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    Spanish company Ferry Group is to invest €42/US$55.2 million in a project for the production of biomass fuel pellets in Bulgaria. The 3-year project consists of establishing plantations of paulownia trees near the city of Tran. Paulownia is a fast-growing tree used for the commercial production of fuel pellets. Dnevnik - Feb. 20, 2007.

    Hungary's BHD Hõerõmû Zrt. is to build a 35 billion Forint (€138/US$182 million) commercial biomass-fired power plant with a maximum output of 49.9 MW in Szerencs (northeast Hungary). Portfolio.hu - Feb. 20, 2007.

    Tonight at 9pm, BBC Two will be showing a program on geo-engineering techniques to 'save' the planet from global warming. Five of the world's top scientists propose five radical scientific inventions which could stop climate change dead in its tracks. The ideas include: a giant sunshade in space to filter out the sun's rays and help cool us down; forests of artificial trees that would breath in carbon dioxide and stop the green house effect and a fleet futuristic yachts that will shoot salt water into the clouds thickening them and cooling the planet. BBC News - Feb. 19, 2007.

    Archer Daniels Midland, the largest U.S. ethanol producer, is planning to open a biodiesel plant in Indonesia with Wilmar International Ltd. this year and a wholly owned biodiesel plant in Brazil before July, the Wall Street Journal reported on Thursday. The Brazil plant is expected to be the nation's largest, the paper said. Worldwide, the company projects a fourfold rise in biodiesel production over the next five years. ADM was not immediately available to comment. Reuters - Feb. 16, 2007.

    Finnish engineering firm Pöyry Oyj has been awarded contracts by San Carlos Bioenergy Inc. to provide services for the first bioethanol plant in the Philippines. The aggregate contract value is EUR 10 million. The plant is to be build in the Province of San Carlos on the north-eastern tip of Negros Island. The plant is expected to deliver 120,000 liters/day of bioethanol and 4 MW of excess power to the grid. Kauppalehti Online - Feb. 15, 2007.

    In order to reduce fuel costs, a Mukono-based flower farm which exports to Europe, is building its own biodiesel plant, based on using Jatropha curcas seeds. It estimates the fuel will cut production costs by up to 20%. New Vision (Kampala, Uganda) - Feb. 12, 2007.

    The Tokyo Metropolitan Government has decided to use 10% biodiesel in its fleet of public buses. The world's largest city is served by the Toei Bus System, which is used by some 570,000 people daily. Digital World Tokyo - Feb. 12, 2007.

    Fearing lack of electricity supply in South Africa and a price tag on CO2, WSP Group SA is investing in a biomass power plant that will replace coal in the Letaba Citrus juicing plant which is located in Tzaneen. Mining Weekly - Feb. 8, 2007.

    In what it calls an important addition to its global R&D capabilities, Archer Daniels Midland (ADM) is to build a new bioenergy research center in Hamburg, Germany. World Grain - Feb. 5, 2007.

    EthaBlog's Henrique Oliveira interviews leading Brazilian biofuels consultant Marcelo Coelho who offers insights into the (foreign) investment dynamics in the sector, the history of Brazilian ethanol and the relationship between oil price trends and biofuels. EthaBlog - Feb. 2, 2007.

    The government of Taiwan has announced its renewable energy target: 12% of all energy should come from renewables by 2020. The plan is expected to revitalise Taiwan's agricultural sector and to boost its nascent biomass industry. China Post - Feb. 2, 2007.

    Production at Cantarell, the world's second biggest oil field, declined by 500,000 barrels or 25% last year. This virtual collapse is unfolding much faster than projections from Mexico's state-run oil giant Petroleos Mexicanos. Wall Street Journal - Jan. 30, 2007.

    Dubai-based and AIM listed Teejori Ltd. has entered into an agreement to invest €6 million to acquire a 16.7% interest in Bekon, which developed two proprietary technologies enabling dry-fermentation of biomass. Both technologies allow it to design, establish and operate biogas plants in a highly efficient way. Dry-Fermentation offers significant advantages to the existing widely used wet fermentation process of converting biomass to biogas. Ame Info - Jan. 22, 2007.

    Hindustan Petroleum Corporation Limited is to build a biofuel production plant in the tribal belt of Banswara, Rajasthan, India. The petroleum company has acquired 20,000 hectares of low value land in the district, which it plans to commit to growing jatropha and other biofuel crops. The company's chairman said HPCL was also looking for similar wasteland in the state of Chhattisgarh. Zee News - Jan. 15, 2007.

    The Zimbabwean national police begins planting jatropha for a pilot project that must result in a daily production of 1000 liters of biodiesel. The Herald (Harare), Via AllAfrica - Jan. 12, 2007.

    In order to meet its Kyoto obligations and to cut dependence on oil, Japan has started importing biofuels from Brazil and elsewhere. And even though the country has limited local bioenergy potential, its Agriculture Ministry will begin a search for natural resources, including farm products and their residues, that can be used to make biofuels in Japan. To this end, studies will be conducted at 900 locations nationwide over a three-year period. The Japan Times - Jan. 12, 2007.

    Chrysler's chief economist Van Jolissaint has launched an arrogant attack on "quasi-hysterical Europeans" and their attitudes to global warming, calling the Stern Review 'dubious'. The remarks illustrate the yawning gap between opinions on climate change among Europeans and Americans, but they also strengthen the view that announcements by US car makers and legislators about the development of green vehicles are nothing more than window dressing. Today, the EU announced its comprehensive energy policy for the 21st century, with climate change at the center of it. BBC News - Jan. 10, 2007.

    The new Canadian government is investing $840,000 into BioMatera Inc. a biotech company that develops industrial biopolymers (such as PHA) that have wide-scale applications in the plastics, farmaceutical and cosmetics industries. Plant-based biopolymers such as PHA are biodegradable and renewable. Government of Canada - Jan. 9, 2007.

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Tuesday, September 26, 2006

Jatropha seeds arrive in Namibia

Quicknote bioenergy crops
The Namibian, Windhoek. The first container of Jatropha seed arrived in Namibia last week so that planting for a future biodiesel project can take off. Jatropha curcas is an alien shrub that has spread to southern Africa over the past century.

Its fruit contains a sought-after oil, which can be used in diesel engines to replace conventional fuel, and also for soap-making. The Namibia Agronomic Board recently commissioned a study for a project to plant 63 000 hectares of Jatropha on communal and commercial land over the next few years.

The sealed container was officially opened by representatives of the Ministry of Agriculture for sample taking at the Agra premises in Windhoek, according to Birgit Hoffmann, Senior Marketing Manager of Agra. The company is the sole distributor for Jatropha seed.

Agra agronomist Francois Wahl says he will showcase the seed at the Agra stand during this year's Windhoek Show, which starts this weekend. After the show, the seed will be available for sale at Agra branches countrywide.

The cultivation of Jatropha to produce biodiesel has already proven successful in Zimbabwe and some South American countries. The Jatropha plant requires very little water for survival; hence Namibia's arid climate seems to be suitable. A yield can be expected with as little as 300 mm of rain a year. Jatropha is not eaten by livestock because of its toxicity and hedges are grown around homesteads in rural Namibia to keep animals out. While irrigation is required during the first two years, the plant already starts to bear fruit after the second rainy season. The fruit is harvested in winter when the shrub is leafless [entry ends here].
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Indonesia announces biofuels budget for 2007

We have been reporting regularly on Indonesia's biofuels and bioenergy crash program, with a series of 'big numbers' being thrown around as estimates about funds, hectarages and jobs that would be created. Today, the country's green energy plan, one of the world's most ambitious, became more concrete as the Indonesian government announced its biofuels budget for the year 2007.

