An Ecological Perspective (BIOL 346)

1. An Ecological Perspective (BIOL 346) Talk Eight: Biofuels

2. Introduction Biofuel is a type of fuel whose energy is derived from biological carbon fixation. Biofuels include fuels derived from biomass conversion, as well as solid biomass, liquid fuels and various biogases.  Although fossil fuels have their origin in ancient carbon fixation, they are not considered biofuels by the generally accepted definition because they contain carbon that has been "out" of the carbon cycle for a very long time.

3. What is a Biofuel? • Layman’s Definition: • “A fuel that gains its’ energy through the use of already existing carbon in the atmosphere.” • Unlike other renewable energy sources, biomass can be converted directly into liquid fuels, called "biofuels," to help meet transportation fuel needs. • The two most common types of biofuels in use today are ethanol and biodiesel.

4. Bio-alcohol Bio alcohol is only obtained from biomass or bio-degradable parts of waste and is usually intended for the use as biofuel. But Bio alcohol can not only be made of waste but also wood, straw or whole plants. If Bio alcohol is used as fuel, there are also different mixture ratios. Bio alcoholis also used to activate fireplaces like ethanol fireplaces. It is also used as a disinfectant in a mixture with water and then it is also used as solvent. It is also a part of medical treats and in the industry it is used as solvent. It is also used as fuel or denatured alcohol - therefore other ingredients are mixed with ethanol.

5. Bioalcohol Bioethanol is ethanol fuel (ethyl alcohol, the same stuff we drink) made from plant matter (as with most ethanol). Currently most of it is made from corn, but in the future more and more of it will be made from cheaper organic material such as grass and various plant waste. It can be used as a fuel similar to gasoline, but is commonly used as a gasoline additive to minimize emissions. It can also be used as a total gasoline replacement to power our cars with only minor modifications to a standard gasoline engine.

6. Biogas • Biogas production using anaerobic (oxygen free) digestion is a biological treatment process to reduce odor, produce energy and improve the storage and handling characteristics of manure. • A biogas production system must be specially designed and requires regular attention by someone familiar with the needs and operation of the digester.

7. Bio-diesel Bio-diesel is a form of diesel fuel manufactured from vegetable oils, animal fats, or recycled restaurant greases. It is safe, biodegradable, and produces less air pollutants than petroleum-based diesel. Bio-diesel is meant to be used in standard diesel engines and is thus distinct from the vegetable and waste oils used to fuel converted diesel engines. Bio-diesel can be used alone, or blended with petro diesel. Bio-diesel can also be used as a low carbon alternative to heating oil.

9. The Plant Cell wall The cell wall is the organelle that ultimately controls the shape of plant cells and consequently of organs and whole organisms. It is sometimes naturally strengthened and made considerably more resistant to such abuses as pathogen infection by the release of specific oligosaccharides and enzymes and by overlaying or impregnation with cutin, suberin, waxes or silica

10. Plant pathogens • In order to infect and enter a plant cell, a pathogen must get through the plant cell wall. • How complex this cell wall? • Cellulose • Cross-linking Glucans: • Xyloglucan (XG). • Glucuronoarabinoxylan (GAX). • Mannans, Glucomannans, Starch, Callose Galactomannans. • Pectin : • Homogalacturonan (HGA). • Rhamnogalacturonan-I (RG-I). • Rhamnogalacturonan-II (RG-II). • Proteins and lignin?

11. How does the pathogen do this? • Substrate induction: • Pathogen always produces very low levels of cell wall degrading enzymes (CWDE). • Mainly pectinases • Upon initial contact with plant, a small number of pectin related monomers are released • These induce gene expression in the pathogen to make more CWDE • The additional enzymes release more monomers which also act as inducers of gene expression

12. How does the pathogen do this? • Catabolite Repression: • At high enough concentrations, the monomers released from the continued breakdown of the Plant Cell Wall repress the synthesis of CWDE. • This reduces the production of the enzymes by the pathogen • Mostly, when this occurs, the pathogen has successfully degraded the plant cell wall.

