Development and application of a membrane cyclone reactor for in vivo NMR spectroscopy with high microbial cell densities

Abstract

A new bioreactor system has been developed for in vivo NMR spectroscopy of microorganisms under defined physiological conditions. This cyclone reactor with an integrated NMR flow cell is continuously operated in the magnet of a 400‐MHz wide‐bore NMR spectrometer system. The residence times of medium and cells are decoupled by a circulation‐integrated cross‐flow microfiltration module to achieve higher cell densities as compared to continuous fermentations without cell retention (increase in cell density up to a factor of 10 in steady state). Volumetric mass transfer coefficients kLa of more than 1.0 s−1 are possible in the membrane cyclone reactor, ensuring adequate oxygen supply [oxygen transfer rate >15,000 mg O2 ·(L h)−1] of high cell densities. With the aid of the membrane cyclone reactor we were able to show, using continuous in vivo 31P NMR spectroscopy of anaerobic glucose fermentation by Zymomonas mobilis, that the NMR signal intensity was directly proportional to the cell concentration in the reactor. The concentration profiles of intracellular inorganic phosphate, NAD(H), NDP, NTP, UDP‐sugar, a cyclic pyrophosphate, two sugar phosphate pools, and extracellular inorganic phosphate were recorded after a shift from one steady state to another. The intracellular cyclic pyrophosphate had not been detected before in in vitro measurements of Zymomonas mobilis extracts due to the high instability of this compound. Using continuous in vivo 13C NMR spectroscopy of aerobic glucose utilization by Corynebacterium glutamicum at a density of 25 gcell dry weight · L−1, the membrane cyclone reactor served to measure the different dynamics of labeling in the carbon atoms of L‐lactate, L‐glutamate, succinate, and L‐lysine with a time resolution of 10 min after impressing a [1‐13C]‐glucose pulse.

NMR bioreactor development for live in-situ microbial functional analysis

“…Several NMR-compatible bioreactors have been developed [11][12][13][14][15]. These bioreactor-in-magnet designs allow for NMR detection in or immediately adjacent to the reaction vessel, thus they provide metabolism measurements under controlled-environment conditions.…”

“…These bioreactor-in-magnet designs allow for NMR detection in or immediately adjacent to the reaction vessel, thus they provide metabolism measurements under controlled-environment conditions. Of particular note is the membrane-cyclone NMR bioreactor [13,16], a hydrocyclone bioreactor promoted for its simplicity of construction and operation [17]. Introduced [18] as an alternative to stirred-tank reactors operating in the sub-liter volume range, hydrocyclones are ideal for NMR because they have no moving parts-a recirculation pump provides the power for both gas exchange and mixing functions.…”

“…Introduced [18] as an alternative to stirred-tank reactors operating in the sub-liter volume range, hydrocyclones are ideal for NMR because they have no moving parts-a recirculation pump provides the power for both gas exchange and mixing functions. The Hartbrich reactor [13] incorporates these features plus enhanced NMR sensitivity of their earlier continuous-flow bioreactor [12]. Increased NMR sensitivity involves using reactor-suspension flow to constantly replenish the NMR test section with fully-polarized spins [19], thus allowing for rapid signal averaging.…”

“…Hydrocyclone bioreactors function as liquid-gas separators and are highly efficient due to the large density contrast [23]. Very high gas transfer rates can be obtained by introducing the influent gas as bubbles either upstream of the cyclone inlet [13] or at the chamber wall [24], leading to high cell densities and product yields [25,26].…”

“…The membrane-cyclone reactor has been applied for metabolic studies of bacterial and yeast suspensions [13,27,28]. These studies employed 31 P (and sometimes 13 C and 15 N) NMR methods.…”