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5. Engineering poplar for bioethanol production. The road toward a field trial.
Frederik Coppens (Ghent University, Belgium)

Introduction

Why bioenergy?
In our current society, the demands for energy are constantly increasing. This results in an enormous increase in oil prices. Therefore, there is a strong stimulus to develop new, sustainable energy sources. Plants can capture solar energy and store it as sugar and biomass. This process is carbon-neutral and renewable. This ‘bioenergy’ could become an important replacement of classical energy sources in the future.

Biomass to bioethanol
The process of producing bioethanol from dried wood first requires a pretreatment with 1 N HCl at 80°C for 2 hours. This procedure breaks the lignin “glue” of the cell wall. This step is necessary to soften the cell wall so cellulase can access the cellulose microfibrils more easily. Afterward, an enzymatic hydrolysis using cellulase releases glucose from the plant material. This can then be fermented to ethanol with baker’s yeast.

CCR-downregulated poplar
The energy-demanding step in the production of bioethanol is the pretreatment. This could be made easier if less lignin and more cellulose were in the cell wall. Cinnamoyl-CoA-reductase (CCR) is an essential enzyme in the biosynthesis of monolignol, the building block of lignin. Downregulation of this enzyme using RNAi yields poplar trees with 20% less lignin and 17% cellulose under lab conditions. This results in 50% more ethanol being released compared to wild-type poplar trees.

A field trial
Growth under laboratory conditions does not resemble growth under natural conditions; the changing of, for example, the seasons, wind, frost, fungi, and insects, are all factors that are not taken into account in the lab. It is also not possible to grow enough plant material in the lab to test the bioethanol production process at the semi-industrial scale. Therefore, an application was filed for a field trial.

The proposal
The field trial includes a wild-type poplar and two CCR-downregulated lines. They will be grown under short rotation coppice for 8 years. In short rotation coppice, the trees are cut about 20 cm above the ground after 1 year. This causes sprouts to form, which are harvested after 3 years. After 3 more years, a second harvest of sprouts can be attained. This can go on for 15 to 20 years.

Measures for containment
Several measures to avoid spread of the transgenic trees to the wild population are taken.
One mechanism of spreading is through adventitious root formation. When a wild-type poplar branch falls on the ground, it can form adventitious roots and establish a new tree. This is avoided by putting up a fence, which will contain most of the branches within the fenced area. In addition, a border of wild-type trees will surround the transgenics. Moreover, the cultivar that was transformed does not easily form adventitious roots. Branches placed in soil, or even artificial growth media, do not form roots and thus do not establish new trees. Crossing of the transgenics with the wild population through pollen can also cause contamination of the natural population. This is avoided by the short rotation coppice. Poplar shoots only flower after 8 years. As the shoots are harvested every 3 years, flowers (and thus also pollen) will never form.
A final method of spreading is through saplings that sprout from the root system. These saplings will be killed within and around the fence. No poplars will be in the immediate vicinity of the fenced area, so spotting and killing these saplings will be very easy.
If transgenic trees should, despite all measures taken, “escape” in nature, they will be contained by negative selective pressure, as they grow more slowly.

Regulatory processes
The field trial underwent the whole regulatory process. First, a consultation of the public was made, followed by the advice of the Biosafety Council and the advice of the regional minister. The final decision was made by the federal ministers for Health Care and Environment.
Public concerns were expressed, but all of them could be easily addressed. The Biosafety Council and the regional minister both supported the application. The federal ministers, however, refused to approve the application.
One of the reasons for refusal was the lack of a protocol to study the effect of the transgenic trees on the soil microflora and microfauna. This protocol is indeed not in the application as it is not demanded by the legislation. However, if this is a concern, this can of course be developed. Another concern was the presence of an antibiotic resistance marker as these are to be phased out by the end of 2008. The marker used, hygromycin, is allowed by the European Food Safety Authority (EFSA). Finally, 40 public comments doubted the relevance of biofuels to society. This is, however, a completely different discussion.

Take home messages
・ Genetically modified (GM) plants can be grown safely in a field trial when the appropriate precautions are taken.
・ The general public needs to be informed correctly about the benefits and dangers of GM plants.
・ An urgent need exists for objective regulation on a European or even world scale.

≫ Audience comments and questions

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