Use of green fluorescent protein to monitor fungal growth in biomass hydrolysate

Authors: Nichols, Nancy; Quarterman, Joshua; Frazer, Sarah

Submitted to: Biology Methods and Protocols
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/24/2017
Publication Date: 1/29/2018
Citation: Nichols, N.N., Quarterman, J.C., Frazer, S.E. 2018. Use of green fluorescent protein to monitor fungal growth in biomass hydrolysate. Biology Methods and Protocols. 3(1)bpx012. doi: 10.1093/biomethods/bpx012.
DOI: https://doi.org/10.1093/biomethods/bpx012

Interpretive Summary: This work developed a fluorescent microbe that is helpful for monitoring experiments involving biomass sugars. Fibrous biomass such as corn stover is a potential feedstock for bio-based production of fuels and chemicals. A hardy soil microbe, a fungus, can be used to “clean up” the sugars so they can be used to make products; it does this by metabolizing inhibitory compounds that interfere with bio-based conversion. However, it is not convenient to measure growth of the fungus in biomass sugars because the sugar mixtures comprise a dark-colored and fibrous suspension. We introduced the green fluorescence protein into the fungal strain and showed that fluorescence can be used to monitor both growth and inhibitor metabolism. This means that, for factors that are important but laborious to measure, fluorescence can be used as a convenient proxy.

Technical Abstract: Green Fluorescent Protein (GFP) was introduced into the Ascomycete Coniochaeta ligniaria NRRL30616, and fluorescence of cultures was monitored as a measure of cell growth. Fluorescence in the GFP-expressing strain was measured during growth of cells in defined and complex media as well as in the liquor derived from pretreatment of corn stover, an agricultural residue. Fluorescence mirrored growth of cultures, as measured by optical density and counts of colony forming units. Because traditional methods to monitor growth cannot be used in biomass liquors due to its fibrous, dark-colored nature, the speed and convenience of using GFP to monitor growth is advantageous. Fluorescence of cultures in biomass hydrolysate also correlated with the concentration of furfural in hydrolysate. Furfural and other compounds, present in hydrolysate due to physical-chemical pretreatment of biomass, are inhibitory to fermenting microbes. Therefore, measurement of fluorescence in GFP-expressing C. ligniaria is a proxy for measures of microbial growth and furfural consumption, and serves as a convenient indicator of metabolism of fermentation inhibitors in biomass hydrolysate.