipid). The wave reactor had high CO2 mass transfer coefficients (10 – 140 h-1) in comparison to the airlift (2.7 – 40 h-1) and oscillatory baffled reactors (6.3 – 37 h-1). Sufficient biomass productivity (0.18 g L- -1 d-1) and higher lipid productivity (0.045 g L-1 d-1) at lower power input in the wave reactor resulted in higher energy efficiency compared to the airlift reactor. Life cycle analysis of simulated algal biodiesel production showed that bioreactor energy contributed 99% of total energy consumption. Therefore, the global warming potential was reduced by 73% when the airlift reactor was operated at the critical minimum CO2 supply (with gas compression to 2 bar) and a further 19% when the wave reactor was used. This work offers an energy efficient alternative to sparging, through the generation of a well-mixed wave in a surface aerated bioreactor. It also offers methods for optimisation of energy usage with respect to mixing and aeration. Reducing bioreactor energy consumption is key to feasibility, and was demonstrated here to reduce energy-related environmental burdens.

Orientadores: Maria Regina Wolf Maciel, Reinaldo Gaspar Bastos
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
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Resumo: A busca por uma maior sustentabilidade tem levado a uma mudança em direção à utilização de fontes renováveis para geração de energia em detrimento do uso de combustíveis fósseis, visando a uma modificação na matriz energética global. A utilização da biomassa de microalgas para produção de biocombustíveis vem sendo vista como uma alternativa promissora, uma vez que o seu cultivo proporciona produtividades em carboidratos e lipídios superiores às matérias-primas vegetais convencionalmente utilizadas na obtenção de etanol e biodiesel. Neste contexto, o objetivo da presente dissertação de mestrado foi avaliar a produção de biomassa da microalga clorofícea Chlorella vulgaris em fotobiorreator de placa plana em diferentes condições de fluxo luminoso, concentração de CO2 na alimentação gasosa e concentração de NaNO3 no meio de cultivo, visando o acúmulo de carboidratos para obtenção de bioetanol de terceira geração. As influências das variáveis nutricionais e de processo sobre a eficiência fotossintética das microalgas também foram estimadas para determinação do estado fisiológico das culturas. A produtividade média de biomassa e a concentração máxima final das microalgas foram significativamente afetadas pela incidência de radiação luminosa e pela suplementação de CO2 gasoso, obtendo-se maiores produtividades de carboidratos em cultivos com alto fluxo luminoso e concentrações de CO2 intermediárias (7,5%). Também foi observado o efeito positivo do aumento do fotoperíodo sobre o crescimento das microalgas. Através de hidrólise ácida foi possível atingir concentrações de até 2 g L-1 de açúcares fermentescíveis no hidrolisado a partir de biomassa de microalgas cultivadas em meio com baixo teor de nitrogênio. A fermentação etanólica foi então conduzida com a levedura Dekkerabruxellensis capaz de converter diferentes hexoses e pentoses em bioetanol, dada a presença de ambos os tipos de açúcares no hidrolisado
Abstract: The search for industrial processes with higher sustainability has led to a change towards the utilization of renewable sources for energy generation in substitution of fossil fuels, aiming the modification of the global energy matrix. The utilization of microalgal biomass for the production of biofuels is viewed as a promising alternative, since its cultivation yields carbohydrate and lipid productivities superior to those of conventional sources used in the obtention of bioethanol and biodiesel. In this context, the goal of this master thesis was to evaluate the biomass production of the chlorophycean microalga Chlorella vulgaris in a flat plate photobioreactor under different conditions of light flux, CO2 concentration in the gas feed and NaNO3 concentration in the culture medium, aiming carbohydrate accumulation for the production of third generation bioethanol. The influences of both process and nutritional variables on the photosynthetic efficiency of microalgae were estimated for the determination of the physiological condition of the cultures. The mean biomass productivity and the maximum final microalgae concentration were significantly affected by the incidence of light radiation and by the supplementation of gaseous CO2, the highest carbohydrate productivities being obtained in cultivations with high light flux and intermediate CO2 concentrations (7,5%). It was also observed the positive effect of increasing the photoperiod over microalgae growth. Through acid hydrolysis, it was possible to attain fermentable sugar concentration of up to 2 g L-1 from biomass of microalgae cultivated in low-nitrogen medium. The ethanolic fermentation was then carried out with the Dekkerabruxellensis yeast, capable of converting different hexoses and pentoses into ethanol, due to the presence of both sugar types in the hydrolysate
Mestrado
Desenvolvimento de Processos Químicos
Mestre em Engenharia Química

Design of Experiments was applied in this study to identify the effects of interaction between environmental conditions on Chlorella vulgaris growth in flat panel photo-bioreactors. Three mathematical regression models were developed to illustrate growth rate, biomass productivity and lipid productivity. The optimum lipid productivity of 1.75 mg/L•day was obtained under optimized process setting of 5 g/L bicarbonate concentration, 0.97 g/L nitrate concentration and light irradiance of 4500 LUX.

This thesis investigates the treatment of sewage water by using microalgae coupled with the membrane technology. Membrane technology was used to serve dual purpose of sewage water treatment as well as to study the harvesting of microalgae. However, harvesting of algal biomass remains a major challenge for the industrial researchers. The present study provides an evidence that backwashing which is currently being practiced worldwide can be a productive way to reduce fouling or blockage of the membrane and increase its harvesting potential, still no ultimate solution for the complete prevention of biofouling of membranes have been developed. In the present research, study was carried out to reduce the residence time for the treatment of sewage water using microalgae by using local microalgae isolates and starving them for the nutrients. Immobilization of standard culture was studied for the sewage water treatment to make the process feasible on large scale. Studies on different membrane materials as well as operating parameters were investigated to choose the best membrane for harvesting the grown algal biomass. Economic analysis for the whole process was examined by considering a village as a case study
“Microalgae in combination with membrane technology is an emerging process to combat the ever increasing pollution in the water bodies (rivers, lakes and sea) along with CO2 sequestration in a eco-friendly way without using the chemicals. As a microalga cultivates under four different conditions (photoautotrophic, heterotrophic, mixotrophic and photohetrotrophic), it can uptake nutrients, organic compounds, inorganic carbon (in the form of CO2)in the presence of bacteria and uses natural sunlight as a energy source for their growth. In short, microalgae are versatile unicellular species which not only prevents the eutrophication of the water bodies but also helps to increase the dissolved oxygen concentration thereby, helping the aquatic habitat to flourish in a natural way. As, microalgae were generally dispersed and suspended in the water, its harvesting is one of the bottleneck issue of the microalgal industries to grow forits mass production for various applications like fertilizers, biofuel, animal feed etc. Recently membrane technology shows sustainable solution for the harvesting of microalgae from water. Membrane filtration in combination with microalgal treatment for sewage water not only reduces the water footprints but also reduce the energy requirement as it does not require extensive oxygen like the conventional sewage water treatment plants. Therefore, microalgae in combination with membrane technology will be the futuristic technology for the treatment of sewage water. Conventional sewage water treatment plant required huge amount of air for aeration which is costly and also space required for plant is very large. The main objective of this dissertation is to (1) Screen and isolate microalgae from its natural habitat having potential to remove nutrients from sewage water.(2) Sewage water treatment using starved isolated microalgae species for the efficient removal of nutrients (TN and TP) (3)Optimization of process parameters for improving growth rate of microalgae and effective removal of nutrients.(4) Study the kinetics of nutrients uptake from the sewage water by immobilizing microalgae as well as to make effort for the reduction in the residence time. (5) Harvesting of microalgal biomass by applying membrane technology using different types of membranes. (6) Study the effects of various operational parameters such as Tran-membrane pressure (TMP), fluxes and membranes physicochemical properties parameters to get higher fluxes with maximum biomass recovery. (7) Economical analysis of the whole process for the treatment of sewage water using microalgae in combination with membrane technology for a small village. The microalgae-membrane based technology has a huge potential for the treatment of sewage as well as industrial wastewater in the near future. However, efficient design of photo-bioreactors or raceway pond using artificial radiation or solar radiation is essential. The commercial viability for the treatment of sewage water/industrial wastewater using microalgae-membrane based process will be depend upon the efficiency of microalgae for uptake of nutrients, design of photo-bioreactor for growth of microalgae and its harvesting using suitable membrane technology.”
CSIR and Goverment of India. project code #ESC0306
AcSIR

Les microalgues sont des organismes photosynthétiques considérés pour la production d'énergie. Les photobioréacteurs sont des systèmes fermés avec des productivités plus importantes que les bassins ouverts. Cette étude concerne l'expérimentation et la modélisation d'un réacteur du type « airlift à circulation interne». Des microparticules sont ajoutées pour modéliser les concentrations d'algues dans le réacteur. Les fractions du gaz et les vitesses du liquide ne sont pas affectées par la présence de microparticules. La distribution de la lumière dans le réacteur est calculée en considérant les concentrations de la biomasse et les propriétés optiques des algues. La lumière est atténuée de la paroi jusqu'au centre du réacteur et cette atténuation augmente avec la concentration de la biomasse. Les productivités de biomasse, estimées en utilisant deux modèles biologiques, montrent que celles obtenues dans les photobioréacteurs sont plus élevées que celles obtenues dans les bassins ouverts. La capacité du réacteur pour absorber du CO2 et produire de l'O2 est évaluée selon les productivités obtenues. A intensités lumineuses modérées, l'O2 dissout n'atteint pas le niveau d'intoxication, même pour de faibles débits d'air. Le CO2 et le CIT deviennent limitant si de l'air naturel est injecté dans le bioréacteur. Il est donc nécessaire d'injecter de l'air enrichi en CO2. L'hydrodynamique d'un réacteur airlift hélicoïdal est également présentée. Deux diamètres de tuyaux sont testés dans la section hélicoïdale. Une corrélation est proposée pour estimer le coefficient de frottement en fonction du Reynolds et le rapport entre le diamètre d'enroulement et le diamètre de tuyau. Pour réaliser la culture de microalgues à l'échelle industrielle, il est indispensable d'obtenir un bilan énergétique positif et de faibles émissions de GES. Pour cela, il faudra intégrer la culture dans des procédés de transformation tels que la production de biodiesel et la digestion anaérobique
Microalgae are photosynthetic organisms considered today for energy production. Photobioreactors are closed systems that present higher productivities than open ponds. In this study, a hydrodynamic model is developed for an internal airlift reactor and validated experimentally. Microparticles are added to the reactor at concentrations found in current microalgae cultures. Results show that gas hold-up and liquid velocities are not affected by the presence of particles. Light distribution and availability in the internal airlift reactor is calculated, taking into account biomass concentrations and algae optical properties. Light is attenuated from the wall to the reactor center while this attenuation increases with biomass concentrations. Based on two biological models, biomass productivities achieved in photobioreactors are higher than in open ponds. From biomass productivities, the reactor capacity to absorb CO2 and to release O2 is estimated. Results show that at moderate irradiances, dissolved O2 levels do not reach intoxication at low air flow rates. If natural air is injected into the reactor, CO2 and TIC become limiting therefore, it is necessary to inject CO2-enriched air. The hydrodynamics of a helical airlift reactor is also presented. Two pipe diameters are tested in the helical section. A mathematical correlation is proposed to estimate the friction factor as a function of the Reynolds number and curvature ratios. To perform microalgae culture at large scale, it is necessary to obtain a positive energy and GHGs balance. Therefore, microalgae culture has to be integrated in a system where conversion processes such as biodiesel production and anaerobic digestion are performed

