Doctor of Philosophy / Department of Biological & Agricultural Engineering / Lisa R. Wilken / Microalgae are a potential feedstock for renewable and sustainable bioproducts and energy but there are significant scientific and engineering challenges to address before widespread acceptance of this platform. In particular, biorefining microalgae serves to maximize biomass valorization and minimize waste to improve process economics. The overall goal of this dissertation was the development of a biological-based microalgae biorefinery to enhance the economic feasibility of Chlamydomonas reinhardtii as a source of multiple products including native proteins and lipids. Specific objectives included accumulating biomass enriched in target biomolecules and determining processing strategies that eliminated the need to dry biomass, employed mild conditions to maintain extractability and quality, and minimized application of petroleum-derived and toxic solvents during extraction. The microalgae biorefinery developed included biomolecule accumulation, biomass harvesting, and targeted enzymatic degradation of the cell wall and organelles for release of native proteins and lipids. Biomass was cultivated, and kinetic studies indicated that 48 h nitrogen deprivation was adequate for protein and lipid accumulation. Four lytic enzymes were screened for their ability to permeate the C. reinhardtii cell wall and the C. reinhardtii-produced enzyme, autolysin, led to >85% cell disruption. TEM imaging confirmed cell disruption and retention of lipid droplets in organelle remnants indicating that protein, lipids, and starch could be distinctly partitioned and recovered. A design of experiments optimization study determined that incubation of disrupted biomass at pH 12 for 4 h at 45°C resulted in up to 65% of total protein released from disrupted biomass followed by 40-50% protein recovery with isoelectric precipitation. The cell disruption and protein extraction steps were subsequently integrated to minimize unit operations, processing time, and energy inputs. Secondary application of trypsin led to release of ~73% of total lipids (enriched in triacylglycerols) from the disrupted biomass. Characterization by thin layer chromatography and GC-FID of released lipids revealed similar profiles of enzymatically released lipids as compared to those released by conventional extraction procedures. Finally, the composition of released lipids indicated favorable combustion behavior, high oxidation stability, and suitability as biodiesel. The developed biological-based biorefinery is a promising step towards adoption of microalgae as a source of bioproducts to provide energy and food to meet the needs of a growing population. The second focus of the work was mitigation strategies for isolation of critical impurities (or potential co-products) while processing microalgae and plant hosts. Specific emphasis was placed on evaluating the impact of proteases, polysaccharides, phenolic compounds and pigments, phytic acid, and host cell proteins on the processing of microalgae and other plant hosts for extraction, recovery, and purification of therapeutic proteins. This review served as evaluation of the broader implications of application of the biorefinery to transgenic microalgae and other plants.

The continued increase in the demand for fossil fuels combined with their ever dwindling supply has prompted the search for a suitable alternative fuel. The research contained within this dissertation seeks to increase the lipid content of cellular feedstocks, improve extraction efficiencies of lipids, and understand the pathways involved in the production of fatty alcohols and triacylglycerides from microbial feedstocks. As part of this research the diatom, Cheatoceros gracilis, was grown at small and large scale to determine optimal growing conditions. No apparent nutrient stress trigger was required to initiate the accumulation of the biodiesel precursor triacylglyceride, unlike other documented algal strains. A follow-up to this project demonstrated that the microalga C. gracilis may utilize light intensity as a trigger for lipid production. A major difficulty in the production of biofuels from microorganisms is the expensive process of dewatering, drying, and extracting the lipid compounds from the cells. As part of this research, a process has been developed that allows for lipid extraction to occur in the presence of water at a point as low as 2 percent solids or 98 percent water. This process utilizes a single organic solvent that mixes well with microbial lipids, but poorly with water allowing for efficient extraction of lipids and fast solvent to water separation. This process greatly decreases the cost of the microbial biofuels production associated with the removal of water from cell slurries. Triacylglycerides and fatty alcohols are oleochemicals that are commonly used in industrial, pharmaceutical, and consumable processes. A predicted fatty acyl CoA reductase enzyme was cloned into an E. coli vector, expressed, characterized and shown to be active as a dual reductive enzyme reducing a fatty acyl CoA to its respective fatty alcohol, constituting the first enzyme of this type discovered in a bacterium. The process of triacylglyceride production in microbes is fairly well understood; however, the process that regulates this production has not yet been fully explored. As part of this research, the model yeast organism, Yarrowea lipolytica, is utilized to identify essential genes for citrate transport that if removed could result in increasing triacylglyceride production in vivo.

