SEB — Texte — Cornelia Eisenach

Lunch made by Stefan Jansson using kale that was genome-editide with Crispr/Cas9. Image: University of Umeå

In a post-truth era, how do we communicate controversial science? The Science with Impact event at the SEB Annual Meeting 2017 in Gothenburg will address this question, and discuss the good, the bad and the ugly of past approaches.

Arguably, one of the ugliest communicat ion wars was fought over genetically modified organisms (GMOs), especially food crops. While some might still lick their wounds, the next generation of crops, bred through New Plant Breeding Technologies such as the Crispr/ Cas9 system, are already on our door step. But can these new crops be classified as GMOs? How they should be regulated is currently being discussed across the globe.

GMO OR NOT GMO, THAT IS THE QUESTION

CRISPR/Cas9 is the new revolution in genetic engineering and many scientists are using it in their day-to-day research. CRISPR stands for clustered regularly interspaced short palindromic repeats, and Cas9 is a CRISPRassociated nuclease. The system is often described as molecular scissors. It can be used to precisely target a certain position in a genome where it cuts the DNA and during subsequent repair, base pairs can be deleted, modified or inserted (see Graphic I). While a moratorium has been advocated for its use on the human germ line (1), it is being applied to modify plants and fungi used in food. Only last year, a white button mushroom engineered with CRISPR/Cas9 to reduce browning was approved by the US Department of Agriculture (USDA) for commercial use and without it being subject to regulation (2).

With the CRISPR/Cas9 system, a crop genome can be modified to breed beneficial traits. It can be used to introduce foreign DNA, rendering the resultant crop a GMO. If it is used to induce point-mutations, the resultant crop cannot be distinguished from a conventional breed, because the CRISPR/ Cas9 system is removed from the crop after it has done its job. The mutation could have arisen spontaneously, or by other means of conventional breeding such as treatment with chemicals or radiation (see Graphic II). This is why scientists from China, USA and Germany have called to exempt so-called gene-edited crops from regulations that currently apply to GMOs (3). The researchers argue that there is no scientific reason to distinguish between two mutated plants based on how the mutations were induced, i.e. either via conventional breeding or via gene-editing. Furthermore, using CRISPR/Cas9 to induce a mutation would lead to fewer unwanted effects in the genome than mutagenesis by chemicals or radiation.

GETTING CONSENSUS

Regulatory authorities across the globe now need to decide on how to legislate gene-edited crops and, more generally, New Plant Breeding Technologies (NPBT, see explainer). And time is running out. Already in 2015, six EU member states judged that a herbicide-tolerant oilseed canola that had been modified using an older gene-editing technique was not to be classed as GMO under EU law. The German Federal Office of Consumer Protection and Food Safety allowed the US firm Cibus, which created the crop, to go ahead with field trials.

At the time the EU commission asked these countries to halt their approval and wait for their legal interpretation of EU law. Will it be interpreted to state that any organism that was created using genetic modification techniques is a GMO, i.e. a process-based interpretation, or does it also allow for a product-based interpretation, i.e. that any organism containing foreign DNA is a GMO? The European Commission already instated an expert group on NPBTs in 2007, and although the Commission’s interpretation was announced for the end of 2015, then postponed to spring 2016, an official document has yet to be released. Current events in France have pushed the ball even further afield: in October 2016, the French Government asked the European Court of Justice to rule whether NPBTs fall under EU law on GMOs and whether countries could ban these technologies (4). A ruling, and therefore a judgement of the EU Commission, cannot be expected before 2018.

MAKING A MEAL OF IT

Someone who did not want to wait so long is Stefan Jansson. Professor at the University of Umeå in Sweden, he demonstrated how scientists can communicate the risks and benefits of New Plant Breeding Technologies. In the tradition of self-experimentation, he cooked and ate what was probably the world’s first CRISPR lunch using gene-edited kale. Jansson is one of the organisers of a session on ‘New breeding technologies in the plant sciences’ at the SEB meeting in Gothenburg 2017. “We succeeded in convincing a Swedish authority to state that, according to their interpretation, the plants could not be considered genetically modified in accordance to EU regulations as they do not contain any ‘foreign DNA’,” he writes in a blog documenting the experiment (5). He grew the kale, which had been modified using the CRISPR/Cas9, in his own vegetable garden and when it came to harvest time, he invited the host of a Swedish radio station and cooked a CRISPR-kale pasta dish, the recipe of which can be found on his blog.

