1. Plant Cell Walls to Alcohols

2. This lesson looks at the break down of plant material to demonstrate the production of biofuels.

3. VOCABULARY: Biofuel- A fuel derived, directly or indirectly, from organic material

4. Biomass- Biological material derived from living, or recently living organisms

5. Cellulose- An insoluble substance that is the main constituent of plant cell walls; it is a polysaccharide, or complex sugar, consisting of chains of glucose monomers

6. Distillation- The action of purifying a liquid by the process of heating and cooling; distillation can be used to purify substances or to remove one component from a complex mixture

7. Hemicellulose- A polysaccharide constituent of plant cell walls

8. Hydrolysis- The chemical breakdown of a compound due to reaction with water

9. Lignin- A complex organic polymer found in plant cell walls, making them rigid and woody

10. Lignocellulosic recalcitrance- The natural resistance of woody plant cell walls to chemical and biological degradation

11. Pretreatment- The process to overcome lignocellulosic recalcitrance and expose the cellulose and hemicellulose so that individual sugars can be released

12. Background: After wood residues have been packaged and transported from logging sites to conversion facilities, the next step of the biofuel production process, CONVERSION, can begin. The conversion process involves three distinct steps: Pretreatment, Enzymatic Hydrolysis, and Fermentation.

13. The process to overcome lignocellulosic recalcitrance and expose the cellulose and hemicellulose so that individual sugars can be released is called PRETREATMENT.

14. Over the last four years, the NARA conversion team evaluated and streamlined four different pretreatment methods, in order to determine the process that would be the most sustainable and economically viable to incorporate into the biojet fuel production pipeline.

15. 1. SPORL (sulfite pretreatment to overcome recalcitrance of lignocellulose): This pretreatment method was developed at the USDA Forest Service, Forest Products Laboratory, a NARA affiliate. The process relies on heat, chemicals (sodium bisulfite) and mechanical grinding.

16. 2. Mild bisulfite (MBS): This process was developed at Catchlight Energy and USDA Forest Service, Forest Products Laboratory. The process is similar to SPORL.

17. 3. Wet Oxidation (WO): Developed at Washington State University’s Bioproducts, Sciences and Engineering Laboratory (WSU-BSEL), this process relies on pressure and oxygen.

18. 4. Dilute Acid (DA): This method uses sulfuric acid and heat and has been widely studied and used to pretreat crop residues like wheat straw and corn stover.

19. Over the last two years, researchers at the USDA Forest Service Forest Products Laboratory and Catchlight Energy made modifications to the SPORL protocol to create a hybrid protocol termed the mild bisulfite (MBS) pretreatment. MBS differs from SPORL by employing calcium bisulfite instead of sodium bisulfite and a lower cook temperature, which were instrumental to improved conditions for downstream isobutanol production and for adoption into existing biorefinery infrastructure.

20. In September 2014, the NARA team decided to select a single pretreatment method to use in the conversion process, in order to streamline the production pipeline. The mild bisulfite protocol was unanimously chosen as the preferred pretreatment method and work to scale the process and produce 1000 gallons of bio-jet fuel is currently underway.

21. Why is fermentation important?
A substantial element of the NARA project is to ensure that the wood residue to biojet fuel conversion process is sustainable environmentally, socially, and economically. The process engineering team at Gevo has been instrumental in providing unit operation costs for the isobutanol to biojet fuel conversion steps and capital cost estimates for infrastructure development. All of these inputs help establish a basis for a developing techno-economic model used to gage the complete cost of producing biojet fuel from wood residues and insure that the process is economically sustainable.

22. Alcohols, like isobutanol, are not the only commercially valuable products generated from the fermentation process. Volatile fatty acids (VFAs) like acetic acid, propionic acid and butyric acid are produced from fermentation in specific bacteria. NARA member Birgitte Ahring and her team at Washington State University’s Bioproducts, Sciences and Engineering Laboratory (BSEL) are optimizing a process called BioChemCat that uses bacteria to convert cellulosic feedstock into volatile fatty acids.

23. Creating the optimal conditions for yeast and bacteria fermentation is one challenge taken up by Gevo and Dr. Ahring’s group; removing the valuable products from the fermentation broth is another challenge. In a recent paper published in the Journal of Supercritical Fluids and funded by NARA, researchers at BSEL describe a novel method used to extract volatile fatty acids from the fermentation broth.

24. Their work expands opportunities to generate valuable bio-based products from wood residuals, which will provide support to the NARA team’s goal to create an economically viable wood-to-biojet fuel industry.  

25. Fermentation methods
Gevo makes use of their proprietary technology GIFT®, Gevo’s Integrated Fermentation Technology®, to convert those simple sugars into isobutanol and simultaneously separate the isobutanol from the fermentation broth. The GIFT® platform relies on specialized yeast to serve as a biological catalyst. The yeast import the glucose and other simple sugars from the wood residue solution into their cell, generate isobutanol from the simple sugars, and then secrete the isobutanol out of their cell and into the solution. As isobutanol accumulates in the solution, it is separated and collected.

