Activity and electron donor preference of two denitrifying bacterial strains identified by Raman gas spectroscopy

Abstract

Human activities have greatly increased the input of reactive nitrogen species into the environment and disturbed the balance of the global N cycle. This imbalance may be offset by bacterial denitrification, an important process in maintaining the ecological balance of nitrogen. However, our understanding of the activity of mixotrophic denitrifying bacteria is not complete, as most research has focused on heterotrophic denitrification. The aim of this study was to investigate substrate preferences for two mixotrophic denitrifying bacterial strains, Acidovorax delafieldii and Hydrogenophaga taeniospiralis, under heterotrophic, autotrophic or mixotrophic conditions. This complex analysis was achieved by simultaneous identification and quantification of H2, O2, CO2, 14N2, 15N2 and 15N2O in course of the denitrification process with help of cavity-enhanced Raman spectroscopic (CERS) multi-gas analysis. To disentangle electron donor preferences for both bacterial strains, microcosm-based incubation experiments under varying substrate conditions were conducted. We found that Acidovorax delafieldii preferentially performed heterotrophic denitrification in the mixotrophic sub-experiments, while Hydrogenophaga taeniospiralis preferred autotrophic denitrification in the mixotrophic incubation. These observations were supported by stoichiometric calculations. The results demonstrate the prowess of advanced Raman multi-gas analysis to study substrate use and electron donor preferences in denitrification, based on the comprehensive quantification of complex microbial gas exchange processes.

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

“…Open have been previously applied to monitoring NO 3metabolism in bacteria; CERS has been used to follow N 2 O and N 2 production in denitrifying organisms, with the use of 15 NO 3to produce 15 N 2 distinguishable from background 14 N 2 . 19,22 A robust CERS instrument has also been designed for field application to study the gas composition of soil samples. 21 We report a combined approach for characterizing DNRA and N 2 O production in anaerobic E. coli batch cultures using mostly non-invasive spectroscopic techniques.…”

“…Additionally, vibrational spectroscopies can distinguish different isotopologues and isotopomers, allowing online monitoring of isotope labelling experiments. 15,16 Good sensitivities are observed in the condensed phase, but measuring headspace gases often suffers from low sensitivity, and special enhancement techniques are required such as Cavity Enhanced Raman Spectroscopy (CERS) [15][16][17][18][19][20][21][22] or long-path absorption White cells in FTIR spectroscopy. 23 Partial pressures in the headspace can be converted into concentrations in the solution via Henry's law.…”

Comprehensive multi-gas study by means of fiber-enhanced Raman spectroscopy for the investigation of nitrogen cycle processes

“…All sample compositions are listed in Table 2. Gas concentrations in the mixtures were based on previous results from experiments on denitrification, 74,75 BNF 77 and respiration 73 as well as the reaction equations in Table 1. Some errors in concentration determination are caused by mass flow controllers (MFCs) used in sample preparation.…”

“…[55][56][57] Due to the weak scattering processes, enhancement methods such as cavity-enhanced Raman spectroscopy (CERS) [58][59][60][61][62] and fiberenhanced Raman spectroscopy (FERS) [63][64][65][66][67][68][69][70][71][72] were developed for the sensitive detection of analytes. Enhanced Raman spectroscopy was already exploited for the detailed investigations of respiration, 73 denitrification [74][75][76] and to quantify BNF directly at atmospheric concentrations. 77 These studies took advantage of the multi-gas analysis and the use of isotopic labels.…”

Isotopomeric Peak Assignment for N2O in Cross-Labeling Experiments by Fiber-Enhanced Raman Multigas Spectroscopy

“…Recently, cavity-enhanced Raman spectroscopy (CERS) − and fiber-enhanced Raman spectroscopy (FERS) − were developed for highly sensitive gas quantification. FERS was applied in several environmental and laboratory-based studies. − This technique offers a high potential for simultaneous quantification of all relevant gases for the characterization of N-cycle processes, including spectrally resolved measurement of N 2 O isotopomer compositions. Such parallel and comprehensive multigas analysis may offer new insights into soil processes and the interplay between N 2 O production processes and help to develop mitigation strategies for reducing N 2 O emissions and fertilizer losses in agricultural systems.…”