Richard Lab:SSCF: Difference between revisions
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*Xylose | *Xylose | ||
*Yeast extract | *Yeast extract | ||
*HK<sub>2</sub>PO<sub>4</sub> | *HK<sub>2</sub>PO<sub>4</sub> | ||
*Tetracycline Stock (5mg/ml ethanol stock solution) | *Tetracycline Stock (5mg/ml ethanol stock solution) | ||
*Wiley mill or cyclone mill. | *Wiley mill or cyclone mill. |
Revision as of 09:03, 14 September 2011
Introduction
This protocol is for the quantitative simultaneus saccharification and co-fermentation of ensiled ligno-cellulosic biomass using Zymomonas mobilis 8b.
Materials
- Z. mobilis 8b culture (available from the National Renewable Energy Lab (NREL))
- Glucose
- Xylose
- Yeast extract
- HK2PO4
- Tetracycline Stock (5mg/ml ethanol stock solution)
- Wiley mill or cyclone mill.
- Aluminum weigh trays
- Cellulase
- Beta-glucosidase
- 125ml flasks
- Gas traps for 250ml flasks
Procedure
Prepare the RMGXTc Medium
- Make RM media
- 90ml water
- 1g Yeast Extract
- 0.2g HK2PO4
- Make Sugar concentrate
- 10ml water
- 2g glucose
- 2g xylose
- Autoclave liquid medium and sugar concentrate.
- After cooling add sugar concentrate and 400μL tetracycline stock solution to medium.
Growing Zymomonas mobilis 8b
- Inoculate the medium with Z. mobilis from -80°C freezer stock.
- Grow with shaking for ~16 hours at 30°C.
Preparing the Biomass
- Grind (using a wiley mill or a cyclone mill) and collect 15g of ensiled biomass.
- Clean out the mill between samples.
- Add 5g of this biomass to two 125 ml flasks. (record these weights)
- Number and weigh two aluminum weight trays for each sample to be tested. (record these weights)
- Add ~1g of biomass to two aluminum weight trays. (also record these weights)
- Put the aluminum trays in the 105°C oven overnight allow to cool.
- Weight the aluminum trays (with the dried biomass) after cooling.
- Calculate the moisture content by the following equation
- %Moisture content (%wet basis) = 100*[(initial biomass weight + tray weight - final biomass weight and tray weight) / (Initial biomass weight + tray weight)]