Publicação
Biotechnological approaches of crude glycerol use: optimization of citric acid production by Yarrowia lipolytica
| Resumo: | The increase of biodiesel production results in an accumulation of crude glycerol, the main byproduct from biodiesel industry. To take advantage of the glycerol surplus, many biotechnological processes are being studied. Crude glycerol can be used as carbon source to produce citric acid by Yarrowia lipolytica under nitrogen-limited growth conditions. However, other operational and medium conditions directly affect citric acid production. Yield of citric acid depends upon the concurrent production of other organic acids, for instance isocitric acid, which is strongly dependent of the strain used. Although there are some works described in the literature, several factors still need to be completely understood and optimized in the production of citric acid using crude glycerol. To start with, an experimental design, based on Taguchi method was applied to optimize the culture conditions and to evaluate the effect of pH, carbon/nitrogen (C/N) ratio in the medium, oxygen mass transfer rate (OTR) and salts concentration on citric acid production from pure glycerol by two different Y. lipolytica strains (W29 (ATCC 20460) and CBS 2073). OTR and pH were the factors, which had more effect on citric acid production. Moreover, a significant interaction between the factors OTR and salts was observed. The optimal conditions were also validated with crude glycerol and the citric acid production was similar for both strains using this low cost substrate. Since, as shown by the Taguchi approach, a high OTR was crucial for citric acid production, it seemed appropriate to further study this matter. Therefore a model describing oxygen volumetric mass transfer coefficient ( kL a), in a lab-scale stirred tank bioreactor (STR), as a function of operating conditions (stirring and aeration rates) and cellular density in the citric acid bioprocess, was developed. An empirical correlation was established that fit well in a wide range of operating conditions. As a result, it was found that raising kL a from 7 h-1 to 55 h-1 the citric acid concentration increased. On the other hand, the increase of dissolved oxygen concentration (DO) up to 60 % using controlled DO, led to an increase of citric acid concentration, reaching identical concentration as obtained at kL a of 55 h-1. This work demonstrated that kL a is an adequate parameter for the optimization and scale-up of citric acid production from crude glycerol by Y. lipolytica W29. Taking into account that oxygen is a crucial parameter in citric acid production by Y. lipolytica W29 from crude glycerol, a pressurized and an airlift bioreactor, both reactors associated to high mass transfer efficiency, were used for batch cultures. Increasing air pressure from 1 bar to 2 bar led to an improvement of 40 % in citric acid concentration, whereas in the airlift bioreactor, with an increase from 1 vvm to 1.5 vvm of the aeration rate a 30 % enhancement was attained. Both bioreactor types can be used as alternative ways of improving OTR for citric acid production, leading to important operating costs savings due to less power input. The simultaneous production of isocitric acid is the major problem of using Y. lipolytica strains as citric acid producer. In order to isolate improved strains with reduced isocitric/citric acid ratio and/or enhanced citric acid production, Y. lipolytica W29 was treated with ultraviolet (UV)-irradiation and/or ethyl methane sulfonate (EMS). A 76% and 2.2- fold higher concentration yield of citric acid, was obtained with a mutant strain, Y. lipolytica UV/EMS-10, isolated after the combined treatment with UV-irradiation and EMS. |
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| Autores principais: | Ferreira, Patrícia Marlene Alves |
| Ano: | 2016 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | The increase of biodiesel production results in an accumulation of crude glycerol, the main byproduct from biodiesel industry. To take advantage of the glycerol surplus, many biotechnological processes are being studied. Crude glycerol can be used as carbon source to produce citric acid by Yarrowia lipolytica under nitrogen-limited growth conditions. However, other operational and medium conditions directly affect citric acid production. Yield of citric acid depends upon the concurrent production of other organic acids, for instance isocitric acid, which is strongly dependent of the strain used. Although there are some works described in the literature, several factors still need to be completely understood and optimized in the production of citric acid using crude glycerol. To start with, an experimental design, based on Taguchi method was applied to optimize the culture conditions and to evaluate the effect of pH, carbon/nitrogen (C/N) ratio in the medium, oxygen mass transfer rate (OTR) and salts concentration on citric acid production from pure glycerol by two different Y. lipolytica strains (W29 (ATCC 20460) and CBS 2073). OTR and pH were the factors, which had more effect on citric acid production. Moreover, a significant interaction between the factors OTR and salts was observed. The optimal conditions were also validated with crude glycerol and the citric acid production was similar for both strains using this low cost substrate. Since, as shown by the Taguchi approach, a high OTR was crucial for citric acid production, it seemed appropriate to further study this matter. Therefore a model describing oxygen volumetric mass transfer coefficient ( kL a), in a lab-scale stirred tank bioreactor (STR), as a function of operating conditions (stirring and aeration rates) and cellular density in the citric acid bioprocess, was developed. An empirical correlation was established that fit well in a wide range of operating conditions. As a result, it was found that raising kL a from 7 h-1 to 55 h-1 the citric acid concentration increased. On the other hand, the increase of dissolved oxygen concentration (DO) up to 60 % using controlled DO, led to an increase of citric acid concentration, reaching identical concentration as obtained at kL a of 55 h-1. This work demonstrated that kL a is an adequate parameter for the optimization and scale-up of citric acid production from crude glycerol by Y. lipolytica W29. Taking into account that oxygen is a crucial parameter in citric acid production by Y. lipolytica W29 from crude glycerol, a pressurized and an airlift bioreactor, both reactors associated to high mass transfer efficiency, were used for batch cultures. Increasing air pressure from 1 bar to 2 bar led to an improvement of 40 % in citric acid concentration, whereas in the airlift bioreactor, with an increase from 1 vvm to 1.5 vvm of the aeration rate a 30 % enhancement was attained. Both bioreactor types can be used as alternative ways of improving OTR for citric acid production, leading to important operating costs savings due to less power input. The simultaneous production of isocitric acid is the major problem of using Y. lipolytica strains as citric acid producer. In order to isolate improved strains with reduced isocitric/citric acid ratio and/or enhanced citric acid production, Y. lipolytica W29 was treated with ultraviolet (UV)-irradiation and/or ethyl methane sulfonate (EMS). A 76% and 2.2- fold higher concentration yield of citric acid, was obtained with a mutant strain, Y. lipolytica UV/EMS-10, isolated after the combined treatment with UV-irradiation and EMS. |
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