13. Just think about all the different enzymes! • With just pectin: • Pectin Lyase (PL) • Break the chain and release molecules with an unsaturated double bond • Pectin methylesterase (PME) • Remove methyl groups – this alters solubility and thus the rate at which other pectinases work • endoPolygalacturonan (PG) • Break the links between two galacturonan molecules in the chain • ectoPolygalacturonan • Break off terminal galacturonan molecules only

14. Just for pectin – there are more enzymes!

15. Just how many enzymes? • sdc

16. Induction: • Extracellular enzymes expressed at low levels generate metabolites that signal pathogen to dramatically increase the expression level of genes encoding plant cell wall degrading enzymes. • Utilization: • Extracellular enzymes and transporters specific for translocation of cell wall degradation products enable pathogen to use plant cell material for growth. • Some extracellular proteins may generate metabolites that modulate gene expression of cellulases and hemicellulases during the utilization phase.

17. The trouble with Lignin… • Lignin is: • Complex aromatic polymer that’s an important component of plant secondary • cell walls provides rigidity and mechanical support to plant tissues • However… • The highly phenolic polymers in lignin are very degradation resistant. • The complexity of the bonds formed among lignin monomers are less • reactive • Chemical diversity of lignin compared to simple polymers precludes the • ability of any single enzyme to degrade it • Therefore: Lignin is hard to get rid of..

18. The trouble with Lignin… Pre-treatment: Phenol monomers produced by the degradation of Lignin are fermentation inhibitors of growth and ethanol production in S. cerevisiae. pentose-utilizing strains Escherichia coli, Pichia stipititis, and Zymomonas mobilis produce ethanol in concentrated hemicellulose liquors but require detoxification. e

19. How to get rid of Lignin • Breed it out! • Irx4 is a mutant of Arabidopsis Thaliana: • Down-regulation of cinnamoyl-CoA reductase (CCR) gene • CCR is involved in the latter stages of lignin biosynthesis • Irx4 = irregular xylem 4: named for phenotype.

20. Irx4 Arabidopsis mutant Has significantly reduced lignin content Low to No significant reduction in cellulose and hemicellulose content.

21. Irx4 Arabidopsis mutant (b): • Has a dwarfed phenotype compared to WT (a) • Also, because Lignin is an important constituent of the secondary cell wall of the xylem the mutant (d) experiences collapse of vessel elements in the xylem compared to the WT (c) • Lignin deficient mutants are weaker and harder to rear –overall • weakness makes them more susceptible to fungal pathogens • CCR is also part of Defense Response Pathway that leads to reactive • Oxygen species

22. How to get rid of Lignin • 2. Chew it up! • Synergy between fungal pathogen enzymes diverse enough for the complexity of Lignin rich sources like Sugarcane Bagasse

23. SUGARCANE • Sugarcane stalks are crushed to extract their juices. There is a biomass • remaining called bagasse. • Bagasse • Is considered to be lignocellulose (combination of both cellulose and • hemicellulose) • Dry weight of sugarcane bagasse composition: • 42% cellulose, 22% lignin, and 28% hemicellulose • Can be utilized as a fuel source: produce steam or substrate for production • of bioethanol

24. Clostridium celluovorans • This is a microorganism that: • Is an anaerobic bacterium • Is mesophilic (meaning moderate temperature) • Is Cellulytic • Has the ability to utilize carbon sources: cellulose, xylan, and pectin • This bacterium contains a cellusome in which specific enzymes were isolated: • Xylanase A (XynA) • Mannanase A (ManA) • Endoglucancase E (EngE): has ability for some xylanase activity • Study was conducted: • Looked at the enzymes in different ratios • Effectiveness in degrading sugarcane bagasse

25. Recap Fungal pathogens are involved in the production of Biofuels with particular respect to the cell wall in that: • Enzymatic Hydrolysis is a necessary pretreatment before fermentation of the simple sugars can yield biofuels • Pathogens are also involved in the production of the ideal biofuel source: low-lignin mutants are difficult to rear due to increased susceptibility to fungal pathogens • Synergy between the right mixture of enzymes is used to optimize the degradation of the fermentation inhibiting Lignin

26. So what? • Many if not all of these enzymes can be isolated and used to digest isolated biomass – PCWM • Breaking down each of the many components of the cell wall will allow us to find ways to use them all. • So many enzymes, so many pathogens, so many combinations. • Is it even possible?

27. What is a biofuel? • A biofuel is a renewable energy source, unlike other natural resources such as petroleum, coal, and nuclear fuels. • One legal definition of biofuel is "any fuel with an 80% minimum content by volume of materials derived from living organisms harvested within the ten years preceding its manufacture".

