Author:
Wang Ruifei,Lorantfy Bettina,Fusco Salvatore,Olsson Lisbeth,Franzén Carl Johan
Abstract
AbstractCell mass and viability are tightly linked to the productivity of fermentation processes. In 2nd generation lignocellulose-based media quantitative measurement of cell concentration is challenging because of particles, auto-fluorescence, and intrinsic colour and turbidity of the media. We systematically evaluated several methods for quantifying total and viable yeast cell concentrations to validate their use in lignocellulosic media. Several automated cell counting systems and stain-based viability tests had very limited applicability in such samples. In contrast, manual cell enumeration in a hemocytometer, plating and enumeration of colony forming units, qPCR, and in situ dielectric spectroscopy were further investigated. Parameter optimization to measurements in synthetic lignocellulosic media, which mimicked typical lignocellulosic fermentation conditions, resulted in statistically significant calibration models with good predictive capacity for these four methods. Manual enumeration of cells in a hemocytometer and of CFU were further validated for quantitative assessment of cell numbers in simultaneous saccharification and fermentation experiments on steam-exploded wheat straw. Furthermore, quantitative correlations could be established between these variables and in situ permittivity. In contrast, qPCR quantification suffered from inconsistent DNA extraction from the lignocellulosic slurries. Development of reliable and validated cell quantification methods and understanding their strengths and limitations in lignocellulosic contexts, will enable further development, optimization, and control of lignocellulose-based fermentation processes.
Funder
Svenska Forskningsrådet Formas
Energimyndigheten
Chalmers University of Technology
Publisher
Springer Science and Business Media LLC
Reference27 articles.
1. Olsson, L. & Nielsen, J. On-line and in situ monitoring of biomass in submerged cultivations. Trends Biotechnol. 15, 517–522 (1997).
2. Berntsson, T., Sandén, B. A., Olsson, L. & Åsblad, A. What is a biorefinery? In Systems Perspectives on Biorefineries (eds. Sandén, B. & Pettersson, K.) http://www.chalmers.se/en/areas-of-advance/energy/publications-media/systems-perspectives/Pages/Systems-Perspectives-on-Biorefineries.aspx (2014). Accessed 3 Mar 2017.
3. Ragauskas, A. J. et al. The path forward for biofuels and biomaterials. Science 311, 484–489 (2006).
4. Pfaller, M. A., Burmeister, L., Bartlett, M. & Rinaldi, M. Multicenter evaluation of four methods of yeast inoculum preparation. J. Clin. Microbiol. 26, 1437–1441 (1988).
5. Bleve, G., Rizzotti, L., Dellaglio, F. & Torriani, S. Development of reverse transcription (RT)-PCR and real-time RT-PCR assays for rapid detection and quantification of viable yeasts and molds contaminating yogurts and pasteurized food products. Appl. Environ. Microbiol. 69, 4116–4122 (2003).
Cited by
11 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献