First steps of carbon metabolism pathway in continuous cultures of Gluconacetobacter diazotrophicus under different nutritional status.

MARÍA LAURA MOLINARI; JOSÉ LUIS BOIARDI

San Miguel de Tucumán, Tucumán, Argentinaan

Congreso; 7mo Congreso Argentino de Microbiología General. SAMIGE del Bicentenario; 2011

Sociedad Argentina de Microbiología General

An acid tolerant N2-fixing bacterium, Gluconacetobacter diazotrophicus, was reported to be associated with sugarcane. It is thought to provide significant amounts of N to this crop and others. This bacterium is able to grow on high sucrose concentration (10 &%;) but this sugar cannot be transported or respired by G. diazotrophicus. This organism secrets an extracellular enzyme, levansucrase (LsdA), that hidrolyses sucrose into fructose and glucose. Glucose is the main carbon and energy source for G. diazotrophicus. The first spteps of its metabolism involves a periplasmic glucose oxidation mediated by a pyrrolo-quinoline-quinone (PQQ)- linked glucose dehydrogenase (PQQ-GDH). In this work we have studied the influence of diverse growth conditions on the regulation of the oxidative pathway of sucrose metabolism in G. diazotrophicus. Chemostat cultures of G. diazotrophicus PAL 5 were grown using the modified LGIM medium at 30 &º; C. A 2-l fermentations unit with a working volume of 1.0 l was used. The dilution rate was adjusted at 0.05 h-1. The pH was automatically maintained at 6.0 using either 1N NaOH or 1N H2SO4. The dissolved oxygen was continuously measured and maintained at the desired level of air saturation by varying the agitation speed. Different nutritional conditions have been tested; i.e. limited in carbon source (sucrose 20 g/l) and excess of carbon source (sucrose 100 g/l); BNF (N2) under microaerophilic conditions (dissolved O2% ≤ 2%) and no-BNF (3 g/l (NH4)2SO4). Under steady-sated conditions we have measured: biomass, CO2 production, O2 consumption and enzymatic activity. Activity of LsdA increased with sucrose concentration in the culture medium. Surprisingly PQQ-GDH, although considered de main route for glucose catabolism in this organism, was not detected in cultures with 20 g/l of sucrose, but significant activities were found with sucrose 100 g/l. The same was observed for GaDH. These results suggest that, in the presence of sucrose G. diazotrophicus seems to express a different metabolic pathway for C-metabolism in relation to cultures performed with glucose. It seems that PQQ-GDH was expressed only when significant glucose concentration could be detected in culture supernatants (as found in cultures with 100 g/l of sucrose) but not under conditions where free glucose was undetectable (as in cultures with 20 g/l of sucrose). Moreover gluconic acid was only detected in cultures performed with sucrose excess.