In Situ Monitoring of an Escherichia coli Fermentation Using a Diamond Composition ATR Probe and Mid-infrared Spectroscopy

Abstract

A diamond composition ATR probe was used in situ to obtain IR spectra on replicate Escherichia coli fermentations involving a complex medium. The probe showed excellent stability over a 6-month operating period and was unaffected by either agitation or aeration. The formation of an unknown was observed from IR spectra obtained during the sterilization; subsequent experiments proved this to be a reaction product between yeast extract and the phosphates used as buffer salts. Partial-least-squares-based calibration/prediction models were developed for both glucose and acetate using in-process samples. The resulting models had prediction errors of +/-0.26 and +/-0.75 g/L for glucose and acetic acid, respectively, errors which were statistically equivalent to the estimated experimental errors in the reference measurements. Relative concentration profiles for the unknown formed during sterilization could be generated either by tracking peak height at an independent wavelength or by self-modeling curve resolution of the spectral region overlapping that of glucose. These profiles indicated that this compound was metabolized simultaneously with glucose; upon depletion of the glucose, when the microorganism switched to consumption of acetic acid, utilization continued but at a lower rate. The data presented provide an extensive characterization of the performance characteristics of this in situ analysis and clearly demonstrate its utility not just in the quantitative measurement of multiple known species but in the qualitative evaluation of unknown species.

Quantitative Determination of the Biodegradable Polymer Poly(β-hydroxybutyrate) in a Recombinant Escherichia coli Strain by Use of Mid-Infrared Spectroscopy and Multivariative Statistics

“…Because FTIR spectra can be considered chemical profiles of samples, the spectra can be used to predict the concentration of an analyte (e.g., the concentration of PHB). FTIR spectroscopy has been used previously to monitor water-soluble extracellular analytes in fermentation systems for ethanol (13,30), lactic acid (12,30), and glucose and acetic acid (11) and, very recently, for quantification of a recombinant protein (27). In a recent study Schuster et al (35) used FTIR spectroscopy to monitor the physiological state of bacterial cells as an indirect method to determine the stage of fermentation.…”

Novel infrared optical probes for process monitoring and analysis based on next-generation silver halide fibers

“…Much lower concentrations of carbohydrates as found in fruit juices, must be tackled with the monitoring of bioreactor media where solute concentrations of subparts-per-thousand and lower can be expected. Diamond ATR-elements have already been successfully employed for the monitoring of fermentation processes with Escherichia coli over an extended time period [11]. A rigid optical mirror system was necessary for connecting the probe and spectrometer in this work; however, a greater flexibility is certainly gained through fiber-coupled probes.…”

“…This wide spectral region is otherwise only accessible by rigid radiation guiding systems based on mirror optics as found within the ReactIR system from Applied Systems (Annapolis, MD, USA), and employed for process monitoring (see, for example Ref. [11]). Further fiber materials have been used for measurements within other limited spectral ranges.…”

Bioenergetic studies on aerobic growth of Pseudomonas aeruginosa in a single‐substrate media

“…Several online tools have been used to monitor bioprocess systems for the detection of metabolite production, 2 cell division, 3,4 oxygen consumption 5 and gaseous product formation. 6 Most of the online monitoring tools are invasive type and require considerable amounts of sample for analysis. Calorimetric heat-flow rate measurements have a considerable advantage over existing techniques in that it directly measures the time-derivative of heat rather than the total heat produced.…”