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  • Title: Advanced spectroscopic analysis and 15N-isotopic labelling study of nitrate and nitrite reduction to ammonia and nitrous oxide by E. coli.
    Author: Metcalfe GD, Smith TW, Hippler M.
    Journal: Analyst; 2021 Nov 08; 146(22):7021-7033. PubMed ID: 34693414.
    Abstract:
    Nitrate and nitrite reduction to ammonia and nitrous oxide by anaerobic E. coli batch cultures is investigated by advanced spectroscopic analytical techniques with 15N-isotopic labelling. Non-invasive, in situ analysis of the headspace is achieved using White cell FTIR and cavity-enhanced Raman (CERS) spectroscopies alongside liquid-phase Raman spectroscopy. For gas-phase analysis, White cell FTIR measures CO2, ethanol and N2O while CERS allows H2, N2 and O2 monitoring. The 6 m pathlength White cell affords trace gas detection of N2O with a noise equivalent detection limit of 60 nbar or 60 ppbv in 1 atm. Quantitative analysis is discussed for all four 14N/15N-isotopomers of N2O. Monobasic and dibasic phosphates, acetate, formate, glucose and NO3- concentrations are obtained by liquid-phase Raman spectroscopy, with a noise equivalent detection limit of 0.6 mM for NO3- at 300 s integration time. Concentrations of the phosphate anions are used to calculate the pH in situ using a modified Henderson-Hasselbalch equation. NO2- concentrations are determined by sampling for colorimetric analysis and NH4+ by basifying samples to release 14N/15N-isotopomers of NH3 for measurement in a second FTIR White cell. The reductions of 15NO3-, 15NO2-, and mixed 15NO3- and 14NO2- by anaerobic E. coli batch cultures are discussed. In a major pathway, NO3- is reduced to NH4+via NO2-, with the bulk of NO2- reduction occurring after NO3- depletion. Using isotopically labelled 15NO3-, 15NH4+ production is distinguished from background 14NH4+ in the growth medium. In a minor pathway, NO2- is reduced to N2O via the toxic radical NO. With excellent detection sensitivities, N2O serves as a monitor for trace NO2- reduction, even when cells are predominantly reducing NO3-. The analysis of N2O isotopomers reveals that for cultures supplemented with mixed 15NO3- and 14NO2- enzymatic activity to reduce 14NO2- occurs immediately, even before 15NO3- reduction begins. Optical density and pH measurements are discussed in the context of acetate, formate and CO2 production. H2 production is repressed by NO3-; but in experiments with NO2- supplementation only, CERS detects H2 produced by formate disproportionation after NO2- depletion.
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