Richard Lab:SSCF

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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)

1. Make Rich Medium
   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 in separate containers.
4. After cooling add sugar concentrate and 400μL tetracycline stock solution to RM.

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 (OD₆₀₀ should be between 1.2 and 2.0).

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.

Preparing the SSCF Medium

1. Combine the following and autoclave:
   1. 80ml water
   2. 1g Yeast Extract
   3. 0.2g HK₂PO₄
   4. 1.175g trisodium citrate dihydrate
   5. 0.210g citric acid monohydrate
2. Check that the pH is around 6.0
3. Autoclave the medium
4. After autoclaving and cooling add the following:
   1. 40 FPU Cellulase
   2. 240 IU Beta-Glucosidase
   3. 200μL Tetracycline stock

Preparing the Inoculum

1. Centrifuge the entire 100ml culture of Z. mobilis in two 50ml centrifge tubes for 15min at 5,000scf
2. Discard the supernatant and resuspend the pellet (in each tube) in 1ml sterile water.

Running the Fermentation

1. Add 40mL of the SSCF solution to the flasks containing the 2g dry cellulose (10g silage).
2. Add 100μL of Z. mobilis suspension.
3. Seal flasks with gas trap
4. Incubate with shaking for 5 days at 30-37°C

Analysis

Determination of Dry Mass

1. Remove the aluminum trays (with dried biomass) from the oven and allow to cool (1hr) in the dessicator.
2. Weigh each tray (with biomass).
3. 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

1. 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.

1. Using a sterile pipette, remove 500μL of liquid from each fermentation flask.
2. Centrifuge the samples for 1 minute at full speed in a microcentrifuge.
3. Measure ethanol production using a YSI 2700 Select Biochemistry Analyzer.
4. 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