Team:TU Darmstadt/Description
Project description
Diclofenac, a commonly used pain killer, cannot be filtered out in wastewater treatment plants (WWTPs)[1]. Measurements have shown that in some water areas in Germany the concentration is higher than the predicted no effect concentration (PNEC)[2]. Such high concentrations can cause problems for aquatic fauna and flora[3, 4]. To learn more about the effects of diclofenac or other micropollutants we talked to several experts like ecotoxicologist Prof. Dr. Jörg Oehlmann.
To help cleaning the wastewater, we developed a specialized Bacillus subtilis biofilm. Bacterial biofilms are already used in WWTP to help cleaning the wastewater, but diclofenac has remained a problem[1, 5]. There are many approaches to oxidize diclofenac and most of them use an enzyme class called laccase[6, 7, 8, 9]. But how should the laccase be applied in WWTPs? Our solution combines the already used B. subtilis biofilms with the laccases and enables the biofilm to render diclofenac less toxic. As soon as our bacteria are modified, they are able to immobilize the laccase in the extracellular matrix. There is no further purification or immobilization step necessary, which makes the system easy to use.
To make sure our genetically modified organisms would not leave the WWTP we designed a kill switch. This system leads to the death of the bacteria, as soon as they leave the biofilm. We call our project “B-TOX”. It has the potential to reduce wastewater toxicity and to contribute to the fight against global environmental pollution.
To increase the acceptance for our project in society, we focused on science communication. Therefore, we started our podcast “Genomenal”, which we do not only talk about our project, but also about biotechnology in general.
To help cleaning the wastewater, we developed a specialized Bacillus subtilis biofilm. Bacterial biofilms are already used in WWTP to help cleaning the wastewater, but diclofenac has remained a problem[1, 5]. There are many approaches to oxidize diclofenac and most of them use an enzyme class called laccase[6, 7, 8, 9]. But how should the laccase be applied in WWTPs? Our solution combines the already used B. subtilis biofilms with the laccases and enables the biofilm to render diclofenac less toxic. As soon as our bacteria are modified, they are able to immobilize the laccase in the extracellular matrix. There is no further purification or immobilization step necessary, which makes the system easy to use.
To make sure our genetically modified organisms would not leave the WWTP we designed a kill switch. This system leads to the death of the bacteria, as soon as they leave the biofilm. We call our project “B-TOX”. It has the potential to reduce wastewater toxicity and to contribute to the fight against global environmental pollution.
To increase the acceptance for our project in society, we focused on science communication. Therefore, we started our podcast “Genomenal”, which we do not only talk about our project, but also about biotechnology in general.
Bacillus subtilis biofilm
We decided to use B. subtilis as our organism of choice because it is a risk group 1 microorganism and is already used for wastewater
treatment[5]. Moreover, it is a natural biofilm former and thus forms a perfect platform to display enzymes on the biofilm matrix[5].
In the literature, a method is known where the extracellular protein TasA is combined with other proteins to form a displayed fusion
protein[10]. Importantly, the study showed that the fusion protein keeps its function[10].
With this strategy it is possible to modify the biofilm with proteins and also use enzyme cascades,
because the enzymes are brought into spatial proximity to one another.
We use this system to fuse enzymes with the extracellular protein TasA.
By employing a tasA-knockout strain, we can reintroduce tasA and our desired enzymes as a fusion protein.
The bacteria then express the TasA-enzyme fusion protein and immobilize it in the biofilm matrix without any further work step being necessary (Figure 1).
Enzymes
Which enzymes are useful to immobilize in order to reach our aim of reduced wastewater toxicity?
We decided to use the laccase CotA from B. subtilis and CueO from Escherichia coli.
Both are known to degrade phenolic substances through copper-catalyzed oxidation[11].
This includes a number of toxic substances in wastewater, like diclofenac.
The laccases oxidize diclofenac to hydroxydiclofenac (Figure 2), which has been shown to be less toxic[12].
To get an idea of the enzymatic activities, we performed in silico docking experiments with our enzymes and compared them with the significantly better characterised fungal laccase from Trametes versicolor.
It was also shown before that an improved catalytic activity could be realized with site saturation mutagenesis[11].
The mutagenesis was also performed in silico and we were able to extract mutations that should lead to an optimized diclofenac degradation.
But diclofenac is not the only micropollutant that is present in wastewater in too high concentrations: the antibiotic azithromycin
is also a problematic substance[2]. To solve this problem we also want to equip our biofilm with a variant of an erythromycin esterase.
The erythromycin esterase type II (EreB) from E. coli is able to degrade the antibiotic erythromycin and shows promiscuous activity towards azithromycin[13,14].
With the help of site saturation mutagenesis we want to optimize its activity to azithromycin. Since this would generate a new antibiotic resistance, we have given ourselves a lot of thought about safety, which you can follow up here.
Kill switch
Since our project should be applied in a WWTP it is important that the genetically modified organisms will not be able to leave the WWTP.
Therefore, we introduced a kill switch system based on the quorum sensing molecules that are present in the biofilm.
Because our goal is to detox the wastewater we argued that a kill switch based on the release of toxins is not an option.
As an alternative, we sought to bring an essential gene, encoding the ribosomal protein RpsB,
under the control of a quorum sensing molecule promoter (Figure 3).
