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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 chloramphenicol. E. coli strains crp*-gfp and crp*-gfp- grown in modified 1 × M9 minimal media, containing 20% d-glucose, with or without chloramphenicol. Chloramphenicol added at 13 h. (a) Each time point for hours 0 to 12 is the average absorbance of six replications. Each time point for hours 13 to 23 is the average absorbance of three replications. (b) Each time point for hours 0 to 12 is the average fluorescence of six replications of crp*-gfp cultures minus the average fluorescence of six replications of crp*-gfp- cultures. Each time point for hours 13 to 23 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 4: Response to the addition of chloramphenicol. E. coli strains crp*-gfp and crp*-gfp- grown in modified 1 × M9 minimal media, containing 20% d-glucose, with or without chloramphenicol. Chloramphenicol added at 13 h. (a) Each time point for hours 0 to 12 is the average absorbance of six replications. Each time point for hours 13 to 23 is the average absorbance of three replications. (b) Each time point for hours 0 to 12 is the average fluorescence of six replications of crp*-gfp cultures minus the average fluorescence of six replications of crp*-gfp- cultures. Each time point for hours 13 to 23 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: In addition to determining how quickly the microbe can respond to an increase in sugar concentration, it is important to establish the response time to a sudden limitation in sugar. We reasoned that the response to a sudden limitation in sugar would be limited by the rate of decay of existing GFP, so we sought to determine this parameter by halting protein production in mid-log phase and determining the effect on cell density and GFP-fluorescence. This was accomplished by the addition of chloramphenicol (a bacterial translation inhibitor) to the bacterial cultures and measuring absorbance and fluorescence over time. Absorbance measurements showed that the bacteria entered stationary phase 1 h after addition of chloramphenicol, and the fluorescence decreased proportionally to the decrease in rate of growth (Figure 4). Within 1 h, GFP fluorescence decreased dramatically, reaching a minimum after 4 h.


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 chloramphenicol. E. coli strains crp*-gfp and crp*-gfp- grown in modified 1 × M9 minimal media, containing 20% d-glucose, with or without chloramphenicol. Chloramphenicol added at 13 h. (a) Each time point for hours 0 to 12 is the average absorbance of six replications. Each time point for hours 13 to 23 is the average absorbance of three replications. (b) Each time point for hours 0 to 12 is the average fluorescence of six replications of crp*-gfp cultures minus the average fluorescence of six replications of crp*-gfp- cultures. Each time point for hours 13 to 23 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 4: Response to the addition of chloramphenicol. E. coli strains crp*-gfp and crp*-gfp- grown in modified 1 × M9 minimal media, containing 20% d-glucose, with or without chloramphenicol. Chloramphenicol added at 13 h. (a) Each time point for hours 0 to 12 is the average absorbance of six replications. Each time point for hours 13 to 23 is the average absorbance of three replications. (b) Each time point for hours 0 to 12 is the average fluorescence of six replications of crp*-gfp cultures minus the average fluorescence of six replications of crp*-gfp- cultures. Each time point for hours 13 to 23 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: In addition to determining how quickly the microbe can respond to an increase in sugar concentration, it is important to establish the response time to a sudden limitation in sugar. We reasoned that the response to a sudden limitation in sugar would be limited by the rate of decay of existing GFP, so we sought to determine this parameter by halting protein production in mid-log phase and determining the effect on cell density and GFP-fluorescence. This was accomplished by the addition of chloramphenicol (a bacterial translation inhibitor) to the bacterial cultures and measuring absorbance and fluorescence over time. Absorbance measurements showed that the bacteria entered stationary phase 1 h after addition of chloramphenicol, and the fluorescence decreased proportionally to the decrease in rate of growth (Figure 4). Within 1 h, GFP fluorescence decreased dramatically, reaching a minimum after 4 h.

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