The continued increase in the demand for fossil fuels combined with their ever dwindling supply has prompted the search for a suitable alternative fuel. The research contained within this dissertation seeks to increase the lipid content of cellular feedstocks, improve extraction efficiencies of lipids, and understand the pathways involved in the production of fatty alcohols and triacylglycerides from microbial feedstocks. As part of this research the diatom, Cheatoceros gracilis, was grown at small and large scale to determine optimal growing conditions. No apparent nutrient stress trigger was required to initiate the accumulation of the biodiesel precursor triacylglyceride, unlike other documented algal strains. A follow-up to this project demonstrated that the microalga C. gracilis may utilize light intensity as a trigger for lipid production. A major difficulty in the production of biofuels from microorganisms is the expensive process of dewatering, drying, and extracting the lipid compounds from the cells. As part of this research, a process has been developed that allows for lipid extraction to occur in the presence of water at a point as low as 2 percent solids or 98 percent water. This process utilizes a single organic solvent that mixes well with microbial lipids, but poorly with water allowing for efficient extraction of lipids and fast solvent to water separation. This process greatly decreases the cost of the microbial biofuels production associated with the removal of water from cell slurries. Triacylglycerides and fatty alcohols are oleochemicals that are commonly used in industrial, pharmaceutical, and consumable processes. A predicted fatty acyl CoA reductase enzyme was cloned into an E. coli vector, expressed, characterized and shown to be active as a dual reductive enzyme reducing a fatty acyl CoA to its respective fatty alcohol, constituting the first enzyme of this type discovered in a bacterium. The process of triacylglyceride production in microbes is fairly well understood; however, the process that regulates this production has not yet been fully explored. As part of this research, the model yeast organism, Yarrowea lipolytica, is utilized to identify essential genes for citrate transport that if removed could result in increasing triacylglyceride production in vivo.

No description available.

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

Um estudo crítico foi realizado utilizando quatro métodos clássicos de extração de lipídios em aveia em flocos e peito de frango. Foram comparados os rendimentos, o tempo de extração, a repetibilidade de cada método e a qualidade oxidativa da fração lipídica obtida. As metodologias utilizadas foram as de Soxhlet, com n-hexano como solvente, nos períodos de quatro e oito horas de extração; Folch et al., com clorofórmio e metanol na proporção de 2:1 (v/v); Bligh & Dyer, com clorofórmio, metanol e água na proporção de 1:2:0,8 (v/v) e Hara & Radin, com n-hexano e isopropanol, na proporção de 3:2 (v/v). Os resultados obtidos indicaram que para a aveia em flocos, os métodos de Soxhlet e Bligh & Dyer apresentaram um rendimento superior (7,71 % e 7,66 %, respectivamente) em relação ao método de Hara & Radin e Folch et al. (6,61 % e 6,93 %, respectivamente). No estudo do peito de frango, os métodos de Soxhlet, Folch et al. e Bligh & Dyer apresentaram um rendimento satisfatório de lipídios totais, sendo, respectivamente, de 1,56 %, 1,56 % e 1,65 %. Apesar do bom rendimento em lipídios totais, o método de Soxhlet afetou a qualidade da fração lipídica em ambas amostras, demonstrada pela presença de peróxidos (4,39 e 4,19 meq O2/kg no peito de frango; 3,71 e 3,70 meq O2/kg na aveia em flocos) e pela acidez oléica (2,58 % de ácidos graxos livres (AGL) no peito de frango; 2,25 e 2,24 % AGL na aveia em flocos), em ambos os períodos de tempo da extração. Com base nos resultados deste trabalho, indica-se a metodologia de Bligh & Dyer para os casos em que haja interesse em uso posterior das frações lipídicas, em detrimento da de Soxhlet, não recomendável para o fim mencionado. / A critical study was carried through with four clasical methods of extraction of lipids from chicken breast and oat flakes. The yield, the extraction time, the repeatability of each method and the oxidative quality of the lipid fraction were evaluated and compared. The methodologies evaluated were Soxhlet, with n-hexane as the solvent, in periods of four and eight hours of extraction; Folch et al., with chloroform and methanol in the ratio of 2:1 (v/v); Bligh & Dyer, with chloroform, water and methanol, in the ratio of 1:2:0.8 (v/v), and Hara & Radin, with n-hexane and isopropanol, in the ratio of 3:2 (v/v). The results indicated that for oat flakes, the methods Soxhlet and Bligh & Dyer presented higher yields in total lipids (7.71 % and 7.66 %, respectively) than the methods Hara & Radin and Folch et al. (6.61 % and 6.93 %, respectively). In the study with chicken breast, the methods Soxhlet, Folch et al. and Bligh & Dyer presented the highest yields, being respectively 1.56 %, 1.56 % and 1.65 %.Despite the good yields in total lipids, the Soxhlet method affected the quality of the lipid fraction in both samples, demonstrated by the presence of peroxides (4.39 and 4.19 meq O2/kg in chicken breast; 3.71 and 3.70 meq O2/kg in oat flakes) and by the acid values (2.58 % FFA [free fatty acids] in chicken breast for both extractions; 2.25 and 2.24 % FFA in oat flakes,) for both periods of time evaluated. Based on the results obtained in this research, the authors recommend Bligh & Dyer methodology whenever there is interest in further use of the lipid fraction and strongly disencourage Soxhlet method for the purpose mentioned.