The rapid depletion of fossil fuels has raised increasing attention worldwide, and initiated intensive research for sustainable alternatives for energy production. Among these, biodiesel from microalgae has appeared as one of the most promising candidate due to their ability to accumulate large amount of lipids. Indeed, microalgae can achieve a productivity up to 25 higher than other crop sources without need of cultivatable soil, therefore without competing with food production. In the meantime, microalgae have also shown promising results for the treatment of various kind of wastewaters. However, the cultivation of microalgae for energy production suffers from the large costs of harvesting and dewatering of biomass, prior to lipid extraction and biofuel production, which accounts for up to 50% of operating costs. Therefore, the search for cost-effective methods of harvesting and dewatering of microalgae biomass has become necessary to optimize their usage. This study investigates forward osmosis (FO) for the dewatering of microalgae biomass and its implementation within a photobioreactor used for wastewater treatment. FO is a cost-effective filtration process based on the differences of osmotic pressure across a semi-permeable membrane. The use of FO for microalgae dewatering is of high interest, given the high fouling ability of microalgae biomass and the low fouling promises of FO. First, the feasibility of using FO for microalgae dewatering was assessed, focusing on better understanding the fouling mechanisms involved. The filtration performances have been investigated under various operating parameters. It has been found that when Ca2+-containing draw solutions were used, microalgae responded to the back diffusion of calcium ions by an extensive excretion of carbohydrates, accelerating the formation of algal flocs, thus enhancing the rate and extent of flux decline and reducing the algae dewatering efficiency. However, most of the fouling was reversible by simple hydraulic flushing. In addition, substantial adsorption of algal biomass was observed on the feed spacer. Also, Scenedesmus obliquus and Chlamydomonas reinhardtii, with fructose and abundant glucose and mannose in its cell wall, showed strong response to the back diffusion of calcium ions which encouraged S. obliquus to produce more extracellular carbohydrates and formed a stable gel network between algal biomass and extracellular carbohydrates, leading to algae aggregation and severe loss in both water flux and algae biomass during FO dewatering with Ca2+-containing draw solution. Among the species investigated, Chlorella vulgaris without fructose was the most suitable microalgae species to be dewatered by FO with a high algae recovery and negligible flux decline regardless of which draw solution was applied. These findings improve mechanical understanding of FO membrane fouling by microalgae; have significant implications for the algae species selection; and are critical for the development and optimization of FO dewatering processes. Finally, the implementation of FO dewatering with continuous microalgae biomass production and synthetic wastewater treatment was investigated. Two systems (External FO ; Immersed FO) have been studied and compared in order to provide insights on the advantages and disadvantages of each system. Constant parameters have been set identical for both systems: operation time; photobioreactor; hydraulic retention time; biomass production; FO permeate volume. The results reveals that the wastewater treatment efficiency (nutrients removal), as well as the production of biomass were greater with the immersed system due to a greater microalgae growth. However, these may not be sustainable in a long term operation of the immersed system. The external FO system was found better in terms of salinity build-up and FO dewatering performances. Overall, an external FO dewatering is recommended due to its better flexibility and sustainability.

Dunaliella, a genus of unicellular, biflagellate green algae, is one of the most studied microalgae for mass culture and is of commercial importance as a source of natural -carotene. Dunaliella species have the desirable properties of halotolerance and photoautotrophy that makes their large-scale culture simple and cheap using resources unsuitable for conventional agriculture. The ease and cost-effectiveness of culture makes Dunaliella a desirable target for increased production of natural compounds by metabolic engineering or for exploitation as biological factories for the synthesis of novel high-value compounds. However, the lack of efficient genetic transformation systems has been a major limitation in the manipulation of these microalgae. In chapter four we describe the development of a nuclear transformation system for Dunaliella tertiolecta. The gene encoding the phleomycin-binding protein from Streptoalloteichus hindustanus, was chosen as the selectable marker as this protein retains activity at high salt concentrations. To drive expression of the chosen selectable marker, two highly expressed Dunaliella tertiolecta RbcS genes and their associated 5' and 3' regulatory regions were isolated and characterised (chapter three). Dunaliella transformation cassettes containing the RbcS promoter and terminator regions flanking the ble antibiotic resistance gene were constructed. These expression cassettes were tested in Chlamydomonas reinhardtii cells and found to drive expression of the ble gene in this heterologous system. This study also demonstrated that truncation of both the D. tertiolecta RbcS1 and RbcS2 regulatory regions significantly increases the expression of the ble gene in C. reinhardtii cells. To determine if the foreign DNA could stably integrate into the Dunaliella genome, four transformation methods: microprojectile bombardment, glass bead-mediated transformation, PEG-mediated transformation and electroporation were tested and a number of parameters varied. Southern blot analysis revealed that the plasmid DNA transiently entered the Dunaliella cells following electroporation but was rapidly degraded. Following electroporation, one stably transformed Dunaliella line was recovered. This is the first demonstration of the stable transformation of this alga. Chloroplast transformation is becoming a favoured method for the production of recombinant proteins in plants, as levels of heterologous protein are often higher than those achieved by transforming the nucleus. The Dunaliella chloroplast genome has not been genetically characterised, and thus there were no existing promoter and terminator sequences or sequences of intergenic regions that could be used for vectors in transformation of the chloroplast. Therefore, this study aimed to isolate and characterise promoters of highly expressed genes and matching terminators capable of driving transgene expression, and also to characterise intergenic regions that would be suitable insertion sites for the vector construct (chapter five). The complete gene sequence of two highly expressed Dunaliella chloroplast genes psbB and rbcL including the promoter and terminator regions as well as the coding sequence of the psbA gene were cloned and sequenced. In addition, the psbA gene is useful as a selectable marker as introduced mutations confer resistance to the herbicide 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU). Two homologous transformation constructs based on mutated psbA genes were developed and tested using microprojectile bombardment. A number of parameters were tested including: the size of the gold microprojectile particle, the distance of the plates from the point of discharge, plating onto membranes or filter paper, helium pressure, addition of an osmoticum to the medium and recovery time. Although no chloroplast transformants were recovered in this study, these homologous recombination constructs should prove useful in the development of a chloroplast transformation protocol. The other major component of this study was to investigate the use of microalgae as an expression system for the production of recombinant proteins. Transformation of Chlamydomonas reinhardtii, a species related to Dunaliella, is well developed. In chapter six, this study examined the expression of two human proteins, -lactalbumin and IGF-1 in Chlamydomonas reinhardtii. Plasmids containing the C. reinhardtii RbcS2 promoter upstream of the cDNAs of these two proteins were introduced into C. reinhardtii cells using glass-bead mediated transformation. Transgenic C. reinhardtii lines were generated and shown to contain the transgenes by PCR and Southern hybridisation. RT- PCR and northern hybridisation were subsequently used to demonstrate that the transgenes were transcriptionally active. The transcripts however, could only be detected by RT-PCR indicating that the genes were transcribed at low levels. Accumulation of the -lactalbumin protein could not be demonstrated, suggesting that although the transgenes were transcribed, they were either not translated or translated at levels below the sensitivity of western blot analysis or that any protein produced was rapidly degraded. Previous studies have indicated that in microalgae codon usage is vital in translation of the foreign protein. Codon modification of the IGF-I and -lactalbumin genes should lead to higher levels of protein accumulation. This study reports the first successful stable nuclear transformation of Dunaliella tertiolecta. Therefore it is now feasible that Dunaliella can be examined as a bioreactor for the expression of recombinant proteins. In addition, two chloroplast genes (psbB and rbcL) and their corresponding promoters and terminators have been characterised and a selectable marker cassette based on the mutated psbA gene constructed.

