TITLE:  Feedstocks-to-Fuels Pipeline (F2F)
AUTHORS: Venkataramana R Pidatala, Mengziang Lei, Hemant Choudhary, Christopher J Petzold, Hector Garcia Martin, Blake A Simmons, John Gladden, Alberto Rodriguez

[DESCRIPTION]
Sustainably grown biomass is a promising alternative to produce fuels and chemicals and reduce the dependency on fossil energy sources. However, the efficient conversion of lignocellulosic biomass into biofuels and bioproducts often requires extensive testing of components and reaction conditions used in the pretreatment, saccharification, and bioconversion steps. This restriction can result in an unwieldy number of combinations of biomass types, solvents, microbial strains, and operational parameters that need to be characterized, turning these efforts into a daunting and time-consuming task. Here we developed a high-throughput feedstocks-to-fuels screening platform to address these challenges. The result is a miniaturized semi-automated platform that leverages the capabilities of a solid handling robot, a liquid handling robot, analytical instruments, and a centralized data repository, adapted to operate as an ionic-liquid-based biomass conversion pipeline. The pipeline was tested by using sorghum as feedstock, the biocompatible ionic liquid cholinium phosphate as pretreatment solvent, a “one-pot” process configuration that does not require ionic liquid removal after pretreatment, and an engineered strain of the yeast Rhodosporidium toruloides that produces the jet-fuel precursor bisabolene as a conversion microbe.

[STEPS]
SECTION: Fermentation
13. Growth and adaptation of Rhodosporidium toruloides 

Streak Rhodosporidium toruloides strain GB2 on an agar plate prepared using yeast peptone dextrose (YPD) medium and the antibiotics cefotaxime (100 µg/mL) and nourseothricin (50 µg/mL), and incubate it for 2 days at 30 C.
SECTION: Fermentation
14. Inoculation of hydrolysates

Transfer 800 µL of the 0.2 µm filtered hydrolysates into individual wells of a 48-well flower plate (M2P-labs.com, Cat # M2P-MTP-48-OFF) using a multichannel pipette.
SECTION: Fermentation Process
30. Bisabolene sampling
SECTION: Biomass dispensing
2. Transfer milled biomass (1 mm particle size) to scintillation vials, filling approximately 75% of the vial.
 
SECTION: Fermentation
13.2. Adapt the cells to the hydrolysate by preparing a mixture of hydrolysate:YPD (50:50), inoculate with cells from the YPD culture, and grow for 2 days. Use this culture for inoculating the pure hydrolysates.
SECTION: Fermentation
12. pH adjustment

To adjust the pH before microbial cultivation, mix 800 µL of the 0.45 µm filtered hydrolysate with 150 µL of 1 N NaOH and 150 µL of 0.5 M phosphate buffer pH 7.0.
SECTION: Fermentation
12.1. Transfer 1000 µL of the pH-adjusted hydrolysate to a 0.2 µm filter plate, spin at 4000 rpm for 30 min and collect the filtrate in a sterile plate. Use the filtered hydrolysate for the fermentations. 

 

SECTION: Pretreatment
4. Prepare a mixture of 5% IL (w/w) and 80% (w/w) water and add the mixture to each vial by using a liquid handling robot or multichannel pipette.

 

SECTION: Biomass dispensing
3. Dispense biomass from scintillation vials into Micronic vials using the Labman robot by following the protocol listed below.

For 15% biomass loading: Transfer 225 mg of ground biomass into 4 mL Micronic vials (in a 48 vial holder) using the Labman.

 
 


SECTION: Enzymatic hydrolysis
6. Measure the pH of the biomass after pretreatment and adjust it to a range of pH 4.8 - pH 5.2 using 1 Molarity (M) citrate buffer, pH 5.0.

 

SECTION: Enzymatic hydrolysis
7. Prepare a mixture of CTec3:HTec3 enzymatic cocktails at a 9:1 volume ratio. Add the mixture to the pretreated biomass in each vial at a final ratio of 10 mg of enzyme per g of biomass. 

1000 µL of enzyme mix contains 104 mg of enzyme. In this protocol, 20 µL of enzyme mix was used. 

SECTION: Enzymatic hydrolysis
8. Incubate the plate at 50 °C  for 4320 min.
SECTION: Pretreatment
5. Cap the vials and autoclave the plate with the vials at 121 °C  for 180 min. Remove the plate from the autoclave after cooling.

