Team:Purdue/TheProblem

• INTRODUCTION
• BACKGROUND
  • The Problem
  • Organisms
  • Enzymes
• PROJECT
  • Description
  • Project Development
  • Our Solution
• PARTS
  • Team Parts
  • Basic Parts
  • Composite Parts
  • Part Collection
  • Parts Used
  • Parts Submitted
• NOTEBOOK
• ATTRIBUTIONS
• OUR PROCESS
• HUMAN PRACTICES
• SAFETY
• MODELING

The Problem

Biofuels

Biofuels are fuels derived from organic matter making them an available and reliable renewable energy source. Biofuel production is important in terms of energy security and greenhouse gas mitigation. Biofuels are made in a variety of ways using different sources of biomass and yield different fuel sources including ethanol, methane and biodiesel. The different sources for biofuels are used to categorize biofuels into 4 rough categories ("What are Biofuels").

The first category, first generation biofuels includes biodiesel, bioalcohols, ethanol, and biogasses made from simple sugars, starches, oils, and fats. These biofuel production methods have a high yield in a relatively short amount of time ("Biofuels Basics"). The biggest problem with first generation biofuels, however, is their use of food commodities and the chemicals and energy used in the production. First generation biofuels are subject to the "Food versus Fuel" debate, are limited in their impact on greenhouse gas emissions, and have the potential to negatively impact biodiversity by incentivising farmers to monoculture their farmland. First generation biofuels are largely unsustainable for these reasons (Sims).Second generation biofuels solve many of the environmental and social issues involved with first generation biofuel production. Second generation biofuels are produced using lignocellulosic biomasss which includes non-feedstock plant material (such as switch grass, corn stover, and oilseeds) and agricultural waste ("Biofuel Basics"). However, second generation biofuels have economic and logistical problems. The logistic network for second generation biofuel production is largely non-existent creating large barriers to entry in the field (Sims). A large reason for the lack of a logistics network is the difficulty in breaching the lignin barrier, lack of demand for non-feedstock plant material, and the expense involved in doing so either chemically or with high heat and pressure treatment coupled with the relative ease and efficiency of first-generation biofuel production (Naik).

Third and fourth generation production are still in early development (Sims). Third generation production of biofuels involves using quickly growing cellulosic biomass such as algae. Fourth generation involves plants specifically engineered to have less lignin making it easier to use the cellulosic biomass (Putri). However, these technologies are still young, expensive, and largely untested compared to first and second generation biofuels (Sims).

Production of second generation biofuels would be preferable to first generation if the two were equally economically feasible as second generation biofuel production does not have as many environmental and social issues as first generation production. The goal for second generation biofuel produciton then is to lower the cost of ligno-cellulosic biofuels compared to the first generation counterparts by making it easier to bypass the lignin barrier.

Sources:

• "Biofuels Basics." National Renewable Energy Laboratory. National Renewable Energy Laboratory, 3 Feb. 2015. Web. 10 Sept. 2015.
• Naik, S. N., Vaibhav V. Goud, Prasant K. Rout, and Ajay K. Dalai. "Production of First and Second Generation Biofuels: A Comprehensive Review." Science Direct. Renewable and Sustainable Energy Reviews, 9 Nov. 2009. Web. 18 Sept. 2015.
• Sims, Ralph, and Michael Taylor. From 1st to 2nd Generation Biofuel Technologies: An Overview of Current Industry and RD&D Activities. Paris: IEA, 2008. International Energy Agency. International Energy Agency, Nov. 2008. Web. 10 Sept. 2015.
• "What Are Biofuels?" Green Choices. Cornell University, n.d. Web. 10 Sept. 2015.

Biodigestion

Biodigestion involves the decomposition of organic matter by bacteria in an anaerobic environment in order to produce a fuel source (biogas, bioethanol, biodiesel, etc.). Biodigestion can be divided into four main stages: Pre-treatment, Digestion, Processing/Filtration, and Reuse/Disposal of Waste (Putri).

Pretreatment for biodigestion in bioreactors or biodigestors for lignocellulosic biomass can take many forms, but is always energy and/or chemically intensive. Pretreatment can be done using uncatalyzed steam explosion, liquid hot water pretreatments, acid catalyzatation, flow through acid pretreatment, lime and other alkali pretreatment processes, and AFEX (Ammonia Fiber/Freeze Explosion) pretreatment. All of these methods require extremely high temperatures (120 - 300 C), pressures (up to 400 psig), or use expensive chemicals. The high costs of pretreatment for lignocellulosic biomass make second generation biofulel produciton a costly and less efficient process compared to production of first generation biofuels (Mosier).