Dunaliella, a genus of unicellular, biflagellate green algae, is one of the most studied microalgae for mass culture and is of commercial importance as a source of natural -carotene. Dunaliella species have the desirable properties of halotolerance and photoautotrophy that makes their large-scale culture simple and cheap using resources unsuitable for conventional agriculture. The ease and cost-effectiveness of culture makes Dunaliella a desirable target for increased production of natural compounds by metabolic engineering or for exploitation as biological factories for the synthesis of novel high-value compounds. However, the lack of efficient genetic transformation systems has been a major limitation in the manipulation of these microalgae. In chapter four we describe the development of a nuclear transformation system for Dunaliella tertiolecta. The gene encoding the phleomycin-binding protein from Streptoalloteichus hindustanus, was chosen as the selectable marker as this protein retains activity at high salt concentrations. To drive expression of the chosen selectable marker, two highly expressed Dunaliella tertiolecta RbcS genes and their associated 5' and 3' regulatory regions were isolated and characterised (chapter three). Dunaliella transformation cassettes containing the RbcS promoter and terminator regions flanking the ble antibiotic resistance gene were constructed. These expression cassettes were tested in Chlamydomonas reinhardtii cells and found to drive expression of the ble gene in this heterologous system. This study also demonstrated that truncation of both the D. tertiolecta RbcS1 and RbcS2 regulatory regions significantly increases the expression of the ble gene in C. reinhardtii cells. To determine if the foreign DNA could stably integrate into the Dunaliella genome, four transformation methods: microprojectile bombardment, glass bead-mediated transformation, PEG-mediated transformation and electroporation were tested and a number of parameters varied. Southern blot analysis revealed that the plasmid DNA transiently entered the Dunaliella cells following electroporation but was rapidly degraded. Following electroporation, one stably transformed Dunaliella line was recovered. This is the first demonstration of the stable transformation of this alga. Chloroplast transformation is becoming a favoured method for the production of recombinant proteins in plants, as levels of heterologous protein are often higher than those achieved by transforming the nucleus. The Dunaliella chloroplast genome has not been genetically characterised, and thus there were no existing promoter and terminator sequences or sequences of intergenic regions that could be used for vectors in transformation of the chloroplast. Therefore, this study aimed to isolate and characterise promoters of highly expressed genes and matching terminators capable of driving transgene expression, and also to characterise intergenic regions that would be suitable insertion sites for the vector construct (chapter five). The complete gene sequence of two highly expressed Dunaliella chloroplast genes psbB and rbcL including the promoter and terminator regions as well as the coding sequence of the psbA gene were cloned and sequenced. In addition, the psbA gene is useful as a selectable marker as introduced mutations confer resistance to the herbicide 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU). Two homologous transformation constructs based on mutated psbA genes were developed and tested using microprojectile bombardment. A number of parameters were tested including: the size of the gold microprojectile particle, the distance of the plates from the point of discharge, plating onto membranes or filter paper, helium pressure, addition of an osmoticum to the medium and recovery time. Although no chloroplast transformants were recovered in this study, these homologous recombination constructs should prove useful in the development of a chloroplast transformation protocol. The other major component of this study was to investigate the use of microalgae as an expression system for the production of recombinant proteins. Transformation of Chlamydomonas reinhardtii, a species related to Dunaliella, is well developed. In chapter six, this study examined the expression of two human proteins, -lactalbumin and IGF-1 in Chlamydomonas reinhardtii. Plasmids containing the C. reinhardtii RbcS2 promoter upstream of the cDNAs of these two proteins were introduced into C. reinhardtii cells using glass-bead mediated transformation. Transgenic C. reinhardtii lines were generated and shown to contain the transgenes by PCR and Southern hybridisation. RT- PCR and northern hybridisation were subsequently used to demonstrate that the transgenes were transcriptionally active. The transcripts however, could only be detected by RT-PCR indicating that the genes were transcribed at low levels. Accumulation of the -lactalbumin protein could not be demonstrated, suggesting that although the transgenes were transcribed, they were either not translated or translated at levels below the sensitivity of western blot analysis or that any protein produced was rapidly degraded. Previous studies have indicated that in microalgae codon usage is vital in translation of the foreign protein. Codon modification of the IGF-I and -lactalbumin genes should lead to higher levels of protein accumulation. This study reports the first successful stable nuclear transformation of Dunaliella tertiolecta. Therefore it is now feasible that Dunaliella can be examined as a bioreactor for the expression of recombinant proteins. In addition, two chloroplast genes (psbB and rbcL) and their corresponding promoters and terminators have been characterised and a selectable marker cassette based on the mutated psbA gene constructed.

[EN] Anaerobic membrane bioreactors for urban wastewater treatment present interesting advantages when compared with aerobic treatments, such as less sludge production, lower energy demand and biogas generation. However, the generated effluent cannot generally be discharged without further ammonium and phosphate elimination. This thesis studies the removal of these inorganic nutrients by means of microalgae cultivation. The main objective of this work is therefore to obtain an autochthonous microalgal culture and to investigate its ability to grow on an already existing anaerobic effluent, as well as to research the extent to which ammonium and phosphate can be removed. Moreover, this thesis aims at providing the kinetic expressions which reproduce the main processes involved, in order to provide the basis for process simulation and design. Microalgae were isolated from a local wastewater treatment plant and their ability to grow on the anaerobic effluent -while successfully removing ammonium and phosphate- was demonstrated. An excellent water quality was obtained with a semicontinuous cultivation mode under constant illumination. The Scenedesmus and Chlorococcum genus proliferated more efficiently and thus became predominant in the culture. Results also showed that phosphorus was the limiting nutrient in the anaerobic effluent to be treated. The influence of phosphorus limitation on ammonium and phosphate removal, as well as the influence of temperature in ammonium removal, were then studied under laboratory conditions. Kinetic expressions which reproduce the observed effects were proposed and validated, taking also into account the effect of light intensity. Additionally, a Scenedesmus-dominated culture was grown under varying light and temperature in an outdoor flat-plate photobioreactor, with constant monitoring of light intensity, temperature and ammonium concentration. Acceptable results were obtained in the reproduction of the experimental data, albeit with less accuracy than under laboratory conditions. The work here presented demonstrates the feasibility of coupling a microalgal cultivation system to an anaerobic membrane bioreactor for urban wastewater treatment. The basic factors affecting microalgal nutrient removal are researched, and mathematical models are provided which reproduce these effects. This Ph.D. thesis is enclosed in a national research project funded by the Spanish Ministry of Economy and Competitiveness entitled "Estudio experimental de la recuperación como biogás de la energía de la materia orgánica y nutrientes del agua residual, acoplando un AnBRM y un cultivo de microalgas" (MINECO project CTM2011-28595-C02-01/02). This research was also supported by the Spanish Ministry of Education, Culture and Sport via a pre doctoral FPU fellowship to the author (AP2009-4903)
[ES] En el tratamiento de aguas residuales urbanas, los bioreactores anaerobios de membranas presentan ventajas interesantes frente a los tratamientos aerobios. Algunas de estas ventajas son la menor producción de fangos, un menor consumo energético y la producción de biogás. Sin embargo, y generalmente, el efluente obtenido no puede ser vertido al medio sin una etapa previa de eliminación de amonio y fosfato. La presente tesis estudia la eliminación de dichos nutrientes inorgánicos empleando para ello un cultivo de microalgas. El objetivo principal de este trabajo es, por tanto, la obtención de un cultivo autóctono de microalgas y la evaluación de la capacidad que éstas tienen tanto de crecer en un efluente anaerobio como de eliminar el amonio y el fosfato presentes. Asimismo, se pretenden proporcionar las bases para la simulación y el diseño del sistema de depuración propuesto, mediante la obtención de las expresiones cinéticas que reproducen los principales procesos involucrados. En primer lugar se ha demostrado la capacidad de las microalgas, aisladas en una estación depuradora de aguas residuales, de crecer en el efluente anaerobio y de eliminar con éxito el amonio y fosfato en éste presente. El agua tratada, obtenida a mediante un proceso semicontinuo y con iluminación constante, presenta una excelente calidad. Los géneros Scenedesmus y Chlorococcum han proliferado más eficientemente y han llegado a ser los predominantes en el cultivo. Los resultados obtenidos indican que el nutriente limitante en el efluente a tratar es el fósforo, y por tanto la influencia de la limitación de fósforo en la eliminación de nutrientes ha sido estudiada en condiciones de laboratorio, junto con la influencia de la temperatura en la velocidad de eliminación de amonio. Han sido propuestas y validadas las correspondientes expresiones cinéticas que reproducen los efectos observados, teniendo en cuenta en todo momento la influencia de la intensidad de la luz. Por otro lado, un cultivo de Scenedesmus ha sido cultivado en el exterior, bajo condiciones cambiantes de luz y temperatura, que a su vez han sido monitorizadas constantemente, junto con la concentración de amonio. Los datos obtenidos han sido reproducidos mediante modelación matemática con resultados aceptables, aunque la precisión obtenida es menor que en condiciones de laboratorio. La presente tesis demuestra la viabilidad de combinar un cultivo de microalgas con un bioreactor de membranas para el tratamiento de agua residual urbana. Se exponen asimismo los factores básicos que influyen en la velocidad de eliminación de nutrientes, y se presentan los modelos matemáticos necesarios para reproducir los efectos observados. La presente tesis doctoral se incluye en el marco de un proyecto nacional de investigación financiado por el Ministerio de Economía y Competitividad de título "Estudio experimental de la recuperación como biogás de la energía de la materia orgánica y nutrientes del agua residual, acoplando un AnBRM y un cultivo de microalgas" (CTM2011-28595-C02-01/02). La presente tesis doctoral ha sido también financiada por el Ministerio de Educación, Cultura y Deporte a través de una ayuda para contratos predoctorales de Formación del Profesorado Universitario (AP2009-4903).
[CAT] En el tractament d'aigües residuals urbanes, els bioreactors anaerobis de membrana tenen avantatges interessants respecte als tractaments aerobis. Alguns d'aquests avantatges són: menys producció de fangs, menys consum energètic i la producció de biogàs. No obstant això, i en general, l'efluent obtingut no es pot tornar al medi sense una etapa prèvia d'eliminació d'amoni i fosfat. Aquesta tesi estudia l'eliminació d'aquests nutrients inorgànics emprant per a fer-ho un cultiu de microalgues. L'objectiu principal d'aquest treball és, per tant, l'obtenció d'un cultiu autòcton de microalgues i l'avaluació de la capacitat que aquestes tenen tant de créixer en un efluent anaerobi com d'eliminar l'amoni i el fosfat presents. Així mateix, volem proporcionar les bases per a la simulació i el disseny del sistema de depuració proposat, mitjançant l'obtenció de les expressions cinètiques que reprodueixen els principals processos involucrats. En primer lloc, s'ha demostrat la capacitat de les microalgues, aïllades en una estació depuradora d'aigües residuals, de créixer en l'efluent anaerobi i d'eliminar amb èxit l'amoni i el fosfat presents. L'aigua tractada, obtinguda mitjançant un procés semicontinu i amb il·luminació constant, presenta una qualitat excel·lent. Els gèneres Scenedesmus i Chlorococcum han proliferat més eficientment i han arribat a ser els predominants en el cultiu. Els resultats obtinguts indiquen que el nutrient limitant en l'efluent per tractar és el fòsfor, i per tant la influència de la limitació de fòsfor en l'eliminació tant d'amoni com de fosfat ha sigut estudiada en condicions de laboratori, juntament amb la influència de la temperatura en la velocitat d'eliminació d'amoni. S'han proposat i validat les expressions cinètiques corresponents que reprodueixen els efectes observats, tenint en compte en tot moment la influència de la intensitat de la llum. D'altra banda, s'ha cultivat a l'exterior un cultiu predominat per Scenedesmus, sota condicions canviants de llum i temperatura, que al seu torn s'han monitorat constantment, juntament amb la concentració d'amoni. Les dades obtingudes s'han reproduït mitjançant simulació matemàtica amb resultats acceptables, encara que la precisió obtinguda és més baixa que en condicions de laboratori. La nostra tesi demostra la viabilitat de combinar un cultiu de microalgues amb un bioreactor de membrana per al tractament d'aigua residual urbana. La tesi exposa així mateix els factors bàsics que influeixen en la velocitat d'eliminació de nutrients, i presenta els models matemàtics necessaris per a reproduir els efectes observats. Aquesta tesi doctoral s'inclou en el marc d'un projecte nacional de recerca finançat pel Ministeri d'Economia i Competitivitat amb el títol "Estudio experimental de la recuperación como biogás de la energía de la materia orgánica y nutrientes del agua residual, acoplando un AnBRM y un cultivo de microalgas" (CTM2011-28595-C02-01/02). La tesi doctoral ha sigut també finançada pel Ministeri d'Educació, Cultura i Esport a través d'una ajuda per a contractes predoctorals de formació del professorat universitari (AP2009-4903).
Ruiz Martínez, A. (2015). Nutrient removal from an anaerobic membrane bioreactor effluent using microalgae. Study and modeling of the process [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/59409
TESIS