EXCEPTIONS TO THE RULE

While in Europe the waiting game continues, the USA has recently taken a clearer stand. In January 2017, the USDA Animal and Plant Health Protection Service (Aphis) released documents which contain suggestions for new regulatory rules. Aphis advocates exempting crops produced by gene-editing from regulation but go even further: the authority also seems to want to exempt certain transgenic crops. Transgenic crops often contain sequences of plant pests such as the 35S promoter of the cauliflower mosaic virus or the left border sequence of Agrobacterium. But the authority states: “The experience has shown that the use of genetic material from plant pests has not resulted in the creation of plant pest risks in recipient organisms.” It did not refer to any systematic analysis of this experience, but the passage suggests that crops bred through Cisgenesis (see Graphic II) might no longer be regulated in the future.

A ruling in the US is likely to impact on Europe, either politically or through import of potentially unclassified geneedited crops. Many organisations including those representing organic farmers are on the alert. Whether gene-editing results in changes that could be obtained through conventional breeding, or not, is not the crucial question. In the view of many organic farming organisations, manipulation of subcellular material is prohibited due to ethical considerations. So far, IFOAM, the European umbrella organisation of the organic movement, has called for NBPTs to be considered as GMOs, and its membership is expected to vote on the organic sector’s position in November 2017. So, are traditional fault lines of past communication wars set to open again? NBPTs could have benefits for organic farming: geneedited crops that do not contain foreign DNA could help to increase productivity without the use of herbicides and pesticides. It is up to scientists to communicate these benefits in engaging ways.

REFERENCES:

Integrated Constructed Wetland located near Waterford, Ireland. Image by Dr Rory Harrington (Dunhill, Waterfoot County, Ireland). — Integrated Constructed Wetland located near Waterford, Ireland.
Image by Dr Rory Harrington (Dunhill, Waterfoot County, Ireland).

"Water, water, everywhere, And all the boards did shrink; Water, water, everywhere, Nor any drop to drink. "

Samuel Taylor Coleridge, the Ancyent Marinere [1798]

We might not be quite as desperate as our ancient mariner yet, but those fresh drops are running low. And not only in arid regions of the world that are plagued by droughts and restricted access to fresh drinking water. “Water scarcity and therefore access to clean drinking water is particularly important in semi-arid regions such as the Mediterranean area, but also in other regions where water demand approaches, or even exceeds, water availability. This includes large areas of Europe”, says Dr. Nick Voulvoulis, whose research focuses on water and environmental management at Imperial College London, UK. Certainly, as our ancient mariner experienced, the majority of our planet’s water is too salty for us to drink, or feed our plants and animals. In addition, over-use and water pollution “not only threaten our water security but nature itself, with the removal or impairment of aquatic ecosystems and the services that they can provide to us”, explains Dr Voulvoulis, adding that more integrated sustainable solutions are needed.

Saltier bacteria – fresher water

Virtual water balance per country and direction of gross virtual water flows related to trade in agricultural and industrial products over the period 1996-2005. Only the biggest gross flows (> 15 Gm3/ yr) are shown; the fatter the arrow, the bigger the virtual water flow. Reprinted from [1] Water is a limited resource only as fresh water so tapping into the oceans would help address our rising fresh water demands. Countries with access to sea water have been employing a technique called reverse osmosis to desalinate water for use in agriculture and although this technique has been developed and improved over the decades, it has one major flaw: it consumes a lot of energy. “This is where biology comes in, because there is nothing like biology when it comes to exploiting energy”, says Prof. Anna Amtmann, who leads an EPSRC-funded, multi-disciplinary research team in the UK to tackle the problem of fresh water availability. She and her team have come up with a biology-based idea to desalinate without the need for large energy input using photosynthetic cyanobacteria. These bacteria live and grow on sunlight, they survive in seawater and they can grow to large densities. “Sodium salt is toxic to many organisms, even those that live and thrive in sea water”, says Amtmann. However, cyanobacteria possess plasma membrane proteins (an ATP-powered proton pump and a Na+/ H+ exchanger that exploits the pump’s proton gradient) that force out salt and so allow the bacteria to grow and reproduce in salt water. Once the cyanobacteria grow to high densities they run out of the ATP necessary to extrude the salt and, as a result begin to take up sodium and chloride. The team intend to enhance this biological process further using a relatively new synthetic biology technique called ‘optogenetics’, in which a light-powered transport system enhances salt uptake by the bacteria. This optogenetic technique uses halorhodopsin, a specialist protein found in halobacteria that pumps chloride ions inside the cell when it is stimulated by light. Amtmann’s team set out to genetically manipulate the cyanobacteria to express halorhodopsin: the pump will use the sun’s natural energy to drive chloride accumulation inside the cyanobacteria and the chloride-concentration gradient will draw in sodium. “We have already made progress in a proof-of-principle approach, where we were able to manipulate membrane potential by expressing halorhodopsin”, says Amtmann. The team has also shown that simply growing and removing cyanobacteria from sea water already reduces the water’s salt content considerably. Sodium chloride adsorbs to cyanobacteria, possibly due to so-called extracellular polymeric substances that are contained within the cell wall and that act like salt scavengers. “We have characterised the chemical and physical properties of these substances and found that they change depending on the salt concentration”, says Amtmann. “Now it’s a matter of identifying the genes in the pathway that underlie this whole metabolism so as to exploit it further”, [2].