Digestion itself can be divided into two categories: Mesophilic digestion and thermophilic digestion. Mesophilic digestion is more energy efficient as it can be done between 20 and 40 degrees Celsius; however, the entire process takes up to two months. Mesophilic digestion is often done in batch reactors. These batch reactors are single chamber vessels with an agitator at the bottom and a metal jacket used for heating or cooling. For mesophilic biodigestion, there are batch reactors for each step (pretreatment, digestion, and fermentation). These reactors are left alone after material is added until the reaction is completed. Once one step is done the mixture is moved to a different batch reactor for the next step (Kleijntjens).

Meanwhile, thermophilic produces more biofuel in a much shorter time period, but comes with higher energy costs to maintain an environment between 50 and 65 degrees Celsius. Furthermore, bioreactors capable of thermophilic digestion are more expensive and the bacteria are more sensitive to environmental changes (Petri).

The Processing/Filtration step involves preparing the desired biofuel for use after the process is complete. For ethanol bioreactors this step would involve distillation while for biogas this step would involve scrubbers in order to remove CO2 and impurities. This step varies largely for different types of biofuel but is not dependent on the input material.

Reuse or Disposal of solids is the final step in biodigestion. The solids left over are very nutrient rich making them perfect for use as fertilizer. However, if there are toxins present in the byproduct it must be disposed of in biohazard waste containers (Petri).

Sources:

• Kleijntjens, Rene H., and Karel Ch. A, M. Luyben. "Bioreactors." 14 Bioreactors (n.d.): 335-38. Web. 10 Sept. 2015.
• Mosier, Nathan, Charles Wyman, Bruce Dale, Richard Elander, Y. Y. Lee, Mark Holtzapple, and Michael Ladisch. "Features of Promising Technologies for Pretreatment of Lignocellulosic Biomass." Bioresource Technology 96.6 (2005): 673-86. Science Direct. Web. 10 Sept. 2015.
• Putri, Ratna E. "Biodigestion." Biodigestion. Student Energy, n.d. Web. 10 Sept. 2015.

Pretreatments

Current pretreatments in biodigestion range from thermal to chemical to mechanical (Montgomery et al, 2014). Mechanical pretreatment involves the physical cutting up of plant matter to increase surface area and is often coupled with thermal treatment. Thermal treatment, which takes the form of either thermal hydrolysis, extrusion, or steam explosion, uses a combination of heat and pressure to wash the plant matter with water nearing 200°C. This causes the lignin, a hydrophobic molecule, to globuralize into spheres, a means of decreasing its surface area to volume ratio. Chemical pretreatment, which is not used at industrial scales, uses a mix of acid and base at different temperatures to make the lignin layer more susceptible to the biodigesting microorganisms. A biological alternative for pretreatment that would eliminate the large energy input required for thermal pretreatment would greatly benefit the efficiency of biodigestion. However, no effective, scalable solution has been developed to date.

When the lignin polymer is broken down by various natural enzymes, the byproducts include phenolic compounds such as phenol, guaiacol, and syringol (Kleinert and Barth, 2008). It has been shown that these lignin-derived phenols inhibit the activity of cellulases and glucosidases (Ximenes et al, 2010). The extent to which these phenols non-productively bind to the lignin has been further studied and found to be directly proportional to the quantity of residual lignin present after pretreatment (Gao et al, 2014). A different study showed the lignin’s role in blocking the surface of cellulose was the major factor in inhibiting enzymatic activity (Li et al, 2013). Although which plays a larger role is still in debate, the two ways lignin inhibits cellulase activity, surface blocking and nonspecific binding, represent the challenges that must be circumvented to have efficient pretreatment of lignocellulosic materials.

Multiple strategies have been investigated to deal with the issue of cellulase inhibition by lignin residuals. One main strategy is to find molecules that will preferentially bind to lignin and block cellulases from doing so. One candidate for such a molecule is bacterial expansins, which have been found to have a high degree of binding with lignin compounds (Kim et al, 2012). Bacterial expansins are thought to have evolved as a method for soil bacteria to form biofilms around plant roots to participate in symbiotic rhizosphere interactions (Georgelis et al, 2015), which would explain their high affinity for lignin. Another candidate lignin blocker is the compound BSA, which was shown to improve glucose production when applied to an enzymatic mixture (Ko et al, 2015). One possibility for stopping inhibition by lignin is the production of these lignin-blockers in conjunction with production of lignin breaking down enzymes. In a completely different route, protein engineering has been applied to cellulases to lower their affinity to nonproductively bind to lignin. Succinylation and acetylation of T. reesi cellulase resulted in twofold cellulose conversion without lowering the km of the cellulase when placed in an ionic liquid (Nordwald et al, 2014).

Lastly, the interactions of lignin and its byproducts with cellulose breaking down enzymes is still not fully understood. A recent study showed that the addition of laccase, a detoxifying agent that was originally thought to increase cellulose conversion, actually lowered the glucose output (Oliva-Taravilla et al, 2015).