Mestrado em Engenharia Mecânica
Este trabalho visou a elaboração do projecto de construção mecânica dum protótipo de um fotobiorreactor que permita a cultura de microalgas num meio artificial controlado, de forma a obter biomassa com elevado teor de lípidos para posterior extracção do óleo. Numa primeira fase deste trabalho consistiu no estudo das actuais estratégias e tecnologias utilizadas para cultura de microalgas. De forma a definir a linha de projecto. Posteriormente foi elaborado o projecto e dimensionamento final do fotobiorreactor, apresentando soluções mecânicas de forma a melhorar as actuais dificuldades técnicas associadas a este tipo de culturas Por fim realizou-se uma análise energética e análise de um possível scale-up para esta tecnologia de produção biomassa de microalgas. ABSTRACT: This work aimed at drafting the mechanical construction of a photobioreactor prototype to allow the cultivation of microalgae in a controlled artificial medium, in order to obtain biomass with high fat content for subsequent extraction of oil for production of biodiesel. The first phase of this work was the study of existing stategies and technologies used for cultivation of microalgae. In order to define the line of project. Subsequently was prepared the project and final design of fotobiorreactor, presenting mechanical solutions to improve the current technical difficulties associated with this type of cultures. Finally we did an energy and scale-up analysis of this technology of biomass production of microalgae.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The project aimed to carry out the techno-economic modeling of the heterotrophic microalgae bioreactors application in the poultry and swine abattoirs and processing wastewater treatment and possible exploitation of bioproducts from biomass generated in the process. The project focus was directed to the techno-economic modeling of bioprocesses following: (i) agroindustrial wastewater treatment and integral biomass production (ii) obtaining of bulk oil and lipid extracted algal (LEA) as feedstock for biodiesel conversion process and protein production (iii) obtaining of microalgal carotenoid-rich oleoresin solubilized in soybean oil. The results indicated that the agroindustrial wastewater treatment costs and production of microalgal biomass were USD 2.66 per cubic meter and USD 30 per ton of dried biomass respectively. The obtaining of bulk oil and LEA had a production cost of approx. USD 386.5 and 70.4 per ton. Finally, the oleoresin produced had an estimated production cost in USD 146.9 per kilogram. The results evidenced that the techno-economic modeling of the heterotrophic microalgae bioreactors application are an alternative to substantially minimize the production costs, giving economic sustainability to the agroindustry of poultry and swine processing.
O projeto teve por objetivo realizar a modelagem técnico-econômica da aplicação de biorreatores heterotróficos microalgais no tratamento de efluentes provenientes do abate e processamento de aves e suínos e possível exploração dos bioprodutos provenientes da biomassa gerada no processo. O foco do projeto foi direcionado à modelagem técnicoeconômica dos seguintes bioprocessos: (i) tratamento do efluente agroindustrial e produção de biomassa integral (ii) obtenção de óleo a granel e farelo microalgal desengordurado, como matéria-prima para o processo de conversão de biodiesel microalgal e produção de proteína (iii) obtenção de oleorresina de carotenóides mistos solubilizada em óleo de soja a partir de microalgas. Os resultados indicaram que os custos de tratamento do efluente agroindustrial e produção de biomassa microalgal foram US$ 2,66 por m³ de efluente e US$ 30 por tonelada de biomassa seca respectivamente. A obtenção de óleo a granel e farelo de microalgas obtiveram um custo de produção de US$ 386,5 e 70,4 por tonelada de óleo e farelo, respectivamente. Por fim, a oleorresina produzida teve um custo de produção estimado em US$ 146,9 por quilograma. Os resultados evidenciaram que a modelagem técnico-econômica da aplicação do biorreator heterotrófico microalgal, são uma alternativa para minimizar substancialmente os custos de produção, dando sustentabilidade econômica às agroindústrias de processamento de aves e suínos.

This master thesis focuses on reuse of waste water from industrial plant processing yellow phosphorus. Theoretical part summarizes physical and chemical properties of phosphorus, its transport in biosphere and its role in living organisms. Also the production process of the industrial plant is described. Waste water that contains waste byproducts is considered to be useful source of essential nutrients for economical large scale microalgae cultivation and development of biotechnological processes. In practical part, optimization of growth conditions for microalgae Chlorella pyrenoidosa Chick (IPPAS C-2) cultivation in medium based on wastewater from yellow phosphorus warehouse is presented.

