Adaptation of E. coli cell method for micro-scale nitrate measurement withthe Griess reaction in culture media

The E. coli cell method for nitrate measurement consists of two-steps: nitr ate reduction by the E. coli cell usually under anaerobic conditions and su bsequently nitrite measurement with the Griess reaction. It was found that the E. coli DSM 498k wildtype cell can reduce nitrate to nitrite under aero bic conditions. Therefore, the E. coli method for nitrate measurement was a dapted to be performed under aerobic conditions in a microtiter plate. The adapted method is simpler than the original E. coli method and other nitrat e methods such as those with inorganic reductants and with purified enzymes . Furthermore, it was found that for the Griess reaction the pH values of s amples after addition of the Griess reagent A should be lower than 1.8 for a stable absorbance at 540 nm to be reached. It is important to add the two Griess reagents separately and to read the absorbance twice consecutively in a microtiter plate. The adapted E. coli method was successfully applied to measure the traces of nitrate in MRS and other medium components by meas uring the standard curve of a dilution of each individual medium component. It was found that many organic medium components contain traces of nitrate , while none of them contain detectable nitrite. Among these, the extract o f meat and yeast extract contain relatively high amounts of nitrate: 217 mg N/kg and 99 mg N/kg respectively. MRS broth contains nitrate from 0.3 to 0 .6 mg N/l depending on the batch numbers of the product. The adapted E. col i can also be used for nitrate measurement in other matrices. (C) 2000 Else vier Science B.V. All rights reserved.