In plants, change in lipid composition is a common response to various abiotic stresses. Lipid constituents of bryophytes are of particular interest as they differ from that of flowering plants. Unlike higher plants, mosses have high content of very long-chain polyunsaturated fatty acids. Such lipids are considered to be important for survival of nonvascular plants. Here, using abscisic acid (ABA )-induced changes in lipid composition in Physcomitrella patens as an example, a protocol for total lipid extraction and quantification by gas chromatography (GC) coupled with flame ionization detector (FID) is described.

abstract: Lipids and free fatty acids (FFA) from cyanobacterium Synechocystis can be used for biofuel (e.g. biodiesel or renewable diesel) production. In order to utilize and scale up this technique, downstream processes including culturing and harvest, cell disruption, and extraction were studied. Several solvents/solvent systems were screened for lipid extraction from Synechocystis. Chloroform + methanol-based Folch and Bligh & Dyer methods were proved to be "gold standard" for small-scale analysis due to their highest lipid recoveries that were confirmed by their penetration of the cell membranes, higher polarity, and stronger interaction with hydrogen bonds. Less toxic solvents, such as methanol and MTBE, or direct transesterification of biomass (without pre-extraction step) gave only slightly lower lipid-extraction yields and can be considered for large-scale application. Sustained exposure to high and low temperature extremes severely lowered the biomass and lipid productivity. Temperature stress also triggered changes of lipid quality such as the degree of unsaturation; thus, it affected the productivities and quality of Synechocystis-derived biofuel. Pulsed electric field (PEF) was evaluated for cell disruption prior to lipid extraction. A treatment intensity > 35 kWh/m3 caused significant damage to the plasma membrane, cell wall, and thylakoid membrane, and it even led to complete disruption of some cells into fragments. Treatment by PEF enhanced the potential for the low-toxicity solvent isopropanol to access lipid molecules during subsequent solvent extraction, leading to lower usage of isopropanol for the same extraction efficiency. Other cell-disruption methods also were tested. Distinct disruption effects to the cell envelope, plasma membrane, and thylakoid membranes were observed that were related to extraction efficiency. Microwave and ultrasound had significant enhancement of lipid extraction. Autoclaving, ultrasound, and French press caused significant release of lipid into the medium, which may increase solvent usage and make medium recycling difficult. Production of excreted FFA by mutant Synechocystis has the potential of reducing the complexity of downstream processing. Major problems, such as FFA precipitation and biodegradation by scavengers, account for FFA loss in operation. Even a low concentration of FFA scavengers could consume FFA at a high rate that outpaced FFA production rate. Potential strategies to overcome FFA loss include high pH, adsorptive resin, and sterilization techniques. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2011