Microalgae culture is currently receiving considerable attention for its potential in wastewater treatment and production of algal biomass from which high-value bioproducts and bioenergy can be obtained, as well as the consequent carbon dioxide sequestration via photosynthesis process. However, biomass harvesting is one of the bottlenecks in microalgae culture and microalgae-based wastewater treatment systems. Energy intensive technologies are required to separate the solid-liquid phase due the low density of microalgae. Low-cost processes, such as sedimentation, are not efficient enough due to the low settling velocities of the microalgae. Sedimentation coupled to coagulation and flocculation has been widely studied on lab-scale in order to increase the microalgae settling velocity. However, few studies have addressed the scaling up of these experimental results in order to industrializing the process. The thesis has been divided into two main parts. A first study addressed the physical and theoretical principles of sedimentation used for the operation and optimization of biomass harvesting from a microalgae culture for wastewater treatment at demonstration scale in the framework of the INCOVER research project "Innovative Eco-technologies for Resource Recovery from Wastewater" (GA 689242) (https://incover-project.eu/), which aimed to validate innovative technologies at demonstration scale to convert wastewater into an alternative energy source and value-added products. A second study focused on the operation and optimization of the downstream thickening process of biomasspreviously harvested in the same facilities. Finally, the second part consist of a study and optimization of the behavior of mixed liquor in its transit through a high rate algae pond for wastewater treatment using Computational Fluid Dynamics modeling for its implementation in the city of Aligarh. This study has been carried out under the H2020 PAVITR project (http://www.pavitr.net; GA 821410), which aims at validation of sustainable natural and advanced technologies for water and wastewater treatment, monitoring and safe reuse of water in India. In the first study of the first part, the physical and theoretical principles of sedimentation were addressed to be used for the operation and optimization of harvesting biomass in lamella settler (700 L) from a microalgae culture for wastewater treatment on a three semi-closed tubular photobioreactor (11.7 m3 each) at demonstrative scale. During 6 months the inflow (6900 m3·day-1), coagulant dosage (1-12 mg·L-1) and purges of the biomass (60-240 L·day-1) were adjusted in order to achieve a proper separation of the solid-liquid phase. Results in this section evidenced the efficiency of the Lamella in the solid-liquid separation task obtaining an outlet turbidity below of 5 NTU after the optimization period. In the second part, two thickeners were operated and optimized in order to achieve a proper concentration (20 g·L-1) of previous harvested biomass for subsequent anaerobic digestion process at the same installations. The scrapers and purges were optimized in four periods during two months. Results showed an eventually concentrations of 26.5 g·L-1 in last period due a minimized use of scrapers in order to avoid the particles resuspension allowing a proper compression settling. In the second part, demonstrative-scale HRAP system was designed to be implemented in Aligarh (India) with a treatment capacity of 50 m3·day-1. The objective of the study was to assist, verify and optimize the conventional dimensioning of the High Rate Algae Ponds (HRAP) by means of biokinetic modelling and hydrodynamic analysis using Computational Fluid Dynamics (CFD). According to the biokinetic model simulations, 4 days was the optimal hydraulic retention time to enhance nutrient removal. A 3D model of the HRAP was built to analyze the hydrodynamic behavior of 36 different carousel designs. The different combinations of baffle numbers on the reversals, center wall widths and tear-shape sizes were simulated. The presence of low velocity zones as well as the useful area vs. the total occupied area were quantify. Two baffles and tear-shapes with a diameter equal to ¼ of the channel width was the most efficient configuration. In addition, a techno-economic assessment of the system determined an investment cost of € 483 per population equivalent (PE) and an operational cost of € 0.19 per m3 of treated wastewater.
El cultiu de microalgues està rebent actualment una atenció considerable pel seu potencial en el tractament d'aigües residuals i la producció de biomassa d'algues de la qual es poden obtenir bioproductes d'alt valor i bioenergia, així com el segrest consegüent de diòxid de carboni mitjançant el procés de fotosíntesi. Tot i això, la recol·lecció de biomassa és un dels colls d'ampolla del cultiu de microalgues i dels sistemes de tractament d'aigües residuals basats en elles. La separació de la fase sòlida-líquida requereix tecnologies d'alt consum energètic a causa de la baixa densitat de les microalgues. Els processos de baix cost, com la sedimentació, no són prou eficaços a causa de la baixa velocitat de sedimentació de les microalgues. La sedimentació combinada amb la coagulació i la floculació s'ha estudiat àmpliament a escala de laboratori per augmentar la velocitat de sedimentació de la biomassa algal. Tot i això, pocs estudis han abordat l'augment d'escala d'aquests resultats experimentals per industrialitzar el procés. La tesi ha estat dividida en dues parts. La primera està formada per dos estudis i és el tema principal d'aquesta tesi. Un primer estudi va abordar els principis físics i teòrics de la sedimentació que s'utilitzen pel funcionament i optimització de la recol·lecció de biomassa d'un cultiu de microalgues pel tractament d'aigües residuals a escala demostrativa en el marc del projecte de recerca INCOVER "Innovative Eco-technologies for Resource Recovery from Wastewater" (GA 689242) (https://incover-project.eu/), l'objectiu del qual era validar tecnologies innovadores a escala demostrativa per convertir les aigües residuals en una font d’energia alternativa i en productes de valor afegit. Un segon estudi es va centrar en el funcionament i optimització del procés d'espessiment posterior de la biomassa prèviament collida a les mateixes instal·lacions mitjançant dos espessidors treballant en línia. Finalment, la segona part va consistir en l'estudi i l'optimització del comportament del licor barrejat en el trànsit per un estany d'algues d'alta taxa pel tractament d'aigües residuals mitjançant la modelització de la Dinàmica de Fluids Computacional per a la seva implantació a la ciutat d’Aligarh. Aquest estudi s'ha realitzat en el marc del projecte H2020 PAVITR (http://www.pavitr.net; GA 821410), l'objectiu del qual és la validació de tecnologies naturals i avançades sostenibles pel tractament de l'aigua i de les aigües residuals, control i la reutilització segura de l’aigua a l’Índia. Al primer estudi de la primera part, es van abordar els principis físics i teòrics de la sedimentació per utilitzar-los en el funcionament i l'optimització de la collita de biomassa en sedimentador de làmines (700 L) d'un cultiu de microalgues pel tractament d'aigües residuals en un fotobioreactor tubular semitancat de tres (11,7 m3 cadascun) a escala demostrativa. Durant 6 mesos es va ajustar el flux d'entrada (6900 m3-dia-1), la dosi de coagulant (1-12 mg·L-1) i les purgues de la biomassa (60-240 L·dia-1) per aconseguir una adequada separació de la fase sòlid-líquida. Els resultats d'aquest apartat van evidenciar l'eficàcia de les lamel·les en la tasca de separació sòlid-líquid obtenint una terbolesa de sortida inferior a 5 NTU després del període d'optimització. En el segon estudi, es van operar i optimitzar dos espessidors per aconseguir una concentració adequada (20 g·L-1) de la biomassa recol·lectada prèviament pel posterior procés de digestió anaeròbia a les mateixes instal·lacions. Els espessidors i les purgues es van optimitzar en quatre períodes durant dos mesos. Els resultats van mostrar una concentració final de 26,5 g·L-1 a l'últim període a causa d'un ús minimitzat dels rascadors per evitar la resuspensió de les partícules permetent una adequada sedimentació per compressió. A la segona part, es va dissenyar una Llacuna d'Alta Càrrega a escala demostrativa per ser implementada a Aligarh (Índia) amb una capacitat de tractament de 50 m3・dia-1. L'objectiu de l'estudi era assistir, verificar i optimitzar el dimensionament convencional de les llacunes d'alta càrrega mitjançant la modelització biocinètica i l'anàlisi hidrodinàmica mitjançant dinàmica de fluids computacional (CFD). Segons les simulacions del model biocinètic, el temps de retenció hidràulica òptim per millorar l'eliminació de nutrients va ser de 4 dies. Es va construir un model 3D de la llacuna per analitzar el comportament hidrodinàmic de 36 dissenys en forma de carrusel amb diferents configuracions. Es van simular les diferents combinacions de nombres de deflectors en les inversions, amples de paret central i mides de forma de llàgrima als extrems del mur central. Es va quantificar la presència de zones de baixa velocitat, així com l'àrea útil davant de l'àrea total ocupada. La configuració més eficient va ser la composta per dos deflectors i formes de llàgrima amb un diàmetre igual a . de l'amplada del canal. A més, una avaluació tecno-econòmica del sistema va determinar un cost d'inversió de 732 euros per població equivalent (PE) i un cost operatiu de 0,19 euros per m3 d'aigua residual tractada.
El cultivo de microalgas está recibiendo actualmente una atención considerable por su potencial en el tratamiento de aguas residuales y la producción de biomasa de algas de la que se pueden obtener bioproductos de alto valor y bioenergía, así como el consiguiente secuestro de dióxido de carbono mediante el proceso de fotosíntesis. Sin embargo, la recolección de biomasa es uno de los cuellos de botella del cultivo de microalgas y de los sistemas de tratamiento de aguas residuales basados en ellas. La separación de la fase sólida-líquida requiere tecnologías de alto consumo energético debido a la baja densidad de las microalgas. Los procesos de bajo coste, como la sedimentación, no son lo suficientemente eficaces debido a la baja velocidad de sedimentación de las microalgas. La sedimentación combinada con la coagulación y la floculación se ha estudiado ampliamente a escala de laboratorio para aumentar la velocidad de sedimentación de la biomasa algal. Sin embargo, pocos estudios han abordado el aumento de escala de estos resultados experimentales con el fin de industrializar el proceso. La tesis se ha dividido en dos partes principales. La primera está conforma por dos estudios y es el tema principal de esta tesis. Un primer estudio abordó los principios físicos y teóricos de la sedimentación que se utilizan para el funcionamiento y la optimización de la recolección de biomasa de un cultivo de microalgas para el tratamiento de aguas residuales a escala demostrativa en el marco del proyecto de investigación INCOVER "Innovative Eco-technologies for Resource Recovery from Wastewater" (GA 689242) (https://incover-project.eu/), cuyo objetivo era validar tecnologías innovadoras a escala demostrativa para convertir las aguas residuales en una fuente de energía alternativa y en productos de valor añadido. Un segundo estudio se centró en el funcionamiento y optimización del proceso de espesamiento posterior de la biomasa previamente cosechada en las mismas instalaciones mediante dos espesadores trabajando en línea. Por último, la segunda parte consistió en el estudio y optimización del comportamiento del licor mezclado en su tránsito por un estanque de algas de alta tasa para el tratamiento de aguas residuales mediante la modelización de la Dinámica de Fluidos Computacional para su implantación en la ciudad de Aligarh. Este estudio se ha realizado en el marco del proyecto H2020 PAVITR (http://www.pavitr.net; GA 821410), cuyo objetivo es la validación de tecnologías naturales y avanzadas sostenibles para el tratamiento del agua y de las aguas residuales, el control y la reutilización segura del agua en la India. En el primer estudio de la primera parte, se abordaron los principios físicos y teóricos de la sedimentación para utilizarlos en el funcionamiento y la optimización de la cosecha de biomasa en sedimentador de láminas (700 L) de un cultivo de microalgas para el tratamiento de aguas residuales en un fotobiorreactor tubular semicerrado de tres (11,7 m3 cada uno) a escala demostrativa. Durante 6 meses se ajustó el flujo de entrada (6900 m3·día-1), la dosis de coagulante (1-12 mg·L-1) y las purgas de la biomasa (60-240 L·día-1) para conseguir una adecuada separación de la fase sólido-líquida. Los resultados de este apartado evidenciaron la eficacia de las lamelas en la tarea de separación sólidolíquido obteniendo una turbidez de salida inferior a 5 NTU tras el periodo de optimización. En el segundo estudio, se operaron y optimizaron dos espesadores para conseguir una concentración adecuada (20 g·L-1) de la biomasa recolectada previamente para el posterior proceso de digestión anaerobia en las mismas instalaciones. Los espesadores y las purgas se optimizaron en cuatro periodos durante dos meses. Los resultados mostraron una concentración final de 26,5 g·L-1 en el último periodo debido a un uso minimizado de los rascadores para evitar la resuspensión de las partículas permitiendo una adecuada sedimentación por compresión. En la segunda parte, se diseñó una Laguna de Alta Carga a escala demostrativa para ser implementada en Aligarh (India) con una capacidad de tratamiento de 50 m3·día-1. El objetivo del estudio era asistir, verificar y optimizar el dimensionamiento convencional de las Lagunas de Alta Carga mediante la modelización biocinética y el análisis hidrodinámico mediante Dinámica de Fluidos Computacional (CFD). Según las simulaciones del modelo biocinético, el tiempo de retención hidráulica óptimo para mejorar la eliminación de nutrientes fue de 4 días. Se construyó un modelo 3D de la laguna para analizar el comportamiento hidrodinámico de 36 diseños en forma de carrusel con diferentes configuraciones. Se simularon las diferentes combinaciones de números de deflectores en las inversiones, anchos de pared central y tamaños de forma de lágrima en os extremos del muro central. Se cuantificó la presencia de zonas de baja velocidad, así como el área útil frente al área total ocupada. La configuración más eficiente resultó ser la compuesta por dos deflectores y formas de lágrima con un diámetro igual a ¼ de la anchura del canal. Además, una evaluación técnico-económica del sistema determinó un coste de inversión de 732 euros por población equivalente (PE) y un coste operativo de 0,19 euros por m3 de agua residual tratada.
Enginyeria ambiental