26. As simple as this process description sounds, there are significant challenges. The wood residue solution is a complex mixture of many chemicals including the simple sugars. The isobutanol being produced, plus some of the chemicals, called fermentation inhibitors, are toxic to yeast and can significantly reduce growth and isobutanol output. Gevo researchers are using multiple approaches to address this challenge.

27. One approach takes advantage of yeast’s ability to reproduce rapidly and modify its genetic makeup to adapt to varied environments. As yeast reproduce in the wood residue solution, individual yeast strains are isolated. These individual strains are evaluated for their growth and isobutanol output and their ability to withstand the toxic conditions. Over the course of testing many stains, individual strains emerge as superior to other strains. These superior strains are then selected and the cycle of strain selection and testing continues. Ultimately strains are isolated that can resist the toxic elements and produce isobutanol at a high level.

28. Another approach to improving isobutanol yields was to evaluate yeast performance on wood residue solution generated from different upstream processes. Gevo tested yeast performance on pretreated hydrolysate samples, or wood residue solutions, derived from multiple pretreatment protocols developed within the NARA project.

29. Optimizations in upstream processes of conversion pipeline, specifically the NARA team’s decision to utilize mild bisulfite as the preferred pretreatment method, has effectively increased the efficiency of the fermentation process by decreasing the number of fermentation inhibitors and significantly enhancing the efficiency of enzymatic hydrolysis.

30. Gevo has optimized the fermentation process, and performance has reached a level that can enable further scale-up to support the production of 1,000 gallon of biojet fuel. Gevo will continue to provide input to refine the process model in order to support the 1,000 gallon biojet fuel demonstration task.

31. Goals:
What are the steps in the process of converting lignocellulosic biomass to biofuel?
What is the source of energy in plants and where is it stored?
How can we harness the energy stored in plants to produce fuel?
What are some of the challenges and complexities associated with the conversion process?
Objectives:
Students will understand:
where the energy in plants comes from
the three basic steps of the conversion process
the various methods used in each step of the conversion process
that pretreatment is the most difficult step in biomass conversion to fuel representing up to 20% of the cost of fuel production
that there is an ongoing ethical debate about the techno-economic feasibility of a biojet fuel industry
that optimizing the conversion process is a very important step in creating an economically sound and environmentally responsible biojet fuel industry

32. QUESTIONS
What are the steps in the process of converting lignocellulosic biomass to biofuel?
What is the source of energy in plants and where is it stored?
How can we harness the energy stored in plants to produce fuel?
What are some of the challenges and complexities associated with the conversion process?

33. PLEASE note that pretreatment is the most difficult step in biomass conversion to fuel representing up to 20% of the cost of fuel production

34. PLEASE note that there is an ongoing ethical debate about the techno-economic feasibility of a biojet fuel industry

35. PLEASE note: that optimizing the conversion process is a very important step in creating an economically sound and environmentally responsible biojet fuel industry

36. ACTIVITY 1- Materials: visual of a plant tissue that shows cell walls
visual of plant cell wall cross-section showing lignin binding cellulos
and hemicellulose
scrap of paper for each student
approximately 10 sugar cubes for each student or each group of students
mud (or eggless cookie dough or peanut butter)
hot water
small (about 32 oz.) container
cocoa krispies or other cereal that changes the milk color
another piece of scrap paper for each student

37. ACTIVITY 2: Step 1: 1/3 the students will be considered hemicellulous, 1/3 cellulose and the rest lignins. Have them wear name cards of “H, C, or L” Step 2: Have the celluloses form lines of 4-6 students and link arms. Have the hemicelluloses do the same, but in lines separate from the cellulose lines.
Step 3: Have the hemicellulose and cellulose lines stand parallel to each other.
Step 4: Have the lignins surround the hemicellulose and cellulose lines. Then have the lignin students hold hands over and through the cellulose/hemicellulose lines, so that the lignins' arms are reaching through and across the lines.
The visual should be of lines of cellulose and hemicellulose surrounded by a lignin web.

38. Question: Why it is necessary to pretreat lignocellulosic biomass?

39. Ways in which biomass can be pretreated
Ways in which biomass can be pretreated. Compare at least two forms of pretreatment in terms of their advantages and disadvantages.

40. Question: Explain how the first step of the activity was similar to and different from the actual removal of lignin.

41. PLEASE note : That hydrolysis is the breaking up of polysaccharides (starches – in this case cellulose and hemicellulose) into monosaccharides.

42. PLEASE note that hydrolysis can use enzymes or chemicals that break the bonds between individual sugar molecules.

43. Question: How was hydrolysis represented in the activity, and how this representation was accurate or inaccurate.

44. PLEASE note that fermentation is the process by which an organism such as a yeast takes in sugars, uses the energy contained within them, and changes them into alcohols.

45. PLEASE consider why it might be necessary to have individual sugar monomers for the next step in the process, fermentation by a yeast.

46. PLEASE note that different yeasts can have different alcohol outputs (i.e. ethanol or isobutanol)