The numbers look as follows:
  • The total budget for biofuels research and development and production in 2007 amounts to 13 trillion rupiah (€1.1/US$1.4 billion)
  • This package adds 12 trillion rupiah (€1/US$1.3 billion) to the 1 trillion rupiah (€85/US$108 million) already allocated under the 2007 budget to subsidize the interest payments on loans taken out for biofuel-related ventures
  • The budget is divided as follows: (1) 10 trillion rupiah (€854 million/US$1 billion) for the development of infrastructures - such as pipelines, irrigation systems and access roads - in areas earmarked for biofuel plantations (2) with the remaining 2 trillion rupiah (€170/US$216 million) for the procurement of seedlings.
  • A first land allocation takes place whereby 500,000 hectares are set aside for the biofuels industry in 2007, out of a total of 6 million hectares over the course of the program that runs from 2007 until 2010
  • This land is divided over several crops, as follows: 3 million hectares are to be earmarked for oil palm (biodiesel), 1.5 million hectares for cassava (ethanol), 1.5 million hectares for jatropha (biodiesel) and 500,000 hectares for sugarcane (ethanol); the 500,00 hectares for 2007 are distributed along the lines of that same ratio
The head of the Indonesian government's biofuel development committee, Alhilal Hamdi, said during a hearing with the House Commission on Energy that additional financial support for biofuel development will come from the banking sector. The country's private financial institutions were prepared to channel up to 20 trillion rupiah (€1.7/US$2.2 billion) in loans to the industry.

Among the largest biofuel planters will be PT AGB, which plans to cultivate 300,000 hectares in North Maluku province, PT Wilmar Bioenergy (150,000 hectares in Riau and East Kalimantan provinces), and PT Bakrie and Rekayasa Industri (25,500 hectares in Jambi).

The government itself will plant 2,000 hectares, as part of pilot and research projects, including 140 hectares under the Agriculture Ministry and 650 hectares by the Research and Technology Ministry's Technology Assessment and Application Agency (BPPT).

Following the surge in oil prices and the resulting strain on the budget, as well as burgeoning domestic fuel consumption and social tensions over rising fuel prices, the Indonesian government rolled out a national alternative energy program. It wants to see biofuels account for 10 percent of the country's total fuel consumption, which reached 70 million kiloliters last year, by 2010. The program is part of a massive anti-poverty policy, with the biofuels industry estimated to bring 2.5 million jobs to the rural poor [entry ends here].
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Social sustainability crucial for biofuels - Brazilian official

We say: biofuels production offers an opportunity to bring social justice and poverty alleviation to the Global South. Through becoming energy farmers, poor smallholders can diversify their crops and cut their dependence on single cash crops for which markets have crashed so often; in energy crops for biodiesel and ethanol they find a (government-supported) market, with demand growing on a global scale now that petroleum resources are in decline (as some suggest), cannot keep up with rising demand and are plagued by geopolitical troubles.

Brazil understands this best and has a long experience with the mass production of ethanol. The country's president Luiz Inácio Lula da Silva, who has pledged to reduce Brazil's infamous inequality, has now launched a nation wide biodiesel programme (Pro-Biodiesel) aimed at reducing poverty, and he is making sure that the mistakes made under the Pro-Álcool programme are not repeated. Rodrigo Augusto Rodrigues, the federal government’s biodiesel coordinator, reminds us that social sustainability is absolutely crucial in this programme. In a country where the landless have organised into Latin America's largest social movement and are struggling to acquire land, where millions live in endemic poverty, where internal migration of rural people to the mega-cities results in vast slums, and where social inequalities are greater than anywhere else in the world, social sustainability might well be the single most important factor determining the long-term success of the biofuels strategies.

Let us see how this social vision of biofuel production as a means for poverty alleviation plays out for Sebastian Luis de Sousa, an ordinary, poor farmer living in central Brazil.

For the better part of his 64 years, de Sousa has scratched a meager living in the paprika-red soil of the vast plains of the Brazilian cerrado. So when offered a chance to grow castor beans to produce an alternative fuel called biodiesel, the rawboned father of nine reckoned he had nothing to lose. The US$200 he earned this summer from his tiny harvest wasn’t much. But rising demand for renewable fuels has de Sousa wanting to expand his 7 1/2 acre farm:
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"I want to buy more land," he said, rolling a prickly castor bean seed pod in his calloused hand. "This is an important thing that Brazil is doing."

Already the world’s largest producer of ethanol, Brazil is now betting on biodiesel, with an eye to helping small farmers such as de Sousa capitalise on what some see as the next big thing in green energy. Derived from animal fats or vegetable oils, this substitute for petroleum diesel is generating ten of millions of dollars from investors.

Major companies, including US agri-business behemoth Archer Daniels Midland Co, are building production plants, encouraged by a federal mandate requiring every liter of diesel fuel sold in Brazil to contain two per cent biodiesel by 2008, rising to 5 per cent by 2013.

Brazil’s state-owned petroleum giant Petrobras is already selling a fuel blend with 2 per cent biodiesel at hundreds of its retail gas stations. The company is investing in manufacturing facilities. It is also patenting a new fuel known as H-Bio that it says will save million of barrels of oil by using vegetable oil in the refining process to create a low polluting petroleum diesel.

Even McDonald’s has collaborated with Brazilian researchers looking to power vehicles with recycled grease from its restaurants. The involvement of big players is crucial if Brazil hopes to reach its goal of embracing biodiesel on a massive scale.
Current production is modest, but is projected to jump to 840 million litres by 2008, which would put Brazil among the worlds’ large producers. Still, officials are looking to involve more subsistence farmers such as de Sousa, who have yet to profit from the nation’s biofuels bonanza.

No country on the planet has been more successful at displacing fossil fuels with green energy than Brazil. Hammered by the oil shocks of the 1970s, the nation committed itself to developing a domestic ethanol industry to reduce its dependence on imported petroleum. Today, 40 per cent of the fuel that powers passenger cars here is made from homegrown sugar cane. That’s been a boon for Brazilian agriculture. But the economic fruits have been reaped by a small number of large farmers growing a single crop.

With biodiesel, officials see a chance to spread the wealth from a fast growing fuel whose demand in Brazil could top that of ethanol. At present, petroleum diesel accounts for more than half of all the vehicle fuel consumed in Brazil, about 42 billion litres a year, thanks to its heavy dependence on truck and bus transport.
By promoting a cleaner-burning alternative made from Brazilian-grown castor beans, soybeans, palm oil, jatropha and other crops, the government is looking to slash diesel imports and improve air quality in its cities, as well as to generate rural income and employment.

President Luis Inacio Lula da Silva, who is currently running for re-election, has touted biodiesel production as a way to spark development in some of the poorest regions of the country, particularly the rural northeast. Biodiesel producers who want to qualify for hefty federal tax breaks must purchase anywhere from 10 per cent to 50 per cent of their raw materials from small growers, depending on the region. That requirement is how poor farmers get connected with companies which provide them with seed and technical advice in addition to purchasing their crops.

