Having a project that works in the lab is good. Having one that can be used in the field is even better. For RhyzUp to become a real product, there are many obstacles to overcome and many aspects to think about. Most importantly, our project has to be turned into a product that can be used in agriculture. It is crucial to think about what agricultural practices RhyzUp could be used in and what the product would need to look like. In addition, market analysis is necessary to determine the financial potential of our product. Since RhyzUp uses gene technology, which is highly regulated in Switzerland and many other countries, it is also crucial to see what it would take to get our product approved.

To find out how our project could be implemented in agriculture, our exchange with Gebrüder Meier and Vivienne Oggier was central to understanding which agricultural techniques could be combined with RhyzUp. We also contacted Andermatt Biogarten, a company that already sells products based on rhizobacteria. In addition, our call with Dr. Natacha Bodenhausen provided insight into how to detect the presence of our strain in soils and which soils could benefit most from RhyzUp. Modeling the growth costs of our bacterial strain has also given insight into the best composition of a potential storage medium.

Hydroponic systems, also known as hydrocultures or soilless cultures, describe any method of growing plants without soil. In hydroponic systems, plants are most often grown in a controlled environment such as a greenhouse. Hydroponic systems may use only nutrient-enriched water or substrates such as sand, gravel or even rockwool to grow their crops. 1

A visit to Gebrüder Meier helped us gain a better understanding of farming in highly controlled systems, as they produce what are called hydro salads. Hydro salad is produced in a system similar to hydroponics. Although the roots of the lettuce seedlings are planted in soil cubes, the seedlings are then grown in a system with a closed, nutrient-enriched water cycle.

Gebrüder Meier already uses supportive microorganisms to produce some of their crops. The microbial solutions are simply added to the circulating water, which provides a good environment for the bacteria to grow and benefit the young plants. This has shown us that it is quite easy to introduce rhizobacteria into hydroponic-like systems, as they can simply be stored in liquid medium and added to the water cycle.

An advantage of soilless cultures is the avoidance of soil-borne pathogens. For this reason, microbes that are not essential for plant growth in hydrocultures were not of interest when hydroponic systems were first established in the 1970s. As waterborne pathogens became more of a problem and supportive microorganisms were lacking, symbiotic microorganisms and biocontrol bacteria became more of an interest. Microorganisms appear to be important to crops, even in soilless cultures. Several genera and strains of Pseudomonas are known biocontrol agents that protect crops from pathogens. Certain strains have been shown to promote tomato plant growth in rockwool hydroponic systems by reducing pathogen pressure.2

The above benefits and the fact that non-GMO microorganisms are already being used to improve plant growth in greenhouse agriculture have shown us that there is potential for RhyzUp to be used in hydroponic systems and greenhouses in general. The fact that these irrigation systems are more controllable than open field agriculture facilitates the use of beneficial rhizobacteria and allows for easy implementation. Although greenhouse and aquaponic systems account for only a fraction of global crop production, their importance is growing.

A potential problem that remains is that overexpression of biofilm could lead to clogging in closed recirculating systems. This is an issue that would need to be tested if RhyzUp were to be considered as a product for commercial use. However, we believe that introducing a kill switch into our Pseudomonas sp. IsoF that prevents it from surviving without the root exudate xylose may limit this problem.

Approximately 1.6 billion hectares of land are used for crop production worldwide 3, while a recent estimate by the University of Copenhagen found that 1.3 million hectares are used for greenhouse farming.4 This shows that the majority of crops are produced in open fields. Evaluating the potential implementation of RhyzUp in the field is crucial because the problems we want to address, such as drought, nutrient availability and pathogen pressure, are more pressing in the open field.5

Our visit to the farm of Vivienne Oggier's partner was key to our understanding of how to implement RhyzUp in the field. Vivienne suggested several ways to get our modified rhizobacteria to the roots of the plant in an open field system. These methods included:

• spraying our bacteria onto the fields in a liquid medium and then working the substrate into the soil
• inoculating the roots of a first generation of seedlings to enrich the bacteria in the soil
• applying RhyzUp as a seed coating
• applying RhyzUp along with microgranular fertilizers

Of the methods mentioned above, all seem feasible except for the method of enriching our Pseudomonas strain in the soil. The reason enrichment wouldn't work is that there is a kill switch in our project designed to prevent the viability of our Pseudomonas sp. IsoF in case it leaves the plant roots and therefore does not detect xylose anymore.

Dr. Natacha Bodenhausen has used metagenomics in the past to detect mycorrhizae that she was studying in the field. She advised us to find a genetic sequence specific to our P. sp. IsoF strain to detect it in soils. She also told us that healthy soils with high microbial diversity are very difficult to infiltrate and that our bacteria would probably be most useful in systems with low microbial diversity, such as fields next to chemical plants. It is good to know that our engineered rhizobacteria would be most useful where nature is most disturbed. So it would likely be used in places where there is less risk of our strain disrupting the local ecosystem.

Besides the variety of application methods, there are several products based on root-supporting microorganisms that are already used in the field. The Andermatt Group, for example, specializes in biological crop protection and sells products for farmers and gardeners that use the rhizobacteria. We were able to arrange a phone call with Reto Flückiger of Andermatt Biocontrol.

