Advanced spectroscopic analysis and 15N-isotopic labelling study of nitrate and nitrite reduction to ammonia and nitrous oxide by E. coli

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.