La tesi descriu el desenvolupament d’un procés biotecnològic per a l’obtenció d’àcids grassos poliinsaturats (PUFA) omega-3 mitjançant microalgues heterotròfiques. El microorganisme seleccionat és l’Aurantiochytrium limacinum SR21 degut a la seva capacitat per produir PUFA, créixer utilitzant fonts de carboni de rebuig i tolerar medis amb elevades salinitats. Durant la investigació descrita en aquesta tesi, diversos mètodes analítics han estat desenvolupats per tal de monitoritzar el creixement d’ A. limacinum. Diferents eines de disseny d’experiències i modelatge com les matrius ortogonals de Taguchi, les Xarxes neuronals artificials i les superfícies de resposta, han estat utilitzades per desenvolupar el medi de cultiu específicament per A. limacinum. En lloc d’utilitzar les fonts de carboni tradicionals, el procés biotecnològic ha estat desenvolupat utilitzant un subproducte industrial com és el glicerol cru. Les mateixes eines de disseny i modelització han estat utilitzades per determinar les millors condicions d’aeració i agitació per a estimular la producció d’àcid Docosahexaenoic (DHA). S’han estudiat diferents estratègies de cultiu com el batch, fed-batch, continuous and “multi-stage” continuous reactor per determinar quina ofereix una millor productivitat de DHA. Els cultius operats en batch i fed-batch han generat el major rendiment de DHA (g DHA/ g biomassa). Contràriament, els reactors continus han ofert majors productivitats de DHA DHA (g DHA/l·h), especialment els “multi-stage”. Utilitzant l’estratègia “multi-stage”, el primer bioreactor es programa per estimular la producció de biomassa mentre que els següents bioreactors s’orienten a la producció de DHA. El productes de DHA que es comercialitzen actualment, estan formulats com a esters metílics o triglicèrids re-esterificats, però en cap cas com a triglicèrids naturals extrets sense modificar. En aquesta tesis es presenta una proposta de purificació per cromatografia (a escala industrial i de laboratori) per tal d’obtenir triglicèrids naturals amb cadenes de DHA.
La tesis describe el desarrollo de un proceso biotecnológico para la obtención de ácidos grasos poliinsaturados (PUFA) omega-3 mediante microalgas heterotróficas. El microorganismo seleccionado es Aurantiochytrium limacinum SR21 debido a su capacidad para producir diferentes PUFA, crecer utilizando subproductos Industriales y su tolerancia a elevadas salinidades. Durante la investigación descrita en esta tesis, diferentes métodos analíticos fueron desarrollados para monitorizar el crecimiento de A. limacinum. Diferentes herramientas de diseño de experiencias y modelización tales como, matrices ortogonales de Taguchi, redes neuronales artificiales o superficies de respuesta, han sido utilizadas para desarrollar el medio de cultivo específico para A. limacinum. En lugar de utilizar fuentes de carbono tradicionales, el proceso biotecnológico se ha desarrollado utilizando un subproducto industrial como el glicerol crudo. Las mismas herramientas de diseño y modelización han sido utilizadas para determinar las mejores condiciones de aireación y agitación para estimular la producción de ácido docosahexaenoico (DHA). Se han estudiado diferentes estrategias de cultivo como el batch, fed-batch, continuo y “multi-stage” continuo para determinar cual ofrece mayores productividades de DHA. Los cultivos operados en batch y fed-batch han ofrecido mejores rendimientos de DHA (g DHA/ g biomasa). Sin embargo, los cultivos operados en continuo han ofrecido una mayor productividad de DHA (g DHA/l·h). Utilizando la estrategia de cultivo continuo “multi-stage”, el primer biorreactor se orienta a la producción de biomasa y los siguientes biorreactores se programan para la estimulación de producción de DHA. Los productos de DHA que se comercializan actualmente, se formulan como esteres metílicos o triglicéridos re-esterificados, pero en ningún caso como triglicéridos naturales sin modificar. En esta tesis se presenta una propuesta de purificación por cromatografía (a escala industrial i de laboratorio) para obtener triglicéridos naturales con cadenas de DHA.
This dissertation describes the development of a biotechnological process to obtain polyunsaturated fatty acids (PUFA) omega-3 by heterotrophic microalgae. The selected heterotrophic microorganism was Aurantiochytrium limacinum SR21 due to its capacity to produce PUFA, grow with different carbon sources and tolerate high salinity. During the thesis investigation, diverse analytical methods have been developed in order to monitor A. limacinum growth. Moreover, different experimental design strategies/tools, such us Taguchi orthogonal matrices, Artificial neural network, Response surface methodology, etc. have been used to develop a growth medium specifically optimized for A. limacinum. Instead of using traditional carbon sources, the process has been developed to grow A. limacinum with crude glycerol, an industrial by‐product. The same experimental design tools served to find the best oxygen supply conditions to stimulate either biomass or DHA production. Batch, fed‐batch, continuous and multi‐stage continuous bioreactors have been studied to find the most prolific strategy. Batch and fed‐batch reactor generated the highest DHA yields (g DHA /g biomass). However, continuous cultivations produced higher DHA productivity values DHA (g DHA/l·h), especially the multi‐stage strategy. In a multi‐stage, continuous bioreactor, the first tank/s were dedicated to biomass production whereas the following tanks were set to stimulate DHA production. DHA products currently in market are formulated as DHA methyl esters or re‐esterified triglycerides, but not as unmodified triglycerides. In this thesis, an approach for preparative and process scale chromatography purification of unmodified triglycerides containing DHA has been developed.

Dissertação para obtenção do Grau de Mestre em Biotecnologia
A associação de dois bioreactores, um fotoautotrófico e um heterotrófico, ligados entre si através das suas correntes gasosas de entrada e saída com troca de gases, nomeadamente CO2 e O2, pode gerar um benefício em ambas as culturas. A corrente gasosa de saída do fotobioreactor, rica em O2, é usada como corrente de entrada no biorector heterotrófico, fermentador, e em paralelo, a corrente de saída do fermentador, rica em CO2 é usada como corrente de entrada no fotobioreactor, proporcionando um melhor crescimento de biomassa e uma melhor produtividade em biomassa e lípidos para posterior transformação em biodiesel. A microalga Chlorella protothecoides e a levedura Rhodotorula glutinis foram escolhidas como microrganismos modelo uma vez que, aliadas à sua robustez, apresentam elevadas produtividades de biomassa e lípidos em regime autotrófico (microalga) e heterotrófico (microalga e levedura). Neste trabalho foram realizados estudos de optimização sobre o crescimento heterotrófico da microalga em meio simples com diferentes fontes de carbono, no qual a glucose apresentou a mais elevada taxa de crescimento (μ), 0,037 h-1, e a melhor produtividade em biomassa (PXmax), 1,55 g/l.d. A simbiose entre Chlorella protothecoides autotrófica e a levedura heterotrófica Rhodotorula glutinis, revelaram resultados bastante promissores no sentido de fornecimento de ar rico em CO2 ao fotobioreactor, proveniente da fermentação da levedura, aumentando a taxa de crescimento da alga autotrófica 30%, o que leva a um aumento da produtividade em biomassa em 100%, e consequentemente a um aumento na produtividade em lípidos. Este resultado é de interesse biotecnológico e passível de levar a futuros estudos de ampliação de escala com possibilidade de diminuição dos custos de produção de biomassa e seus produtos sem a necessidade de investimentos adicionais.

Thesis (M. Sc.)--University of Alberta, 2010.
Title from pdf file main screen (viewed on Jan. 22, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Process Control, Department of Chemical and Materials Engineering, University of Alberta. Includes bibliographical references.