Rodrigo Augusto Rodrigues, the federal government’s biodiesel coordinator, says the effort could eventually involve 360,000 family farms nationwide, up from about 2,500 at present. He says the varied crops provided by small growers would keep small farmers on the land and provide them a reliable stream of income.

"We don’t want to repeat the same mistakes we made with ethanol," Rodrigues says. "The social aspect is critical." But some energy experts are dubious that peasant farmers toiling on tiny plots will be more than bit players. Large-scale cultivation and ruthless efficiency were crucial to the nation’s success with ethanol. Mass produced soybeans, while not the most efficient feedstock, are fast emerging as the crop with the greatest potential to help producers achieve economies of scale:

"There is a lack of focus in this biodiesel program," says Luiz Augusto Horta Nogueira, former director of the Brazil’s National Agency of Petroleum, National Gas and Biofuels. "One group of stakeholders is looking to substitute large amounts of diesel. Others want rural development. ... It’s a real problem."

Some observers doubt the fuel can be cost competitive without fat government subsidies such as those that propped up Brazil’s ethanol market for years. Others say the environmental benefits may be overblown. Biofuels emit fewer greenhouse gases than fossil fuels when burned in combustion engines. But other factors must be considered when making the comparison, such as how much petroleum was needed to plant, harvest, produce and transport the renewable fuels, and how many native trees and plants were plowed under in the process.

Soybean farming has already destroyed large swaths of Brazil’s Amazon forest. The long standing agricultural practice of burning sugar cane fields prior to harvest is a major pollutant. Renewable fuels such as ethanol and biodiesel are "not as green as we like to think they are," said Joe Ryan, who manages air-quality projects in Brazil for the Menlo Park, Calif.-based William and Flora Hewlett Foundation.

Still, with the government projecting more than three dozen manufacturing plants to be on line by 2008 with a capacity of 1.7 billion litres, producers here, and across the globe, are bullish on biodiesel.

Worldwide production is surging, led by the European Union, which has adopted a goal of substituting 5.75 per cent of petroleum diesel with biodiesel by 2010 as part of its commitments under the Kyoto Protocol to reduce greenhouse gas emissions. The world’s top producer is Germany, where biodiesel made from rapeseed is widely available in gas stations.

Asia is fast becoming a major player, with the cultivation of palm oil for use in biodiesel growing rapidly in Malaysia and Indonesia. In the United States, where soybeans are the primary feedstock, production is projected to more than triple this year to around 250 million gallons or almost 950 million litres. The US already boasts 86 biodiesel plants, with another 62 under construction, according to the National Biodiesel Board.

Just like in the US Midwest, soybeans are the principle feedstock for biodiesel refineries in Brazil’s heartland. On a recent afternoon, near the city of Anapolis about two hours west of the nation’s capital Brasilia, workers with hardhats and torches welded seams on the gleaming steel storage tanks of a $20m biodiesel plant.
The plant, which will be produce up to 100 million litres of biodiesel annually, is one of three production facilities that Brazilian soybean processor Granol plans to have running by next year. Company executives see biodiesel as a lucrative new outlet for its soybeans, with domestic sales of its cooking oil and animal feed stagnating, and exports hurt by Brazil’s strong currency, .

"Renewable fuels are the future," says manager Paulo Donato, explaining his employer’s $45m bet on biodiesel. Hours to the north in Porto Nacional, farmer de Sousa says that he hoped that future would include small farmers like him. "I’m just one man," he said, poking at the soil with his sandal. "But I’m proud to play a part in this."

Original story:
The Peninsula: Brazil wagers on biodiesel - [s.d] Sept. 2006.

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The UN's Global Bioenergy Partnership opens its Secretariat

The Secretariat of the Global Bioenergy Partnership (GBEP), launched at the 14th Session of the UN Commission for Sustainable Development in May 2006 to promote the use of bioenergy, has formally opened its doors and is up and running. The GBEP will promote a global move to bioenergy.

Located at Food and Agriculture Organisation's (FAO) headquarters and supported by the Italian Ministry for the Environment, Land and Sea, the Secretariat's mandate is to facilitate a global political forum to promote bioenergy and to encourage the production, marketing and use of "green" fuels, with particular focus on developing countries.

Current Partners of the Global Bioenergy Partnership are: all G8 Countries (Canada, France, Germany, Italy, Japan, Russia, United Kingdom, U.S.A.), China, Mexico, the International Energy Agency (IEA), the UN Foundation, the European Biomass Industry Association (EUBIA) and FAO.

The Secretariat will be the principal coordinator of Partnership communications and activities and will assist international exchanges of know-how and technology, promote supportive policy frameworks and identify ways of fostering investments and removing barriers to the development and implementation of joint projects.

In the short term, the Secretariat will update the inventory of existing networks, initiatives and institutions dealing with bioenergy and identify any gaps in knowledge. It will also assist the Partners in identifying and implementing bilateral and multilateral projects for sustainable bioenergy development and support the formulation of guidelines for measuring reductions in greenhouse gas emissions due to the use of biofuels:
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The Partnership's overall aim is to respond to the growing need to develop renewable energy sources in the light of high oil prices, global warming and concerns about diminishing fossil fuel reserves.

FAO has always actively promoted biofuels as a means of reducing poverty while producing clean, low-cost energy. Given the right technologies, an abundant energy supply could be tapped by converting biomass such as crop residues, grass, straw and brushwood into fuel, while crops like sugar cane, corn and soybeans are already being used to produce ethanol or bio-diesel.

"We are happy that FAO was chosen to host the GBEP Secretariat," said Alexander Muller, FAO Assistant Director-General for Sustainable Development. "Its presence will stimulate us to continue helping governments and institutions formulate appropriate bioenergy policies and strategies."

"We hope the creation of a Global Bioenergy Partnership will help reduce current dependency on oil. Over the next decades, we will most probably see bioenergy providing an increasing amount of the world's energy needs, but we need to assure that this is done in a sustainable manner. Positive synergies between GBEP and FAO's International Bioenergy Platform (IBEP) will contribute to an expanded and sustainable role of biofuels," said Muller.

Italy and Mexico were respectively appointed as Chair and Vice-Chair of GBEP's Steering Committee for the next two years.

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Breakthrough in biodiesel production from bacteria

After continuous breakthroughs in cellulosic ethanol production processes, a breakthrough has now been achieved on the front of biodiesel production: a new and highly efficient process in the production of biodiesel has been developed by scientists in Germany. Research published in the September 2006 issue of Microbiology describes how specially engineered bacteria could be used to make biodiesel completely from food crops (or non-food oil crops), without relying on fossil fuels (as biodiesel does). (The original article "Microdiesel: Escherichia coli engineered for fuel production" is publicly available online; we reproduce it below in full, for future reference.)

Ordinary biodiesel is made by replacing the glycerol in vegetable oils with toxic methanol which is derived from fossil resources, in a process called transesterification. This biodiesel is therefor not 100% fossil fuel free. The 'microdiesel' process, as the scientists call it, avoids the use of methanol. Instead, they engineered the Escherichia coli bacterium by introducing the ethanol production genes from another bacterium, the ethanol-producing fermentative Zymomonas mobilis in combination with a gene from the A. baylyi bacterium, into the micro-organism. The result is that once the modified E. Coli is allowed to work on the vegetable oil, it produces its own ethanol which acts as a substitute for the toxic methanol. This makes the biodiesel genuinely renewable. The microdiesel process is also much more efficient than classic transesterification with methanol and keeps production costs down.

Professor Steinbüchel of the Westfälische Wilhelms-Universität in Münster who developed the process sums up the rationale behind it:
"Biodiesel is an alternative energy source and a substitute for petroleum-based diesel fuel, and a growing number of countries are already making biodiesel on a large scale, but the current method of production is still costly". But "biodiesel production depends on plant oils obtained from seeds of oilseed crops like rapeseed or soy. The production of these plant oils has a huge demand of acreage which is one of the main factors limiting a more widespread use of biodiesel today. In addition, biodiesel production must compete with the production of food, which also raises some ethical concerns".
"Due to the much lower price of the raw materials used in this new process, as well as their great abundance, the Microdiesel process can result in a more widespread production of biofuel at a competitive price in the future", Steinbüchel adds:
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We reproduce the full article below for future reference:

Rainer Kalscheuer, Torsten Stölting and Alexander Steinbüchel, Microdiesel: Escherichia coli engineered for fuel production, Microbiology 152 (2006), 2529-2536

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Corrensstrasse 3, D-48149 Münster, Germany


Biodiesel is an alternative energy source and a substitute for petroleum-based diesel fuel. It is produced from renewable biomass by transesterification of triacylglycerols from plant oils, yielding monoalkyl esters of long-chain fatty acids with short-chain alcohols such as fatty acid methyl esters and fatty acid ethyl esters (FAEEs). Despite numerous environmental benefits, a broader use of biodiesel is hampered by the extensive acreage required for sufficient production of oilseed crops. Therefore, processes are urgently needed to enable biodiesel production from more readily available bulk plant materials like sugars or cellulose. Toward this goal, the authors established biosynthesis of biodiesel-adequate FAEEs, referred to as Microdiesel, in metabolically engineered Escherichia coli. This was achieved by heterologous expression in E. coli of the Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase and the unspecific acyltransferase from Acinetobacter baylyi strain ADP1. By this approach, ethanol formation was combined with subsequent esterification of the ethanol with the acyl moieties of coenzyme A thioesters of fatty acids if the cells were cultivated under aerobic conditions in the presence of glucose and oleic acid. Ethyl oleate was the major constituent of these FAEEs, with minor amounts of ethyl palmitate and ethyl palmitoleate. FAEE concentrations of 1.28 g l–1 and a FAEE content of the cells of 26 % of the cellular dry mass were achieved by fed-batch fermentation using renewable carbon sources. This novel approach might pave the way for industrial production of biodiesel equivalents from renewable resources by employing engineered micro-organisms, enabling a broader use of biodiesel-like fuels in the future.

Abbreviations: FAEE, fatty acid ethyl ester; FAME, fatty acid methyl ester; TAG, triacylglycerol; WS/DGAT, wax ester synthase/acyl-coenzyme A : diacylglycerol acyltransferase


A major challenge mankind is facing in this century is the gradual and inescapable exhaustion of the earth's fossil energy resources. The combustion of those fossil energy materials lavishly used as heating or transportation fuel is one of the key factors responsible for global warming due to large-scale carbon dioxide emissions. In addition, local environmental pollution is caused. Thus, alternative energy sources based on sustainable, regenerative and ecologically friendly processes are urgently needed.

One of the most prominent alternative energy resources, attracting more and more interest in recent years with the price for crude oil reaching record heights, is biodiesel, which is a possible substitute for petroleum-based diesel fuel. Biodiesel is made from renewable biomass mainly by alkali-catalysed transesterification of triacylglycerols (TAGs) from plant oils (Ma & Hanna, 1999Down). It consists of monoalkyl esters of long-chain fatty acids with short-chain alcohols, primarily methanol and ethanol, resulting in fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs). Biodiesel offers a number of interesting and attractive beneficial properties compared to conventional petroleum-based diesel (for an overview see Krawczyk, 1996Down). Most important, the use of biodiesel maintains a balanced carbon dioxide cycle since it is based on renewable biological materials. Additional environmental benefits are reduced emissions (carbon monoxide, sulphur, aromatic hydrocarbons, soot particles) during combustion. Biodiesel is non-toxic and completely biodegradable. Due to its high flash point, it is of low flammability and thus its use is very safe and non-hazardous. Furthermore, it provides good lubrication properties, thereby reducing wear and tear on engines. Pure biodiesel or biodiesel mixed in any ratio with petroleum-based diesel can be used in conventional diesel engines with no or only marginal modifications, and it can be distributed using the existing infrastructure. Biodiesel is already produced in a growing number of countries on a large scale (e.g. 1 080 000 t biodiesel was produced in Germany in 2004: Bockey & von Schenck, 2005Down).

Despite these positive ecological aspects, however, biodiesel, as currently produced on a technical scale, has also numerous drawbacks and limitations. (1) Production is dependent on the availability of sufficient vegetable oil feedstocks, mainly rapeseed in Continental Europe, soybean in North America and palm oil in South East Asia. Therefore, industrial-scale biodiesel production will remain geographically and seasonally restricted to oilseed-producing areas. (2) Vegetable oils predominantly consisting of TAGs can not be used directly as diesel fuel substitute, mainly because of viscosity problems. Additional problems are the reliability of product quality in bulk quantities and filter plugging at low temperatures due to crystallization. Therefore, plant oils must be transesterified with short-chain alcohols like methanol or ethanol to yield the FAME and FAEE constituents of biodiesel. This transesterification process and the subsequent purification steps are cost intensive and energy consuming, thereby reducing the possible energy yield and increasing the price. (3) FAMEs and FAEEs have comparable chemical and physical fuel properties and engine performances (Peterson et al., 1995Down), but for economic reasons, only FAMEs are currently produced on an industrial scale due to the much lower price of methanol compared to ethanol. Methanol, however, is currently mainly produced from natural gas. Thus, FAME-based biodiesel is not a truly renewable product since the alcohol component is of fossil origin. Furthermore, methanol is highly toxic and hazardous, and its use requires special precautions. Use of bioethanol for production of FAEE-based biodiesel would result in a fully sustainable fuel, but only at the expense of much higher production costs. (4) The major limitation impeding a more widespread use of biodiesel is the extensive acreage needed for production of oilseed crops. The yield of biodiesel from rapeseed is only 1300 l ha–1, since only the seed oil is used for biodiesel production, whereas the other, major part of the plant biomass is not used for this purpose. Furthermore, oilseed crops like rapeseed and soybean are not self-compatible; therefore, their cultivation requires a frequent crop-rotation regime. In consequence, biodiesel based on oilseed crops will probably not be able to substitute more than 5–15 % of petroleum-based diesel in the future.

A recent study assessing the use of bioethanol for fuel came to the conclusion that large-scale use will require a cellulose-based technology (Farrell et al., 2006Down). A substantial increase of biodiesel production and a more significant substitution of petroleum-based diesel fuel in the future will probably only be feasible when processes are developed enabling biodiesel synthesis from bulk plant materials such as sugars and starch, and in particular cellulose and hemicellulose.

