|1315-2||Neisseria lactamica IN BATCH AND FED-BATCH BIOREACTOR CULTIVATION|
|Autores:||Beatriz Isva Gonçalves (IBU - Instituto Butantan / USP - Universidade de São Paulo) ; Giovanna Ferreira Costa Leão Salustiano (IBU - Instituto Butantan / USP - Universidade de São Paulo) ; Rocilda Perazzini Furtado Schenkman (IBU - Instituto Butantan) |
Introduction: Neisseria lactamica is a commensal microorganism whereas Neisseria meningitidis can be occasionally a pathogen invading nasal mucosa to cause septicemia and meningitis. N. lactamica colonizes the human nasopharynx during early childhood. Its presence has been implicated in naturally acquired immunity against N. meningitidis. These bacteria naturally release outer membrane vesicles (OMV) during growth. An alternative approach to a vaccine for serogroup B meningococcal disease has been the development of an OMV vaccine based on the commensal N. lactamica. The focus of this study is to analyze growth and OMV yield produced by N. lactamica in bioreactor, in modified Catlin medium (MC) in discontinuous batch, fed-batch and fed-batch with pulses. Methods: N. lactamica was cultured in 5L bioreactor, on MC with the double concentration of lactate, amino acids and 2 g/L of yeast extract during 12 or 14 hours. The cultivations groups were: A, batch process; B, fed-batch process with lactate feeding from hour 4; C, fed-batch process with lactate feeding and amino acids pulse on 4th hour and yeast extract pulse on 8th hour. Samples were collected every each hour and biomass was determined by dry weight (g/L) and optical density at 540 nm (OD540). OMV were purified from culture medium by centrifugation followed by ultracentrifugation. OMV yield was determined by Lowry’s method. Lactate, acetate and citrate were measured by high performance liquid chromatography (HPLC). Results and discussion: The maximum OD540 was 5.82 at group A, 7.30 at group B, 9.88 at group C and the maximum OMV yield was 175 mg/L, 298 mg/L, and 401 mg/L, respectively. In group A, lactate was consumed until the 6th-8th hour. During cultivations, N. lactamica released citrate earlier than acetate in the medium. The maximum values of acetate were 4.38 g/L at group A, 13.12 g/L at group B, 14.84 g/L at group C. The maximum values of citrate were 3.39 g/L, 9.79 g/L, and 9.06 g/L, respectively. These components participate of the Krebs cycle. The fed-batch with lactate contributed to get a better biomass and OMV yield, and the pulses of amino acids and yeast extract were also important. Conclusion: The lactate, amino acids and yeast extract were fundamental to get a better N. lactamica biomass and a better OMV yield.
Palavras-chave: Cultivation kinetics, Neisseria lactamica, OMV