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Development of a fluorescence-based method for monitoring glucose catabolism and its potential use in a biomass hydrolysis assay.

Haney LJ, Coors JG, Lorenz AJ, Raman DR, Anex RP, Scott MP - Biotechnol Biofuels (2008)

Bottom Line: We found that instantaneous fluorescence is proportional to the bacterial growth rate.We demonstrate that corn stover varieties can be differentiated based on sugar yields in enzymatic hydrolysis reactions using post-hydrolysis fluorescence measurements.Also, it may be possible to monitor fluorescence in real-time during hydrolysis to compare different hydrolysis protocols.

View Article: PubMed Central - HTML - PubMed

Affiliation: Corn Insects and Crop Genetics Research Unit, ARS, USDA, USA. lisa.haney@syngenta.com

ABSTRACT

Background: The availability and low cost of lignocellulosic biomass has caused tremendous interest in the bioconversion of this feedstock into liquid fuels. One measure of the economic viability of the bioconversion process is the ease with which a particular feedstock is hydrolyzed and fermented. Because monitoring the analytes in hydrolysis and fermentation experiments is time consuming, the objective of this study was to develop a rapid fluorescence-based method to monitor sugar production during biomass hydrolysis, and to demonstrate its application in monitoring corn stover hydrolysis.

Results: Hydrolytic enzymes were used in conjunction with Escherichia coli strain CA8404 (a hexose and pentose-consuming strain), modified to produce green fluorescent protein (GFP). The combination of hydrolytic enzymes and a sugar-consuming organism minimizes feedback inhibition of the hydrolytic enzymes. We observed that culture growth rate as measured by change in culture turbidity is proportional to GFP fluorescence and total growth and growth rate depends upon how much sugar is present at inoculation. Furthermore, it was possible to monitor the course of enzymatic hydrolysis in near real-time, though there are instrumentation challenges in doing this.

Conclusion: We found that instantaneous fluorescence is proportional to the bacterial growth rate. As growth rate is limited by the availability of sugar, the integral of fluorescence is proportional to the amount of sugar consumed by the microbe. We demonstrate that corn stover varieties can be differentiated based on sugar yields in enzymatic hydrolysis reactions using post-hydrolysis fluorescence measurements. Also, it may be possible to monitor fluorescence in real-time during hydrolysis to compare different hydrolysis protocols.

No MeSH data available.


Related in: MedlinePlus

Response to the addition of glucose. E. coli strains crp*-gfp and crp*-gfp- grown in modified 1 × M9 minimal media containing 2% glucose for 20 h. 8% glucose was added after 20 h to the indicated cultures. (a) Each time point is the average absorbance of three replications. (b) Each time point is the average fluorescence of three replications of crp*-gfp cultures minus the average fluorescence of three replications of crp*-gfp- cultures. All points are the mean ± s.e.
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Figure 3: Response to the addition of glucose. E. coli strains crp*-gfp and crp*-gfp- grown in modified 1 × M9 minimal media containing 2% glucose for 20 h. 8% glucose was added after 20 h to the indicated cultures. (a) Each time point is the average absorbance of three replications. (b) Each time point is the average fluorescence of three replications of crp*-gfp cultures minus the average fluorescence of three replications of crp*-gfp- cultures. All points are the mean ± s.e.

Mentions: We conducted two experiments to determine the response time of the microbial system to either a flux in sugar concentration or a sudden limitation in sugar during a hydrolysis reaction. The first objective was to determine how quickly the microbe responded to adding glucose to a reaction where glucose was limiting. To test this, after growing several cultures on 2 mg/ml glucose to stationary phase, we spiked half the cultures with 8 mg/ml sugar then monitored the response of the microbe. This spiking caused the absorbance and fluorescence to increase (Figure 3). As observed previously, the fluorescence level changed in proportion to the change in the cell mass with time, dX/dt. Within 2 h the effect of glucose addition was evident and fluorescence reached a maximum after 4 h. Because we conducted a time-course experiment, we were also able to calculate the integrated fluorescence. By plotting the glucose concentrations (2 mg/ml and 10 mg/ml) against the fluorescence units from either GFP-specific fluorescence or integrated fluorescence at 18 h, the R2 values were 0.86 and 0.99 respectively (data not shown), confirming our hypothesis that integrated fluorescence values are the most accurate predictors of sugar concentration.