The 2015 iGEM project has focused around designing a microbial pretreatment system to couple with a recent synthetic yeast biodigestion platform that internalizes nearly all of the biodigesting reactions inside the yeast cell (Wei et al, 2015), including the breakdown of cellobiose to glucose monomers via β-glucosidase. This way, lignin can be broken down without inhibiting the fermentation of glucose. The project centers around expressing termite-based lignin-degrading enzymes in a synthetic yeast cell, which has not been done before in enzymatic pretreatment. Finally, the performance of the individual enzymes and combinations of them will be compared to determine the most effective enzyme cocktail.

Sources:

• Gao, D., Haarmeyer, C., Balan, ., Whitehead, T.A., Dale, B.E., Chundawat, S.P. (2014), Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification. Biotechnology for Biofuels, 7(2014): 175. doi:10.1186/s13068-014-0175-x
• Georgelis, N., Nikolaidis, N., Cosgrove, D.J. (2015), Bacterial expansins and related proteins from the world of microbes. Appl Microbiol Biotechnol 99(9):3807-3823. doi: 10.1007/s00253-015-6534-0
• Kim IJ, Ko HJ, Kim TW, Nam KH, Choi IG, Kim KH (2013) Binding characteristics of a bacterial expansin (BsEXLX1) for various types of pretreated lignocellulose. Appl Microbiol Biotechnol 97(12):5381–5388. doi:10.​1007/​s00253-012-4412-6
• Kleinert, M. and Barth, T. (2008), Phenols from Lignin. Chem. Eng. Technol., 31: 736–745. doi: 10.1002/ceat.200800073
• Ko, J. K., Kim, Y., Ximenes, E. and Ladisch, M. R. (2015), Effect of liquid hot water pretreatment severity on properties of hardwood lignin and enzymatic hydrolysis of cellulose. Biotechnol. Bioeng., 112: 252–262. doi: 10.1002/bit.25349
• Li, H., Pu, Y., Kumar, R., Ragauskas, A. J. and Wyman, C. E. (2014), Investigation of lignin deposition on cellulose during hydrothermal pretreatment, its effect on cellulose hydrolysis, and underlying mechanisms. Biotechnol. Bioeng., 111: 485–492. doi: 10.1002/bit.25108
• Montgomery, L.F.R., Bochman, G. (2014), Pretreatment of feedstock for enhanced biogas production. IEA Bioenergy.
• Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M.. (2005), Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol., 96 (2005):673–686. doi: 10.1016/j.biortech.2004.06.025
• Nordwald, E. M., Brunecky, R., Himmel, M. E., Beckham, G. T. and Kaar, J. L. (2014), Charge engineering of cellulases improves ionic liquid tolerance and reduces lignin inhibition. Biotechnol. Bioeng., 111: 1541–1549. doi: 10.1002/bit.25216
• Oliva-Taravilla, A., Moreno, A.D., Demuez, M., Ibarra, D., Tomas-Pejo, E., Gonzalez-Fernandez, C., Ballesteros, M. (2015), Unraveling the effects of laccase treatment on enzymatic hydrolysis of steam-exploded wheat straw. Bioresource Technology, 175(2015): 209-215. doi:10.1016/j.biortech.2014.10.086
• Wei, N., Oh, E.J., Million, G., Cate, J.H.D., Jin, Y.S.. (2015). Simultaneous Utilization of Cellobiose, Xylose, and Acetic Acid from Lignocellulosic Biomass for Biofuel Production by an Engineered Yeast Platform. ACS Synth Biol. doi: 10.1021/sb500364q
• Ximenes, E., Kim, Y., Mosier, N., Dien, B., Ladisch, M.. (2010), Inhibition of cellulases by phenols. Enzyme Microb Technol, 46 (2010): 170–176. doi:10.1016/j.enzmictec.2009.11.001

Bioreactors

In order to produce biofuels, industries and researchers use what is known as bioreactors. Bioreactors are machines utilized to convert multiple substances into products (such as: pharmaceuticals, chemical detergents, food, and biofuels) through biological or chemical processes.

An image that shows the process of the removal of lignin and the production of ethanol at large industries.

Bioreactors that produce biofuels, such as ethanol, convert biomass into biofuel in batches; they are a part of a class of bioreactors known as batch-reactors. (Batch-reactors gradually transform reactants, step-by-step, through chemical and biological reactions). There are at least three main steps that must be used to produce biofuels: a pretreatment process, liquefaction process, and fermentation process. To efficiently produce biofuels, bioreactors must separate these processes because each microbe and enzyme have different requirements and conditions that must be met in order for them to produce their desired outcomes. (For example, different optimal pHs and temperatures.)

Sources:

• RETSECK, GEORGE. Is Ethanol the Long Haul? Digital image. Nature. Scientific America, 2007. Web. 2015.