Continuous monitoring of nitrogenase activity in Azotobacter vinelandii fermentation using off-gas mass spectrometry

• Published in: Biotechnology and bioengineering 1995-08, Vol.47 (3), p.373-383
• Main Authors: Prior, J.J. (Massachusetts Institute of Technology, Cambridge, MA.), Christie, P.D, Murray, R.J, Orme-Johnson, W.H, Cooney, C.L
• Format: Article
• Language: English
• Subjects: AZOTOBACTER VINELANDII; Biological and medical sciences; Biotechnology; FERMENTACION; FERMENTATION; fermentation monitoring; Fundamental and applied biological sciences. Psychology; mass spectrometry; Methods. Procedures. Technologies; Microbial engineering. Fermentation and microbial culture technology; NITROGENASA; NITROGENASE
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.260470311

This study evaluated the feasibility of monitoring nitrogenase activity in situ through measurement of N2 uptake rate (NUR) using off-gas mass spectrometry. Four 50-L cultures of Azotobacter vinelandii were grown aerobically in nitrogen-free medium to cell densities of 1.0-1.3 g L-1. A magnetic-sector mass spectrometer was used to monitor NUR along with other gas exchange rates. The small specific uptake rate (1.2 mmol g-1 h-1) and low cell density were found to lead to a NUR below the measurement accuracy limits under normal conditions. An operating strategy and feed gas mixture (40% O2, 45% N2, 15% Ar) were designed to improve the signal-to-noise ratio while maintaining dissolved O2 and N2 levels in desired ranges. The fraction of N2 removed from the air stream was increased approximately 5-fold from 0.2% to 1.0% and the measurement noise was reduced 25-fold from a baseline of +/- 5 to +/- 0.2 mmol L-1 h-1. The NUR measurements were compared against in vivo and in vitro acetylene reduction assays as well as on-line cell growth rate measurements. While electron transfer requirements predict an NUR-to-acetylene reduction rate ratio of 0.33 measured ratios for the in vivo and in vitro assays were 0.8 and 0.44, respectively. This suggests that other rate-limiting steps were present in the case of the in vivo assay. In accordance with reports in the literature no concomitant hydrogen evolution was detected. This is the first reported continuous and direct measurement of NUR in fermentation and demonstrates a novel approach for improving measurement accuracy through rational adjustment of operating conditions. The technique has potential to provide useful insight for development and control of microbial nitrogen fixation processes