Xu J, Xu X, Verstraete W
Laboratory of Microbial Ecology and Technology, Department of Biochemical and Microbial Technology, Ghent University, Belgium.
J Microbiol Methods. 2000 Jun;41(1):23-33. doi: 10.1016/s0167-7012(00)00141-x.
The E. coli cell method for nitrate measurement consists of two-steps: nitrate reduction by the E. coli cell usually under anaerobic conditions and subsequently nitrite measurement with the Griess reaction. It was found that the E. coli DSM 498k wildtype cell can reduce nitrate to nitrite under aerobic conditions. Therefore, the E. coli method for nitrate measurement was adapted to be performed under aerobic conditions in a microtiter plate. The adapted method is simpler than the original E. coli method and other nitrate methods such as those with inorganic reductants and with purified enzymes. Furthermore, it was found that for the Griess reaction the pH values of samples 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 measuring 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 of 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. coli can also be used for nitrate measurement in other matrices.
通常在厌氧条件下由大肠杆菌细胞将硝酸盐还原，随后用格里斯反应测定亚硝酸盐。研究发现，大肠杆菌DSM 498k野生型细胞在有氧条件下可将硝酸盐还原为亚硝酸盐。因此，大肠杆菌法测定硝酸盐被调整为在微量滴定板中于有氧条件下进行。调整后的方法比原始的大肠杆菌法以及其他硝酸盐测定方法（如使用无机还原剂和纯化酶的方法）更简单。此外，研究发现，对于格里斯反应，加入格里斯试剂A后样品的pH值应低于1.8，才能在540nm处达到稳定的吸光度。在微量滴定板中分别加入两种格里斯试剂并连续读取两次吸光度很重要。通过测量每种单独培养基成分稀释液的标准曲线，调整后的大肠杆菌法成功应用于测定MRS和其他培养基成分中的痕量硝酸盐。研究发现，许多有机培养基成分含有痕量硝酸盐，但均未检测到亚硝酸盐。其中，肉提取物和酵母提取物含硝酸盐量相对较高，分别为217mg N/kg和99mg N/kg。根据产品批次不同，MRS肉汤中硝酸盐含量为0.3至0.6mg N/l。调整后的大肠杆菌也可用于其他基质中的硝酸盐测定。
