Online monitoring of fermentation processes via non‐invasive low‐field NMR

For the development of biotechnological processes in academia as well as in industry new techniques are required which enable online monitoring for process characterization and control. Nuclear magnetic resonance (NMR) spectroscopy is a promising analytical tool, which has already found broad applications in offline process analysis. The use of online monitoring, however, is oftentimes constrained by high complexity of custom‐made NMR bioreactors and considerable costs for high‐field NMR instruments (>US$200,000). Therefore, low‐field 1H NMR was investigated in this study in a bypass system for real‐time observation of fermentation processes. The new technique was validated with two microbial systems. For the yeast Hansenula polymorpha glycerol consumption could accurately be assessed in spite of the presence of high amounts of complex constituents in the medium. During cultivation of the fungal strain Ustilago maydis, which is accompanied by the formation of several by‐products, the concentrations of glucose, itaconic acid, and the relative amount of glycolipids could be quantified. While low‐field spectra are characterized by reduced spectral resolution compared to high‐field NMR, the compact design combined with the high temporal resolution (15 s–8 min) of spectra acquisition allowed online monitoring of the respective processes. Both applications clearly demonstrate that the investigated technique is well suited for reaction monitoring in opaque media while at the same time it is highly robust and chemically specific. It can thus be concluded that low‐field NMR spectroscopy has a great potential for non‐invasive online monitoring of biotechnological processes at the research and practical industrial scales. Biotechnol. Bioeng. 2015;112: 1810–1821. © 2015 Wiley Periodicals, Inc.

[1]  J. Bailey,et al.  Observations of aerobic, growing escherichia coli metabolism using an on‐line nuclear magnetic resonance spectroscopy system , 1993, Biotechnology and bioengineering.

[2]  Jacobus P. H. van Wyk,et al.  Biotechnology and the utilization of biowaste as a resource for bioproduct development , 2001 .

[3]  J. Shanks,et al.  Influence of Aeration on Cytoplasmic pH of Yeast in an NMR Airlift Bioreactor , 1996, Biotechnology progress.

[4]  H. Sahm,et al.  Continuous-flow NMR bioreactor for in vivo studies of microbial cell suspensions with low biomass concentrations , 1992 .

[5]  J. Callis,et al.  Determination of alcohols in gasoline/alcohol blends by nuclear magnetic resonance spectrometry , 1985 .

[6]  A. Nordon,et al.  Process NMR spectrometry. , 2001, The Analyst.

[7]  Frank Lundby,et al.  Determination of total fat and moisture content in meat using low field NMR. , 2004, Meat science.

[8]  D. W. Ribbons,et al.  In situ proton-NMR analyses of Escherichia coli HB 101 fermentations in 1 H 2 O and in D 2 O , 2022 .

[9]  Bernhard Blümich,et al.  Essential NMR: For Scientists and Engineers , 2005 .

[10]  M. Bölker,et al.  Genetic Analysis of Biosurfactant Production in Ustilago maydis , 2005, Applied and Environmental Microbiology.

[11]  V. Wray,et al.  Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources , 1999, Applied Microbiology and Biotechnology.

[12]  M. Bölker,et al.  Ustilago maydis secondary metabolism-from genomics to biochemistry. , 2008, Fungal genetics and biology : FG & B.

[13]  Determination of ethanol in alcoholic beverages by low-resolution pulsed nuclear magnetic resonance , 1988 .

[14]  D. W. Ribbons,et al.  In situ proton-NMR analyses of Escherichia coli HB101 fermentations in 1H2O and in D2O. , 1999, Microbiology.

[15]  M M Domach,et al.  Performance Trade‐offs in In Situ Chemostat NMR , 1999, Biotechnology progress.

[16]  D. W. Ribbons,et al.  Simple device to monitor aerobic biotransformations by in situ 1H-NMR , 2000, Biotechnology Letters.