None. 
SECTION: Sugar analysis
9. Centrifuge the plates in a benchtop plate centrifuge at 4000 rpm for 10 min to precipitate the solid fraction from the aqueous hydrolysate. Transfer 50 µL of hydrolysate from each vial to a 96-well 0.45 µm filter plate using a liquid handler or multichannel pipette. Place the filter plate on top of a collection plate and spin the plate in a benchtop centrifuge at 4000 rpm for 10 min. 
SECTION: Sugar analysis
10. Prepare samples for HPLC by diluting the filtered hydrolysates 20 times with water (10 µL sample + 190 µL water) in a Bio-Rad hard shell 96-well plate (Cat # HSP9601). Samples can also be stored at -20 °C until analysis.
SECTION: Fermentation
14.6. Spin the plate containing the 1.4 mL tubes at 400 rpm for 10 min to pellet the cells and separate the aqueous and organic phases. 
SECTION: Fermentation
14.5. Remove the plate from the incubator. Transfer all the contents into clean 1.4 mL micronic tubes. Allow the aqueous phase and Durasyn layers to separate by leaving on the bench for about 15 min. Transfer 0.1 mL of the aqueous layer into 900 µL of water, measure the OD 600nm of the mixture to determine the microbial growth after cultivation.
SECTION: Fermentation
14.4. Incubate the plate at 30 °C and 900 rpm in a Multitron incubator (Infors HT, USA) with humidity control (>70% humidity) for 7 days.
SECTION: Fermentation
14.3. Add 200 µL of Durasyn 164 (INEOS Oligomers, USA) to the hydrolysates as overlay (25% of the culture volume). Cover the plate with a gas permeable sealing film (M2P-labs.com, product # F-GPR48-10).
SECTION: Fermentation
14.2. Measure the OD 600nm of the adapted R. toruloides GB2 culture using a non-inoculated hydrolysate as blank. Calculate the volume of the culture required to have a starting O.D. of 0.5 and inoculate the hydrolysates. 
SECTION: Fermentation
14.1. Prepare a 50X stock of filter-sterilized ammonium sulfate (250 g/L). Add ammonium sulfate to the hydrolysates to reach a final concentration of 5 g/L. Add cefotaxime 100 µg/mL (Sigma cat # C7039) to the hydrolysates.
SECTION: Fermentation
13.1. Inoculate a colony from the plate in 2 mL of liquid YPD medium and grow at 30 °C  for 2880 min at 200 rpm shaking speed.
SECTION: Sugar analysis
11. Analyze the samples in an Agilent Inifinity 1260 HPLC equipped with an Aminex HPX-87H column, using 4 mM sulfuric acid as mobile phase, 0.6 mL/min flow rate, 60 ºC column temperature, 20 min run time and a refractive index detector kept at 35 ºC during analysis. Integrate the peak areas and compare to calibration curves made with pure glucose and xylose standards.

SECTION: Bisabolene analysis
15. Mix 10 µL of the overlay with 990 µL of ethyl acetate with napthalene (5 mg/L) as internal standard in GC-MS vials.
SECTION: Data processing
17. Compile results from sugar, bisabolene and OD 600 nm measurements and upload to the Experiment Data Depot following the instructions in the protocol below.
 


SECTION: Bisabolene analysis
16. Analyze samples on a GC-MS Intuvo 9000 instrument using the following method. Column: HP-5ms, inlet temperature: 250 °C, column initial temperature: 80 °C, ramp 20 °C per min until 300 ºC and hold for 3 min. Calculate bisabolene concentrations by integration of the peak area values and compare to the areas obtained from a calibration curve made with pure bisabolene.

SECTION: Ionic liquid synthesis
1. Synthesize the ionic liquid (IL) cholinium phosphate by titrating cholinium hydroxide (Sigma-Aldrich CAS # 123-41-1) with phosphoric acid (Sigma-Aldrich CAS # 7664-38-2) at a 3:1 mole ratio inside a fume hood. Place cholinium hydroxide in a round bottom flask with a magnetic stirrer on an ice bath attached to a burette containing phosphoric acid. Use slow dripping to complete the reaction overnight. Lyophilize the IL for 3 days at -50 ºC to reach a semi-solid consistency.
Cholinium hydroxide and phosphoric acid come mixed with water and the percentage may vary based on the manufacturer. Calculate the mole ratio based on the specific amounts.