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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.
- Z. mobilis 8b culture (available from the National Renewable Energy Lab (NREL))
- Yeast extract
- Tetracycline (5mg/ml ethanol stock solution)
- Wiley mill or cyclone mill (optional)
- Aluminum weigh trays
- 125ml flasks
- Gas traps for 125ml flasks
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
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)
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.
- 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).
- 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