A Nuclear Magnetic Resonance (NMR) Platform for Real-Time Metabolic Monitoring of Bioprocesses

Abstract

We present a Nuclear Magnetic Resonance (NMR) compatible platform for the automated real-time monitoring of biochemical reactions using a flow shuttling configuration. This platform requires a working sample volume of ∼11 mL and it can circulate samples with a flow rate of 28 mL/min., which makes it suitable to be used for real-time monitoring of biochemical reactions. Another advantage of the proposed low-cost platform is the high spectral resolution. As a proof of concept, we acquire 1H NMR spectra of waste orange peel, bioprocessed using Trichoderma reesei fungus, and demonstrate the real-time measurement capability of the platform. The measurement is performed over more than 60 h, with a spectrum acquired every 7 min, such that over 510 data points are collected without user intervention. The designed system offers high resolution, automation, low user intervention, and, therefore, time-efficient measurement per sample.

Keywords: NMR-compatible bioreactor; automated NMR; bioprocess monitoring; microbial bioprocess; waste degradation.

Figure 1

Conceptual diagram of the real-time Nuclear Magnetic Resonance (NMR) measurement system using a flow-through capillary arrangement. The processing of waste orange peel, with the help of (T. reesei) in minimal medium, was the bio-reaction to demonstrate the system functionality.

Figure 2

(a) Experimental setup. The bioreaction is performed using the personal reaction station (PRS). A peristaltic pump is mounted on the PRS to flow the sample periodically through the capillary-based flow cell (inside the NMR magnet) for NMR measurement and subsequently return it to the bioreactor. The peristaltic pump is controlled by a microcontroller using a relay and a motor driver, with a liquid crystal display (LCD) used to display the time to the user. A TTL (transistor-transistor logic)signal is sent to the NMR spectrometer after the sample has been transferred to the NMR detector to initiate measurement. Note: tubing length not to scale. (b) NMR spectrometer with flow-through capillary system in the center.

Figure 3

${}^1$H NMR spectra obtained with real-time NMR platform. The initial spectrum (Day 0) shows the response obtained before the addition of T. reesei to the dried orange peel waste. The final spectrum (Day 3) shows the response obtained on day 3 after the addition of fungus. Inset, left: zoom of the spectral region between −0.5–4.5 ppm, where signals were observed to evolve during the experiment. Inset, right: zoom of the ethanol triplet signal. Abbreviations: (a)—acetate; (b)—alanine; (c)—ethanol; (d)—glycine; (e)—lactate; and, (f)—saccharide.

Figure 4

(a) Area under the curve for significant peaks seen at the output over three days of continuous real-time NMR monitoring of the bio-reaction of orange peel fed to T. reesei. Note: only 80 data points are plotted for clarity. (b) Real-time acquired spectra of the 516 runs plotted in 3D with water peak and stationary peaks (except 3-(trimethylsilyl)propionic-2,2,3,3-d${}_4$ acid sodium salt (TSP)) removed. Note: only 52 in 516 runs are plotted for better visualization. X-axis is chemical shift (ppm) axis, y axis is time point and z axis is normalized value with respect to TSP. Abbreviations: (a)—acetate; (b)—alanine; (c)—ethanol; (d)—glycine; (e)—lactate; and, (f)—saccharide, (g)—TSP.