Um estudo crítico foi realizado utilizando quatro métodos clássicos de extração de lipídios em aveia em flocos e peito de frango. Foram comparados os rendimentos, o tempo de extração, a repetibilidade de cada método e a qualidade oxidativa da fração lipídica obtida. As metodologias utilizadas foram as de Soxhlet, com n-hexano como solvente, nos períodos de quatro e oito horas de extração; Folch et al., com clorofórmio e metanol na proporção de 2:1 (v/v); Bligh & Dyer, com clorofórmio, metanol e água na proporção de 1:2:0,8 (v/v) e Hara & Radin, com n-hexano e isopropanol, na proporção de 3:2 (v/v). Os resultados obtidos indicaram que para a aveia em flocos, os métodos de Soxhlet e Bligh & Dyer apresentaram um rendimento superior (7,71 % e 7,66 %, respectivamente) em relação ao método de Hara & Radin e Folch et al. (6,61 % e 6,93 %, respectivamente). No estudo do peito de frango, os métodos de Soxhlet, Folch et al. e Bligh & Dyer apresentaram um rendimento satisfatório de lipídios totais, sendo, respectivamente, de 1,56 %, 1,56 % e 1,65 %. Apesar do bom rendimento em lipídios totais, o método de Soxhlet afetou a qualidade da fração lipídica em ambas amostras, demonstrada pela presença de peróxidos (4,39 e 4,19 meq O2/kg no peito de frango; 3,71 e 3,70 meq O2/kg na aveia em flocos) e pela acidez oléica (2,58 % de ácidos graxos livres (AGL) no peito de frango; 2,25 e 2,24 % AGL na aveia em flocos), em ambos os períodos de tempo da extração. Com base nos resultados deste trabalho, indica-se a metodologia de Bligh & Dyer para os casos em que haja interesse em uso posterior das frações lipídicas, em detrimento da de Soxhlet, não recomendável para o fim mencionado. / A critical study was carried through with four clasical methods of extraction of lipids from chicken breast and oat flakes. The yield, the extraction time, the repeatability of each method and the oxidative quality of the lipid fraction were evaluated and compared. The methodologies evaluated were Soxhlet, with n-hexane as the solvent, in periods of four and eight hours of extraction; Folch et al., with chloroform and methanol in the ratio of 2:1 (v/v); Bligh & Dyer, with chloroform, water and methanol, in the ratio of 1:2:0.8 (v/v), and Hara & Radin, with n-hexane and isopropanol, in the ratio of 3:2 (v/v). The results indicated that for oat flakes, the methods Soxhlet and Bligh & Dyer presented higher yields in total lipids (7.71 % and 7.66 %, respectively) than the methods Hara & Radin and Folch et al. (6.61 % and 6.93 %, respectively). In the study with chicken breast, the methods Soxhlet, Folch et al. and Bligh & Dyer presented the highest yields, being respectively 1.56 %, 1.56 % and 1.65 %.Despite the good yields in total lipids, the Soxhlet method affected the quality of the lipid fraction in both samples, demonstrated by the presence of peroxides (4.39 and 4.19 meq O2/kg in chicken breast; 3.71 and 3.70 meq O2/kg in oat flakes) and by the acid values (2.58 % FFA [free fatty acids] in chicken breast for both extractions; 2.25 and 2.24 % FFA in oat flakes,) for both periods of time evaluated. Based on the results obtained in this research, the authors recommend Bligh & Dyer methodology whenever there is interest in further use of the lipid fraction and strongly disencourage Soxhlet method for the purpose mentioned.