Intracytoplasmic storage lipid accumulation in the Gram-negative bacterium Acinetobacter baylyi strain ADP1 (formerly Acinetobacter sp. strain ADP1: Vaneechoutte et al., 2006Down) is mediated by the wax ester synthase/acyl-coenzyme A : diacylglycerol acyltransferase (WS/DGAT; the atfA gene product). This unspecific acyltransferase simultaneously synthesizes wax esters and TAGs by utilizing long-chain fatty alcohols or diacylglycerols and fatty acid coenzyme A thioesters (acyl-CoA) as substrates (Kalscheuer & Steinbüchel, 2003Down). Biochemical characterization of WS/DGAT revealed that this acyltransferase exhibits an extremely low acyl acceptor molecule specificity in vitro. The remarkably broad substrate range of WS/DGAT comprises short chain-length up to very long chain-length linear primary alkyl alcohols; cyclic, phenolic and secondary alkyl alcohols; diols and dithiols; mono- and diacylglycerols as well as sterols (Kalscheuer et al., 2003Down, 2004Down; Stöveken et al., 2005Down; Uthoff et al., 2005Down). By expression of WS/DGAT in different recombinant hosts, this substrate promiscuity has already been exploited to synthesize various fatty acid ester molecules in vivo. The type of fatty acid ester synthesized by WS/DGAT was determined by the physiological background of the expression host regarding the provision of substrates accomplished by natural metabolism, medium supplementation or genetic engineering. Examples of those recombinantly synthesized fatty acid ester derivatives are wax esters in recombinant Pseudomonas citronellolis (Kalscheuer & Steinbüchel, 2003Down), wax esters and fatty acid butyl esters (FABEs) in recombinant Escherichia coli (Kalscheuer et al., 2006Down), wax diesters and wax thioesters in the mutant A. baylyi strain ADP1acr1{Omega}Km (Kalscheuer et al., 2003Down; Uthoff et al., 2005Down), and TAGs, FAEEs and fatty acid isoamyl esters (FAIEs) in recombinant Saccharomyces cerevisiae (Kalscheuer et al., 2004Down). Although only trace amounts were produced, recombinant biosynthesis of FAEEs and FAIEs in yeast as well as FABEs in E. coli indicated that production of biodiesel-appropriate fatty acid monoalkyl esters might in principle be feasible by using recombinant WS/DGAT-expressing micro-organisms. The objective of our present study was thus the development of a microbial process for the production of FAEEs for use as biodiesel from simple and renewable carbon sources. For this approach, the natural WS/DGAT host A. baylyi strain ADP1 was not a suitable candidate since it is a strictly aerobic bacterium not able to form ethanol. We therefore established FAEE biosynthesis in recombinant E. coli by coexpression of the ethanol production genes from the ethanol-producing fermentative bacterium Zymomonas mobilis in combination with the WS/DGAT gene from A. baylyi strain ADP1.


Strains, plasmids and cultivation conditions.
Escherichia coli TOP10 (Invitrogen) was used in this study. The plasmids used are pLOI297 harbouring the Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) cloned in pUC18 collinear to the lacZ promoter (Alterthum & Ingram, 1989Down), and pKS : : atfA and pBBR1MCS-2 : : atfA harbouring the WS/DGAT gene from A. baylyi strain ADP1 collinear to the lacZ promoter in pBluescript KS– or pBBR1MCS-2, respectively (Kalscheuer & Steinbüchel, 2003Down). The construction of plasmid pMicrodiesel is described in Results.

Recombinant strains of E. coli were cultivated in LB medium (0.5 %, w/v, yeast extract, 1 %, w/v, tryptone and 1 %, w/v, NaCl) containing 1 mM IPTG and 2 % (w/v) glucose at 37 °C in the presence of ampicillin (75 mg l–1) and kanamycin (50 mg l–1) for selection of pLOI297, pKS : : atfA and pMicrodiesel or pBBR1MCS-2 : : atfA, respectively. Where indicated, sodium oleate was added from a 10 % (w/v) stock solution in H2O to a final concentration of 0.1 or 0.2 % (w/v). Cells were grown aerobically in 300 ml baffled Erlenmeyer flasks containing 50 ml medium on an orbital shaker (130 r.p.m.).

Bioreactor cultivation.

Fermentation experiments were done in a 2 litre stirred bioreactor (B. Braun Biotech International) with an initial volume of 1.5 l LB medium containing 0.2 % (w/v) sodium oleate, 2 % (w/v) glucose, 1 mM IPTG and appropriate antibiotics for plasmid selection (see above). Cultivations were done at 37 °C and at a stirring rate of 200 r.p.m. If not stated otherwise, the pH was controlled at 7.0 by automated addition of 4 M HCl or NaOH. Cells were cultivated either aerobically (aeration rate 3 vvm), under restricted oxygen conditions (aeration rate 0.75 vvm), or anaerobically. Inoculum was 5 % (v/v) of saturated overnight cultures.

Thin-layer chromatography.
TLC analysis of lipid extracts from whole cells was done as described previously (Kalscheuer & Steinbüchel, 2003Down) using the solvent system hexane/diethyl ether/acetic acid (90 : 7.5 : 1, by vol.). Lipids were visualized by spraying with 40 % (v/v) sulfuric acid and charring. Ethyl oleate was purchased from Sigma-Aldrich Chemie and used as reference substance for FAEEs.

GC and GC/MS analysis of FAEEs.

For quantification of FAEEs, 5 ml culture broth was extracted with 5 ml chloroform/methanol (2 : 1, v/v) by vigorous vortexing for 5 min. After phase separation, the organic phase was withdrawn, evaporated to dryness, and redissolved in 1 ml chloroform/methanol (2 : 1, v/v). FAEEs were analysed by GC on an Agilent 6850 GC (Agilent Technologies) equipped with a BP21 capillary column (50 mx0.22 mm, film thickness 250 nm; SGE) and a flame-ionization detector (Agilent Technologies). A 2 µl portion of the organic phase was analysed after split injection (1 : 20); hydrogen (constant flow 0.6 ml min–1) was used as carrier gas. The temperatures of the injector and detector were 250 and 275 °C, respectively. The following temperature programme was applied: 120 °C for 5 min, increase of 3 °C min–1 to 180 °C, increase of 10 °C min–1 to 220 °C, 220 °C for 31 min. Identification and quantification were done by using authentic FAEE standards.

For coupled GC/MS analysis, FAEEs were purified by preparative TLC. GC/MS analysis of FAEEs dissolved in chloroform was done on a Series 6890 GC system equipped with a Series 5973 EI MSD mass-selective detector (Hewlett Packard). A 3 µl portion of the organic phase was analysed after splitless injection on a BP21 capillary column (50 mx0.22 mm, film thickness 250 nm; SGE). Helium (constant flow 0.6 ml min–1) was used as carrier gas. The temperatures of the injector and detector were 250 °C and 240 °C, respectively. The same temperature programme as described for GC analysis was applied. Data were evaluated by using the NIST-Mass Spectral Search Program (Stein et al., 1998Down).

Ethanol quantification.
Ethanol in cell-free aqueous culture supernatants was determined by GC essentially as described above for FAEE quantification, but applying a modified temperature programme: 70 °C for 20 min, increase of 10 °C min–1 to 180 °C, increase of 10 °C min–1 to 220 °C, 220 °C for 25 min.

General molecular biological techniques.
Standard molecular biological techniques were applied according to Sambrook et al. (1989)Down.


