STUDY ON CONTINUOUS CULTURES OF GLUCONACETOBACTER DIAZOTROPHICUS USING SUCROSE AS CARBON AND ENERGY SOURCE

MOLINARI M.L.; BOIARDI J.L.

MAR DEL PLATA

Congreso; VIII Congreso Argentino de Microbiología General. Sociedad Argentina de Microbiología General (SAMIGE).; 2012

SAMIGE (Sociedad Argentina de Microbiología General)

Gluconacetobacter diazotrophicus is an acid tolerant, N2–fixer bacterium and endophyte of sugarcane. G. diazotrophicus secrets an extracellular enzyme, levansucrase (LsdA), that hydrolyses sucrose and it has levan biosynthetic activities. Glucose is metabolized by a periplasmic PQQ-GDH to gluconic acid and fructose is channeled to the synthesis of levans. G. diazotrophicus growth with glucose has been extensively studied, but there are not reports about sucrose metabolic. In this work we studied the stoichiometry of continuous culture under different nutritional conditions. G. diazotrophicus PAL 5 were grown at 30 °C in a 2-l fermentations unit with a working volume of 1 l. The dilution rate was adjusted at 0.05 h-1. The pH was maintained at 6.0. G. diazotrophicus were grown using LGIM medium. Different nutritional conditions have been tested were: sucrose 20 g/l (C-limited) and source 100 g/l (C-excess); BNF (N2 fixed) under microaerophilic conditions and No-BNF (3 g/l (NH4)2(SO4)). Under steady-sated conditions we have measured: O2; biomass, CO2, exo-polysaccharides, glucose and gluconic acid productions; LsdA, GDH-PQQ, GaDH activity. We were able to determine the stoichiometry of G. diazotrophicus grown under continuous culture with sucrose as main carbon and energy source. The activity of LsdA increased with sucrose concentration in the culture medium. Surprisingly PQQ-GDH, although considered de main pathway for glucose, was not detected in cultures under C-limited, but significant activities were found with C-excess as glucose could be detected in culture. So, in presence of sucrose G. diazotrophicus seems to express a different carbon pathway. Moreover gluconic acid and keto-acid were only detected in culture performed with C-excess and increased under BNF conditions. The exo-polysaccharides production was detected in all of cultures conditions. The levans production increased under C-limited and BNF that could be used in nitrogenase protection. The data of stoichiometry of growth under C-limited and BNF shows a higher oxygen demand and decreased in the synthesis of extracellular products. However cultures under BNF increased in metabolic flux to substrate oxidation and ATP production, which increased the production of CO2. Thus, under C-excess and BNF shows a lower biomass production that No-FBN. The biomass and CO2 production decreased under C-excess. Consequently, the carbon source should be directed to the formation of other products not detected. In addition a low demand for O2 and low CO2 production, we assumed that the cell metabolism was directed to the synthesis of products with an oxidation states similar to sucrose.