81 resultados para Biodiesel production
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Dissertation presented at Faculdade de Ciências e Tecnologia from Universidade Nova de Lisboa to obtain the degree of Master in Chemical and Biochemical Engineering
Employment of the side product of biodiesel production in the formation of surfactant like molecules
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Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia Química e Bioquímica
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Dissertação para obtenção do Grau de Mestre em Biotecnologia
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Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica
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Different oil-containing substrates, namely, used cooking oil (UCO), fatty acids-byproduct from biodiesel production (FAB) and olive oil deodorizer distillate (OODD) were tested as inexpensive carbon sources for the production of polyhydroxyalkanoates (PHA) using twelve bacterial strains, in batch experiments. The OODD and FAB were exploited for the first time as alternative substrates for PHA production. Among the tested bacterial strains, Cupriavidus necator and Pseudomonas resinovorans exhibited the most promising results, producing poly-3-hydroxybutyrate, P(3HB), form UCO and OODD and mcl-PHA mainly composed of 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) monomers from OODD, respectively. Afterwards, these bacterial strains were cultivated in bioreactor. C. necator were cultivated in bioreactor using UCO as carbon source. Different feeding strategies were tested for the bioreactor cultivation of C. necator, namely, batch, exponential feeding and DO-stat mode. The highest overall PHA productivity (12.6±0.78 g L-1 day-1) was obtained using DO-stat mode. Apparently, the different feeding regimes had no impact on polymer thermal properties. However, differences in polymer‟s molecular mass distribution were observed. C. necator was also tested in batch and fed-batch modes using a different type of oil-containing substrate, extracted from spent coffee grounds (SCG) by super critical carbon dioxide (sc-CO2). Under fed-batch mode (DO-stat), the overall PHA productivity were 4.7 g L-1 day-1 with a storage yield of 0.77 g g-1. Results showed that SCG can be a bioresource for production of PHA with interesting properties. Furthermore, P. resinovorans was cultivated using OODD as substrate in bioreactor under fed-batch mode (pulse feeding regime). The polymer was highly amorphous, as shown by its low crystallinity of 6±0.2%, with low melting and glass transition temperatures of 36±1.2 and -16±0.8 ºC, respectively. Due to its sticky behavior at room temperature, adhesiveness and mechanical properties were also studied. Its shear bond strength for wood (67±9.4 kPa) and glass (65±7.3 kPa) suggests it may be used for the development of biobased glues. Bioreactor operation and monitoring with oil-containing substrates is very challenging, since this substrate is water immiscible. Thus, near-infrared spectroscopy (NIR) was implemented for online monitoring of the C. necator cultivation with UCO, using a transflectance probe. Partial least squares (PLS) regression was applied to relate NIR spectra with biomass, UCO and PHA concentrations in the broth. The NIR predictions were compared with values obtained by offline reference methods. Prediction errors to these parameters were 1.18 g L-1, 2.37 g L-1 and 1.58 g L-1 for biomass, UCO and PHA, respectively, which indicates the suitability of the NIR spectroscopy method for online monitoring and as a method to assist bioreactor control. UCO and OODD are low cost substrates with potential to be used in PHA batch and fed-batch production. The use of NIR in this bioprocess also opened an opportunity for optimization and control of PHA production process.
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Due to the prospective partial replacement of fossil fuels by biodiesel, its production has continuously grown in the last decade. The increase in global biodiesel production demands the development of sustainable applications of its main by-product, crude glycerol. In this thesis the feasibility of producing polyhydroxyalkanoates (PHA) by a mixed microbial community using crude glycerol as feedstock was investigated. Several incubation conditions were studied in order to maximize PHA production. The microbial population selected under aerobic dynamic feeding conditions had the ability to consume both major carbon fractions present in the crude, glycerol and methanol. Two biopolymers were stored, poly-3-hydroxybutyrate (PHB) and glucose biopolymer (GB), apparently using glycerol as the only carbon source for their production. The microbial enrichment obtained was able to accumulate up to 47% PHB of cell dry weight with a productivity of 0.24 g HA/L d. The overall PHA yield on total substrate consumed (0.32 g COD HB/g COD crude glycerol) was in the middle range of those reported in literature (0.08–0.58 g COD PHA/g COD real waste). The increase of temperature from 23ºC to 30ºC favored the culture fraction that accumulates glucose biopolymer with a maximum accumulation value of 25% of cell dry weight, which is an interesting value but not the main goal of this thesis. The fact that crude glycerol can be used to produce PHA without any pre-treatment step, makes the overall production process economically more competitive, reducing polymer final cost. This was the first study that demonstrates the valorization of the glycerol fraction present in the crude glycerol into PHA using an aerobic mixed microbial consortium.
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Dissertação apresentada à Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção do grau de Mestre em Bioenergia
Valorization of olive pomace through combination of biocatalysis with supercritical fluid technology
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A supercritical carbon dioxide (scCO2) based oil extraction method was implemented on olive pomace (alperujo), and an oil yield of 25,5 +/- 0,8% (goil/gdry residue) was obtained. By Soxhlet extraction with hexane, an oil extraction yield of 28,9 +/- 0,8 % was obtained, which corresponds to an efficiency of 88,4 +/- 4,8 % for the supercritical method. The scCO2 extraction process was optimized for operating conditions of 50 MPa and 348,15 K, for which an oil loading of 32,60 g oil/kg CO2 was calculated. As a proof of concept, olive pomace was used as feedstock for biodiesel production, in a process combining the use of lipase as a catalyst with the use of scCO2 as a solvent, and integrating the steps of oil extraction, oil to biodiesel transesterification and subsequent separation of the latter. In the conducted experiments, FAME (fatty acid methyl ester) purities of 90% were obtained, with the following operating parameters: an oil:methanol molar ratio of 1:24; a residence time of 7,33 and 11,6 mins; a pressure of 40 MPa; a temperature of 313,15 K; and Lipozyme (Mucor miehei; Sigma-Aldritch) as an enzyme. However, oscillations of FAME purity were registered throughout the experiments, which could possibly be due to methanol accumulation in the enzymatic reactor. Finally, the phenolic content of olive pomace, and the effect of the drying process – oven or freeze-drying – and the extraction methods – hydro-alcoholic method and supercritical method – on the phenolic content were analysed. It was verified that the oven-drying process on the olive pomace preserved 90,1 +/- 3,6 % of the total phenolic content. About 62,3 +/- 5,53% of the oven-dried pomace phenolic content was extracted using scCO2 at 60 MPa and 323,15 K. Seven individual phenols – hydroxytyrosol, tyrosol, oleuropein, quercetin, caffeic acid, ferulic acid and p-coumaric acid – were identified and quantified by HPLC.