[17]  A. Nordon,et al.  Comparison of in-line NIR, Raman and UV-visible spectrometries, and at-line NMR spectrometry for the monitoring of an esterification reaction , 2002 .

[18]  J. Büchs,et al.  Effect of oxygen supply on passaging, stabilising and screening of recombinant Hansenula polymorpha production strains in test tube cultures. , 2003, FEMS yeast research.

[19]  N. Shimba,et al.  In vivo NMR system for evaluating oxygen-dependent metabolic status in microbial culture. , 2002, Journal of microbiological methods.

[20]  A. Fluharty,et al.  A mannose- and erythritol-containing glycolipid from Ustilago maydis. , 1969, Biochemistry.

[21]  J. Büchs,et al.  The oxygen mass transfer, carbon dioxide inhibition, heat removal, and the energy and cost efficiencies of high pressure fermentation. , 2005, Advances in biochemical engineering/biotechnology.

[22]  Ae Ja Kim,et al.  High-resolution NMR process analyzer for oxygenates in gasoline , 1994 .

[23]  D. L. Botlan,et al.  Proton Low‐Field NMR Measurements on Crackers , 1994 .

[24]  M. Maiwald,et al.  Process and reaction monitoring by low-field NMR spectroscopy. , 2012, Progress in nuclear magnetic resonance spectroscopy.

[25]  H. Blanch,et al.  Quantitative in vivo nuclear magnetic resonance studies of hybridoma metabolism , 1994, Biotechnology and bioengineering.

[26]  Charles Tellier,et al.  Monitoring alcoholic fermentation by low-resolution pulsed nuclear magnetic resonance , 1989 .

[27]  E. Danieli,et al.  Small magnets for portable NMR spectrometers. , 2010, Angewandte Chemie.

[28]  T. Nakane,et al.  Intracellular accumulation of mannosylerythritol lipids as storage materials by Candida antarctica , 1992, Applied Microbiology and Biotechnology.

[29]  J. Scholten,et al.  NMR bioreactor development for live in-situ microbial functional analysis. , 2008, Journal of magnetic resonance.

[30]  D Weuster-Botz,et al.  Development and application of a membrane cyclone reactor for in vivo NMR spectroscopy with high microbial cell densities. , 2000, Biotechnology and bioengineering.

[31]  A. Koretsky,et al.  NMR‐Observed Phosphate Trafficking and Polyphosphate Dynamics in Wild‐Type and vph1–1 Mutant Saccharomyces cerevisae in Response to Stresses , 1999, Biotechnology progress.

[32]  J. V. van Wyk Biotechnology and the utilization of biowaste as a resource for bioproduct development. , 2001, Trends in biotechnology.

[33]  M M Domach,et al.  Cultivator for NMR studies of suspended cell cultures , 1992, Biotechnology and bioengineering.

[34]  P. N. Gambhir Applications of low-resolution pulsed NMR to the determination of oil and moisture in oilseeds , 1992 .

[35]  Philipp M. Grande,et al.  Biomass pretreatment affects Ustilago maydis in producing itaconic acid , 2012, Microbial Cell Factories.

[36]  V. Pomin Unravelling Glycobiology by NMR Spectroscopy , 2012 .

[37]  G. Pielak,et al.  A bioreactor for in-cell protein NMR. , 2010, Journal of magnetic resonance.

[38]  H. Weber,et al.  Online NMR for monitoring biocatalysed reactions. , 2000, Current opinion in biotechnology.

[39]  E. Danieli,et al.  Mobile Low-Field 1H NMR Spectroscopy Desktop Analysis of Biodiesel Production , 2013 .

[40]  B. Buckland,et al.  Toward consistent and productive complex media for industrial fermentations: studies on yeast extract for a recombinant yeast fermentation process. , 2003, Biotechnology and bioengineering.

[41]  T. Tabuchi,et al.  Accumulation of Itaconic, 2-Hydroxyparaconic, Itatartaric, and Malic Acids by Strains of the Genus Ustilago , 1990 .