Orientador: Neura Bragagnolo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-26T13:23:17Z (GMT). No. of bitstreams: 1 Costa_DeboradosSantosVenancio_M.pdf: 659453 bytes, checksum: 771dfa7781b70db92d56d782ce00e5e2 (MD5) Previous issue date: 2014 / Resumo: A utilização da energia de micro-ondas tem sido explorada para extração de compostos orgânicos, inorgânicos e para aceleração de reações químicas em diferentes matrizes. Os métodos convencionais utilizados para extração de lipídeos são métodos laboriosos e que utilizam grandes quantidades de solventes, com a possibilidade de degradar os compostos de interesse. A técnica de extração assistida por micro-ondas (microwave assisted extraction - MAE) combina a energia de micro-ondas gerada pela condução iônica e rotação de dipolos e a extração com solventes, sob freqüência de 2,45 GHz. O aquecimento das moléculas ocorre de forma mais rápida, o que aumenta a eficiência da extração em um tempo menor que as técnicas convencionais. Nesse trabalho foi desenvolvido um novo método de extração de lipídeos para peixes a partir de um delineamento composto central rotacional utilizando material de referência certificado de tecido de peixe homogeneizado (1946 NIST). Os parâmetros avaliados foram massa de amostra, massa de sulfato de sódio e volume de solvente extrator. De acordo com os resultados obtidos, as melhores condições para a MAE foram 0,60 g de amostra; 0,8 g de sulfato de sódio e 9 mL de solvente extrator (acetato de etila:metanol 2:1 (v/v)), a 54°C por 15 minutos (Medina, 2013). O método foi validado com material de referência de peixe homogeneizado e filé de tilápia (Oreochromis niloticus). O teor de lipídeos obtido pela MAE no material certificado foi 10,07 ± 0,25 g/100g e o valor declarado no material certificado de peixe é 10,17 ± 0,48 g/100g. No filé de tilápia o teor de lipídeos obtido pela MAE foi 2,28 ± 0,14 g/100g enquanto pelo método de extração sólido-líquido (Folch et al., 1957) foi 2,33 ± 0,09 g/100g. Esses valores não apresentaram diferença estatística ao nível de significância de 95% pelo Test t, além disso, não foi observada alteração na composição de ácidos graxos. Foram realizadas análises de produtos de oxidação lipídica (dienos conjugados, peróxidos, substâncias reativas ao ácido tiobarbitúrico e compostos voláteis) nos extratos lipídicos provenientes da MAE e do método de extração sólido-líquido. Os valores obtidos para estes parâmetros por MAE foram menores que os valores obtidos pelo método de extração sólido-líquido. Para avaliar o efeito das micro-ondas no tecido de filé de tilápia foi realizada microscopia eletrônica de varredura. O tecido do peixe foi totalmente destruído nas amostras irradiadas por micro-ondas enquanto que o tecido proveniente do método de extração sólido-líquido manteve-se íntegro. O método foi aplicado em amostras de peixes pacu (Piaractus mesopotamicus), pescada (Cynoscion jamaicensis) e atum (Thunnus thynnus), cujos valores de lipídeos totais variaram de 1 a 13 g/100g. Os valores de lipídeos totais e de ácidos graxos obtidos não apresentaram diferença estatística quando comparados aos valores obtidos pelo método de extração sólido-líquido. Dessa forma, a MAE pode ser aplicada para extração de lipídeos em peixes, uma vez que os resultados demonstraram boa exatidão e precisão, além de ser uma técnica rápida, robusta e de baixo consumo de solvente comparada à extração sólido-líquido / Abstract: The use of microwave energy has been explored for extracting organic and inorganic compounds and accelerating chemical reactions in different matrices. The conventional methods used for extracting lipids are laborious and use a large amount of solvent, possibly degrading the compounds of interest. Microwave assisted extraction (MAE) combines microwave energy generated by ionic conduction and dipole rotation and the solvent extraction, at a 2.45 GHz frequency. Molecule heating occurs more rapidly, which increases extraction efficiency in a shorter time than conventional techniques. In the present study, a new lipid extraction method assisted by microwave was developed for fish meat by way of a rotational composite design using lake superior fish tissue certified reference material (1946, NIST). According to the results, the best conditions for for MAE were 0.60 g of sample; 9 mL of extraction solvent (ethyl acetate:methanol 2:1 (v/v)), 0.8 g de of sodium sulfate, at 54°C for 15 min (Medina, 2013). The method was validated using the lake superior fish tissue certified reference material and tilapia fillet (Oreochromis niloticus). Lipid content of the certified reference material by MAE was 10.07 ± 0.25 g/100g while the value declared in the certificate is 10.17 ± 0.48 g/100g. Lipid content of tilapia fillet by MAE was 2.28 ± 0.14 g/100g while by solid-liquid extraction (Folch et al., 1957) it was 2.33 ± 0.09 g/100g. These values were not statistically different by the t test at a 95% significance level; in addition, no changes in fatty acid composition were noticed. Lipid oxidation products (conjugated dienes, peroxides, TBARS and volatile compounds) were determined in the lipids extracted by MAE and solid-liquid extraction, being the values obtained for MAE lower than those obtained by solid-liquid extraction. Scanning electron microscopy was carried out in the microwave irradiated samples to evaluate the effects of microwave on the tilapia fillet tissue. The fish tissue submitted to microwave irradiation was completely destroyed in comparison to the tissue from the fish tissue submitted to solid-liquid extraction, which remained intact. The method was applied to fish samples, i.e., pacu fish (Piaractus mesopotamicus), hake (Cynoscion jamaicensis) and tuna (Thunnus thynnus), whose lipid contents varied between 1 and 13 g/100g. No significant differences were found between MAE and solid-liquid extraction for total lipid content and fatty acid composition of these three fishes. Therefore, MAE can be applied for lipid extraction from fish since the results showed good accuracy and precision, moreover it is a fast and robust technique with low solvent consumption as compared to solid-liquid extraction / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos