Richard Lab:SSCF: Difference between revisions
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==Introduction== | ==Introduction== | ||
This protocol is for the quantitative simultaneus saccharification and co-fermentation of ensiled ligno-cellulosic biomass using ''Zymomonas mobilis 8b''. | This protocol is for the quantitative simultaneus saccharification and co-fermentation of ensiled ligno-cellulosic biomass using ''Zymomonas mobilis 8b''. The amounts given in this protocol are for fermenting one biomass sample in duplicate, so you should scale-up this protocol to process as many samples as you need. | ||
==Materials== | ==Materials== | ||
*Z. mobilis 8b culture (available from the National Renewable Energy Lab (NREL)) | *Z. mobilis 8b culture (available from the National Renewable Energy Lab (NREL)) | ||
Line 8: | Line 10: | ||
*Yeast extract | *Yeast extract | ||
*HK<sub>2</sub>PO<sub>4</sub> | *HK<sub>2</sub>PO<sub>4</sub> | ||
*Tetracycline | *Tetracycline (5mg/ml ethanol stock solution) | ||
*Wiley mill or cyclone mill | *Wiley mill or cyclone mill (optional) | ||
*Aluminum weigh trays | *Aluminum weigh trays | ||
*Cellulase | *Cellulase | ||
Line 17: | Line 19: | ||
==Procedure== | ==Procedure== | ||
====Prepare the | ====Prepare the RMGXT (Rich Medium with Glucose, Xylose, and Tetracycline) ==== | ||
#Make | #Make Rich Medium | ||
##90ml water | ##90ml water | ||
##1g Yeast Extract | ##1g Yeast Extract | ||
Line 26: | Line 28: | ||
##2g glucose | ##2g glucose | ||
##2g xylose | ##2g xylose | ||
#Autoclave liquid medium and sugar concentrate. | #Autoclave liquid medium and sugar concentrate in separate containers. | ||
#After cooling add sugar concentrate and 400μL tetracycline stock solution to | #After cooling add sugar concentrate and 400μL tetracycline stock solution to RM. | ||
====Growing ''Zymomonas mobilis'' 8b==== | ====Growing ''Zymomonas mobilis'' 8b==== | ||
#Inoculate the medium with ''Z. mobilis'' from -80°C freezer stock. | #Inoculate the medium with ''Z. mobilis'' from -80°C freezer stock. | ||
#Grow with shaking for ~16 hours at 30°C. | #Grow with shaking for ~16 hours at 30°C (OD<sub>600</sub> should be between 1.2 and 2.0). | ||
====Preparing the Biomass==== | ====Preparing the Biomass==== | ||
#Collect 25g of biomass. | |||
##If you want you can grind the samples using a wiley mill or a cyclone mill | |||
##The samples will have to be less than 20% moisture to grind (i.e. they will have to be pretty dry). | |||
##If analyzing multiple different samples the mill should be cleaned out between samples to avoid cross contamination. | |||
#Add ≈2g of dry cellulose to two 125 ml flasks. (record these weights) | |||
##This usually works out to be ≈10g of silage | |||
#Number and weigh two aluminum weigh trays. (record these weights) | |||
#Add ≈1g of biomass to each aluminum tray. (also record these weights) | |||
#Put the aluminum trays in the 105°C oven overnight. | |||
# | ====Preparing the SSCF Medium==== | ||
## | #Combine the following and autoclave: | ||
# | ##80ml water | ||
# | ##1g Yeast Extract | ||
#Add | ##0.2g HK<sub>2</sub>PO<sub>4</sub> | ||
# | ##1.175g trisodium citrate dihydrate | ||
# | ##0.210g citric acid monohydrate | ||
#Calculate the moisture content | #Check that the pH is around 6.0 | ||
#Autoclave the medium | |||
#After autoclaving and cooling add the following: | |||
##40 FPU Cellulase | |||
##240 IU Beta-Glucosidase | |||
##200μL Tetracycline stock | |||
====Preparing the Inoculum==== | |||
#Centrifuge the entire 100ml culture of ''Z. mobilis'' in two 50ml centrifge tubes for 15min at 5,000scf | |||
#Discard the supernatant and resuspend the pellet (in each tube) in 1ml sterile water. | |||
====Running the Fermentation==== | |||
#Add 40mL of the SSCF solution to the flasks containing the 2g dry cellulose (10g silage). | |||
#Add 100μL of ''Z. mobilis'' suspension. | |||
#Seal flasks with gas trap | |||
#Incubate with shaking for 5 days at 30-37°C | |||
==Analysis== | |||
====Determination of Dry Mass ==== | |||
#Remove the aluminum trays (with dried biomass) from the oven and allow to cool (1hr) in the dessicator. | |||
#Weigh each tray (with biomass). | |||
#Calculate the moisture content using the following equation: | |||
:::*%Moisture content (%wet basis) = 100*[(initial biomass weight + tray weight - final biomass weight and tray weight) / (Initial biomass weight + tray weight)] | :::*%Moisture content (%wet basis) = 100*[(initial biomass weight + tray weight - final biomass weight and tray weight) / (Initial biomass weight + tray weight)] | ||
:::*This will allow you to determine the exact amount of dry biomass you added to each flask | |||
#Calculate the dry mass added to each flask using the following equation: | |||
:::*Dry mass = recorded wet mass * (1-moisure content) | |||
====Monitoring Fermentation==== | |||
This step can be done at any time during the fermentation, but should especially be done at the end. | |||
#Using a sterile pipette, remove 500μL of liquid from each fermentation flask. | |||
#Centrifuge the samples for 1 minute at full speed in a microcentrifuge. | |||
#Measure ethanol production using a [[YSI 2700 Select Biochemistry Analyzer]]. | |||
#If desired, also measure glucose in the same sample using the same device (this will require a different membrane). | |||
::*If your ethanol is leveling off and there is residual glucose it means that your organism is being inhibited by something. | |||
::*Only do this step if you suspect inhibition. | |||
==Notes== | |||
*On the development of ''Z. mobilis'' 8B | |||
**''Z. mobilis'' 8B is an acetic acid tolerant strain of ''Z. mobilis'' ZM4 (Mohagheghi et al., 2004). | |||
**''Z. mobils'' ZM4 is an ethanol tolerant mutant of ''Z. mobilis'' CP4 (Joachimsthal et al., 1999). | |||
**''Z. mobilis'' CP4 contans the xylose fermentation genes from ''E. coli'' (Zhang et al. 1995). | |||
== | ==References== | ||
*Zhang M, Eddy C, Deanda K, Finkelstein M, Picataggio S. 1995. '''Metabolic engineering of a pentose metabolism pathway in ethanologenic zymomonas mobilis'''. ''Science'' 267(5195):240–243. | |||
*Joachimsthal E, Haggett KD, Rogers PL. 1999. '''Evaluation of recombinant strains of Zymomonas mobilis for ethanol production from glucose/xylose media'''. ''Appl Biochem Biotechnol'' 77–79:147–157. | |||
*Mohagheghi A, Dowe N, Schell D, Chou Y, Eddy C, Zhang M. 2004. '''Performance of a newly developed integrant of Zymomonas mobilis for ethanol production on corn stover hydrolysate.''' ''Biotechnol Lett'' 26:321–325. | |||
*Zhang, J. and L. Lynd. 2010. '''Ethanol Production From Paper Sludge by Simultaneous Saccharification and Co-Fermentation Using Recombinant Xylose-Fermenting Microorganisms'''. ''Biotechnology and Bioengineering'' 107, 2:235-244 |
Latest revision as of 23:17, 28 September 2011
Back to Richard Lab:protocols
Introduction
This protocol is for the quantitative simultaneus saccharification and co-fermentation of ensiled ligno-cellulosic biomass using Zymomonas mobilis 8b. The amounts given in this protocol are for fermenting one biomass sample in duplicate, so you should scale-up this protocol to process as many samples as you need.
Materials
- Z. mobilis 8b culture (available from the National Renewable Energy Lab (NREL))
- Glucose
- Xylose
- Yeast extract
- HK2PO4
- Tetracycline (5mg/ml ethanol stock solution)
- Wiley mill or cyclone mill (optional)
- Aluminum weigh trays
- Cellulase
- Beta-glucosidase
- 125ml flasks
- Gas traps for 125ml flasks
Procedure
Prepare the RMGXT (Rich Medium with Glucose, Xylose, and Tetracycline)
- Make Rich Medium
- 90ml water
- 1g Yeast Extract
- 0.2g HK2PO4
- Make Sugar concentrate
- 10ml water
- 2g glucose
- 2g xylose
- Autoclave liquid medium and sugar concentrate in separate containers.
