SSF Ethanol production

Introduction

These notes are for 10% solid loading with high enzyme loading: 1 g of dry biomass and 10 mL liquid per sample

Pretreatment Note

Due to moisture In the pretreated biomass more than one gram of biomass will need to be fermented. After pretreatment about 30% of the mass will be water but this percentage will vary so one needs determine the amount of water that was in the sample so you can calculate the exact amount of dry mass that was fermented. For example, fill the pretreatment cells with 1.6 grams (if using dried, milled material this will be about 10% water, pretreat (after which 30% will be water)), then take out 1.43 g to ferment and dry the rest to get actual moisture percent.

Fermentation Note

SSF can be done in Erlenmeyer flasks with air trap stoppers, serum bottles with rubber stoppers or 50 mL centrifuge tubes. NREL procedure is written for serum bottles. If you use bottles or centrifuge tubes, you should let out the CO2 after a day using a needle in the case of serum bottles or by briefly loosening the tops of the centrifuge tubes. This procedure is written for using an antibiotic but they are not needed if an autoclave and sterile conditions are used following guidelines in NOTE 1 at the end of this document.

Procedure

• Prep Fermentation 1

SSF can be done in Erlenmeyer flasks with air trap stoppers, serum bottles with rubber stoppers or 50 mL centrifuge tubes. NREL procedure is written for serum bottles. If you use bottles or centrifuge tubes, you should let out the CO2 after a day using a needle in the case of serum bottles or by briefly loosening the tops of the centrifuge tubes. This procedure is written for using an antibiotic but they are not needed if an autoclave and sterile conditions are used following guidelines in NOTE 1 at the end of this document.

• Pretreatment

1. Check seals and put filters into cells
2. Tare a cell without lid
3. Place biomass into cell with funnel- make sure you have at least 1.60 grams
4. Fill each cell with the same mass of biomass

Pretreatment condition using the ASE 350: 190°C for 15 min static time. This will result in a total run time of about 30 min for each cell; 9 min to heat biomass to set temperature +15 min to retain biomass at set temperature (the static time or actual pretreatment time) + 3 minutes to purge the cell (get out excess water) + 2 minutes to release pressure in the cell. NOTE 2 contains more info on pretreatment

• Fermentation Prep 2

The growth medium (YPD) is poured out of the yeast culture and replaced with the sterile PBS buffer. A spectrophotometer is used to produce an optical density of about 30. The yeast is now kept in the refrigerator. See angel doc Methods_Irene_Darku for steps.

• After Pretreatment

1. Weigh and label a tin for each sample.
2. Refrigerate (freeze if you will be storing longer than a week) at least 1.43g of each or ferment immediately.
3. Tare tin and record mass of remaining biomass from each cell after sample for fermentation has been removed.
4. Place tin and biomass in 105 C for 24 hours (or even 15 hrs – these samples will be very small)
5. See NOTE 3 if you will not be fermenting the extracted liquid. Or NOTE 4 if you are not.

• Fermentation

1. To sterilize air trap stoppers, rinse in bleach. Use about 1:10 to 1:5 bleach to DI water. Leave in solution for about 20 minutes
2. Weigh biomass into flasks
3. Add reagents to biomass
4. Add enzymes and yeasts aseptically to sterilized flasks, preferable under laminar flow hood
5. Cover with sterilized air trap stopper

Table to determine amounts needed based on the solid loading you have selected

Solid loading = solid/liquid * 100	This example is for 10% solid loading so the 10 mL of liquid used in all of the calculations is based on 1 gram of solid being 10% of the 10mL of liquid.	For each sample
Antibiotic	1% of total volume if using Penicillin-Strptomycin, (10mL total liquid)(0.01)= x mL Or 0.4% if using tetracycline	0.1mL
Buffer	Refer to reagent preparation. Citric acid prepared has a concentration of 1M. targeted concentration in broth is 0.05M (1M) (x) = (0.05M) (10mL total liquid)	0.55 mL
Cellulases (Cellulose degrading enzyme)	15 FPU per gram cellulose (NOTE 5), and cellulose is 34% of the dry mass of corn stover so (.34)(1)(15)=5.1 FPU per sample (currently the activity is 135 FPU/mL)(x mL) = 5.1 FPU, x=0.038 Note: the activity of enzymes will change over time	0.04 mL
Beta glucosidase	60 IU per gram dry matter is used for Enzymatic Hydrolysis, so 60 IU for 1 gram (current activity around 330 IU/mL) (x mL) = 60 IU Or see NOTE 6 below	0.18 mL
Yeast	We prepare the yeast inoculums (yeast cells in PBS) to have an OD of around 30. Our target is to get an initial OD of 0.5 in fermentation broth. (current OD of yeast) (x) = (.5) (10mL total liquid)	X=0.2 mL
Nutrient (Yeast Peptone, YP)	Refer to reagent preparation. YP is prepared with a 10X concentration. Our target is to get 1X concentration in fermentation broth. (10X) (x) = (1X) (10 mL total liquid)td>	Use 1 mL of the 10X YP
Nano pure water	10 mL – (use total from all of the above +water in the sample) If you are using the extracted water from pretreatment you will need to adjust all the amounts	NA

Note 1

When using the autoclave method for sterilization and no antibiotics are used, the biomass and reagents [everything except the enzymes and yeast] are added to the flask before autoclaving. Because weighing of each component into flask exposes flask to contamination.

Note 2

The total time will depend on the set temperature [which determines how long the heating time will be. This cannot be manipulated by the user] the purge and relief time (usually chosen by user with guidelines based on cell size from equipment manual) and the pretreatment time defined by the user.

Note 3

If you are not using the liquid extracted during the pretreatment for your fermentation, you should save some extracts to determine the amount of sugars removed- xylose and glucan in particular. This will help in more accurate determination of enzymes to add as well as calculation of yield as fraction of theoretical. Remember to get total volume so the numbers the IC gives you for concentration can be turned into an amount. Or for more precision, place the extract into a 50 mL volumetric flask and fill the rest with water. Then mix and filter some of this to be analyzed on the IC.

Note 4

If you are using the extracted water from the pretreatment you will need to adjust all of the amounts so all liquid can be included. For example, after pretreating a cell of rye biomass, 11mL of liquid extracted but only 7.33 mL of water of liquid needs to be added to reach 10% solids loading for 1 g of DM so one would be better off achieving 10% loading by using 1.5 g DM and 15mL

Note 5

FPU/g cellulose is chosen as the minimum to give the best yields based on the conditions of the pretreatment and fermentation applied in our lab. Different labs have used different enzyme loadings

Note 6

The protocol used in the BE 497 lab is simply using Beta glucosidase 4X of cellulases. We have seen articles that used 3X as much as others with no obvious relationship. 4X was found to work best with past fellows in the lab. The idea is to have sufficient Beta glucosidase so that cellobiose is not present to inhibit the hydrolysis.