Abstract
An in situ nuclear magnetic resonance (NMR) bioreactor was developed and employed to monitor microbial metabolism under batch growth conditions in real time. We selected Moorella thermoacetica ATCC 49707 as a test case. M. thermoacetica (formerly Clostridium thermoaceticum) is a strictly anaerobic, thermophilic, acetogenic, gram-positive bacterium with potential for industrial production of chemicals. The metabolic profiles of M. thermoacetica were characterized during growth in batch mode on xylose (a component of lignocellulosic biomass) using the new generation NMR bioreactor in combination with high-resolution NMR (HR-NMR) spectroscopy. In situ NMR measurements were performed using water-suppressed H-1 NMR spectroscopy at 500 MHz, and aliquots of the bioreactor contents were taken for 600-MHz HR-NMR spectroscopy at specific intervals to confirm metabolite identifications and expand metabolite coverage. M. thermoacetica demonstrated the metabolic potential to produce formate, ethanol, and methanol from xylose, in addition to its known capability of producing acetic acid. Real-time monitoring of bioreactor conditions showed a temporary pH decrease, with a concomitant increase in formic acid during exponential growth. Fermentation experiments performed outside of the magnet showed that the strong magnetic field employed for NMR detection did not significantly affect cell metabolism. Use of the in situ NMR bioreactor facilitated monitoring of the fermentation process, enabling identification of intermediate and endpoint metabolites and their correlation with pH and biomass produced during culture growth. Real-time monitoring of culture metabolism using the NMR bioreactor in combination with HR-NMR spectroscopy will allow optimization of the metabolism of microorganisms producing valuable bioproducts.
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References
Alam KY, Clark DP (1989) Anaerobic fermentation balance of Escherichia coli as observed by in vivo nuclear magnetic resonance spectroscopy. J Bacteriol 171:6213–6217
Angelidaki I, Petersen SP, Ahring BK (1990) Effects of lipids on thermophilic anaerobic digestion and reduction of lipid inhibition upon addition of bentonite. Appl Microbiol Biotechnol 33:469–472
Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF et al (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1462
Bullis K (2008) Creating ethanol from wood more efficiently. MIT technology review
Caspi R, Altman T, Dale JM, Dreher K, Fulcher CA, Gilham F, Kaipa P, Karthikeyan AS, Kothari A, Krummenacker M et al (2010) The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Res 38:D473–D479
Drake HL, Daniel SL (2004) Physiology of the thermophilic acetogen Moorella thermoacetica. Res Microbiol 155:869–883
Fontaine FE, Peterson WH, McCoy E, Johnson MJ, Ritter GJ (1942) A new type of glucose fermentation by Clostridium thermoaceticum. J Bacteriol 43:701–715
Hartbrich A, Schmitz G, Weuster-Botz D, de Graaf AA, Wandrey C (1996) Development and application of a membrane cyclone reactor for in vivo NMR spectroscopy with high microbial cell densities. Biotechnol Bioeng 51:624–635
Henstra AM, Sipma J, Rinzema A, Stams AJ (2007) Microbiology of synthesis gas fermentation for biofuel production. Curr Opin Biotechnol 18:200–206
Hwang TL, Shaka AJ (1995) Water suppression that works. Excitation sculpting using arbitrary wave-forms and pulsed-field gradients. J Magn Reson Series A 112:275–279
Inokuma K, Nakashimada Y, Akahoshi T, Nishio N (2007) Characterization of enzymes involved in the ethanol production of Moorella sp. HUC22-1. Arch Microbiol 188:37–45
Kobayashi K, Ehrlich SD, Albertini A, Amati G, Andersen KK, Arnaud M, Asai K, Ashikaga S, Aymerich S, Bessieres P et al (2003) Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A 100:4678–4683
Kopke M, Mihalcea C, Liew FM, Tizard JH, Ali MS, Conolly JJ, Al-Sinawi B, Simpson SD (2011) 2,3-Butanediol production by acetogenic bacteria, an alternative route to chemical synthesis, using industrial waste gas. Appl Environ Microbiol 77:5467–5475
Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessieres P, Bolotin A, Borchert S et al (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249–256
Lakhal R, Auria R, Davidson S, Ollivier B, Dolla A, Hamdi M, Combet-Blanc Y (2010) Effect of oxygen and redox potential on glucose fermentation in Thermotoga maritima under controlled physicochemical conditions. Int J Microbiol 2010:896510