- After cooling add sugar concentrate and 400μL tetracycline stock solution to RM.
Growing Zymomonas mobilis 8b
- Inoculate the medium with Z. mobilis from -80°C freezer stock.
- Grow with shaking for ~16 hours at 30°C (OD600 should be between 1.2 and 2.0).
Preparing the Biomass
- Collect 25g of biomass.
- If you want you can grind the samples using a wiley mill or a cyclone mill
- The samples will have to be less than 20% moisture to grind (i.e. they will have to be pretty dry).
- If analyzing multiple different samples the mill should be cleaned out between samples to avoid cross contamination.
- Add ≈2g of dry cellulose to two 125 ml flasks. (record these weights)
- This usually works out to be ≈10g of silage
- Number and weigh two aluminum weigh trays. (record these weights)
- Add ≈1g of biomass to each aluminum tray. (also record these weights)
- Put the aluminum trays in the 105°C oven overnight.
Preparing the SSCF Medium
- Combine the following and autoclave:
- 80ml water
- 1g Yeast Extract
- 0.2g HK2PO4
- 1.175g trisodium citrate dihydrate
- 0.210g citric acid monohydrate
- Check that the pH is around 6.0
- Autoclave the medium
- After autoclaving and cooling add the following:
- 40 FPU Cellulase
- 240 IU Beta-Glucosidase
- 200μL Tetracycline stock
Preparing the Inoculum
- Centrifuge the entire 100ml culture of Z. mobilis in two 50ml centrifge tubes for 15min at 5,000scf
- Discard the supernatant and resuspend the pellet (in each tube) in 1ml sterile water.
Running the Fermentation
- Add 40mL of the SSCF solution to the flasks containing the 2g dry cellulose (10g silage).
- Add 100μL of Z. mobilis suspension.
- Seal flasks with gas trap
- Incubate with shaking for 5 days at 30-37°C
Analysis
Determination of Dry Mass
- Remove the aluminum trays (with dried biomass) from the oven and allow to cool (1hr) in the dessicator.
- Weigh each tray (with biomass).
- Calculate the moisture content using the following equation:
- %Moisture content (%wet basis) = 100*[(initial biomass weight + tray weight - final biomass weight and tray weight) / (Initial biomass weight + tray weight)]
- This will allow you to determine the exact amount of dry biomass you added to each flask
- Calculate the dry mass added to each flask using the following equation:
- Dry mass = recorded wet mass * (1-moisure content)
Monitoring Fermentation
This step can be done at any time during the fermentation, but should especially be done at the end.
- Using a sterile pipette, remove 500μL of liquid from each fermentation flask.
- Centrifuge the samples for 1 minute at full speed in a microcentrifuge.
- Measure ethanol production using a YSI 2700 Select Biochemistry Analyzer.
- If desired, also measure glucose in the same sample using the same device (this will require a different membrane).
- If your ethanol is leveling off and there is residual glucose it means that your organism is being inhibited by something.
- Only do this step if you suspect inhibition.
Notes
- On the development of Z. mobilis 8B
- Z. mobilis 8B is an acetic acid tolerant strain of Z. mobilis ZM4 (Mohagheghi et al., 2004).
- Z. mobils ZM4 is an ethanol tolerant mutant of Z. mobilis CP4 (Joachimsthal et al., 1999).
- Z. mobilis CP4 contans the xylose fermentation genes from E. coli (Zhang et al. 1995).
References
- Zhang M, Eddy C, Deanda K, Finkelstein M, Picataggio S. 1995. Metabolic engineering of a pentose metabolism pathway in ethanologenic zymomonas mobilis. Science 267(5195):240–243.
- Joachimsthal E, Haggett KD, Rogers PL. 1999. Evaluation of recombinant strains of Zymomonas mobilis for ethanol production from glucose/xylose media. Appl Biochem Biotechnol 77–79:147–157.
- Mohagheghi A, Dowe N, Schell D, Chou Y, Eddy C, Zhang M. 2004. Performance of a newly developed integrant of Zymomonas mobilis for ethanol production on corn stover hydrolysate. Biotechnol Lett 26:321–325.
- Zhang, J. and L. Lynd. 2010. Ethanol Production From Paper Sludge by Simultaneous Saccharification and Co-Fermentation Using Recombinant Xylose-Fermenting Microorganisms. Biotechnology and Bioengineering 107, 2:235-244