Wired up bacteria

The power of bacteria has been harnessed in the waste-water treatment industry for a long time. However, as with conventional desalination plants, waste-water treatment plants consume a lot of energy. So water engineers have been looking to biology to clean water without a large expense of energy. “Many of these energy-efficiency treatments rely on anaerobic processes involving complex microbial communities”, says Dr. Federico Aulenta of the Water Research Institute, IRSA-CNR in Monterotondo, Italy. He explains that one biological approach to clean waste water is indeed called anaerobic digestion. If you have ever seen images of bubbles arising from lake floors or from water-logged boggy land, you have an idea of what this process looks like. Anaerobic digestion involves distinct metabolic steps and each step is carried out by a different class of microbe such as acetogenic or methanogenic bacteria. These different microbes live in what is called syntrophic association, that is they link up their specialised metabolisms to form a conversion chain that breaks down the organic matter all the way to methane. The electrons arising from the initial oxidation of organic matter are passed on between microbes through a process called interspecies electron transfer. Ultimately, these electrons are used to reduce carbon dioxide to methane. Aulenta and his team have recently discovered that this process can be boosted by the addition of conductive nanoparticles. These nanoparticles, made up of the natural mineral magnetite, enhance the transformation of organic matter to methane by boosting interspecies electron transfer, essentially ‘wiring up’ the different bacteria involved in the process. Reported in a recent publication, the researchers demonstrated that if they added the magnetite nanoparticles to anaerobic sludge, the production rate of methane from a model substrate could be increased by up to 33% [3].

Hungry for electrons

As well as microorganisms being able to transfer electrons from the oxidation of organic matter to other microbes, they can also transfer them to extracellular electron acceptors, for example, an electrode. This means they can generate electric current from fuel, e.g. organic matter, a principle exploited in microbial fuel cells. However, Aulenta is actually interested in reversing this process to tackle the problem of ground water contamination by industrial pollutants. “Chlorinated solvents are priority pollutants”, he says. “It is estimated that 80% of our contaminated ground water contains these solvents, due to improper storage, handling and accidental spills”. This is where ‘electron transfer’ comes in: The electrodes donate electrons to the microbes which in turn pass them on to chlorinated solvents, as they use them as a substrate for their respiration. “These anaerobic microorganisms, such as Dehalococcoides, ‘respire’ the solvent, reducing it to ethylene, which is non-hazardous”, explains Aulenta. But how easy is it to take what works in the laboratory to a real-life problem? Prof. Mark Riley, who heads the Biological Systems Engineering Department at the University of Lincoln-Nebraska, U.S.A., answers: “To develop a technology for a broad application the process has to be taken from a laboratory scale, via a small pilot scale to full scale. However, a system that works well on a small scale does not necessarily perform with the same efficiency or outcome when scaled-up. This challenge is often seen in the pharmaceutical industry which by necessity must put much effort into this scale-up process”. This is an issue for Aulenta who says: “The key challenges of these technologies is loss of efficiency when you scale up electrochemical reactors. The larger the reactor, the poorer the performance”. To address this challenge Aulenta and his collaborators are going to take a small step towards scaling up their chlorinated solvent decontamination and will soon run a demonstrative test at a real contaminated site in northern Italy.

Safe haven for microbes

Wetlands, which are a natural habitat for water-cleaning biodegrading bacteria, are fast disappearing from the planet. In Ireland, for example, 90% of the wetlands have been transformed into agricultural land since the industrial revolution. Prof. Miklas Scholz from The University of Salford, UK, explains: “The transformation of wetlands into agricultural land is having a detrimental effect on the environment and climate change as carbon that had been locked away has now been released as CO2 and methane.” Scholz chairs the Civil Engineering Research Group at Salford and focuses on the use of so-called ‘Integrated Constructed Wetlands’ in waste water treatment. The principle of this technique is simple: you need a large area of land, divide it into wetland areas, apply a substrate, and seed or allow plants to settle on it and flood it with waste water. The surface will be covered by about 20 cm of waste water. The natural degradation processes performed partly by plants and mostly by microbes remove contaminants from farmland run-off, agricultural or domestic waste water [4].

Surprisingly, it is even possible to build wetlands in the form of artificial floating islands. “BioHaven® is a commercially available floating wetland”, explains Leela O’Dea, an ecologist and founding partner at the aquatic environmental consulting firm frog environmental. “It is about six inches deep and made up of a recycled plastic matrix, which allows plants to grow on it”. The floating island allows plants to grow with their roots dangling down into the water. “The advantage of this system is that the roots offer a really high surface area for microbial and sedimentation processes to take place”, says O’Dea. “There are a lot of microscopic particles or colloids suspended in the water and the dangling roots, which are covered with a sticky biofilm of microbes, help the sedimentation of these colloids”. Once the roots become heavy they drop off and carry the contaminants away into the sediment at the bottom of the water. “The sedimentation process is especially important for the removal of phosphorus”, says O’Dea, “because phosphorous often adsorbs to soil particles and so gets trapped in colloids.”

Microbial processes such as nitrification, denitrification and ammonification contribute the lion’s share to a floating wetland’s efficiency. “We estimate that plants manage 6-8% of the treatment efficiency, either through direct uptake or transformation processes. From microbes, however, we get a 61-63% treatment value”, says O’Dea. Despite the huge workload on the microbes, plants can be used in integrated or floating wetland systems depending on their ability to accumulate certain contaminants, for example heavy metals. “We do look at the phytoremediation a plant can offer. For example, we can add Iris to the island which aids treatment of water with lead contamination”, says O’Dea. Miklas Scholz has been involved in a project examining the use of constructed wetlands in arsenic removal [5]. Scholz says: “If you use plants known to hyperaccumulate certain contaminants in a freshly set-up wetland system, you can actually see differences in treatment efficiency. But as the system matures, this effect lessens as other natural species out compete the hyperaccumulators”. Scholz believes that other benefits of constructed wetlands, such as attracting natural flora and fauna, creating biodiversity and offering room for recreational activities, should be factored into the equation of cost and energy efficiency when considering treating and decontaminating our water: “Research should not only compare and contrast the most advanced treatment methods but also consider alternative techniques”.

[1] Mekonnen, M.M. and Hoekstra, A.Y. (2011) National water footprint accounts: the green, blue and grey water footprint of production and consumption, Value of Water Research Report Series No. 50, UNESCO-IHE, Delft, the Netherlands.

[2] Amezaga, J. M., Amtmann, A., Biggs, C. A., Bond, T., Gandy, C. J., Honsbein, A., Karunakaran, E., Lawton, L., Madsen, M. A., Minas, K., Templeton, M. R. (2014) Biodesalinatio: A Case Study for Applications of Photosynthetic Bacteria in Water Treatment. Plant Physiology, 164 (4), 1661-1676

[3] Viggi, C. C., Rossetti, S., Fazi, S., Paiano, P., Majone, M., Aulenta, F. (2014). Magnetide Particles Trigger a Faster and More Robust Syntrophic Pathway of Methanogenic Proprionate Degradation. Environ. Sci. Technol., 2014, 48 (13), pp 7536–7543

[4] Dzakpasu M., Scholz M., McCarthy V. and Jordan S. (2015) Assessment of Long-term Phosphorus Retention in an Integrated Constructed Wetland Treating Domestic Wastewater. Environm. Sci. Pollut. Res., 22(1), pp. 305–313.

[5] Wu M., Li Q., Tang X., Huang Z., Lin L. and Scholz M. (2014) Arsenic(V) Removal in Wetland Filters Treating Drinking Water with Different Substrates and Plants. Int J Environ Anal Chem. 94(6): 618–638.

New plant breeding technologies - A regulatory conundrum

Liquid Assets - capital ways to save water