Presentation Information
[P02-277]TOWARD PILOT-SCALE BIOPRODUCTION OF GRAMICIDIN S ANALOG EXTRACTS: IMPACT OF HEADSPACE TO WORKING VOLUME RATIO
○Jose M. Cruz1, María Garrrido1, Ksenia Lvova1, Andrea Chiara2, Letizia Fracchia2, Noelia González-Fernández1, Ana B. Moldes1 (1. Chemical Engineering Department, School of Industrial Engineering – CINTECX, University of Vigo. (Spain), 2. Department of Pharmaceutical Sciences, University of Piemonte Orientale (Italy))
Keywords:
sustainability,bioprocess,green extractants,antibiotics,Bacillus
Gramicidin S (GS) is a potent cyclic antimicrobial peptide first isolated in 1942 from the soil bacterium Aneurinibacillus migulanus. Despite its strong activity, clinical use has largely remained topical due to hemolytic toxicity, although newly reported analogs are reopening prospects for systemic applications.
Sustainable bioproduction demands process intensification (fewer unit operations, safer auxiliaries, lower energy use) and robust scale-up. Usually biotechnologically produced GS is obtained by fermentation followed by several extraction and purification process. During scale-up, the balance between headspace (air volume) and working volume is frequently not considered, despite its relevance for maintaining adequate oxygen transfer and mixing, which can impact metabolite production. Here, it is reported a preliminary study about the effect of reactor volume headspace with fermentation broth (Vreactor/Vbroth) in the framework of a proof-of-concept toward pilot-scale production of a Gramicidin S (GS). This is a based antibiotic extract (mixture of GS analogs) produced by Aneurinibacillus aneurinilyticus and extracted involving a simplified downstream step consisting of a single extraction process carried out at room temperature using a green solvent.
Fermentations were performed at 37 °C under established operating conditions using a commercial medium supplemented or not with Corn Steep Liquor (CSL) at 100 rpm. To probe scale-up-relevant hydrodynamics and aeration constraints, different working volumes were tested while keeping whole volume reactor constant and varying the ratio Vreactor/Vbroth to 5.0, 2.0, and 1.25 (v/v), therefore modifying the relative air volume above the broth. After fermentation, A. aneurinyilyticus cells were recovered by centrifugation, washed with deionized water, and the biomass was subjected to one-step green-solvent extraction at room temperature adjusting the amount of solvent to the volume of fermented broth following the conditions stablished in previous works.
Across the tested Vreactor/Vbroth range, the GS-based extract showed stable qualitative composition. Mass spectrometry revealed that the analogs with m/z corresponding to 1141 were consistently predominant, whereas minor GS analogs were detected with masses compromising 1155, 1169, 1126, and 1112 Da. Raw extraction yields almost did not vary with the Vreactor/Vbroth ratio in high extend within the evaluated range, indicating that headspace and working volume did not affect significantly the concentration of raw GS extract in the extractant and without substantially altering the main analog GS profile. Regarding yields, under the most favorable conditions evaluated in this work, 1 g/L of raw GS extract can be obtained depending on the nutritional medium. After this study it can be concluded that in the range tested for Vreactor/Vbroth, it is recommended to work at the lowest ratio Vreator/Vbroth, with the aim to reduce operational costs.
Sustainable bioproduction demands process intensification (fewer unit operations, safer auxiliaries, lower energy use) and robust scale-up. Usually biotechnologically produced GS is obtained by fermentation followed by several extraction and purification process. During scale-up, the balance between headspace (air volume) and working volume is frequently not considered, despite its relevance for maintaining adequate oxygen transfer and mixing, which can impact metabolite production. Here, it is reported a preliminary study about the effect of reactor volume headspace with fermentation broth (Vreactor/Vbroth) in the framework of a proof-of-concept toward pilot-scale production of a Gramicidin S (GS). This is a based antibiotic extract (mixture of GS analogs) produced by Aneurinibacillus aneurinilyticus and extracted involving a simplified downstream step consisting of a single extraction process carried out at room temperature using a green solvent.
Fermentations were performed at 37 °C under established operating conditions using a commercial medium supplemented or not with Corn Steep Liquor (CSL) at 100 rpm. To probe scale-up-relevant hydrodynamics and aeration constraints, different working volumes were tested while keeping whole volume reactor constant and varying the ratio Vreactor/Vbroth to 5.0, 2.0, and 1.25 (v/v), therefore modifying the relative air volume above the broth. After fermentation, A. aneurinyilyticus cells were recovered by centrifugation, washed with deionized water, and the biomass was subjected to one-step green-solvent extraction at room temperature adjusting the amount of solvent to the volume of fermented broth following the conditions stablished in previous works.
Across the tested Vreactor/Vbroth range, the GS-based extract showed stable qualitative composition. Mass spectrometry revealed that the analogs with m/z corresponding to 1141 were consistently predominant, whereas minor GS analogs were detected with masses compromising 1155, 1169, 1126, and 1112 Da. Raw extraction yields almost did not vary with the Vreactor/Vbroth ratio in high extend within the evaluated range, indicating that headspace and working volume did not affect significantly the concentration of raw GS extract in the extractant and without substantially altering the main analog GS profile. Regarding yields, under the most favorable conditions evaluated in this work, 1 g/L of raw GS extract can be obtained depending on the nutritional medium. After this study it can be concluded that in the range tested for Vreactor/Vbroth, it is recommended to work at the lowest ratio Vreator/Vbroth, with the aim to reduce operational costs.
Comment
To browse or post comments, you must log in.Log in