To highlight the mechanism of this kill switch,
we developed a conceptual model which helped us to understand our kill switch more deeply and fine tune different aspects of it.
In case the bacterium leaves the biofilm the concentration of the quorum sensing molecules diminish and the essential gene is no longer expressed.
This will lead to the death of the bacteria and makes the bacterial survival biofilm dependent. With this system, we sought to ensure that the bacteria do not leave the WWTP.
When all these components come together, we create a bacterium that – on its own – is able to form a biofilm with immobilized laccases.
Moreover the presence of our kill switch makes the bacterial viabililty dependent on the high cell density of the biofilm. Initially,
the biofilm is grown in the presence of inducing substances where it also produces the quorum sensing molecules.
Once the biofilm is grown, it is ready to be applied in a wastewater treatment plant.
To find out which strategy is the most suitable for the implementation of our project we talked to experts of different WWTPs.
Thereby we realized that our biofilm should grow on a so called floating body[15].
With this you could just put the biofilm in a clarifier and it will help to clean the wastewater.
If a bacterium leaves the biofilm because of the water flow, the kill switch should ensure that this bacterium dies.
If this is not working, there are additional security steps.
All in all, this makes our project save and helps the environment by reducing the toxicity of wastewater.
References
[1] S. Tewari, R. Jindal, Y.L. Kho et al., Major pharmaceutical residues in wastewater treatment plants and receiving waters in Bangkok, Thailand, and associated ecological risks, Chemosphere, 2013, Volume 91, Issue 5: 697-704, https://doi.org/10.1016/j.chemosphere.2012.12.042 [2] https://www.umweltbundesamt.de/themen/wasser/fluesse/zustand/arzneimittelwirkstoffe#eu-watch-list-und-nationale-beobachtungsliste, accessed on September 30th 2020 [3] https://www.usgs.gov/special-topic/water-science-school/science/wastewater-treatment-water-use?qt-science_center_objects=0#qt-science_center_objects, accessed on September 30th 2020 [4] Lucian Copolovici, Daniela Timis et al., Diclofenac Influence on Photosynthetic Parameters and Volatile Organic Compounds Emission from Phaseolus vulgaris L. Plants, Revista de Chimie (Rev. Chim.), 2017, Volume 68, Issue 9: 2076-2078, DOI: 10.37358/RC.17.9.5826 [5] Vlamakis, H., Chai, Y., Beauregard, P. et al., Sticking Together: Building a Biofilm the Bacillus Subtilis Way. Nature Reviews Microbiology. NIH Public Access March 2013, pp 157–168. https://doi.org/10.1038/nrmicro2960 [6] Linson Lonappan, Tarek Rouissi, Mohamed Amine Laadila et al., Agro-industrial-Produced Laccase for Degradation of Diclofenac and Identification of Transformation Products, ACS Sustainable Chemistry & Engineering, 2017, 5 (7): 5772-5781, DOI: 10.1021/acssuschemeng.7b00390 [7] Mateja Primožič, Gregor Kravanjaa, Željko Knez et al., Immobilized laccase in the form of (magnetic) cross-linked enzyme aggregates for sustainable diclofenac (bio)degradation, Elsevier, 2020: https://doi.org/10.1016/j.jclepro.2020.124121 [8] Sultan K. Alharbi, Long D. Nghiem, Jason P. van de Merwe et al., Degradation of diclofenac, trimethoprim, carbamazepine, and sulfamethoxazole by laccase from Trametes versicolor: Transformation products and toxicity of treated effluent, Biocatalysis and Biotransformation, 2019, 37:6: 399-408, DOI: 10.1080/10242422.2019.1580268 [9] Jakub Zdartaa, Katarzyna Jankowskaa, Marta Wyszowskaa et al., Robust biodegradation of naproxen and diclofenac by laccase immobilized using electrospun nanofibers with enhanced stability and reusability, Elsevier, 2019: https://doi.org/10.1016/j.msec.2019.109789 [10] Huang, J., Liu, S., Zhang, C. et al., Programmable and printable Bacillus subtilis biofilms as engineered living materials, Nat Chem Biol, 2019, 15: 34–41, https://doi.org/10.1038/s41589-018-0169-2 [11] Mate, D. M., & Alcalde, M., Laccase engineering: from rational design to directed evolution, Biotechnology advances, 2015, 33(1): 25-40, doi: 10.1016/j.biotechadv.2014.12.007 [12] Yu, H., Nie, E., Xu, J. et al., Degradation of diclofenac by advanced oxidation and reduction processes: kinetic studies, degradation pathways and toxicity assessments, Water research, 2013, 47(5): 1909-1918, doi: 10.1016/j.watres.2013.01.016. [13] Michel Arthur, Denise Autissier and Patrice Courvalin, Analysis of the nucleotide sequence of the ereB gene encoding the erythromydn esterase type II, Nucleic Acids Research, 1986, Volume 14, Issue 12: 4987–4999, https://doi.org/10.1093/nar/14.12.4987 [14] Mariya Morar, Kate Pengelly, Kalinka Koteva et al., Mechanism and diversity of the erythromycin esterase family of enzymes, Biochemistry, 2012, 51(8): 1740-51, doi: 10.1021/bi201790u. [15] https://www.rvtpe.com/fileadmin/documents/print_and_publications/RVT_BiologicalCarrierMedia_130207.pdf, accessed on September 30th 2020