Richard Lab:SSCF

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.

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 125ml 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. Collect 25g of biomass.
   1. If you want you can grind the samples using a wiley mill or a cyclone mill
   2. The samples will have to be less than 20% moisture to grind (i.e. they will have to be pretty dry).
   3. If analyzing multiple different samples the mill should be cleaned out between samples to avoid cross contamination.
2. Add ≈2g of dry cellulose to two 125 ml flasks. (record these weights)
   1. This usually works out to be ≈10g of silage
3. Number and weigh two aluminum weigh trays. (record these weights)
4. Add ≈1g of biomass to each aluminum tray. (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 (this will be done after your fermentation has started).
7. 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)]
• This will allow you to determine the exact amount of dry biomass you added to each flask

Preparing the SSCF Medium

1. Combine the following and autoclave:
   1. 80ml water
   2. 1g Yeast Extract
   3. 0.2g HK₂PO₄
   4. 1g trisodium citrate dihydrate
   5. 0.33g citric acid monohydrate
2. Chaekc that the pH is around 5.5
3. Autoclave the medium
4. After autoclaving and cooling add the following:
   1. 40FPU Cellulase
   2. 240IU Beta-Glucosidase
   3. 200μL Tetracycline stock

Running the Fermentation

1. Add 40mL of the SSCF solution to the flasks containing the 2g dry cellulose (10g silage).
2. Seal flasks with gas trap
3. Incubate with shaking for 5 days at 30-37°C
4. Measure ethanol production using a YSI 2700 Select Biochemistry Analyzer.

References

• 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