Establishment of FAEE biosynthesis in recombinant E. coli TOP10 by metabolic engineering
The unspecific acyltransferase WS/DGAT from A. baylyi strain ADP1 has been shown to be capable of utilizing ethanol to some extent as an acyl acceptor substrate (Kalscheuer et al., 2004Down; Stöveken et al., 2005Down). However, heterologous expression of the WS/DGAT-encoding atfA gene alone from pBBR1MCS-2 : : atfA did not result in FAEE formation in E. coli TOP10 during cultivation in LB medium containing 2 % (w/v) glucose, 1 mM IPTG and 0.1 % (w/v) sodium oleate under either aerobic or anaerobic conditions (data not shown). Although E. coli is known to form ethanol during mixed acid fermentation, obviously ethanol synthesis and/or uptake of oleic acid from the medium and activation to the acyl-CoA thioester were too inefficient to support detectable FAEE formation under anaerobic conditions. However, increased ethanol production has been achieved in E. coli upon heterologous expression of pyruvate decarboxylase (the pdc gene product) and alcohol dehydrogenase (the adhB gene product) from the strictly anaerobic ethanologenic Gram-negative bacterium Zymomonas mobilis. Using this system, efficient ethanol biosynthesis was achieved from glucose via the glycolysis product pyruvate even under aerobic conditions (Ingram et al., 1987Down; Alterthum & Ingram, 1989Down).

We therefore attempted to establish FAEE biosynthesis in a recombinant E. coli by combining expression of the Z. mobilis genes pdc and adhB and of the atfA gene from A. baylyi strain ADP (Fig. 1Down) using plasmids pLOI297 (pdc and adhB) and pBBR1MCS-2 : : atfA. Recombinant strains carrying either plasmid alone did not exhibit FAEE levels detectable by TLC (Fig. 2aDown, lanes 1 and 2). However, coexpression of all three relevant genes in a strain carrying both plasmids resulted in significant FAEE formation (Fig. 2aDown, lane 3). FAEE biosynthesis was strictly dependent on the presence of sodium oleate in the medium (data not shown). Growth of strains harbouring plasmid pLOI297 was very poor in LB medium without glucose addition, and FAEE synthesis was not observable in E. coli TOP10 harbouring both plasmids under these conditions (data not shown). The FAEEs formed were accumulated intracellularly, and no significant extracellular lipids were found in cell-free culture supernatants (data not shown).

Fig. 1. Pathway of FAEE biosynthesis in recombinant E. coli. FAEE formation was achieved by coexpression of the ethanolic enzymes pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) from Z. mobilis and the unspecific acyltransferase WS/DGAT from A. baylyi strain ADP1.

Fig. 2. Chemical analysis of FAEEs produced by recombinant E. coli TOP10. (a) TLC analysis of intracellular lipids accumulated by recombinant E. coli TOP10. Cells were cultivated aerobically in shake flasks for 24 h at 37 °C in LB medium containing 2 % (w/v) glucose, 0.1 % (w/v) sodium oleate, 1 mM IPTG and appropriate antibiotics as described in Methods. A,oleic acid; B, ethyl oleate; C, oleyl oleate; 1, E. coli TOP10(pLOI297); 2, E. coli TOP10(pBBR1MCS-2 : : atfA); 3, E. coli TOP10(pBBR1MCS-2 : : atfA+pLOI297). Total lipid extracts each obtained from 1.5 mg lyophilized cells were applied in lanes 1–3. (b) Total ion profile of GC/MS analysis of FAEEs isolated from E. coli TOP10(pBBR1MCS-2 : : atfA+pLOI297). Cells were cultivated as described above. FAEEs were purified by preparative TLC. Identified substances: 1, ethyl palmitate (C16 : 0-ethyl ester, m/z=284 [C18H36O2]+); 2, ethyl palmitoleate (C16 : 1-ethyl ester, m/z=282 [C18H34O2]+); 3, ethyl oleate (C18 : 1-ethyl ester, m/z=310 [C20H38O2]+).

GC/MS analysis of FAEE isolated from E. coli TOP10(pBBR1MCS-2 : : atfA+pLOI297) cultivated in medium supplemented with sodium oleate revealed a mixture of esters mainly consisting of ethyl oleate plus minor amounts of ethyl palmitate and ethyl palmitoleate (Fig. 2bUp). The presence of ethyl palmitate indicated that also some fatty acids derived from de novo fatty acid biosynthesis were channelled into FAEE production. When technical-grade sodium oleate (content ~80 %) was used for cultivations at a larger scale, low amounts of ethyl myristate (C14 : 0-ethyl ester, m/z=256 [C16H32O2]+), ethyl myristoleate (C14 : 1-ethyl ester, m/z=254 [C16H30O2]+) and ethyl linoleate (C18 : 2-ethyl ester, m/z=308 [C20H36O2]+) were also observed due to the presence of the corresponding fatty acid impurities (data not shown).

Batch fermentations of E. coli TOP10(pBBR1MCS-2 : : atfA+pLOI297) for FAEE production
The shake-flask experiments under aerobic conditions described above clearly proved the concept that FAEE biosynthesis is feasible in recombinant E. coli. Oxygen availability might have a great influence on the ethanol synthesis rate in this recombinant system, with low-oxygen conditions supposed to favour ethanol formation, and thus might also have a profound impact on the FAEE biosynthesis rate. We therefore cultivated E. coli TOP10(pBBR1MCS-2 : : atfA+pLOI297) under conditions permissive for FAEE formation with different controlled oxygen conditions (Fig. 3Down). Although ethanol production was slightly higher under anaerobic conditions (maximal 4.39 g l–1 after 17 h), only a very low FAEE content was observed, plateauing already after 18 h at a concentration of 0.05–0.07 g l–1 (Fig. 3bDown). In contrast, FAEE biosynthesis was significantly higher under aerobic conditions (aeration rate 3 vvm). FAEE formation was not restricted to a certain growth phase but continued throughout the cultivation period, finally reaching 0.26 g l–1 after 48 h (Fig. 3aDown). With a final cellular dry biomass of 4.3 g l–1 obtained by aerobic cultivation this corresponds to a cellular FAEE content of 6.1 % (w/w). When the cells were cultivated under oxygen-restricted conditions (aeration rate 0.75 vvm) a final FAEE concentration of 0.16 g l–1 was obtained after 48 h (data not shown). Under all three cultivation conditions ethanol concentration reached a maximum after 15–20 h cultivation, after which a rapid decrease was unexpectedly observed (Fig. 3a, bDown), which has not to our knowledge been described before for ethanologenic E. coli strains employing the Z. mobilis pdc and adhB genes for recombinant ethanol synthesis.

Fig. 3. FAEE production during batch fermentations of E. coli TOP10(pBBR1MCS-2 : : atfA+pLOI297). Cultivations were done in a 2 litre stirred bioreactor initially filled with 1.5 l LB medium containing 0.2 % (w/v) sodium oleate, 2 % (w/v) glucose, 1 mM IPTG, 75 mg ampicillin l–1 and 50 mg kanamycin l–1 as described in Methods. Sodium oleate causes turbidity of the medium, explaining the high initial optical densities. {blacksquare}, OD600; {blacktriangleup}, ethanol concentration; bullet, FAEE concentration. (a) Cultivation under aerobic conditions (aeration rate 3 vvm). (b) Cultivation under anaerobic conditions.