Rapidly depleting fossil fuel reserves with the burgeoning demand, associated escalating GHG footprint, and changes in the climate necessitated the exploration of alternate sustainable energy resources that are renewable, economically viable, and environmentally sound. Among renewable energy feedstocks, microalgae possess an intrinsic ability to convert atmospheric CO2 into renewable bioproducts at a shorter cycling period. Microalgae-based biofuel is emerging as a viable feedstock as it possesses faster growth rates and higher lipid productivity. Sustainable biofuel production from microalgae entails i) appropriate strain selection, ii) economic harvesting, and iii) eco-friendly transesterification. However, attaining economic viability in the production of microalgae-derived biofuels as potential drop-in fuels depend on minimising energy costs involved in its production. Detailed literature review reveals multiple research gaps in i) strain selection, ii) optimal harvesting, and iii) sustainable and environmentally friendly transesterification for achieving economic viability with the technical feasibility of microalgal biodiesel. A significant drawback of the current approaches is inappropriate strain selection of either random isolation or repository procured microalgal strains and inducing variations in growth conditions. Estimates indicate out of 72,500 strains of microalgae identified, about 44,000 species have been investigated with characteristics. Commonly researched species from a biofuel perspective belong to the genera Chlorella sp., Scenedesmus sp., Botryococcus sp., Nanochloropsis sp., Tetraselmis sp. Haematococcus sp. Cyanobacteria (blue-green algae) and the model pennate (bilaterally symmetrical) diatom Phaeodactylum tricornutum (Bacillariophytes). Among the investigated algal groups, green microalgae (class: Chlorophyceae) predominates (64%) while diatoms (Bacillariophyceae), picoplankton (Esutigmatophyceae), and cyanobacteria constitute only 8% necessitating further exploration to prioritise microalgal species through inventorying, habitat mapping, and monitoring to understand the factors responsible for species occurrence and its variability, which would provide vital insights on tolerance and sensitivity levels of different microalgae at the local level leading to resilient strain selection. This would help address the problems frequently encountered in the open cultivation of microalgae, such as difficulty in acclimatisation of microalgae, resistance to contamination, and prevalence of invasive taxa (pests/pathogens). Biomass harvesting continues to be significant energy-intensive process among various downstream operations, which not only escalates production costs but also enhances carbon footprints. Estimates reveal an approximately 30 – 40 % of the total costs involved in microalgal biodiesel production are expended in the harvesting of the algal biomass. Thus, to realise cost-effective production of microalgal biodiesel, the implementation of optimal harvesting strategies to minimise fuel production costs is crucial. Substrate-based attached algal cultivation systems are gaining prominence as a viable alternative to the conventional algal cultivation in open ponds and photobioreactors (PBRs’) owing to ease of harvesting, minimal water requirement per unit biomass production, lesser footprint area requirement, efficient light transmission, utilisation without adverse effects of cell shading and light saturation, higher biomass productivity and also scope for the use of wastewater with the remediation potential. Conventional transesterification using acids and alkalis poses environmental pollution consequences during disposal with large-scale production. In this context, the objectives of the current research were: Inventorying and habitat mapping of benthic diatom in the Aghanashini estuary, situated on the central west coast of India covering all seasons, revealed the occurrence of 27 tolerant diatoms species belonging to genera Amphora, Cyclotella, Navicula, Nitzschia, and Pleurosigma. During monsoon, species such as Amphora, Nitzschia, and Navicula were found to be dominant, while Achnanthes sp., Cyclotella sp., and Melosira sp., were predominant in monsoon. The post-monsoon season was dominated by diatoms Achnanthes, Navicula and Pleurosigma. Assessment of habitat conditions with species occurrence highlights the role of nutrient composition on species richness. Out of the 27 species found to be predominant during field studies, Nitzschia sp., Amphora sp. and Navicula sp. were prioritised due to their dominance in terms of species richness recorded during field investigations. Further to understand their lipid responses to varying nutrient and salinity conditions, lab-scale experimental studies were carried out using water equivalent to field conditions. The biomass productivity and lipid accumulation results of this study revealed the scope of using aquaculture wastewater for diatom cultivation. nutrient removal efficiencies of diatoms (N: 89.1 ± 0.85 %, P: 90.8 ± 0.12 %) from aquaculture wastewater highlights the scope for bioremediation and biofuel production. Comparative assessment of lipid accumulation in the prioritised diatom Nitzschia sp. under different trophic modes of photo, hetero, and mixotrophy revealed significant morphological variations, biomass yield (95.2 to 139.1 mg/L), fatty acid profiles, and biodiesel quality was observed when diatom cells were subjected to mixotrophic nutrition mode. Elemental composition analysis using scanning electron microscopy revealed the highest C (~ 48 %) in diatoms under mixotrophy compared to other trophic modes. This exercise also involved the comparison of biocatalyst over the conventional acid catalyst in the effective transesterification of diatom lipids into biodiesel. The results revealed a higher FAME conversion efficiency of biocatalyst-based transesterification (~ 87 %) than that of a conventional acid catalyst (~ 83%), thus demonstrating biocatalyst’s potential in large-scale sustainable production of biodiesel. A prototype low-cost substrate based bioreactor deployed in the flood plains of the Aghanashini estuary, based on the insights obtained from field investigations and lab-scale experiments, showed the highest areal biomass yield (22.23 g m-2), demonstrating its technical feasibility under actual field conditions. Techno-economic assessment of prototype bioreactor scaled up to one hectare exhibited economic viability with an estimated yearly profit ranging between ₹ 35,296 INR/ha and 2,09,190 INR/ha based on the level and type of inputs during cultivation. The payback period was the least (0.98 years) with the lowest biodiesel production cost (30.1 – 31.8 INR/kg biodiesel). The production cost of biofuel in a hectare (bioreactor) varied between 30.08 INR/kg to 59.52 INR/kg of biodiesel. Lifecycle assessment of microalgal biofuel to assess environmental footprint under different scenarios (no nutrient input, wastewater input, and fertiliser input) revealed a fossil energy requirement variation between 3.6 – 5.7 MJ/kg and the greenhouse gas emission (as kg equivalent CO2 emissions) of 0.85 – 1.46 kg CO2eq.kg-1 of biodiesel. This highlights a reduction in fossil energy requirement of about 87.3 % in the pilot substrate-based microalgal bioreactor. Wastewater – biocatalyst scenario exhibited the highest net energy ratio (NER) of 18.8 with an additional benefit of low-cost remediation of wastewater along the coastal regions of India which would enhance the job opportunities for rural women, while ensuring the energy security of the nation.

碩士
國立成功大學
化學工程學系
103
This study presents the cultivation and monitoring of Chlorella vulgaris and Scenedesmus abundans GH-D11 on a microfluidic platform, which is compatible to commercially and readily available plate readers. The properties of cell, including microalgae cell density (O.D.682) and total pigment (O.D.440), were determined by absorbance read by a plate reader during cultivation. Conventional screening methods for optimization of microalgae culture are time-consuming and complicated. Therefore, this research aims for developing prompt culture and rapid quantification of microalgae cellular contents using microfluidic bioreactor. In this study, the effects of carbon source and electric field on microalgae cultivation are investigated. The results show microalgae were cultivated better, indicated by more abundant pigment and cell density, by applying suitable carbon source and electric field. Chlorella vulgaris had high cell density (O.D.682=2.57, biomass = 2.71 g/L) and abundant pigment (O.D.440=2.71) after 120 hour of cultivation with sodium acetate as carbon source and 10 V/cm electric field. Scenedesmus abundans had the absorbance value of 2.25 and 1.92 in pigment and cell density after 120 hour of cultivation with glucose and 10 V/cm electric field and the biomass was 2.23 g/L. In summary, our microfluidic platform provides not only fast and convenient way to determining microalgae cellular contents but also considerably decreases the amount of microalgae cell culture size for screening cultivation parameters.

碩士
國立清華大學
工程與系統科學系
105
This study presents the cultivation and monitoring of Scenedesmus abundans GH-D11 on a microbioreactor, which is compatible to commercially and readily available plate reader. The properties of cell, including microalgae cell density (O.D.682) and carotenoids (O.D.440), were determined by absorbance read by a plate reader during cultivation. Conventional screening methods for optimization of microalgae culture are time-consuming and complicated. Therefor, this research aims for developing prompt culture and rapid quantification of microalgae cellular contents using microbioreactor. In this study, the effects of nitrogen resource, pH, salinity stress and spectral light on microalgae cultivation are investigated. The results show microalgae were cultivated better, indicated by more carotenoids and cell density, by applying suitable nitrogen source, adjusting a suitable pH value and using suitable light source. Scenedesmus abundans had high cell density (biomass = 2.126 g/L after 96 hour of cultivation) and carotenoids (total carotenoids = 1.014 mg/L) with sucrose as carbon source and ammonia as nitrogen source. The suitable pH value is 7 for Scenedesmus abundas cultivation and any shift of pH value may decrease the cell density and carotenoids. Salinity stress also decreased the cell density and carotenoids.The cultivation with salinity stress had the low cell density (biomass = 2.087 g/L after 96 hour of cultivation) and carotenoids (total carotenoids = 0.3580 mg/L) compared to the cultivation without salinity stress (biomass = 2.437 g/L after 96 hour of cultivation, total carotenoids = 0.504 mg/L). From the experimental results of effects of spectral light on microalgae cultivation, green LED exhibited the highest cell density (biomass = 2.412 g/L after 96 hour of cultivation) and red LED exhibited the highest carotenoids (total carotenoids = 0.2185 mg/L).In summary, our microbioreactor provides not only fast and convenient way to determining microalgae cellular contents but also considerably decreases the amount of microalgae cell culture size for screening cultivation parameters.

Dissertação de Mestrado em Energia para a Sustentabilidade apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
As microalgas caracterizam um grupo grande e diversificado de organismos fotossintéticos. Presentemente, o cultivo de microalgas é tido como uma opção viável em lagoas e fotobiorreatores fechados. Embora os fotobiorreatores possam possuir certos benefícios, o seu custo pode revelar um enorme impedimento para um desenvolvimento em larga-escala. As microalgas apresentam um desenvolvimento rápido, precisando apenas para o seu crescimento de, água, luz solar, dióxido de carbono (CO2) e alguns nutrientes adicionais. Neste trabalho, o custo de produção assumido para os fotobiorreatores foi aproximadamente 24€/kg biomassa seca. Durante a análise de custo, foi possível identificar que os custos relativos à mão-de-obra, logo seguidos dos encargos com depreciações, correspondem aos elementos com maior dimensão na estrutura de custos final. A metodologia de análise de cenários foi aplicada para avaliar o nível de custos de um sistema de produção em larga escala. Foi possível identificar uma possível redução de custo de aproximadamente de 16 €/kg, obtendo um preço final de biomassa de, aproximadamente, 7,70 €/kg. Para o cenário de média escala, o preço final aproximado de biomassa foi de 14 €/kg. Através de um cenário em que se adoptaram diferentes níveis de produtividade, foi possível demonstrar que para um "cenário produtividade máxima", a redução é excecionalmente elevada, evidenciando-se valores próximos dos 5,25 €/kg. Já quando consideramos um "cenário de alta produtividade", o valor cai para cerca de 10,50€/kg.
Microalgae represent a large and diverse group of photosynthetic organisms. The cultivation of microalgae is currently feasible in ponds raceway (open system) and closed photo-bioreactors. Microalgae are characterized by rapid growth. The only elements that they need to grow are water, sunlight, carbon dioxide (CO2) emissions and some nutrients. This work performs a cost analysis for the production of biomass. The production cost for the Photobioreactors system was about 24 €/kg of dry biomass with labor and depreciation representing the major elements contributing to the final production cost of the microalgae biomass. Three different scenarios of microalgae biomass cultivation with different volumes were evaluate, for the large scale production system, a cost reduction of 16 €/kg is possible to achieve, with a final price of biomass of approximately, 7.70 €/kg. For the medium scale scenario, the final price of microalgae biomass was 14 €/kg. A further significant scenario is the possibility of assuming different productivities, for a “maximum productivity scenario”, the reduction is exceptionally high, to values close to 5.25 €/kg, for the “high productivity scenario” the value decreases to 10.50 €/kg.

碩士
東海大學
環境科學與工程學系
103
Membrane ultrafiltration (UF) method is a simply separation method which is usually used for microalgae harvesting. However, membrane fouling is an important problem that decreases the performance of microalgae harvesting. The aim of this research is study of different disc-type UF membrane filtrations (400B and 400HB) on harvesting of two different microalgae (Spirulina maxima and Scenedesmus obliquus), to investigate the optimum operating parameters for filtration-remediation process. Moreover, cause of membrane fouling was also studied in this research. The result indicated that 400HB membrane predominantly shows higher efficiency of membrane filtration than 400B membrane, corresponded with initial resistance, reversible resistance and irreversible resistance owning to higher porosity. Scenedesmus obliquus causes slower membrane fouling because of its smaller sizes and can be easily remediated than Spirulina maxima. The membrane fouling can also be caused by the polysaccharides and proteins from broken cells also not only for the cells, confirmed by FTIR study. It was found that fouling process can be temporized up to 20 % when applied aeration system during filtration process. Backwash cleaning method is the most effective cleaning method and the flux decline can be completely recovered when combining aeration with backwash cleaning method. Keywords: microalgae, UF, harvesting

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.

