Cultivation of Mycobacteria in Submerged Fermentation: Towards a Reproducible Method for Vaccine Production

Carolina Viana da Costa Araújo

Key information

Authors:

Carolina Viana da Costa Araújo (Carolina Viana da Costa Araújo)

Supervisors:

Pedro Carlos De Barros Fernandes (Pedro Carlos De Barros Fernandes); Carla da Conceição Caramujo Rocha de Carvalho (Carla da Conceição Caramujo Rocha de Carvalho)

Published in

November 16, 2021

Abstract

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis that to this day continues to affect millions of people. The only vaccine available to prevent the development of the disease is the Bacille Calmette-Guérin vaccine, which is made from a live attenuated M. bovis, the causing agent of bovine TB. However, BCG has ranging efficacies, one of the reasons being the cell aggregates formed, resulting in a defective quantification and quality control of the vaccine. In this study, the aggregative phenotype was studied to find conditions allowing smaller cell clusters and to obtain cells in planktonic state, using M. smegmatis as model organism. To this end, the effect of culture media composition and growth temperature were evaluated. Cells exposed to either 0.25% (v/v) ethanol, methanol, or DMSO presented a decrease in aggregation velocity, meaning that the cell aggregates formed were smaller. Cell aggregation also decreased after seven 15-min cold shocks without agitation were executed. Moreover, carbon starvation led to a decrease in the size of the cell aggregates. The effect of nitrogen supplementation of the culture medium via ammonium-containing compounds in cell aggregation was studied, with cells in planktonic state being obtained during supplementation with 50 mM ammonium acetate, ammonium chloride, or 100 mM ammonium sulphate. The fatty acid composition of the phospholipids of the membrane was assessed for every experiment, with mycobacteria responding either by increasing or decreasing the fluidity of the membrane. Surprisingly, increased fluidity was obtained through the introduction of polyunsaturated fatty acids in the cellular membrane.