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| Author(s): |
César Augusto Piedrahíta Aguirre
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: | 2013-11-19 |
| Examining board members: |
Ranulfo Monte Alegre;
Alexandre Nunes Ponezi;
Vanildo Luiz Del Bianchi;
Reinaldo Gaspar Bastos;
Crispin Humberto Garcia Cruz
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| Advisor: | Ranulfo Monte Alegre |
| Abstract | |
This work covers a study of the production of iturin A by Bacillus by solid-state fermentation in packed bed bioreactors. The study was conducted in four parts. At first a screening was conducted with wild strains and their mutants obtained from exposure to UV light and mutagenic agent acridine orange. The strain Bacillus subtilis subsp subtilis NRRL NRS 1270 showed the highest antagonistic activity against Aspergillus fumigatus NRRL 164, Aspergillus fumigatus NRRL 166 and Aspergillus flavus var . oryzae NRRL 484. A methanolic extract obtained by solid state fermentation of Bacillus subtilis subsp subtilis NRRL NRS 1270 was analyzed with mass spectrometry showing lipopeptides with molecular mass between m/z 1021.43 and m/z 1087.48, but without the presence of iturin A. In the second stage, the strain Bacillus Iso 1 was isolated from soybean roots. Given the difficulty of identifying iturin A by high performance liquid chromatography (HPLC), a iturin A purification methodology was developed using glass column chromatography packed with activated Silica gel 60 and alumina. This methodology involved three solvent systems for elution of the iturin A from the column. A first fraction consisted of 20 ml of chloroform-methanol-water (65:25:4 , v/v/v) and was followed by 20 ml of chloroform - methanol- water (30:50 : 10, v/v/v), that was then followed by a final fraction consisting of 10 ml of chloroform-methanol-water (20:60:15, v/v/v). The fractions obtained of fermentation were analyzed by both HPLC and mass spectrometry, identifying five iturin A isomers (C13-C16). In the third stage of the study, an experimental design was constructed in the form of a central composite rotational design (CCRD) to evaluate the effect of rice husk as an inert support and air flow rates to the iturin A production, using defatted soybean meal and wheat bran as substrate. Although none of the studied variables showed statistical significance, the operational importance of reduction of oxygenation of the Bacillus Iso 1 fermentation due to the low concentration of rice husk and air flow rate was observed to favor the production of iturin; in these conditions high productivity was obtained reaching 6.88 g/kg-dry substrate of iturin A. Concluding from available literature, this is the highest concentration of iturin A ever produced in packed bed bioreactor (column) with forced aeration to date. In the fourth stage, in order to understand the behavior of the fermentation under aeration conditions between 0.4 L/min and 0.8 L/min, the following process parameters were studied, based on the results obtained from the CCRD: pressure drop, oxygen consumption and temperature profiles. The maximum production of iturin obtained was 5.58 g/kg-dry substrate with the air flow rate at 0.4 L/. The increase of the pressure gradients is caused not only by increasing the volumetric air flow rate but also by the production of biopolymer ?-PGA by Bacillus iso 1, which occupies the free interparticle space, hindering or preventing the normal flow of air through the bed and thus leading to reduced oxygen consumption. The low oxygenation favored the high iturin A production and resulted in low metabolic heat generation (5.75 W/kg-dry substrate.min). The results of this work are expected to be conducive for designing strategies to scale up the process in aerated packed bed bioreactors (AU) | |