28. What is a biofuel? Like coal and petroleum, biomass is a form of stored solar energy. The energy of the sun is "captured" through the process of photosynthesis in growing plants. One of the major advantages of biofuel over most other fuel types is that it is biodegradable, and so relatively harmless to the environment if spilled.

29. Predicted increase in global mean temperature due to CO2 accumulation The carbon in biofuels was recently extracted from atmospheric carbon dioxide by growing plants, so burning it does not result in a net increase of carbon dioxide in the Earth's atmosphere. Therefore, many people believe that a way to reduce the amount of carbon dioxide released into the atmosphere is to use biofuels to replace non-renewable sources of energy. www.metoffice.com/research/hadleycenter

30. Ethanol from Cornstarch Courtesy of Bruce Ferguson, Edenspace

31. Switchgrass • A warm season grass and is one of the dominant species of the central North American tallgrass prairie. • Switchgrass can be found in remnant prairies, along roadsides, pastures and as an ornamental plant in gardens.

32. Switchgrass • ethanol fuel — production due to its hardiness against poor soil and climate conditions, rapid growth and low fertilization and herbicide requirements. • Switchgrass is also perennial, unlike corn and sugarcane, and has a huge biomass output, the raw plant material used to make biofuel, of 6-10 tons per acre

33. Other Steam Transport Biomass Grinding Electricity The challenge is efficient conversion • Burning switchgrass (10 t/ha) yields 14.6-fold more energy than input to produce* • But, converting switchgrass to ethanol calculated to consume 45% more energy than produced Energy consumption *Pimentel & Patzek, Nat Res Res 14,65 (2005)

34. Sugar beet • Sugar beet is a hardy biennial plant that can be grown commercially in a wide variety of temperate climates. • During its first growing season, it produces a large (1–2 kg) storage root whose dry mass is 15–20% sucrose by weight.

35. Sugar beet • If not harvested, during its second growing season, the plant uses the nutrients in this root to produce flowers and seeds. • In commercial beet production, the root is harvested after the first growing season, when the root is at its maximum size.

36. Biochemical Composition in Sugar Beet Pulp vs. ‘Typical’ Dicot Sugar Beet • 30% cellulose and hemicellulose • 19% pectin • 70% RGA-I • 0.8% ferulic acid • 50% sugar (arabinose, galactose, rhamnose, etc) • Increased pectin concentration important • Feruloyl esters ‘Typical’ Dicot • 20-30% cellulose • 20% hemicellulose • 11-15% pectin

37. What can be done • Pyrolysis heats the biomass to temperatures of 300oC – 500oC. in the absence of air. • The biomass “melts” and vaporizes, producing petroleum-like oil called bio-crude. • This bio-crude can be converted to gasoline or other chemicals or materials.

38. Pyrolysis • The chemical decomposition of a condensed substance by heating and is a special case of thermolysis. • Geologists view crude oil and natural gas as the product of compression and heating of ancient organic materials over geological time. • Formation of petroleum occurs from hydrocarbon pyrolysis, in a variety of mostly endothermic reactions at high temperature and/or pressure

39. crude oil composition • Mostly alkanes, cycloalkanes and various aromatic hydrocarbons while the other organic compounds contain nitrogen, oxygen and sulfur, and trace amounts of metals such as iron, nickel, and copper. • As crude oil is made from plant material, it is reasonable to suggest that pyrolysis of sugar beet would result in the formation of the same components.

40. Fuel from crude • Crude oil is separated into fractions by fractional distillation. The fractions at the top of the fractionating column have lower boiling points than the fractions at the bottom. • The heavy bottom fractions are often cracked into lighter, more useful products. All of the fractions are processed further in other refining units.

41. Conclusions • Up to 2005 biofuels were more costly than fossil fuels. • 2012 estimates: • Estimated ethanol production cost in 2012 was $0.46 per gasoline energy equivalent L. • Wholesale gasoline prices averaged $0.44/L in 2012 • Estimated soybean biodiesel production cost in 2012 was $0.55 per diesel EEL, • Diesel wholesale prices averaged $0.46/L in 2012 • Recently: • Decrease in fossil-fuel prices • Increase in corn prices

42. Conclusions Biofuels can not replace fossil fuels without having impact on food supplies. Even if all corn grown in U.S.A were dedicated to produce biofuels it would be far from meeting the energy demand of U.S alone. Because it would meet only 12% of gasoline demand and 6% of diesel demand.

43. The End! Any Questions?