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james_wagner_-_fp_biofuels.pptxGenetic engineering in Biofuels
Empire State College
Biology I
James Wagner
Why Biofuels?
It’s easy to see our dependence on fossil fuels in every day life. Just by driving down the road, powering some of our homes or even cooking on a gas range or charcoal grill, we are burning through the fossil fuels contained in our planet. With a growing population and increased production and manufacturing, we will need to find a replacement for these non-renewable resources, preferably sooner than later. There are many options for renewable energy sources. However, if you look at our current situation, the transition to renewable fuel would be easiest if it could be utilized in our current equipment with maybe just a few modifications instead of a total replacement. Biofuels seem the likely candidate for this scenario.
What types of biofuel are there?
Biofuels fall into two basic categories:
First Generation
Biofuels which are not sustainable as they are produced from a limited feedstock (the material used for their manufacture). Many of these types of biofuel are derived from the food we eat meaning feedstock is expensive and also widespread use could affect our food supply. Examples of these include biodiesel, typically derived from vegetable oil, some ethanol types commonly manufactured from cane sugar or even whale oil used for generations as lamp oils.
Second Generation
Biofuels considered “Second Generation” are more sustainable in that their feedstocks are, or can be, widely available and would have a much lesser environmental impact than those considered “First Generation”. These “green” biofuels are just now coming into production where as the first generation variety have been used for centuries. Examples include algae based fuels which can have a broad range of uses, anything from methane to jet fuel, these also have the benefit of large production for a small use of land. Another example, and my main focus for this presentation, is cellulosic ethanol.
Good listing of biofuels and types available here : biofuel.org.uk
Vegetable oil biodiesel manufacturing plant
©istockphoto.com/Banks Photos
Biodiesel manufacture from algae
History channel, “Modern Marvels”, 2006
Why aren’t all feedstocks sustainable?
As was mentioned on the previous slide, some biofuels are not sustainable. It may seem that any renewable alternative to fossil fuels would be beneficial when in fact, the production of many of them, especially in large scale, could have a hugely negative impact on our environment. For instance, centuries of whale harvesting for lamp oil production has created a huge decline in their population. In the same way, ethanol produced from corn or sugarcane couldn’t possibly replace gasoline. It is estimated that, “2.5 gallons of ethanol from a bushel of corn (56 pounds or 25.5 kg)” (Pimentel, 2003) is the approximate yield from a large production plant. 1 acre of land yields around 7,110 lbs of corn meaning an acre could produce about 317 gallons of ethanol. In 2015, the US alone used 140.43 billion gallons of gasoline meaning we would need to harvest 443 million acres of corn. It is estimated that we farm approximately 17% of the land in the US (http://data.worldbank.org/indicator/AG.LND.ARBL.ZS). We would need to increase that to 18%, and all of it would need to be corn, just in order to meet our need for gasoline or ethanol.
In the above picture, the area in red represents 1 acre of land area laid over an American football field (blue is a soccer field). For someone who owns a fuel efficient vehicle, corn grown on this land area could supply enough fuel for half of a year to the average driver. It is also estimated this land area could feed a family of 6 for an entire year.
Picture: Wikipedia.
Why cellulosic ethanol is important?
So why is cellulosic ethanol more sustainable than ethanol made from the harvest of the corn? Its all in the amount of available feedstock. In the corn example, through production of ethanol derived from cellulose, we would be able to use the entire corn plant to supply the glucose necessary for ethanol production and not just the easy to get at sugars found in the corn itself. Cellulose is one of the most abundant materials found on earth, it comprises a large portion of all plants since it is a main component of the cell wall in plant cells. As we have learned, cellulose is a polysaccharide made up of a chain of glucose monomers, very similar to a starch. Unlike a starch, however, it has a slightly different structure making hydrolysis slightly more difficult. As we use corn as food and livestock feed, it’s really not a viable source for cellulose, so lets stop looking at that example. Trees however, or even possibly recycled paper, could be a fantastic source of cellulose for the manufacture of ethanol. That, is what makes cellulosic ethanol an exciting alternative fuel!
Shown in the picture are examples of different plants being looked at in specific areas which are candidates for cellulosic ethanol production.
Cellulosic Ethanol Production
Lets start by taking a look at what makes up cellulosic materials.
There are 3 main components to cellulosic material:
Cellulose – ( Cellulose is composed of a long chain of glucose monomers. Cellulose requires some sort of a catalyst in order to undergo hydrolysis for the harvest of the contained 6-carbon sugars. From there, these sugars can undergo fermentation producing ethanol.
Hemicellulose – Hemicellulose differs from cellulose in that along with the 6-carbon sugars, there are also 5-carbon sugars in the long chain of monomers. 5-carbon sugars need to undergo different fermentation from the 6-carbon variety. In order to effectively and efficiently use our feedstock, these will need to be fermented into ethanol as well.
Lignin – Lignin is a very complex organic polymer. It adds to the rigidity of the cell wall. For example, a tree grows tall and therefore needs to be quite rigid. The cells of a tree have a higher lignin content than those of grass or another plant which lays flat. The cellulose and hemicellulose are weaved into a matrix of this lignin, it must first be broken down in order to release the chains of sugars which will be used for ethanol production.
Cellulosic Ethanol Production: Lignin degradation
The major hurdle to production of ethanol from cellulosic feedstocks would be the fact that the lignin acts almost like a glue, effectively trapping our usable materials. The lignin will degrade naturally, but for effective production we need to speed the process up. There are many fungi and bacteria which produce enzymes, particularly lignin peroxidase, which can speed up the oxidation of lignin causing it to break down. These enzymes are currently quite expensive to produce which adds to our cost and thereby viability of cellulosic ethanol.
Small segment of the Lignin polymer
Photo: Wikipedia
Cellulosic Ethanol Production: Hydrolysis
The second step in our production process is hydrolysis. As we have learned, the sugars needed for ethanol are contained in our cellulose and hemicellulose. Hydrolysis needs to occur in order to break down these polysaccharides into simple sugars. There are two main types of hydrolysis which can be used to cause this breakdown.
Acid hydrolysis – By catalyzing hydrolysis of the cellulose with a low concentration acid along with a high temperature, the breakdown into useable sugar if very quick. “0.22 minutes and a temperature of 237°C (458°F) with pure cellulose provided a yield over 50% sugars. In this case, 0.9 t (1 ton) of dry wood would yield about 189 L (50 gallons) of pure ethanol.” (Badger, P.C., 2002) This unfortunately can also result in 5- carbon sugar degradation as it breaks down quicker than the 6-carbon sugars. This decreases total sugar yield. A second way, which slows the process a bit, uses a higher concentration acid at normal temperatures over a longer time. This can result in a higher sugar yield, however more time is required. Especially on a large scale, this can get quite expensive.
Enzymatic hydrolysis – For enzymatic hydrolysis, cellulase can be used to break down our cellulose into sugars. Cellulase can be produced by bacteria and other micro organisms, which will catalyze our hydrolysis reaction. Unfortunately, the cellulase can take a long time to work, several days compared to fractions of a minute. This, once again, results in a high cost of production.
Enzymatic Hydrolysis of Cellulose
Cellulosic Ethanol Production: Fermentation
Our final step in production is fermentation, where our sugars are turned into usable ethanol. Earlier in the course we studied fermentation by yeast. Our glucose molecule underwent a reaction which yielded 2 ethanol () and 2 carbon dioxide (). We can distill this ethanol and we have good a good useable product, our ethanol yield from the 6-carbon glucose molecules of cellulose is complete. What about the 5-carbon sugars contained in the hemicellulose? As with the other steps in our production, we would have a high cost to benefit ratio if we just considered the hemicellulose material as waste.
Ethanol fermentation and distillation is not a new thing. Moonshiners have been distilling ethanol from fermenting glucose obtained from a mash of fruit for hundreds of years.
The costs of Cellulosic Ethanol
Now that the process of creating cellulosic ethanol has been outlined it is easy to see that the entire process, from harvest of feedstock to distillation of ethanol, can get quite expensive. Even using easily harvestable materials, such as in our corn example, the cost of ethanol production is still estimated around $2.15 per gallon (energy.gov, 2015). This doesn’t leave much room for profit most likely placing the cost to consumer in the neighborhood of $5.00 per gallon. In order to make ethanol a viable replacement for it’s fossil fuel counterparts, we will need to substantially decrease this production cost. Fortunately, through genetic modification and biological engineering, we are developing some very significant methods to begin cutting the costs of the processes and also increasing efficiency throughout production.
Genetic Modification and Biological Engineering
As you can see, there are many costly biological processes involved in the production of viable cellulosic ethanol. In order to reduce the production costs we are looking to genetic modification of our many biological variables involved. Over the next few slides I will be taking a look at a few of the many advances we are making, particularly in genetic modification of bacteria, fungi and our initial feedstock, how these processes work, and how they will begin to cut the associated costs.
Photo: earthtimes.org
Genetic modification of feedstock: Lessening lignin production
In production, we saw that a major hurdle to overcome in our process was the separation of the cellulose and hemicellulose from the lignin matrix. What if we were able to modify our initial feedstock so that less lignin is produced? With less lignin, the treatment methods which increase the oxidation of lignin would themselves have less material to work on therefore speeding up the process to free our useable materials.
By modifying the genes responsible for the lignin biosynthetic pathway, this has been accomplished. For instance, by downregulating 4- coumarate:coenzyme A ligase (Pt4CL1), an enzyme in lignin biosynthesis, there was a 45% decrease in lignin along with a 15% increase in cellulose contained in the lignocellulosic matrix of transgenic aspen trees. (Sticklen, 2006) Not only was the decrease in lignin beneficial, but the increase in cellulose also increases our potential sugar yield.
Genetic modification of feedstock: Increasing available polysaccharides in biomass
As shown in the previous example, an increase in available polysaccharide would be very beneficial to the production of biofuel. Through genetic modification of the cellulose biosynthetic pathway, an increase in total biomass for several different plant species was experienced.
One example given involved the tobacco plant. By the addition of a gene known to delay flowering, the Flowering Locus C ( flc) gene, the energy usually spent for reproduction was put into the synthesis of biomass yielding a more robust plant. (Sticklen, 2006)
This photo is of two poplar leaves. The one on the right is from a genetically modified tree.
Photo by: Steven Strauss
Genetic modification of feedstock: Cellulase production in biomass
From start to finish, several enzymes catalyze the reactions required to make ethanol from cellulosic material. Plants naturally need to make these enzymes in order to repair themselves and also to make the sugars available for their own use when needed. Increasing the amount of the enzyme these plants produce should do some of our work for us as far as breaking down the material into useable components. There would also be a decreased need for the production of these enzymes elsewhere.
Commonly, bacteria or other microorganisms are genetically modified to produce the enzymes needed for this type of process. This leads to types of genetic modification outside of our feedstock.
Genetic modification of Microorganisms for use in cellulosic ethanol production
We have found that lignin peroxidase is needed in order to free our cellulose and hemicellulose from the lignocellulosic material. Lignin peroxidase (LiP) is produced by many fungi, one of which, white rot fungus (Phanerochaete chrysosporium) is commonly used to produce LiP for processing of lignocellulosic material. In 1994, Gaskell identified 10 genes responsible for the production of LiP’s and labeled them LipA through LipJ. By causing a rate limiting enzyme to be over expressed, the production of LiP’s by the fungus can be increased, while genes can also be deleted to limit the production of other compounds. The result is fungi which has a significantly increased production of LiP to assist with the oxidation of lignin. (Paudel, 2014)
Photo of White Rot Fungi. This fungus naturally produces LiP’s which allow it to utilize cellulosic material as an energy source.
Photo: Joseph OBrien , USDA Forest Service, Bugwood.org
Genetic modification of yeast and bacteria for the fermentation of 5-carbon sugars.
Yeast has been used for centuries for the production of ethanol from glucose molecules by fermentation. This is great for ethanol production from cellulose, but what about the 5-carbon sugars contained in hemicellulose? Scientists have begun modifying bacteria and yeast in order to allow for the fermentation of these 5-carbon sugars as the 6- carbon glucose is fermenting. Research is still being done as to which is the most effective organism for this process, whether yeast or bacteria, however progress is being made in these efforts. This will significantly increase the final yield of ethanol as, until this point, hemicellulose was considered waste material along with the lignin. This example of genetic modification could also lead to the ability to use waste paper as a viable biomass for ethanol production. (NREL)
Why biofuel?
So far, I’ve given examples of biofuels, an outline of the production process of cellulosic ethanol and examples of advances we are making through genetic modification and biological engineering toward wide spread availability of the process of biofuel manufacture. But still the question remains, why do we need biofuel? Why is it so beneficial when compared to fossil fuel and other means of energy production?
Why biofuel?
To answer this question we need to look at the negatives associated with fossil fuels and compare these to those of biofuels. As this could be a paper in itself, I’d like to just touch on a few subjects that I believe make biofuels a stand out favorite over fossil fuel.
Biofuel is renewable.
Biofuel can reduce greenhouse gas emissions and the growth of feedstock can even help convert existing greenhouse gasses into oxygen.
Reduced cost of fuel.
Increase of fuel availability
Renewability
Fossil fuels are formed over millennia of heat and pressure deep in the earth. Although it is tough to say how much there actually is available, we can tell that we are burning through it way faster than it is being made. At the risk of running out, we need to find an alternative energy source that can be manufactured as quickly as it is being used. Streamlined biofuel production using the procedures we’ve outlined in this presentation could be a great start to meeting this production : consumption ratio.
Greenhouse Gases
Greenhouse gases are defined as gasses which absorb infrared radiation contributing to the greenhouse effect. Carbon dioxide is a big one of these and is produced not only by the burning of fossil fuels but also their production. By using plant based feedstocks, this existing carbon dioxide will be turned into oxygen and sugars as the plants perform photosynthesis. The fermentation process does produce some carbon dioxide, but this can even be re-used to create more sugars if the process is streamlined. Burning of ethanol does also still produce carbon dioxide, however, the amount per kg of ethanol compared to gasoline is substantially reduced.
Reduced Fuel Cost
As our knowledge of the process of biofuel production increases, our cost related to the production is decreasing. Through genetic modification it is estimated that the cost of cellulosic ethanol has been reduced to between $.30 and $.50 per gallon (Sticklen, 2006) The cost of crude oil, before gasoline manufacture even begins, is about $.93 a gallon. (energy almanac) As the process is refined farther and farther we will only see decreases in the cost of ethanol production.
Availability of fuel
Fossil fuels aren’t easily available. Through advances in transportation and pipelines throughout the world, we have made it possible to move oil and fossil fuels to where they are needed. Manufacture of biofuels, however, can be accomplished pretty much anywhere. For instance, there are advances being made with algae which allows it to directly produce just about any form of fuel needed: jet fuel, methane, ethanol. Granted, the technology isn’t quite there yet, but if there is water, sunlight and a way to harvest the products, the algae could theoretically run everything we have.
Conclusion
In the not so distant future, we are going to need to significantly reduce our dependence on fossil fuels. There is a need for an alternative fuel source to step up and fill that hole. Through advances in genetic engineering, we may have already found a sustainable, available and viable candidate. Where the costs of fossil fuels rise as our supplies begin to dwindle, the cost of biofuels should decrease as they become more and more available. Hopefully, in the near future, we will be able to start cleaning up the mess we have made over the relatively short time we have been burning fossil fuels.
Works cited
David Pimentel, “Ethanol Fuels: Energy Balance, Economics, and Environmental Impacts are Negative”, Natural Resources Research, Vol. 12, No. 2, June 2003
http://www.energyjustice.net/files/ethanol/pimentel2003.pdf
Mariam Sticklen , “Plant genetic engineering to improve biomass characteristics for biofuels”, Current Opinion in Biotechnology 2006, 17:315–319
https://www.msu.edu/~stickle1/Sticklen_Curr%20Opinion_paper.pdf
Paudel, Sudip and Menze, Michael A., "Genetic engineering, a hope for sustainable biofuel production: review" (2014). Faculty Research & Creative Activity. Paper 273.
http://thekeep.eiu.edu/bio_fac/273
Badger, P.C. 2002. Ethanol from cellulose: A general review. p. 17–21. In: J. Janick and A. Whipkey (eds.), Trends in new crops and new uses. ASHS Press, Alexandria, VA.
https://www.hort.purdue.edu/newcrop/ncnu02/v5-017.html
Jason Hill, Erik Nelson, David Tilman, Steven Polasky, Douglas Tiffany, “Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels”, Departments of Ecology, Evolution, and Behavior and Applied Economics, University of Minnesota; and Department of Biology, St. Olaf College, Northfield June 2, 2006
http://www.pnas.org/content/103/30/11206.full#ack-1
NREL, “Ethanol from Biomass: The Five-Carbon Solution”
http://www.nrel.gov/docs/legosti/old/5682.pdf
Energy.gov
http://energy.gov/eere/articles/energy-department-helping-lower-biofuel-costs-nation
Biofuel.org
http://biofuel.org.uk/types-of-biofuels.html
