Fossil fuels create a huge burden on the environment, and we need to make a switch to greener alternatives to combat this. Utilizing fermentations for bio-based products has become part of this solution; they are however not problem free. They tap into feed we use for animals, and require large fields of land for production which also compete with our food sources. At the same time, increased crop production also leads to deforestation and loss of biodiversity. With our A. niger strain, we seek to use underutilized waste sources from agriculture and forestry for fermentations, while removing a common inhibitor called furfural in said waste. In our journey to create this strain, we got in contact with representatives from several companies, professors and experts, all of which granted valuable input for our project. An overview of our human practices work, and how different people affected different aspects of our project can be seen below.

Overview of our integrated human practices

Challenges in fermentation

When we first started meeting as a team, we had many discussions as to what our project was going to be. Some early ideas included creating synthetic horseshoe crab blood, enabling PET degradation using fungal enzymes, microbial batteries, probiotic bacteria that kills parasites and many more. However, one idea stood out the most to us, both in regards to the feasibility and applicability to the biotech industry and the synthetic biology community in general: creating synthetic regulators capable of detecting any compound of interest.

This is a demanding challenge, but one that, if successful, would benefit not only many companies and researchers, but would also help create technology that many people in the world would benefit from.
Now comes the big question, something you may already be thinking (and also something we spent a considerable amount of time discussing): How do we reach our goal in a way that will benefit society the most?

Our point of entry

Before our work to create a synthetic transcription factor started, we needed to select a compound that we were interested in sensing. Said compound needed to fulfill a couple different requirements:

  • Easily accessible (for easy testing of synthetic regulator)
  • Few to no compound sensing regulators already existing
  • The compound should be of industrial and/or environmental relevance

From these requirements, we created a long list of compounds. We chose to focus on inhibitory compounds found in media used for fermentations, as sustainable fermentation was of interest to the team.

Furfural, a small toxic derivative of pentose, is a compound that stood out to us. The compound is produced during pretreatment of biomass in preparation for fermentations. Given the ever-increasing demand for sustainable alternatives to petrochemical products, detecting and degrading common toxins found in renewable energy sources makes for a good story and a great challenge that can potentially change the way we think about media for fermentation.

Fermentation-based solutions are not problem-free

An area that is sometimes touched upon when discussing the sustainability of fermentation-based products is the issue of which growth medium to use. As the production of biobased chemicals increases, so does the amount of crops needed to sustain said production. The unfortunate reality of today is that the production of biobased chemicals taps into the feed used for livestock (ICCT (2017)), while also increasing the necessity for farms to produce enough biomass. While some of the byproducts from e.g. production of ethanol can be used to supplement the feed, it still creates the need for farmers to further supplement their animal feed, increasing cost for farmers.

Yearly, large amounts of lignocellulosic biomass are left unused due to the difficulty of utilization. Think; rice husks, sawdust, etc. Germany, France and the UK alone produced over 100 million dry tons of lignocellulosic biomass in 2014 (ICCT (2015)), highlighting the huge potential resource that the biomass represents. Making use of this biomass for fermentations would help increase sustainability of industries that create lignocellulosic waste.

Why A. niger?

As our project has a large industrial focus, it was important to make sure that the organism we worked on could easily be used by companies. Our organism of choice, Aspergillus niger, is generally regarded as safe (GRAS) and used to create a wide variety of compounds (such as organic acids) and enzymes. These advantages have caused it to become a standard production organism in several industries (Cairns et al. (2018)). A. niger is also a rot fungus, meaning it is capable of growing in a wide variety of environments and on lignocellulosic waste. The aforementioned properties of the fungus make it an opportune organism to use for biorefinery use.

Mette Lübeck, an associate professor at the University of Aalborg is an expert in organisms used for biorefineries that we were happy to come into contact with Mette Lübeck . Mette has experience in overcoming the burden of acetic acid, another common inhibitor found in lignocellulosic hydrolysate. In our talks with her, we learned that furfural has not yet been a target for A. niger strains to combat, however it still makes up a large hurdle for lignocellulose based fermentations.

Scoping out the landscape - contacting the industry

We knew early on that our project would be largely industry focused. Thus, it was important for us to be in contact with companies that could be interested in applying our product.
We visited Novozymes to discuss with Carsten Hjort, a senior research director for the company. Carsten has many years of experience working with lignocellulosic waste-based fermentations, especially regarding ethanol production. Carsten helped highlight how furfural was a problematic compound that required a workaround to properly utilize lignocellulosic biomass, enforcing our belief that furfural was a relevant target compound. At the same time, his advice helped us decide that ethanol production would not be our primary objective with our production strain due to it being difficult to reach the same ethanol production levels as with yeast.

