2010-4-20 Session on Lignin modification in Panicum virgatum L. by Fu, Mielenz, Xiao, Hamilton, Chen, Bouton, Dixon, and Wang

The 2:00 session on Tuesday (2010-4-20) was on lignin modification in order to increase ethanol production efficiency in switchgrass (Panicum virgatum L.). The rationale for researching lignin was that lignin is a structural and physical barrier to polysaccharide degradation and they looked at switchgrass because it is a native species that would not harm biodiversity. Fu, Mielenz, Xiao, Hamilton, Chen, Bouton, Dixon, and Wang used transgenic methods to modify the biosynthesis of lignin and, by this, increase the bioconversion properties in switchgrass. They isolated the primary genes involved with lignin production (using PCR) and found that down regulation of a particular gene resulted in decreased lignin content. This, in turn, improved sugar recovery. They found that a lower S/G ratio was linked with carbohydrate conversion efficiency.

Summary of the 1:00 session on the Genetic dissection of bioenergy traits in Sirghum by Vermerris, Saballos, Kresovich, Murray, Rooney, Pederson, Sattler, and Xin

There has recently been a substantial amount of research done on sorghum as a biofuel feedstock because of its low water usage and fertilizer requirements. In the first session after lunch on Tuesday April 20, 2010, Vermerris, Saballos, Kresovich, Murray, Rooney, Pederson, Sattler, and Xin research on the genetics of sorghum was presented. They said that Sorghum is best known as a grain crop, but sweet sorghum is very similar to sugar cane in chemical composition. There is also forage sorghum. Sorghum plants are diploid, C-4, annuals that are produced from seeds; they have low-input requirements and a high yield potential. The genome has been sequenced. When sugar production is examined, sorghum “stacks up favorably against sugar cane and switchgrass,” according to the presentor. According to Vermerris et al., the ultimate goal in sustainable biofuel production is to minimize inputs and maximize outputs—this applies to grains, biomass, and sugar. They would ideally like to optimize crops to produce food, fodder, heat, biofuels, and biproducts.They said that sugar accumulation is poorly understood and that it depends on juice volume and cell wall modification. Their research is being done on several groups of Brown Rib Mutants; two of these mutants are the Bmr6 and the Bmr 2 mutants. Of the 19 Bmr mutants, Bmr 2 and Bmr 6 are the most promising for genetic modification. The mutants are being used because they are easier to convert to higher sugar yielding plants. There are 14 different copies of this one particular gene related to sugar production in sorghum, so it has taken them a long time to actually isolate it. Eventually, they succeeded. Vermerris et al. used several mapping techniques and then different cloning techniques in their research. The results will be published later this year.

Algenol session at Clearwater conference (Blog post 1 from conference)

At the 8:30 session on Tuesday morning (2010-4-20), a representative of the company Algenol presented information about a new technique for producing ethanol from algae with carbon dioxide. Algenol is a developmental stage company, founded by Paul Woods, Ed Legere, and Craig Smith, that will be opening a pilot scale refinery next year. It is largely based around several patents owned by Woods. They have several partners involved with their research including Dow Chemical Company that is involved with photobioreactors, Linde Gas AG that works on CO2 supply for the algae, Biofields S.A. that deals with commercial development in Mexico, and Georgia Tech. that has been researching gas separation between water and ethanol. The company was originally based out of Switzerland but has moved its headquarters to Baltimore. The company is now moving its lab to Naples (near the homes of Woods and Smith). The pilot biorefinery will be in Texas. This new biorefinery is being government funded and will be built on 17 acres. They are working on genetically engineering several hybrids of marine algae species to produce ethanol efficiently when supplied with carbon dioxide. Many of their current species are cyanobacteria that they have modified by adding pyruvate decarboxylase and alcohol dehydrogenase to the genomes. What is unique about their photobioreactor is that it does not require the algae to be dead for harvesting the ethanol. These modified algae produce ethanol that diffuses through the cell wall. It then evaporates with water. The biorefinery captures this once it condenses using some unique channels along the walls. The mixture of ethanol and water is then distilled. At the moment, these algae are only producing abou 1.5 moles of ethanol per meter squared per week. This is a relatively small amount adding up to about 5,000 gallons per acre per year. Through their genetic engineering processes, Algenol intends to improve this.

11:00 session at Clearwater conference on microalgae feedstocks

The 11:00 session on Tuesday (2010-4-20) was about the development of microalgae as a biomass for biofuels. Micro aquatic crops such as duckweed, macroalgae, and microalgae are all currently being researched; this group of researchers, A. Darzins, L. Elliot, L. Laurens, E.J. Wolfrum, and M. Posewitz, is focused on microalgae. They mention the 30 year government study that researched algae based biofuels. This is the same government study that Ian Woertz talks about in his research article on algae based biofuels grown on wastewater streams. The government study ended in 1996. However, the program was essentially revived by the NREL in 2006. This study is continuing the research of that 30 year study. This new projects goal was to isolate and characterize microalgal strains utilizing high throughput techniques. They have research locations in Colorado, New Mexico, and several other locations elsewhere. Freshwater, brackish water, and saline/brine waters were brought back to the lab and they put one alga cell and 1 droplet into 96 well plate and allowed them to propagate themselves. High speed FACS sorting was utilized. They did clonal isolation and then used image positive brightfield microscopy. The green algae were found to be the most prominent, but many were fast growing. They currently think that the water vacuole in diatoms is lipids, but this remains a hypothesis for the time being. They are now working on a “bioprospecting project” using FACS, or florescence activated cell sorting. They are also studying high throughput methods for lipid extraction and identificaltion.

The use of algae and duckweed for wastewater treatment (Shi et al., 2010)

Estrogen is a female hormone that many people take in the form of medication. However, the human body never fully uses any medication and some of it passes through the body and is excreted. This estrogen, not used by humans, is carried in the waste stream to wastewater treatment plants. Unfortunately, most wastewater treatment plats have no way of removing. Without treatment, the estrogen flows through the plat, into streams or lakes where it is consumed by fish, frogs, and other amphibians. The animals that consume this estrogen are, not surprisingly affected by it; over time, these communities of fish and amphibians become more feminine—either by having higher percentages of females in the communities or by having feminine characteristics develop in males (this can occur to the extent of producing androgenous individuals).

A study conducted by Shi et al. was published in March of this year in Environmental Science and Pollution Research; they examined the fate of endocrine disrupting compounds (EDCs) in algae and duckweed wastewater treatment ponds (Shi et al., 2010). The EDCs 17α-ethinylestradiol (a synthetic estrogen compound), estrone (a naturally occurring estrogen form), and 17 β-estradiol (also naturally occurring) are commonly found in domestic sewage and were hence chosen for Shi et al.’s study (Shi et al., 2010). They used a species of Lemna duckweed (Shi et al., 2010). Several species of algae were used for the study including the following species: Anabaena cylindrical, Chlorococcus sp., Spirulina platensis, Chlorella sp., Scenedesmus quadricauda, and another Anaebena variant (Shi et al., 2010).

They conducted continuous-flow studies and batch tests (Shi et al., 2010). The batch test was carried out for 6 days; this time period, they found that algae or duckweed greatly helped with the removal of estrogens (Shi et al., 2010). The duckweed removed a higher percentage of estrogens than did the algae, but both were effective (Shi et al., 2010). Similar results were observed in the continuous-flow model. The continuous-flow system consisted of an initial pond and then two following tanks (Shi et al., 2010). The first pond was the most effective of these three: the duckweed first pond removed 85.4% of estrogens and the algae pond removed 76.8% (Shi et al., 2010). The following tanks removed 7.1 and 8.9 % for algae and duckweed respectively (Shi et al., 2010). Hence, the continuous-flow algae system removed a total of 83.9% of estrogens and the continuous-flow duckweed system removed 95.4% (Shi et al., 2010).

Some of the genera of algae studied here are also being studied by biofuel researchers. This system of wastewater treatment could be coupled with a biofuel production system.

Clearwater Conference

The program started with an opening speech given by the program Chairman Thomas who gave a tribute to the father of Biofuels, Ray Katzen whom the 32^nd Symposium on Biotechnology for fuels and chemicals was dedicated. He said that according to Dr. Katzen, black crude is bad, green biomass is good!; this is the first law of motor fuel according to Ray Katzen. In 1978, the US government was desperate to establish a new road map for biofuels. Ray Katzen conducted a study and sold 30,000 copies; one of the maximum copies sold ever by the US government. Ray is also known as “Doctor who turned on the lights”. He established industries in different parts of the world such as Cuba, and Switzerland to name a few. His proposed Project 20 aims to produce 20 billion gallons of biofuels by 2020. I was highly inspired by the works done by Dr. Katzen and thought that this opening speech was a great way to start the symposium.

Blog #1: Monday, Session 1 @ 1 pm, Increasing ethanol productivity from xylose in recombinant Saccharomyces cerevisiae by protein engineering.

This talk was based on the mechanism of pre-treatment that reduces inhibitors such as furfural, which comes from hemicelluloses. The purpose of their study was to engineer a protein involved in the metabolism in order to maximize yields and to increase rates. Cellulose is broken down into glucose (hexose) and hemicellulose is broken down into pentoses. Their research was focused mainly on the intermediate product, pyruvate. Metabolic evolution of E.coli was done to get the desired E. Coli, mutants that were resistant to furfural from xylose and arabinose and hydroxy methyl furfural (HMF) from glucose, fructose, mannose, galactose, etc. Betaine was used to prevent the evolved E. coli cells from bursting. Furfural resistant strains of E. coli were cross-resistant to HMF.

They did a micro-array analysis of RNA of an E. coli to check for the expression analysis of the E. coli. Proteins that are NADPH-dependent oxido-reductases that reduce furfural and HMF were studied. Expression of both genes is reduced in furfural-resistant E. coli. The researchers then deleted certain genes and looked at the expression of certain genes as well as mutanized genes done by evolution. They detected the expression of these genes through microarrays. They also checked the activity of the promoter and the expression and regulation of genes via real-time PCR. Firefly luciferase promoter activity was used to detect the promoter. This presentation was mostly about changing the pathway that makes pyruvate and looking at the effect on the product formation. Normally, amino acids such as cysteine and thionine are not made in large quantity. Genes that were able to reduce furfural and HMF were determined. Expression of such genes from the determined promoter (yqhD) was aldehyde inducible.

Blog #2: Tuesday, Session 4 @ 8:30-9am, A novel, cost-effective method for producing ethanol from carbon dioxide in hybrid algae.

Scientific basis of direct to ethanol process: This talk was from a person representing Algenol Biofuels Inc., which was founded in 2006 and has collaborations with multiple countries across the world such as Germany, Spain, Switzerland, USA- Baltimore and Florida where the headquarter is located. The speaker based his study from Coleman and Deng (1999) who studied linking the Calvin cycle with exogenous fermentation pathway. According to the speaker, the biggest challenge is in producing ethanol in a commercial scale. Their goal was to produce ethanol from carbon dioxide using hybrid algae that are grown in seawater and contained in enclosed bioreactors. The main approach in doing so was to channel as much pyruvate as possible into the fermentation pathway in algae especially targeting the alcohol dehydrogenase. Their study used large flexible plastic bag into which seawater and algae are placed. Nutrients were then introduced periodically into the culture and during the course of the day; the bioreactor was heated, where ethanol is produced from the algae. Heat transfer across the plastic occurs and condensate forms in the surface when the temperature drops during the day. The condensate that is produced is 2-3 times more concentrated ethanol. The condensate is then collected which is run through a processor, which will boost ethanol yield. In order to maintain the salinity within the tolerance of the algae, fresh water must be returned back into the bioreactor. According to the speaker, this utilizes less gallons of fresh water than ethanol produced which is therefore a water saving process.Their goals to be achieved are as follows: Ethanol produced of 6,000-10,000 ga/acre/yr; Capital costs under 5$/ga capacity; Operating cost less than $1/ga/yr, initially hope to be around 1.25$/ga/yr. Another advantage of their method was that there was no competition with feedstock, and marginal or abandoned lands were used.

Blog #3: Tuesday, Session 5 @ 1:30pm, Molecular factors associated with altered cell wall chemistry in Populus.

In order to better understand how plant cell walls are formed and how the composition and properties of lignocellulosic feedstock materials are controlled, a research strategy was developed. The speakers talked about this study that consisted the use of –omics, spectroscopy, sugar release assay, imaging and bioinformatics approaches to characterize a selected set of Populus samples. Populus trees vary greatly in their cell wall compositions and salient individuals were used in sugar release assays. They also characterized a wild-type Populus plants under tension stress in controlled greenhouse conditions; such usage of wood would help in understanding the wood’s tension capabilities, lignin content, cellulose content and xylem composition. New genes in cellulose biosynthesis pathway were determined via the Illumina-based transcriptome and LC-MS/MS proteome profiling datasets. The speaker then presented preliminary results from functional genomics investigations using transgenic Populus plants.

Blog #4: Wednesday, Session 7 @ 10 am, An integrated analysis tool to guide sustainable biomass production.

Iowa used to be the maid of prairie during 1833-1890. According to the speaker, land use change responds to increase in agricultural commodity prices in ways such as: 1. Extensification, 2. Substitution, and 3. Intensification. Extensification relates to bringing new land into production. CRP acres are present in a lot of quantity in Iowa. Although CRP land is less good land for corn, it is not horrible especially those in Iowa. Hill slopes in Iowa that are more prone to erosion were studied. Soybean is more susceptible to erosion than corn. If crops that are planted in the land are changed, you change the chances of erosion in those lands too. E.g. if soybean is replaced with corn or switch grass, erosion is greatly reduced. Substitution means changing the crops planted in the land, e.g. putting perennial grasses into land. If a landscape is re-shaped into one way, it affects the moisture and rainfall in other way in different landscape. The ethanol plants and the corn-corn plants do not coincide. Soybeans do not take up much nitrogen and so you take corn-corn rotation with distance from ethanol plants. This relates to intensification where the nitrogen in the soil is increased. The speaker also discussed some of the integrated tools to guide land use choices. Soil carbon model was presented as well as the social and economic impacts of land use changes were discussed.

One of the statements said by the speaker drew my attention: “Land use is driven by complex social-economic-technological changes.” It made me realize that it is a vicious circle as to how people use land for different purposes at the same time, different social changes affects people’s attitudes regarding land use changes as well.

Blog #5: Thursday, Session 9 @ 9am, Biomass supply and logistics: A Tennessee perspective.

The talk by Mr. Jackson, representing Genera Energy in Knoxville, Tennessee was about Tennessee and the people’s perspective for biofuels. He collaborated with a company that works with local framers to develop switch grass production, which is a potential biofuel feedstock. In order to provide sufficient feedstock, the state has a statewide commitment of $70.5 million. The speaker then talked about how they would go about in working with the farmers to grow ample switchgrass that can make sufficient biofuels. Their current crop is corn but they are planning to use switchgrass pretty soon. The speaker also mentioned about how the farmers are educated and taught through seminars; the farmers are paid $450/acre/year for costs of production and opportunity costs while the switchgrass seeds and the technical expertise are provided to them for free. The drawbacks of using switchgrass are that their cost is increasing as well as the seeds are hard to find. Moreover, the right herbicide for switchgrass hasn’t been found and so trying to figure this out would increase the cost of the switchgrass since the yield might be low. His talk pointed out that much of the research is needed to make the use of biofuels possible which starts with trying to find the right feedstock and in adequate quantity.

Novel mechanism to generate biofuel from bacterium

The Department of Energy’s Advanced Research Projects Agency-Energy (ARPA-E) has again given a grant of $1million to another project led by Derek Lovley and his team at the University of Massachusetts (Amherst).  The team is working with Geobacter bacterium to produce butanol costing as little as $2 per gallon.  The interesting part is that the fuel is not produced through conventional photosynthesis from sugar, but through electricity.  Another team at Harvard Medical School’s Wyss Institute is working with Shewanella oneidensis to produce octanol.   Because this new mechanism has no official name, they explain it as an equivalent of a reverse fuel cell.

They grow these bacteria on a surface of a graphite electrode by using solar panel energy, which is 100times more efficient at capturing sunlight. Naturally, these bacteria generate electricity through their long protein tubes.  But the researchers can reverse-engineer this to make the bacteria take up electrons from the electrode.  And with an addition of a photosynthetic pathway modification n these bacteria allow them to generate butanol or octanol.

New York Times, April 30, 2010

100% Biodiesel to be Tested in Cars in Finland

Neste Oil, the world’s leading supplier of renewable diesel, is a refining and marketing company located in Finland and focuses on low-emission, high–quality traffic fuels.  They have announced that they’ll be doing a trial of Neste Green 100 diesel produced from 100% renewable raw materials.  The test drivers will fill up at certain Neste oil gas stations and will continue from mid-May till Midsummer.  Previously they have shown that the fuel’s GHG over its life cycle is 40-80% lower than conventional diesel fuels.  Currently, Neste Oil provides a blend of minimum of 10% renewable diesel with regular diesel all over Finland, but hope to make it 100% renewable diesel in the future.

Making Bio-petroleum

Chemical engineers, Savage with his team, at University of Michigan are doing research to produce bio-petroleum from microalgae.  They are applying heat and pressure, the method Nature uses to make petroleum naturally, to make oil from microalgae faster and to also use all the by-products as feedstock for more biofuel.  Algal biofuel looks very promising because it could be efficient (one company promise to produce 1billion gallons of algal fuel annually by 2025), it’s carbon neutral, and compatible with existing infrastructure.  But one concern is the quality of this fuel and their performance in cold temperatures.  Microscopically, the high temperature and pressure allow the algae to break down in the water, yielding crude oil.  This is exactly the same process petroleum goes under over millennia under ground, except it’s much faster.

(Wired.com, Making Oil in Minutes, Not Millennia, April 30, 2010)

Federal and Local Grants allow Research on Extremophiles

North Carolina State University has won a grant of $2.7 for three years from the U.S. Department of Energy’s Advanced Research Projects Agency to do research on extremophiles for biofuel production.  Dr. Robert Kelly, the principal investigator, will be working with Dr. Michael Adams of University of Georgia to genetically engineer these extremophiles,specifically Metallosphaera sedula and Pyrococcus furiosus.  Extremophiles are microbial organisms that live in high-temperature environments (75-100C) in freshwater and saltwater. They take CO2 from the environment and produce acetyl-coA, building blocks for biofuels. The researchers are interested in combining the two microbes’ abilities together to produce butanol.  These microbes are also good candidates because their extremophile nature allow them to withstand the high temperatures of distillation process and the fact that they don’t require light allow facilities to be set up anywhere.

Another local energy initiative is also happening in Kentucky. The Kentucky Agrilcultural Development Board is allowing funds from Agricultural Development and American Recovery and Reinvestment Acts to assist energy-related projects by making $100,000 available. (Portsmouth Daily Times:  Agricultural energy initiative announced, April 4, 2010)