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| Author(s): |
Cristiano José de Andrade
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia de Alimentos |
| Defense date: | 2016-09-03 |
| Examining board members: |
Glaucia Maria Pastore;
Carlos Ricardo Soccol;
Helia Harumi Sato;
Marcos Rogério Tótola;
Marta Cristina Teixeira Duarte
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| Advisor: | Francisco Fabio Cavalcante Barros; Glaucia Maria Pastore |
| Abstract | |
The set of biotechnological processes including the production of enzymes, peptides, bioflavours, biosurfactants, among other, is significantly increasing. In general, the purification process represents ? 60% of production cost of biosurfactants, whereas the culture medium ? 30%. This study describes, for the first time, the ultrafiltration of two biosurfactants (independent studies), which were produced using an industrial waste as culture medium, that is, surfactin by Bacillus subtilis LB5a and mannosylerythritol lipids by Pseudozyma tsukubaensis. Surfactin was produced by Bacillus subtilis LB5a at top-bench bioreactor scale (3 liters of working volume). The foam (high concentration of surfactin) was collected by the top of bioreactor and used for the calculations of yield of process and evaluation of purification by ultrafiltration. The yield was ? 366.66 mg of surfactin by liter of culture medium. The ultrafiltration of surfactin was carried out in two-steps (i) the micelles were retained and, (ii) the adition of organic solvent (ethanol) destabilized the surfactin micelles, allowing the free surfactin (unaggregated) be recovered in the permeate. For the process of ultrafiltration, polyethersulfone membranes with two molecular weight cut-off, 100 kDa and 50 kDa, were used. The best strategy was the use of membrane of 100 kDa in the first step of ultrafiltration and 50 kDa in the second step of ultrafiltration. The ultrafiltration of crude biosurfactant was associated with fouling and/or concentration polarization. However, the ultrafiltration of semi-purified biosurfactant resulted in high recovery of surfactin (78.25%), high sepration from proteins and reduced effects of fouling and/or concentration polarization. Thus, on one hand the use of cassava wastewater for the production of surfactin decreases the production costs. On the other hand, makes harder the purification process. Since the steps of production, purification and application should be evaluated sequentially, the use of cassava wastewater has to be integrated to a treatment for remove the proteins before the fermentation process, or before the ultrafiltration steps (lower concentration of proteins), for instance acid precipitation and extraction by organic solvent, or even alternative process of purification, for instance bubble column. The chemical structure identification of surfactin was carried out by two analyses: (i) matrix assisted lazer desorption ionization followed by the detection using analyzer of time of flight and, (ii) nuclear magnetic resonance spectroscopy. By the analyses of these two techniques were identified 11 potential isoforms of surfactin, in which are composed by two sequences of amino acids (Glu1-Leu2-Leu3-Val4-Asp5-Leu6-Leu7) and (Glu1'-Leu2'-Leu3'-Val4'-Asp5'-Leu6'-Val7'). Mannosylerythritol lipids were produced by Pseudozyma tsukubaensis at top-bench bioreactor scale (3 liters of working volume) using cassava wastewater as culture medium. The foam (high concentration of mannosylerythritol lipids) was collected by the top of bioreactor and used for the calculations of yield of process and evaluation of purification by ultrafiltration. The yield was ? 1.23 g of mannosylerythritol lipids by liter of culture medium, which demonstrates that cassava wastewater is a good culture medium for the production of mannosylerythritol lipids by Pseudozyma tsukubaensis. The experiments of ultrafiltration with mannosylerythritol lipids removed ? 95% of proteins and retained (vesicles) ? 80% of mannosylerythritol lipids. Therefore, only one step of ultrafiltration was needed for the purification of mannosylerythritol lipids. The process of ultrafiltration was scaled-up from 20 mL (ultrafiltration device) to 500 mL (top-bench ultrafiltration equipment), and the results were similar. The production of mannosylerythritol lipids-B by Pseudozyma tsukunbaensis was confirmed by gas chromatography coupled to mass spectrometry, matrix assisted lazer desorption ionization followed by the detection using analyzer of time of flight and nuclear magnetic resonance spectroscopy. It was also identified a second stereoisomer (? 9%) related to erythritol. The recovery of mannosylerythritol lipids-B by the foam overflow on the top of bioreactor integrated to ultrafiltration is a remarkable alternative of purification, instead of the traditional extraction using ethyl acetate followed of silica column. After the production and purification of biosurfactants, their potentials for enhanced oil recovery were evaluated. The experiments were carried out with 3 sorts of oils, light, medium and heavy. According to the results obtained of oil displacement and emulsification index tests, mannosylerythritol lipids-B are more efficient on microbial enhanced oil recovery, em particular for heavy oil (AU) | |
| FAPESP's process: | 11/22776-5 - Utilization of cassava waste water dehydrated and whey as culture medium to production of biossurfactant and buteno-2,3-diol by Bacillus subtilis. |
| Grantee: | Cristiano José de Andrade |
| Support Opportunities: | Scholarships in Brazil - Doctorate |