Development of a fluorescence-based method for monitoring glucose catabolism and its potential use in a biomass hydrolysis assay.

Haney LJ, Coors JG, Lorenz AJ, Raman DR, Anex RP, Scott MP - Biotechnol Biofuels (2008)

Response to the addition of glucose. E. coli strains crp*-gfp and crp*-gfp- grown in modified 1 × M9 minimal media containing 2% glucose for 20 h. 8% glucose was added after 20 h to the indicated cultures. (a) Each time point is the average absorbance of three replications. (b) Each time point is the average fluorescence of three replications of crp*-gfp cultures minus the average fluorescence of three replications of crp*-gfp- cultures. All points are the mean ± s.e.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602992&req=5

Figure 3: Response to the addition of glucose. E. coli strains crp*-gfp and crp*-gfp- grown in modified 1 × M9 minimal media containing 2% glucose for 20 h. 8% glucose was added after 20 h to the indicated cultures. (a) Each time point is the average absorbance of three replications. (b) Each time point is the average fluorescence of three replications of crp*-gfp cultures minus the average fluorescence of three replications of crp*-gfp- cultures. All points are the mean ± s.e.
Mentions: We conducted two experiments to determine the response time of the microbial system to either a flux in sugar concentration or a sudden limitation in sugar during a hydrolysis reaction. The first objective was to determine how quickly the microbe responded to adding glucose to a reaction where glucose was limiting. To test this, after growing several cultures on 2 mg/ml glucose to stationary phase, we spiked half the cultures with 8 mg/ml sugar then monitored the response of the microbe. This spiking caused the absorbance and fluorescence to increase (Figure 3). As observed previously, the fluorescence level changed in proportion to the change in the cell mass with time, dX/dt. Within 2 h the effect of glucose addition was evident and fluorescence reached a maximum after 4 h. Because we conducted a time-course experiment, we were also able to calculate the integrated fluorescence. By plotting the glucose concentrations (2 mg/ml and 10 mg/ml) against the fluorescence units from either GFP-specific fluorescence or integrated fluorescence at 18 h, the R2 values were 0.86 and 0.99 respectively (data not shown), confirming our hypothesis that integrated fluorescence values are the most accurate predictors of sugar concentration.

Bottom Line: We found that instantaneous fluorescence is proportional to the bacterial growth rate.We demonstrate that corn stover varieties can be differentiated based on sugar yields in enzymatic hydrolysis reactions using post-hydrolysis fluorescence measurements.Also, it may be possible to monitor fluorescence in real-time during hydrolysis to compare different hydrolysis protocols.

View Article: PubMed Central - HTML - PubMed

Affiliation: Corn Insects and Crop Genetics Research Unit, ARS, USDA, USA. lisa.haney@syngenta.com

ABSTRACT

Background: The availability and low cost of lignocellulosic biomass has caused tremendous interest in the bioconversion of this feedstock into liquid fuels. One measure of the economic viability of the bioconversion process is the ease with which a particular feedstock is hydrolyzed and fermented. Because monitoring the analytes in hydrolysis and fermentation experiments is time consuming, the objective of this study was to develop a rapid fluorescence-based method to monitor sugar production during biomass hydrolysis, and to demonstrate its application in monitoring corn stover hydrolysis.

Results: Hydrolytic enzymes were used in conjunction with Escherichia coli strain CA8404 (a hexose and pentose-consuming strain), modified to produce green fluorescent protein (GFP). The combination of hydrolytic enzymes and a sugar-consuming organism minimizes feedback inhibition of the hydrolytic enzymes. We observed that culture growth rate as measured by change in culture turbidity is proportional to GFP fluorescence and total growth and growth rate depends upon how much sugar is present at inoculation. Furthermore, it was possible to monitor the course of enzymatic hydrolysis in near real-time, though there are instrumentation challenges in doing this.

Conclusion: We found that instantaneous fluorescence is proportional to the bacterial growth rate. As growth rate is limited by the availability of sugar, the integral of fluorescence is proportional to the amount of sugar consumed by the microbe. We demonstrate that corn stover varieties can be differentiated based on sugar yields in enzymatic hydrolysis reactions using post-hydrolysis fluorescence measurements. Also, it may be possible to monitor fluorescence in real-time during hydrolysis to compare different hydrolysis protocols.

No MeSH data available.


Related in: MedlinePlus