Aerobic activated sludge membrane bioreactors (AS-MBR) in municipal wastewater treatment are compact systems that can efficiently perform biological organic oxidation. However, aerobic processes require mechanical aeration accounting for over 40% of total expenditure of a wastewater facility. Additionally, a global urgency for nutrient (Nitrogen/Phosphorus) removal strategies due to surges of eutrophication events requires complex MBR configurations. An innovative and cost-effective process was developed with a dual income-stream: high-quality treated effluent and value-added microalgal biomass for several applications. The proposed process involved several integrated components; an ultrafiltration AS-MBR for organic oxidation followed by a microalgal membrane photobioreactor (MPBR) to remove nutrients (N/P) through assimilation while simultaneously photosynthetically generating dissolved oxygen effluent that was recirculated back into the AS-MBR, thereby reducing the need for mechanical aeration for oxidation. A lab-scale system was fed with a synthetic medium-strength municipal wastewater. The microalgal species C. vulgaris was initially tested in batch trials as a proof-of-concept study on its potential as a photosynthetic oxygenator for the AS-MBR and identify its nutrient utilization efficiencies. The MPBR and MBR were later constructed for continuous operation, with the aim to identify an optimal process configuration. The unit processes were subsequently isolated, where the AS-MBR was subjected to a modelled algal effluent to assesses the impact of varying influent characteristics and effluent recycle rates. A microbial community analysis was performed by high-throughput sequencing and a statistical data-driven modeling approach to assess treatment performances. The MPBR stage was then subjected to the effluent achieved by the AS-MBR stage under varying operating conditions to assess its treatment performance and the resulting algal biomass biochemical composition to identify its suitability for bioethanol, biodiesel, or animal feed production. The findings of this study ultimately confirmed the ability of C. vulgaris to support the AS-MBR for organic removal and fractional nutrient removal by supplying the oxygen demand, and further achieve an effluent polish stage for nutrient removal. The process configuration also demonstrated the ability to achieve a high microalgal biomass production with the potential of extracting valuable products as an added benefit of the wastewater treatment.

[ES] La combinación de reactores anaerobios de membranas (AnMBRs) con el cultivo de microalgas en un fotobiorreactor de membranas (MPBR) aparece como una opción ideal dentro del marco de tecnologías sostenibles para la depuración de aguas residuales. Con esta combinación de tecnologías, se puede obtener biogás a partir de la materia orgánica presente en el agua residual, mientras que los nutrientes del efluente de AnMBR se recuperan con la biomasa algal. Además, la tecnología de membranas permite obtener un efluente limpio y apto para su reutilización. Estudios previos han demostrado la capacidad de un cultivo de microalgas para recuperar los nutrientes presentes en el efluente de un sistema AnMBR a escala laboratorio. Sin embargo, el traslado de esta tecnología a condiciones controladas de laboratorio a condiciones ambientales variables puede suponer una limitación en su aplicación industrial. Este trabajo consiste en la evaluación del proceso de cultivo de microalgas en una planta piloto MPBR alimentada con el efluente de un sistema AnMBR. Para ello se han evaluado las condiciones óptimas de operación de la planta, teniendo en cuenta tanto el proceso biológico de microalgas como la velocidad de ensuciamiento de las membranas. También se ha estudiado el efecto de otros parámetros que influyen en el proceso, como la intensidad de luz aplicada a los fotobiorreactores (PBRs), temperatura, concentración de materia orgánica, presencia de otros organismos, etc.; así como el peso específico de cada parámetro dentro del proceso. Otro objetivo consiste en la búsqueda de nuevos parámetros de control del proceso que faciliten la operación en continuo del sistema. El sistema MPBR utilizado en este estudio se mostró capaz de tratar un efluente de AnMBR, cumpliendo con los límites legales de vertido. Sin embargo, esta operación se consiguió únicamente cuando se cumplían una serie de condiciones: i) El espesor de los fotobiorreactores era estrecho (10 cm). ii) Las condiciones de operación (BRT y HRT) se mantenían dentro del rango adecuado. iii) Temperatura se mantenía habitualmente debajo del límite máximo de 30 ºC. iv) No existía acumulación de nitrito. v) La fuente principal de nitrógeno era amonio. vi) La materia orgánica presente en el cultivo no era excesiva.
[CAT] La combinació de reactors anaerobis de membranes (AnMBRs) amb el cultiu de microalgues en un fotobioreactor de membranes (MPBR) apareix com una opció ideal dins el marc de tecnologies sostenibles per a la depuració d'aigües residuals. Amb aquesta combinació de tecnologies, es pot obtenir biogàs a partir de la matèria orgànica present en l'aigua residual, mentre que els nutrients de l'efluent de AnMBR es recuperen amb la biomassa algal. A més, la tecnologia de membranes permet obtenir un efluent net i apte per a la seua reutilització. Estudis previs han demostrat la capacitat d'un cultiu de microalgues per recuperar els nutrients presents en l'efluent d'un sistema AnMBR a escala laboratori. No obstant això, el trasllat d'aquesta tecnologia de condicions controlades de laboratori a condicions ambientals variables pot suposar una limitació en la seua aplicació industrial. Aquest treball consisteix en l'avaluació del procés de cultiu de microalgues en una planta pilot MPBR alimentada amb l'efluent d'un sistema AnMBR. Per a això s'han avaluat les condicions òptimes d'operació de la planta, tenint en compte tant el procés biològic de microalgues com la velocitat d'embrutiment de les membranes. També s'ha estudiat l'efecte d'altres paràmetres que influeixen en el procés, com la intensitat de llum aplicada als fotobioreactors (PBRs), temperatura, concentració de matèria orgànica, presència d'altres organismes, etc .; així com el pes específic de cada paràmetre dins del procés. Un altre objectiu consisteix en la recerca de nous paràmetres de control del procés que facilitin l'operació en continu del sistema. El sistema MPBR utilitzat en aquest estudi es va mostrar capaç de tractar un efluent de AnMBR, complint amb els límits legals d'abocament. No obstant això, aquesta operació es va aconseguir únicament quan es complien una sèrie de condicions: i) El gruix dels fotobioreactors era estret (10 cm). ii) Les condicions d'operació (BRT i HRT) es mantenien dins del rang adequat. iii) La temperatura es mantenia habitualment baix del límit màxim de 30 ºC. iv) No existia acumulació de nitrit. v) La font principal de nitrogen era amoni. vi) La matèria orgànica present en el cultiu no era excessiva.
[EN] The combination of anaerobic membrane reactors (AnMBRs) and microalgae membrane photobioreactor (MPBR) appears as an ideal option within the framework of sustainable technologies for wastewater treatment. This combination enables to produce biogas from the organic matter present in wastewater, while the nutrient content of the AnMBR effluent can be recovered from microalgae biomass. In addition, membrane technology allows obtaining a water effluent which can be suitable for reclamation. Previous studies have proved the capability of a microalgae culture to recover the nutrients present in AnMBR effluent at lab scale. However, up-scaling from controlled lab conditions to varying outdoor conditions could limit the industrial applications of this technology. This study consists of the assessment of a microalgae culture in an MPBR pilot plant fed by effluent of an AnMBR system. For this, optimal operating conditions of the MPBR plant were evaluated, considering both the microalgae biological process and the membrane fouling rate. The effect of other parameters that have an influence on the process such as light intensity applied to the photobioreactors (PBRs), temperature, organic matter concentration, presence of other organisms, etc., was also studied; as well as the specific weight of each parameter on the process. Another goal consisted of finding new controlling parameters that ease the continuous operation of the system. The MPBR system used in this study showed appeared to be capable of treating AnMBR effluent, successfully accomplishing legal discharge limits. However, this was only achieved when the following conditions were reached: i) PBR light path was as narrow as 10 cm. ii) Operating conditions (BRT and HRT) were in the appropriate range. iii) Temperature was under the máximum limit of around 30 ºC. iv) Nitrite was not accumulated. v) Ammonium was the main nitrogen source. vi) Organic matter concentration in the culture was not high.
González Camejo, J. (2019). Assessment of the flat-pannel membrane photobioreactor technology for wastewater treatment: Outdoor application to treat the effluent of an anaerobic membrane bioreactor [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/133056
TESIS

Microalgae cultivation has received much research attention in recent decades due to its high photosynthetic productivity and ability to produce biofuel feedstocks as well as high value compounds for the health food, cosmetics, and agriculture markets. Microalgae are conventionally grown in open pond raceways or closed photobioreactors. Due to the high water contents of these cultivation systems, they require large energy inputs for pumping and mixing the dilute culture, as well as concentrating and dewatering the resultant biomass. The energy required to operate these systems is generally greater than the energy contained in the resultant biomass, which precludes their use in sustainable biofuel production. To address this challenge, we designed a novel photobioreactor inspired by higher plants. In this synthetic leaf system, a modified transpiration mechanism is used which delivers water and nutrients to photosynthetic cells that grow as a biofilm on a porous, wicking substrate. Nutrient medium flow through the reactor is driven by evaporation, thereby eliminating the need for a pump. This dissertation outlines the design, construction, operation, and modeling of such a synthetic leaf system for energy positive biofuel production. First, a scaled down synthetic leaf reactor was operated alongside a conventional stirred tank photobioreactor. It was demonstrated that the synthetic leaf system required only 4% the working water volume as the conventional reactor, and showed growth rates as high as four times that of the conventional reactor. However, inefficiencies in the synthetic leaf system were identified and attributed to light and nutrient limitation of growth in the biofilm. To address these issues, a modeling study was performed with the aim of balancing the fluxes of photons and nutrients in the synthetic leaf environment. The vascular nutrient medium transport system was also modeled, enabling calculation of nutrient delivery rates as a function of environmental parameters and material properties of the porous membrane. These models were validated using an experimental setup in which the nutrient delivery rate, growth rate, and photosynthetic yield were measured for single synthetic leaves. The synthetic leaf system was shown to be competitive with existing technologies in terms of biomass productivity, while requiring zero energy for nutrient and gas delivery to the microorganisms. Future studies should focus on utilizing the synthetic leaf system for passive harvesting of secreted products in addition to passive nutrient delivery.
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