Construction of plasmid pMicrodiesel
To simplify the process by reducing the number of antibiotics required for plasmid stabilization and to potentially increase FAEE yield by providing all three relevant genes on a high-copy-number vector, plasmid pMicrodiesel was constructed. For this, a 3.2 kbp DNA fragment was amplified from plasmid pLOI297 by tailored PCR using the oligonucleotides 5'-AAAGGATCCGCGCAACGTAATTAATGTGAGTT-3' (forward primer) and 5'-TTTGGATCCCCAAATGGCAAATTATT-3' (reverse primer) introducing BamHI restriction sites (underlined). This 3.2 kbp BamHI fragment, which comprised the Z. mobilis genes pdc and adhB and the upstream lacZ promoter region, was cloned into BamHI-linearized pKS : : atfA, a derivative of the high-copy-number plasmid pBluescript KS– (Kalscheuer & Steinbüchel, 2003Down), yielding pMicrodiesel (Fig. 4Down). The orientation of atfA, pdc and adhB was determined by EcoRI restriction and DNA sequence analysis. Plasmid pMicrodiesel carried all three genes relevant for FAEE synthesis in a collinear orientation, with atfA driven by a lacZ promoter and with pdc and adhB controlled by a second lacZ promoter, thereby ensuring effective transcription of all three genes.

Fig. 4. Map of plasmid pMicrodiesel. Relevant characteristics: rep, origin of replication; AmpR, ampicillin-resistance gene; PlacZ, lacZ promoter; pdc, pyruvate decarboxylase gene from Z.mobilis; adhB, alcohol dehydrogenase gene from Z. mobilis; atfA, WS/DGAT gene from A. baylyi strain ADP1.

Fed-batch fermentation of E. coli TOP10(pMicrodiesel) for FAEE production

Shake-flask experiments with E. coli TOP10 harbouring either pMicrodiesel alone or pLOI297 plus pBBR1MCS-2 : : atfA revealed a more than twofold higher FAEE production using the newly constructed plasmid pMicrodiesel (0.64 g l–1 compared to 0.26 g l–1) whereas ethanol concentrations were similar. This indicated the positive influence of provision of all three relevant genes on a high copy-number vector and, as consequence, potentially higher expression rates on FAEE yield.

We then aspired to further optimize FAEE production by E. coli TOP10(pMicrodiesel), employing an aerobic fed-batch fermentation regime. Initial optimization experiments revealed that no regulation of medium pH during cultivation, resulting in a slightly acidic pH of 6.0–6.5 at the end, rather than a strict regulation at pH 7.0, might be favourable for FAEE biosynthesis (data not shown). Thus, the pH value was only roughly regulated automatically between 6.0 and 8.5 during the following fed-batch fermentation experiment (Fig. 5Down). To avoid carbon limitation, glucose was fed several times during the cultivation period. FAEE concentration continuously increased throughout the fermentation process, whereas its composition remained relatively constant (similar to the results shown in Fig. 2bUp). Employing this fed-batch strategy, a final FAEE content of 1.28 g l–1 was achieved after 72 h, which was about five times higher compared to aerobic batch fermentation of the E. coli TOP10 strain harbouring pLOI297 plus pBBR1MCS-2 : : atfA (Fig. 3aUp). With a final cellular dry biomass of 4.9 g l–1 this corresponds to an impressive cellular FAEE content of 26 % (w/w). Referred to the initial amount of 2 g l–1 present in the medium at the beginning of the cultivation, sodium oleate was converted to FAEEs with an efficiency of 62.7 % on a molar basis.

Fig. 5. FAEE production during fed-batch fermentation of E. coli TOP10(pMicrodiesel). Cultivation was done in a 2 litre stirred bioreactor initially filled with 1.5 l LB medium containing 0.2 % (w/v) sodium oleate, 2 % (w/v) glucose, 1 mM IPTG and 75 mg ampicillin l–1 under aerobic conditions (aeration rate 3 vvm) as described in Methods. The pH was kept between 6.0 and 8.5 by automated addition of 4 M HCl or NaOH. To prevent carbon limitation, 1 g glucose l–1 was fed several times during cultivation (indicated by arrows). Sodium oleate causes turbidity of the medium, explaining the high initial optical density. {blacksquare}, OD600; {blacktriangleup}, ethanol concentration; bullet, FAEE concentration.


Biodiesel is an interesting alternative energy source and is used as substitute for petroleum-based diesel. Offering numerous environmental benefits, it has attracted broad public interest and is being produced in increasing amounts (see Introduction). However, a broader use of biodiesel and a more significant substitution of petroleum-based fuels in the future will only be possible if production processes are developed that are not solely based on oilseed crops but on more bulk plant materials like cellulose. Toward this goal, we report here on a novel approach to establish biotechnological production of biodiesel using metabolically engineered micro-organisms, which we refer to as Microdiesel. The early optimization studies described here revealed FAEE yields of up to 26 % of the bacterial dry biomass. Although these yields are still far below the needs for an industrial process, this study has clearly proved the feasibility, in principle, of this novel approach. Therefore, the present study might open new avenues potentially enabling microbial production of fuel equivalents from cheap and readily available renewable bulk plant materials like sugars, starch, cellulose or hemicellulose in the future.

Microbial FAEE biosynthesis for Microdiesel production is based on the exploitation of the extraordinarily low substrate specificity of the acyltransferase (WS/DGAT) of A. baylyi strain ADP1, which in its natural host mediates wax ester and TAG biosynthesis from acyl-CoA thioesters plus long chain-length fatty alcohols or diacylglycerols (Kalscheuer & Steinbüchel, 2003Down). E. coli does not produce such substances by its natural metabolism; however, recombinant strains enabled to produce large amounts of ethanol and simultaneously expressing WS/DGAT provided an unusual, alternative substrate for this acyltranferase. This resulted in production of substantial amounts of FAEEs utilizing WS/DGAT's substrate promiscuity.

E. coli forms ethanol, among other fermentation products, during mixed acid fermentation under anaerobic conditions from acetyl-CoA via two sequential NADH-dependent reductions catalysed by a multifunctional alcohol dehydrogenase (the adhE gene product) (Goodlove et al., 1989Down; Kessler et al., 1992Down). However, ethanol levels naturally occurring in E. coli under anaerobic conditions are probably not sufficient to support formation of significant amounts of FAEE. In addition, several other fermentation products besides ethanol occur in substantial amounts. By using a recombinant system employing Z. mobilis pyruvate decarboxylase and alcohol dehydrogenase, this limitation was circumvented, resulting in substantial amounts of ethanol under aerobic conditions, which is in accordance with previous reports (Ingram et al., 1987Down; Alterthum & Ingram, 1989Down). In fed-batch fermentations conducted under controlled aeration rates, the highest FAEE levels were observed in recombinant E. coli under aerobic conditions (approximately five times higher compared to anaerobic conditions) although ethanol levels were similar. This indicates that uptake of exogenous fatty acids from the medium and their activation to the corresponding acyl-CoA thioesters is probably another factor limiting Microdiesel production in E. coli under anaerobic conditions.