Lambert C, Weuster-Botz D, Weichenhain R, Kreutz EW, De Graaf AA, Schoberth SM (2002) Monitoring of inorganic polyphosphate dynamics in Corynebacterium glutamicum using a novel oxygen sparger for real time P-31 in vivo NMR. Acta Biotechnol 22:245–260
Majors PD, McLean JS, Scholten JC (2008) NMR bioreactor development for live in-situ microbial functional analysis. J Magn Reson 192:159–166
Maki M, Leung KT, Qin W (2009) The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int J Biol Sci 5:500–516
Metz TO, Zhang QB, Page JS, Shen YF, Callister SJ, Jacobs JM, Smith RD (2007) Future of liquid chromatography-mass spectrometry in metabolic profiling and metabolomic studies for biomarker discovery. Biomark Med 1:159–185
Mou DG, Cooney CL (1983) Growth monitoring and control through computer-aided online mass balancing in a fed-batch penicillin fermentation. Biotechnol Bioeng 25:225–255
Nakano MM, Dailly YP, Zuber P, Clark DP (1997) Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth. J Bacteriol 179:6749–6755
Neves AR, Ramos A, Nunes MC, Kleerebezem M, Hugenholtz J, de Vos WM, Almeida J, Santos H (1999) In vivo nuclear magnetic resonance studies of glycolytic kinetics in Lactococcus lactis. Biotechnol Bioeng 64:200–212
Neves AR, Pool WA, Kok J, Kuipers OP, Santos H (2005) Overview on sugar metabolism and its control in Lactococcus lactis—the input from in vivo NMR. FEMS Microbiol Rev 29:531–554
Phillips JR, Clausen EC, Gaddy JL (1994) Synthesis gas as substrate for the biological production of fuels and chemicals. Appl Biochem Biotechnol 45–6:145–157
Pierce E, Xie G, Barabote RD, Saunders E, Han CS, Detter JC, Richardson P, Brettin TS, Das A, Ljungdahl LG et al (2008) The complete genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum). Environ Microbiol 10:2550–2573
Remize F, Cambon B, Barnavon L, Dequin S (2003) Glycerol formation during wine fermentation is mainly linked to Gpd1p and is only partially controlled by the HOG pathway. Yeast 20:1243–1253
Sakai S, Nakashimada Y, Inokuma K, Kita M, Okada H, Nishio N (2005) Acetate and ethanol production from H2 and CO2 by Moorella sp. using a repeated batch culture. J Biosci Bioeng 99:252–258
Schiel-Bengelsdorf B, Durre P (2012) Pathway engineering and synthetic biology using acetogens. Febs Letters 586:2191–2198
Sommer P, Georgieva T, Ahring BK (2004) Potential for using thermophilic anaerobic bacteria for bioethanol production from hemicellulose. Biochem Soc Trans 32:283–289
Taylor MP, Eley KL, Martin S, Tuffin MI, Burton SG, Cowan DA (2009) Thermophilic ethanologenesis: future prospects for second-generation bioethanol production. Trends Biotechnol 27:398–405
Weidenmaier C, Peschel A (2008) Teichoic acids and related cell-wall glycopolymers in Gram-positive physiology and host interactions. Nat Rev Microbiol 6:276–287
Weljie AM, Newton J, Mercier P, Carlson E, Slupsky CM (2006) Targeted profiling: quantitative analysis of H-1 NMR metabolomics data. Anal Chem 78:4430–4442
Zaldivar J, Nielsen J, Olsson L (2001) Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 56:17–34
Acknowledgments
We acknowledge the financial support from Washington State STARS researcher program given to Prof. Ahring 2008–2010, the Danish Strategic Research Council ENMI which supported the stay of Jens Iversen at Washington State University (WSU), and the Environmental Molecular Sciences Laboratory (EMSL) Intramural program, which funded the development and implementation of NMR instrumentation and methods for this project. The NMR spectroscopy portion of the research was performed at EMSL, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory (PNNL). Finally, we thank EMSL Machine Shop for construction of the in situ bioreactor custom components, EMSL’s Instrument Development Lab (IDL) for helping to integrate the controller and in situ NMR, and the EMSL Crafts personnel for their assistance in bioreactor assembly, adjustment, and installation. We are very grateful to Weiqun Zhong (WSU) for her technical assistance.
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Xue, J., Isern, N.G., Ewing, R.J. et al. New generation NMR bioreactor coupled with high-resolution NMR spectroscopy leads to novel discoveries in Moorella thermoacetica metabolic profiles. Appl Microbiol Biotechnol 98, 8367–8375 (2014). https://doi.org/10.1007/s00253-014-5847-8
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DOI: https://doi.org/10.1007/s00253-014-5847-8