Biodegradation of 2,4-Dinitrotoluene(DNT) by a recombinant Pseudomonas fluorescens strain

LARA, J.A; LUCCA, M.E.; SIÑERIZ, F.; FERRERO M.A

Baltimore Waterfront Marriott Hotel

Congreso; Society for Industrial Microbiology Annual Meeting; 2006

Industrial Microbiology and Biotechnology

Biodegradation of 2,4-Dinitrotoluene (DNT) by a recombinant Pseudomonas fluorescens strain. Lara ,J.A. (1) ; Lucca , M.E.(1,2), Siñeriz, F* . (1,2) and Ferrero, M. (1)  (1) PROIMI (Planta Piloto de Procesos Industriales Microbiológicos),CONICET, (2) Microbiología Superior, Bioquímica, Qca y Farmacia, UNT, Tucumán, Argentina. The aim of this work is the study of production of biomass and degradation of 2,4- DNT by Pseudomonas fluorescens recombinant (PFR) in submerged cultivation. Strains: Burkholderia cepacea strain DNT (BC) and Pseudomonas fluorescens recombinant (dntA, dntB, dntC, dntD) (PFR). Batch fermentation assays were performed in Erlenmeyer flasks of 250 ml with 90 ml of MMB (Minimum Medium Bhrum), different carbon and nitrogen sources were combined with different concentration of DNT, at shaker (200 rpm) at 30 ºC. An stock solution of 200 mM of DNT was dissolved in acetonitrilo (99,5 %, Sintorgan), added into the medium and incubated (3 h) previously to be inoculated. All assays were made by triplicate. Optical density, residual carbon source concentration, residual DNT concentration, duplication time (td), maximum growth rate (µ) were determined. DNT was determined in the culture supernatant by HPLC: Mobile phase: 70% Methanol / 30% water; caudal: 1 ml / min; temperature: 25 ºC; manual injection with valve RHEODYNE  (7125); spectrophotometer detector: 254 nm; register/integrator SHIMASZU; column SPHERISORB  ODS 2 (Waters). Results¨  PFR strain is unable to grow with DNT as a sole carbon and nitrogen source. Yeast extract, glucose and glycerol improved biomass yield. Both, PFR and BC strains degraded completely 2,4-DNT (1mM) in presence of with yeast extract. BC strain  degradaded 100% DNT between 12-18 h  while PFR strain degradation time was slower (19- 37,5 h) . Consuming rate of 2,4- DNT by PFR strain is maximal with 200 μM de DNT while 1mM of DNT produce the maximal specific consuming  rate.  Specific consuming rate (μmol DNT consumed /g cells) is directly proportional to concentration of DNT.