Although an impressive FAEE content as high as 26 % of the cellular dry weight was finally obtained, E. coli is not ideal for Microdiesel production for various reasons. Although the occurrence of ethyl palmitate as a minor constituent indicated that fatty acids derived from de novo fatty acid biosynthesis were channelled into FAEE production, substantial FAEE biosynthesis was strictly dependent on supplementation of exogenous fatty acids. This indicates that de novo fatty acid biosynthesis, in contrast to fatty acid beta-oxidation, can not provide sufficient intracellular acyl substrates for WS/DGAT-mediated FAEE synthesis. Therefore, it will be challenging to establish Microdiesel production solely from simple bulk plant materials like sugars, cellulose or hemicellulose in the future using E. coli as a production platform. As an alternative, storage-lipid-accumulating bacteria, in particular those of the actinomycete group, may be used; these bacteria are capable of synthesizing from simple carbon sources like glucose under growth-restricted conditions remarkably high amounts of fatty acids (up to ~70 % of the cellular dry weight) and accumulate them intracellularly as TAGs (Alvarez & Steinbüchel, 2002Down). If the flux of fatty acids could be directed from TAG towards FAEE biosynthesis by genetic manipulation, storage-lipid-accumulating bacteria might be promising candidates for more simplified Microdiesel production processes in the future. Establishment of recombinant ethanol biosynthesis in these aerobic, non-fermentative bacteria would be a prerequisite for this purpose. In this regard, a recently developed heterologous ethanol production system for Gram-positive bacteria could become of great value and utility (Talarico et al., 2005Down). Future optimization of biotechnological Microdiesel production will also benefit from the progress made in recent years in lignocellulose utilization as feedstock for bioethanol production by recombinant micro-organisms (Dien et al., 2003Down; Zaldivar et al., 2001Down).

A further bottleneck in the path towards optimized FAEE levels is the relatively low reaction rate of WS/DGAT with ethanol in comparison with longer chain-length fatty alcohols (C10–C18) (Kalscheuer et al., 2004Down; Stöveken et al., 2005Down). Numerous genes encoding WS/DGAT homologues have been identified in several other bacteria (Kalscheuer & Steinbüchel, 2003Down). One of those acyltransferases might be more suitable for FAEE production since it may exhibit a higher specificity for ethanol. Alternatively, increase of the reaction rate of WS/DGATs with ethanol may be achieved by enzyme engineering.

Optimized Microdiesel production by engineered micro-organisms could finally offer some major advantages over established conventional production processes. Biotechnological Microdiesel production could be significantly less expensive than conventional biodiesel production if plant products like starch or lignocellulose are used for its production. These plant polymers are not only much cheaper than plant oils, but are also much more abundant, and Microdiesel production will not be restricted to oilseed-producing regions of the world. In contrast to conventional FAME-based biodiesel, Microdiesel is a fully sustainable biofuel completely derived from renewable materials, also avoiding the use of highly toxic methanol. In conclusion, this study provides a basis to achieve more competitive production costs, and therefore a more substantial substitution of petroleum-derived fuels by biofuels in the future.


The authors would like to thank Nicole Tessmer for skilful technical assistance in fermentation experiments.


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Received 27 March 2006; revised 12 June 2006; accepted 26 June 2006.

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Ted Turner: biofuels "key" to revive WTO trade negotiations

Very important news. Earlier we hinted at the fact that biofuels might revive the collapsed WTO trade negotiations known as the Doha Development Round which is aimed at creating a global trade regime that is more fair to the developing world. No major news source however looked at this potential role of biofuels in the negotiations. As we have written on numerous occasions, our mission at the Biopact is to make the case that it is time to get rid of EU/US agricultural subsidies and trade barriers (which are the cause of the deadlock), and to open the market to biofuels produced by developing countries. The resulting 'win win' situation would bring millions of jobs to the world's poorest, cut their energy bill, increase energy security in the West, bring the EU/US abundant and competitive biofuels (unlike their own which need subsidies) and bring a more equitable distribution of capital and wealth on a global scale.

Now CNN/AOL Time Warner boss Ted Turner thinks biofuels are indeed the key to resolve the deadlocked trade negotiations and he sums up the exact same reasons as we have been doing here. He urges negotiators to save Doha, using bioenergy as the lever.

Turner told a public forum at the World Trade Organization yesterday that biofuels — liquid fuels made from plants and trees, including biodiesel for trucks and generators and ethanol for cars and cooking — can do more than fight global scourges like pollution and global warming.

They can also solve the bitter dispute that scuttled the commerce body’s trade liberalization talks two months ago by providing rich countries a means of keeping their farmers in business, instead of doggedly subsidizing products that can be farmed more cheaply in poor nations, such as cotton, sugar beets or cane and rice.

The Doha round of trade talks was launched in Qatar’s capital in 2001 with the aim of boosting the global economy by lowering trade barriers across all economic sectors, with a particular focus on helping developing countries by boosting their export growth.

The talks came to a screeching halt in July, largely over the unwillingness of rich countries like the United States, the 25-nation European Union and Japan to offer deeper cuts in subsidies paid to farmers or ease access to their agricultural markets for foreign goods. Recent meetings in Brazil and Australia have only confirmed the deep divisions among the organization’s 149 members.

"I would have preferred to stand in front of you under different, more encouraging circumstances since it is always easier to find your way to the door with the lights on," WTO Director-General Pascal Lamy told the forum of academics, activists and government officials. "We missed an important opportunity to advance our plea for a stronger multilateral trading system," Lamy said.

Turner talked of the promising opportunities in corn, sugar beets and sugar cane that can be converted into ethanol, and palm, soy and rapeseed oil that can be transferred into biodiesel. These sources, he said, would provide poor countries with local jobs through substituting the fuels for oil imports:

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He said subsidies and tariffs should be replaced by support for biofuels. "Farmers have always grown crops for food and fibre," Mr Turner said. "Today, farmers can grow crops for food, fuel and fibre. The global demand for biofuels is huge and rising. "That's why I'm confident that in the near future, farmers' incomes will be assured, not by subsidies and tariffs, but by market forces."

Mr Turner was speaking in his role as chairman of the United Nations Foundation, which was set up in 1998 after he gave $1bn to support UN causes and activities.

The UN Foundation is promoting the production and use of biofuels in developing countries and wants to attract more foreign and domestic investment in the area.

"By investing in biofuels, developing countries can produce their own domestic transportation fuels, cut their energy costs, improve public health, create new jobs in the rural economy and ultimately build export markets," Mr Turner said:

It is very interesting to hear Mr Turner's ideas, but of course many questions remain: Turner rejected a question later at a media conference concerning how biofuel production can be safeguarded against new forms of subsidies. It is unclear what protection poor countries would have under WTO rules for protecting biofuel producers from competitors in rich countries aided by government subsidies.

Moreover, nothing would change if the West were merely to replace its agricultural subsidies for food and fibre crops, and transfer them to energy farming. So it remains to be seen how this proposal will be worked out technically.

But one thing is certain, if tariffs and subsidies in the EU and the US are removed, and if a global level playing field for trade is created, then the developing world has an enormous opportunity to benefit and lift millions out of poverty by producing and exporting biofuels to these markets. The global South has the competitive advantage: plenty of unused arable land, suitable agro-ecological conditions, where tropical crops with high yields can be grown.

We will follow up very closely on this issue and report back as soon as more details emerge.

More information:

Forbes: Turner Sees Biofuels As Key To Trade - Sept. 25, 2006

International Herald Tribune / Associated Press: Ted Turner tells WTO of benefits in biofuel use - Sept. 25, 2006

BBC: Biofuels 'answer' to trade talks - Sept. 25, 2006

MercoPress: Biofuels could be key at WTO - Sept. 25, 2006

South Africa Business Report: Let green energy rescue Doha negotiations, says Ted Turner - Sept. 25, 2006

India Business Domain: Biofuels can break WTO deadlock, says Ted Turner - Sept. 25, 2006

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