Reto Flückiger from Andermatt Biocontrol confirmed the different methods of application of rhizobacteria in agriculture suggested by Vivienne Oggier. According to him, the Andermatt Biocontrol product RhizoSol can be applied by pouring, while the product RhizoVital 42 can be applied by pouring, spraying, adding to irrigation systems, coating seeds before planting or adding to microgranular fertilizers. In all cases, the bacteria are part of a liquid medium. Both of the above products use the soil bacterium Bacillus amyloliquefaciens.

The same process could be done with our engineered Pseudomonas bacteria, as the kill switch that requires xylose for the bacteria to survive is only activated once a bacterium has been exposed to xylose. Choosing a suited application of our product is highly dependent on the equipment and needs of each individual farmer. What we were really amazed by, however, were the methods of seed coating and adding bacteria to microgranular fertilizers. This method lets farmers apply the fertilizers directly when they sow their seeds and drop the microgranules next to the seeds. Using said methods would allow our bacteria to be locally applied without huge risk of spreading to the environment.

One problem that Reto Flückiger mentioned is that Pseudomonas strains are generally more difficult to store than B. amyloliquefaciens. Their products containing B. amyloliquefaciens can be stored for several years. This has shown us the importance of the shelf life of a product which would have to be considered and tested in the development of a product.

The bacteria used in Andermatt Biocontrol's products are said to survive between three and six weeks in agricultural systems. Finding out how long your product lasts is very important for proper application.

When asked if the products mentioned above are used more often in greenhouses or in the field, Reto Flückiger replied that they are used in both systems, but are more popular in greenhouses. Like Dr. Natacha Bodenhausen, he told us that the addition of rhizobacteria is most useful in systems that are simple. This means that soils with high microbial diversity would benefit less than systems with lower microbial diversity. For us, this means that in addition to greenhouses, RhyzUp could be most useful in fields with poor soil quality and low microbial diversity.

During our discussion with the Andermatt Group, we also realized that agriculture may not be the only application for RhyzUp, as there are several Andermatt Biogarten products for gardeners that also contain B. amyloliquefaciens to strengthen plants. This opens up new entrepreneurial opportunities.

It is difficult to estimate the demand for RhyzUp because it is not entirely clear whether a potential product would be marketed for its ability to reduce pathogen pressure (as a biocontrol agent), for its ability to improve nutrient uptake by the plant root (as a form of co-fertilizer), or for improved drought resistance. At present, products like RhyzUp do not fully replace fertilizers or pesticides and are more often used in combination with chemical methods. Therefore, we decided that a good measure for a product like RhyzUp would be to check the market development of agricultural biologicals instead of pesticides and fertilizers. Agricultural biologicals are products made from living or naturally occurring products and are commonly used as biocontrol agents, biofertilizers, and biostimulants.6

The agricultural biologicals market is estimated to be worth $16.7 billion in 2024 and is expected to reach more than $30 billion by 2030. This represents a compound annual growth rate of approximately 13.8%. Organic food is becoming increasingly popular, especially in Europe and North America. In addition, European policies are becoming more restrictive on synthetic pesticides and fertilizers. These two developments, together with the current progress in biotechnology, are the main reasons for the growing sales of agricultural biologicals as substitutes for synthetic pesticides and fertilizers7.

With regard to Andermatt's B. amyloliquefaciens-based products, Reto Flückiger emphasized the importance of markets outside Europe and North America for us. He mentioned that their bio-stimulants, which target the roots of crops, gained popularity in South America and Africa before they did in Europe and North America when the Andermatt Group started selling these products about 20 years ago.

Looking at the market projections for agricultural biologicals shows the potential of RhyzUp as a commercial product. However, there are many players in the market who are already selling and developing agricultural biologicals with great influence. Key players include BASF SE, Syngenta Group, Bayer AG, and many others. To see how RhyzUp would compete with similar products, we conducted a SWOT (strengths, weaknesses, opportunities, threats) analysis.

Strengths:

• Hyperexpression of biofilm: potentially stronger effects than wild-type strains in other products
• Sensing root xylose prior to biofilm formation: no wasted energy prior to actual application
• Kill switch when bacteria leave roots: reduced risk of spillover into ecosystems when used in open fields
• Broadly applicable: potential use in all crops, applicable with existing technology, applicable in greenhouses and open fields
• Promising benefits: improved drought resistance, improved nutrient uptake, improved pathogen resistance

Weaknesses:

• Genetic engineering: issue for consumers and subject to stricter regulation (in many countries)
• Worse storage properties than e.g. Bacillus amyloliquefaciens
• Mechanism doesn't work yet

Opportunities:

• Increased pressure to withstand changing climate conditions (and increased risk of drought)
• Change in pathogen dispersal
• Current debate on facilitating the use of GMOs in agriculture (EU, Switzerland)

Threats:

• Strict regulation of GMOs in agriculture (EU, Switzerland)

In summary, we believe that RhyzUp has the potential to compete in the marketplace. However, this would require much more research. Also, the biggest challenge in Switzerland at the moment is the regulation of GMOs in agriculture.