Richard Lab:SSCF

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

1. Make RM media
   1. 90ml water
   2. 1g Yeast Extract
   3. 0.2g HK₂PO₄)
2. Make Sugar concentrate
   1. 10ml water
   2. 2g glucose
   3. 2g xylose
3. Autoclave liquid medium and sugar concentrate.
4. After cooling add sugar concentrate and 400μL tetracycline stock solution to medium.

Growing Zymomonas mobilis 8b

1. Inoculate the medium with Z. mobilis from -80°C freezer stock.
2. Grow with shaking for ~16 hours at 30°C.

=Preparing the Biomass

1. Grind (using a wiley mill or a cyclone mill) and collect 15g of ensiled biomass.
   1. Clean out the mill between samples.
2. Add 5g of this biomass to two 125 ml flasks. (record these weights)
3. Number and weigh two aluminum weight trays for each sample to be tested. (record these weights)
4. Add ~1g of biomass to two aluminum weight trays. (also record these weights)
5. Put the aluminum trays in the 105°C oven overnight allow to cool.
6. Weight the aluminum trays (with the dried biomass) after cooling.
7. Calculate the moisture content by the following equation

• %Moisture content (%wet basis) = 100*[(initial biomass weight + tray weight - final biomass weight + tray weight)/(Initial biomass weight + tray weight)]