Hydrogen production in the presence of oxygen by Escherichia coli K-12.

Escherichia coli is a facultative anaerobe that can grow in a variety of environmental conditions. In the complete absence of O2, E. coli can perform a mixed-acid fermentation that contains within it an elaborate metabolism of formic acid. In this study, we use cavity-enhanced Raman spectroscopy (CERS), FTIR, liquid Raman spectroscopy, isotopic labelling and molecular genetics to make advances in the understanding of bacterial formate and H2 metabolism. It is shown that, under anaerobic (anoxic) conditions, formic acid is generated endogenously, excreted briefly from the cell, and then taken up again to be disproportionated to H2 and CO2 by formate hydrogenlyase (FHL-1). However, exogenously added D-labelled formate behaves quite differently from the endogenous formate and is taken up immediately, independently, and possibly by a different mechanism, by the cell and converted to H2 and CO2. Our data support an anion-proton symport model for formic acid transport. In addition, when E. coli was grown in a micro-aerobic (micro-oxic) environment it was possible to analyse aspects of formate and O2 respiration occurring alongside anaerobic metabolism. While cells growing under micro-aerobic conditions generated endogenous formic acid, no H2 was produced. However, addition of exogenous formate at the outset of cell growth did induce FHL-1 biosynthesis and resulted in formate-dependent H2 production in the presence of O2.

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

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.

On-line analysis and in situ pH monitoring of mixed acid fermentation by Escherichia coli using combined FTIR and Raman techniques.

We introduce an experimental setup allowing continuous monitoring of bacterial fermentation processes by simultaneous optical density (OD) measurements, long-path FTIR headspace monitoring of CO2, acetaldehyde and ethanol, and liquid Raman spectroscopy of acetate, formate, and phosphate anions, without sampling. We discuss which spectral features are best suited for detection, and how to obtain partial pressures and concentrations by integrations and least squares fitting of spectral features. Noise equivalent detection limits are about 2.6 mM for acetate and 3.6 mM for formate at 5 min integration time, improving to 0.75 mM for acetate and 1.0 mM for formate at 1 h integration. The analytical range extends to at least 1 M with a standard deviation of percentage error of about 8%. The measurement of the anions of the phosphate buffer allows the spectroscopic, in situ determination of the pH of the bacterial suspension via a modified Henderson-Hasselbalch equation in the 6-8 pH range with an accuracy better than 0.1. The 4 m White cell FTIR measurements provide noise equivalent detection limits of 0.21 µbar for acetaldehyde and 0.26 µbar for ethanol in the gas phase, corresponding to 3.2 µM acetaldehyde and 22 µM ethanol in solution, using Henry's law. The analytical dynamic range exceeds 1 mbar ethanol corresponding to 85 mM in solution. As an application example, the mixed acid fermentation of Escherichia coli is studied. The production of CO2, ethanol, acetaldehyde, acids such as formate and acetate, and the changes in pH are discussed in the context of the mixed acid fermentation pathways. Formate decomposition into CO2 and H2 is found to be governed by a zeroth-order kinetic rate law, showing that adding exogenous formate to a bioreactor with E. coli is expected to have no beneficial effect on the rate of formate decomposition and biohydrogen production.

Cavity-Enhanced Raman and Helmholtz Resonator Photoacoustic Spectroscopy to Monitor the Mixed Sugar Metabolism of E. coli.

We introduce and compare two powerful new techniques for headspace gas analysis above bacterial batch cultures by spectroscopy, Raman spectroscopy enhanced in an optical cavity (CERS), and photoacoustic detection in a differential Helmholtz resonator (DHR). Both techniques are able to monitor O2 and CO2 and its isotopomers with excellent sensitivity and time resolution to characterize bacterial growth and metabolism. We discuss and show some of the shortcomings of more conventional optical density (OD) measurements if used on their own without more sophisticated complementary measurements. The spectroscopic measurements can clearly and unambiguously distinguish the main phases of bacterial growth in the two media studied, LB and M9. We demonstrate how 13C isotopic labeling of sugars combined with spectroscopic detection allows the study of bacterial mixed sugar metabolism to establish whether sugars are sequentially or simultaneously metabolized. For E. coli, we have characterized the shift from glucose to lactose metabolism without a classic diauxic lag phase. DHR and CERS are shown to be cost-effective and highly selective analytical tools in the biosciences and in biotechnology, complementing and superseding existing conventional techniques. They also provide new capabilities for mechanistic investigations and show a great deal of promise for use in stable isotope bioassays.