Next we decided to get in contact with a smaller company, which might be a bit more willing to try alternative sources of energy and carbon - a biotech startup called Chromologics ApS. This company focuses on the production of natural food dye through fermentation using filamentous fungi and has a large focus on sustainability, thus we hoped they would have some valuable input in regards to our project. Anders Ødum, the co-founder gave great advice in regards to the applicability of our strain - especially in regards to why some industries can’t use lignocellulosic waste as a feedstock; Some industries (especially food and medicine) have very high quality control and require very high purity for their product, something that is difficult to achieve with lignocellulosic waste.

The applications for our strain had now been narrowed down to two applications: production of bulk chemicals and enzymes. A. niger is already being used for these cases, making us optimistic for the capabilities of our production strain.

Designing our biosensor

Creating a biosensor capable of detecting a specific compound of choice is not an easy task. Our first step in attempting to create a biosensor was to talk about feasibility with several different professors at DTU; Fabiano Contesini (Assistant professor, DTU Bioengineering) and Gerd Seibold (Associate professor, DTU Bioengineering), as well our supervisors Henrik Toft Simonsen (Associate professor, DTU Bioengineering) and Christopher Workman (Associate professor, DTU Bioengineering).

Gerd Seibold helped us in determining how we would test our biosensor. His research group works with stress response in organisms, which is something we were interested in looking at for furfural. Due to it being difficult to determine why a certain stress response is activated, Gerd advised us to use a powerful reporter gene to determine if the desired response is achieved by our strain.

A large obstacle for us to overcome was the design of our biosensor. How would we know if any mutations we introduced could increase the binding affinity of our binding site to furfural, and if it could even bind furfural at all? Using software to model a binding site and the docking of furfural into said site required the advice and support of experts such as Fabio Parmeggiani (Group leader) and Georgie Hau Sørensen (PhD. student) from the University of Bristol, who have a lot of experience with the softwares Rosetta and AlphaFold. Inducing sensible mutations to our transcription factor greatly increased the likelihood of us potentially creating transcription factors that could detect furfural.

We discussed experimental design with Verena Siewers, a research professor at the department of biology and biological engineering at Chalmers University. Fluorescence-activated cell sorting (FACS) combined with error prone PCR is an approach that can be used for biosensor development. We investigated the possibility of using this approach for our biosensor, however this was not possible partly due to FACS being difficult to utilize for filamentous fungi, and partly due to time restraints.

What is to come?

While we managed to talk to a lot of people and corporations for our project, there are still some inputs that we would have greatly enjoyed receiving.
We got to talking with Thomas Fruergaard Astrup, a professor at DTU Sustain. He was interested in helping us create a Life cycle analysis (LCA) of our project idea, but we were unfortunately not able to create one due to overlapping schedules and time restraints. We believe a LCA would have helped us in determining further stakeholders for our project and in highlighting the sustainable impact of our project compared to other conventional production methods. Many articles (Donini et al (2015), Corona et al (2018)) discuss the use of lignocellulosic biomass as feed for biorefineries, which show that utilizing lignocellulosic biomass generally is environmentally beneficial.

We were unable to get in contact with a biorefinery to discuss our project, which might have given some valuable input as to our process design. We would also like to seek out more companies that use A. niger as their production organism, to hear if they would benefit from using our furfural detecting and degrading strain. Furthermore, it would have been interesting to talk to producers of lignocellulosic waste such as foresters or farmers, to see what would be needed for them to see the benefit in collecting and selling the waste.


  • Cairns, T. C., et al. How a fungus shapes biotechnology: 100 years of Aspergillus niger research. Fungal biology and biotechnology, 5, 13 (2018)
  • Corona A. et al. Techno-environmental assessment of the green biorefinery concept: Combining process simulation and life cycle assessment at an early design stage. Science of The Total Environment 635 (2018)
  • Donini T. et al. Environmental implications of the use of agro-industrial residues for biorefineries: application of a deterministic model for indirect land-use changes. GCB Bioenergy 8, 4 (2015)
  • The international council on Clean Transportation (ICCT), (2017a) - IF WE USE LIVESTOCK FEED FOR BIOFUELS, WHAT WILL THE COWS EAT? Accessed: 09-10-2022
  • The international council on Clean Transportation (ICCT), (2015) - WASTED: EUROPE'S UNTAPPED RESOURCE. Accessed: 09-10-2022