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Succinic acid (SA) is a highly versatile building block that is used in a wide range of industrial applications. The biological production of succinic acid has emerged in the last years as an efficient alternative to the chemical production based on fossil fuels. However, in order to fully replace the competing petro-based chemical process from which it has been produced so far, some challenges remain to be surpassed. In particular, one main obstacle would be to reduce its production costs, mostly associated to the use of refined sugars. The present work is focused on the development of a sustainable and cost-e↵ective microbial production process based on cheap and renewable resources, such as agroindustrial wastes. Hence, glycerol and carob pods were identified as promising feedstocks and used as inexpensive carbon sources for the bioproduction of succinic acid by Actinobacillus succinogenes 130Z, one of the best naturally producing strains. Even though glycerol is a highly available carbon source, as by-product of biodiesel production, its consumption by A. succinogenes is impaired due to a redox imbalance during cell growth. However, the use of an external electron acceptor such as dimethylsulfoxide (DMSO) may improve glycerol metabolism and succinic acid production by this strain. As such, DMSO was tested as a co-substrate for glycerol consumption and concentrations of DMSO between 1 and 4% (v/v) greatly promoted glycerol consumption and SA production by this biocatalyst. Aiming at obtaining higher succinic acid yield and production rate, batch and fed-batch experiments were performed under controlled cultivation conditions. Batch experiments resulted in a succinic acid yield on glycerol of 0.95 g SA/g GLY and a production rate of 2.13 g/L.h, with residual production of acetic and formic acids. In fed-batch experiment, the SA production rate reached 2.31 g/L.h, the highest value reported in the literature for A. succinogenes using glycerol as carbon source. DMSO dramatically improved the conversion of glycerol by A. succinogenes and may be used as a co-substrate, opening new perspectives for the use of glycerol by this biocatalyst. Carob pods, highly available in Portugal as a residue from the locust bean gum industry, contain a significant amount of fermentable sugars such as sucrose, glucose and fructose and were also used as substrate for succinic acid production. Sugar extraction from raw and roasted carobs was optimized varying solid/water ratio and extraction time, maximizing sugar recovery while minimizing the extraction of polyphenols. Kinetic studies of glucose, fructose and sucrose consumption by A. succinogenes as individual carbon sources till 30 g/L were first determined to assess possible metabolic diferences. Results showed no significant diferences related to sugar consumption and SA production between the diferent sugars. Carob pods water extracts were then used as carbon source during controlled batch cultivations. (...)
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Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica
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This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)
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Polysaccharides are gaining increasing attention as potential environmental friendly and sustainable building blocks in many fields of the (bio)chemical industry. The microbial production of polysaccharides is envisioned as a promising path, since higher biomass growth rates are possible and therefore higher productivities may be achieved compared to vegetable or animal polysaccharides sources. This Ph.D. thesis focuses on the modeling and optimization of a particular microbial polysaccharide, namely the production of extracellular polysaccharides (EPS) by the bacterial strain Enterobacter A47. Enterobacter A47 was found to be a metabolically versatile organism in terms of its adaptability to complex media, notably capable of achieving high growth rates in media containing glycerol byproduct from the biodiesel industry. However, the industrial implementation of this production process is still hampered due to a largely unoptimized process. Kinetic rates from the bioreactor operation are heavily dependent on operational parameters such as temperature, pH, stirring and aeration rate. The increase of culture broth viscosity is a common feature of this culture and has a major impact on the overall performance. This fact complicates the mathematical modeling of the process, limiting the possibility to understand, control and optimize productivity. In order to tackle this difficulty, data-driven mathematical methodologies such as Artificial Neural Networks can be employed to incorporate additional process data to complement the known mathematical description of the fermentation kinetics. In this Ph.D. thesis, we have adopted such an hybrid modeling framework that enabled the incorporation of temperature, pH and viscosity effects on the fermentation kinetics in order to improve the dynamical modeling and optimization of the process. A model-based optimization method was implemented that enabled to design bioreactor optimal control strategies in the sense of EPS productivity maximization. It is also critical to understand EPS synthesis at the level of the bacterial metabolism, since the production of EPS is a tightly regulated process. Methods of pathway analysis provide a means to unravel the fundamental pathways and their controls in bioprocesses. In the present Ph.D. thesis, a novel methodology called Principal Elementary Mode Analysis (PEMA) was developed and implemented that enabled to identify which cellular fluxes are activated under different conditions of temperature and pH. It is shown that differences in these two parameters affect the chemical composition of EPS, hence they are critical for the regulation of the product synthesis. In future studies, the knowledge provided by PEMA could foster the development of metabolically meaningful control strategies that target the EPS sugar content and oder product quality parameters.
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Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia Electrotécnica e de Computadores
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Dissertation presented in partial fulfillment of the requirements for the degree of Master in Biotechnology
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Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia