Meganucleases, the End of GMO plants?

By Daniela Sciaky, Ph.D.

Advancements in DNA sequencing technology have produced a large library of completed plant genome sequences. These sequences are an important step to the introduction of desirable characteristics into plants such as soybean and corn, including those traits important to the production of biofuels. Coupled with these sequences are advancements in the technology to breed plants for these traits.

Cellectis plant sciences, a subsidiary of Cellectis (Alternext: ALCLS) designs meganucleases that can be used to make targeted gene modifications. Recently the company announced the licensing of PureIntro® Agrobacterium transformation technology for monocotyledonous plants such as corn and rice from Japan Tobacco Inc. and Aerosol Beam Injector technology from Midwest Oilseeds Inc. These tools will be used by Cellectis plant sciences to modify plant cells by helping to deliver meganucleases into plant cells. Likewise, Precision BioSciences has also developed meganuclease technology for plant genome modification.

Meganucleases are enzymes that recognize specific strings of DNA sequence containing more than 12 nucleotides. These specific sequences occur in the plant genome rarely, no more than two times. Cellectis plant sciences, as well as Precision BioSciences, design meganucleases that can make targeted gene modifications without deleteriously affecting other genes. The plant’s DNA replication apparatus is used in conjunction with the meganucleases to introduce modification of specific genes.

The advantages of this technique are enormous and have implications for the development of GMO crops. Because endogenous genes are targeted for modification, no foreign sequences are retained or can be removed after the modification has taken place. Expression of the modified gene is not affected by any random placement in the genome as might occur with other modification systems. The time to achieve regulatory approval of the modified plant can potentially be reduced.

Many of the commercial plant sciences companies have become interested in the technology. Precision BioSciences is collaborating with DuPont, Bayer CropSciences and BASF Plant Sciences while BASF, Bayer CropSciences, Pioneer, Limagrain and Monsanto are reported to use Cellectis plant sciences technology. However these two technology companies appear to be embroiled in a series of patent disputes. On October 28^th Precision BioSciences announced that the US Patent and Trademark Office (PTO) had issued final decisions rejecting the claims of four Cellectis patents. Whatever the outcome, expect to see more companies interested in this technology.

Big Oil, Big Food, Small Biofuels

By Robin J. Johnson, Ph.D.

In her excellent analysis of biofuels' prospects in the 112th Congress, biofuels blogger Joanna Schroeder quotes Renewable Fuels Association head, Bob Dinneen as saying, "The ethanol industry, and the domestic biofuel industry as a whole, ought not be asked to unilaterally disarm while extensive government support continues for petroleum companies."

More and more, the line between the biofuels and petroleum industries is becoming blurred. Companies such as Valero*, Petrobras, and Sunoco* own outright, or hold partial stakes in, a number of existing ethanol plants. (Many of these plants were acquired at bargain basement prices following the collapse of the original owners' businesses). In addition, investment in biofuels technology development has become de rigueur for oil companies such as Exxon Mobil*, Chevron*, Shell, ConocoPhillips*, and Marathon Oil*.

Not to be left behind by big oil, big food is part of the biofuels scene. Actually, big food preceded big oil into the biofuels arena. Companies such as Archer Daniels Midland*, Tyson*, and Cargill** have major presences in the biofuels industry, having developed technologies and established production facilities for biofuels. ADM was once the world's largest ethanol producer, a position now held by POET of South Dakota.

With these giants mixing it up, is it any surprise the food versus fuel debate continues to rage and oil and food execs get richer?

Government subsidies for petroleum companies dwarf those for biofuels. The famous volumetric ethanol excise tax credit (VEETC), or blenders' credit, pays oil companies to blend ethanol into gasoline and biodiesel into diesel. (The American Jobs Creation Act of 2004 created the blenders' tax credit for both ethanol and biodiesel. The credit for biodiesel expired December 31, 2009, seriously harming the US biodiesel industry. So much for jobs!)

With big oil claiming three of the top ten spots on the 2010 Fortune 500 list, it's time to revamp biofuels subsidies so they benefit producers who take the lion's share of the risk. Let's reward big oil and big food for innovation and support of biofuels instead of simply paying them for obeying mandates on using it.

* indicates member of the Fortune 100 list for 2010

** indicates member of the Forbes top 100 private companies list for 2010

Drop-in Fuels from Butanol

By Robin J. Johnson, Ph.D.

Despite a 55% drop in U.S. venture capital investment in the cleantech sector, new technologies in biofuels continue to appear on the horizon. One of these, drop-in fuels from biobutanol, has gained air time as players such as the U.S. Navy, and French oil giant TOTAL throw their support behind biobutanol development.

On November 3, 2010, Cobalt Technologies of California announced that it signed a Cooperative Research and Development Agreement (CRADA) with the U.S. Navy to develop jet and diesel fuels from biobutanol. Cobalt has developed a process for converting biomass to n-butanol (a butanol isoform) using a Clostridium bacterium that can metabolize both C5 and C6 sugars found in lignocellulosic materials. The technology for converting n-butanol (which has 4 carbons) to jet fuel (a mixture of hydrocarbons containing 6-16 carbons) and diesel fuel (a mixture of hydrocarbons containing 8-21 carbons) was developed by the U.S. Naval Air Warfare Center Weapons Division. Cobalt's biobutanol process employs a chemical process to extract sugars from biomass followed by continuous fermentation. Converting n-butanol to larger hydrocarbons involves a dehydration reaction that converts n-butanol to butene (a.k.a. butylene). Butene reacts with itself to form oligomers. Cobalt opened its pilot facility in Mountain View California in January of this year.

A similar story (though without the U.S. Navy support and with support from TOTAL) exists for a Colorado company, Gevo. Gevo produces isobutanol (another isoform of butanol) from sugar and grain crops using genetically modified yeast for the fermentation step. The company has demonstrated technology to convert isobutanol into aliphatic and aromatic hydrocarbons. Like Cobalt Technologies, Gevo's chemistry depends on an initial dehydration step where isobutanol is converted to isobutylene. Isobutylene can in turn be oligomerized to jet and diesel fuel blend stocks. In August of this year, Gevo filed a registration statement on form S-1 with the Securities and Exchange Commission for a proposed initial public offering of shares of its common stock. In September, the company acquired Agri-Energy's ethanol production plant in Luverne, MN in order to retrofit it for isobutanol production.

While many may decry the widespread use of biofuels in automobiles in the U.S., preferring development of electric cars, it is clear that certain modes of transport (airplanes, trucks, freight trains, freighters) will depend for quite some time upon the energy density contained in chemical bonds. It is for these applications that drop-in biofuels hold the most promise.

Pumping Out the Biofuel

By Daniela Sciaky, Ph.D.

A $50,000 ConocoPhillips Energy Prize was recently awarded to the research team headed by Gregory Stephanopoulos of the Massachusetts Institute of Technology for engineering of a microbe that converts carbohydrates into lipids. The significance of this award is that the patent-pending technology has resulted in the creation of a heterotrophic microbe that, according to Stephanopoulos, has remarkable yields. These yields, which are reported to be close to the theoretical maximum, will help to reduce the cost of producing oil-based biofuels such as biodiesel.

The prize from ConocoPhillips is to be used by the MIT team to explore the use of hydrogen and carbon dioxide as feedstocks for the microbe. One of the schemes proposed by the team sends the carbon dioxide produced by the aerobic (organisms that grow in the presence of oxygen) heterotrophic oil-producing microbe from fermentation to a separate fermentor for use by anaerobic bacteria in the presence of hydrogen, resulting in the production of ethanol. This scheme converts carbohydrate and hydrogen to both oil and ethanol.

In another scheme anaerobic bacteria or bacteria that grow in the absence of oxygen are used to fix carbon dioxide with hydrogen to produce the feedstock for use by the aerobic heterotrophic oil-producing microbe. This scheme converts hydrogen to oil for production of high-density biofuels. The technology proposed in these schemes has been proven in different contexts to varying degrees. Existing facilities can be used to test the scalability of the technology.

Are there any companies proposing or commercializing a similar approach? As discussed in a previous post “Algae, the Dark Side of Solazyme’s Success”, Solazyme has successfully used the heterotrophic concept to produce algal-based renewable naval distillate fuel for the US Navy. The company continues to explore the use of cheap sources of fixed carbon such as conversion of biomass to sugars for fermentation by heterotrophic microorganisms. Qteros has developed the Q Microbe also known as Clostridium phytofermentans, capable of digesting a variety of cellulosic feedstocks as a source of fermentable sugars. The organism can then ferment these sugars to produce ethanol. Qteros reports that it has significantly improved the yield and titre of ethanol produced by optimizing the microbe’s metabolism.

The key to success is that the heterotrophic microbe developed by the MIT team has yields close to theoretical maximum.  Historically, production of oil from oleaginous microorganisms, organisms whose lipid content is more than 20% of their biomass, has been prohibitively expensive, ranging from $1000 to $2400 per ton. High costs result from the need to supply microorganisms with a carbon source and the slow cell growth rate during fermentation resulting in high lipid accumulation with very few cells. Only when these costs become competitive with production of biofuels from oilseeds or sugar/starch-based feedstocks will production of biofuels from heterotrophic microorganisms gain favor.

Ethanol, Why Brazil and Not the United States ….Yet?

By Daniela Sciaky, Ph.D.

Ethanol as a transportation fuel can be a dirty word in the United States when it comes to fuel efficiency. The energy content of ethanol is less than gasoline resulting in lower gas mileage. However ethanol has a higher octane rating than gasoline and if engines are tuned to use ethanol then much better performance is delivered by the use of ethanol.

What technology is available to take into account ethanol’s advantage? Sturman Industries hosted a technology summit in late October that brought the renewable fuel industry together with engine designers. Sturman proposed to replace the camshaft-controlled valves traditionally found in internal combustion engines with computer-controlled valves whose performance can be adjusted on the fly. This technology results in maximizing the performance of any fuel used by the engine.

Scania, a European engine and vehicle manufacturer, has pioneered many efforts to develop engines for ethanol and other renewable fuel powered vehicles. In October the company was awarded a 3.3 million EUR grant by the Strategic Vehicle Research and Innovation Initiative in Sweden to develop an engine for heavy commercial vehicles capable of running on biofuels. The company will focus on developing technology for both alcohol and methane gas-based fuels.

However, in case people haven’t noticed Kia Motors just unveiled the Kia Soul Flex, a car that can run on 100% ethanol, 100% gasoline or an ethanol-gasoline mixture. According to Kia the Soul Flex exhibits a 44% improvement in fuel efficiency. Power and torque are also improved compared to the existing gasoline model. The Soul Flex has a newly adapted fuel injection and catalyst system as well as a fuel tank assistance system that aids engine ignition at low temperatures. Unfortunately for the US, the car is only being introduced to Brazil.

Acceptance of ethanol in the US is still a long way off. Brazil, because of its adoption of alternative fuels has become a magnet for the adoption of new technologies that are still in the proposal/testing stages in the US. Ethanol will fail to reach its full potential in the US unless these innovative technologies are also adopted in the US.

Petrobras Cellulosic Ethanol Project - Who's on First?

By Robin J. Johnson, Ph.D.

In the ongoing quest by Petrobras to enter the cellulosic ethanol field, at least two companies appear to be involved. Novozymes of Denmark and KL Energy of South Dakota have both signed agreements to develop cellulosic ethanol technology for sugarcane bagasse.

The relationship between the three companies isn't clear. Petrobras' ethanol production activities are handled through its subsidiary, Petrobras Biocombustivel, which plans to hold a 45.7% equity interest in Acucar Guarani, the fourth largest sugarcane processor in Brazil, and a 49% equity stake in Nova Fronteira Bioenergia S.A., a joint venture with Grupo Sao Martinho.

Novozymes is a major supplier of enzymes for (among other things) biofuel production. The company is a leader in cellulosic ethanol enzyme technology.

KL Energy claims to have a proprietary process for producing cellulosic ethanol. Their technology includes thermal and mechanical pretreatment and enzymatic hydrolysis of cellulosic materials and fermentation of both C5 and C6 sugars.

Is Novozymes supplying enzymes for KL Energy's process to be integrated in a Petrobras sugar mill? Here's what we know.

In August of 2010, Petrobras, and KL Energy Corporation signed a joint development agreement for technology to turn sugarcane bagasse into ethanol. The 18-month project includes an investment by Petrobras of $11 million to adapt KL Energy’s demonstration facility in Upton, Wyoming to use bagasse. The agreement provides Petrobras with the option to license KL Energy's technology for use within Petrobras Group assets. Integration of KL Energy technology into one of Petrobras’ Brazilian sugarcane mills is scheduled to go online sometime in 2013.

On October 15, 2010 Novozymes and Petrobras announced an agreement to develop a process to make ethanol from sugarcane bagasse.

On October 21, 2010, KL Energy announced it had re-acquired all third party technology licensing agreements in order to facilitate commercialization of its technology with its strategic partners. On September 1, 2010 KL Energy signed a Master Coillaboration Agreement with add blue Consultoria Ltda. of Brazil. The two companies plan to set up a new company to handle business in Brazil.

KL Energy lists Petrobras as one of its partners on its website, but websites aren't always kept current. The Novozymes press release doesn't mention any other partners, though any cellulosic ethanol plant would have to use some form of pretreatment and fermentation technology. It will be interesting to see how all these arrangements shake out and are revealed over time.

Brazil's Braskem Invests in "Green" Plastics to Supply J&J, P&G, Others

THE VIEW FROM BRAZIL

By Henrique Oliveira

Braskem, the Brazilian petrochemical company with 29 operations in the US and Brazil, started production on its lines of “green plastics” barely a month ago and has already announced the construction of a new plant, scheduled to commence operations in 2013. Braskem plans to invest USD 100 million to produce 30,000 tonnes of plastic per year.

The project for the new plant will be officially presented today at the International Trade Fair for Plastics and Rubber, known as the “K Trade Fair", held in Germany. According to Brazilian media, Braskem has plans to become the world leader in sustainable chemistry by 2020.

The company kicked off R&D efforts in the field in 2005 and, two years later, announced the construction of a plant in the city of Triunfo, in Brazil’s southernmost state, Rio Grande do Sul. The plant, which cost USD 500 million, was inaugurated in September and has the capacity to produce 200,000 tonnes of polyethylene.

Even before the plant opened, Braskem already had twenty contracts with companies interested in its “green” plastics. The first products made from the resin are expected to reach consumers by January 2011. Johnson & Johnson, for instance, will use it in its Sundown sunscreen packaging, while P&G will produce bottles for its Pantene product line.