Team:TU Darmstadt/Description
To help cleaning the wastewater, we developed a specialized Bacillus subtilis biofilm. Such bacterial biofilms are already used in WWTP[5] to help cleaning the wastewater, but diclofenac has remained a problem[1]. 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 WWTP? 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, what 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, when 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 the society we focused on science communication. Therefore, we started our podcast “Genomenal” where we do not only talk about our project, but also about biotechnology in general.
We decided to use B. subtilis as our organism of choice because it is a risk group 1 microorganism and is already used for the wastewater treatment.[5] Moreover, it is a natural biofilm former[5] and thus forms a perfect platform to display enzymes on the biofilm matrix. 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 remains 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 on the biofilm matrix (Fig. 1) without any further work step being necessary.
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 (Fig. 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 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 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 to Azithromycin. [13][14] With the help of site saturation mutagenesis we want to optimize its activity to Azithromycin.
Since our project should be applied in 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(Fig. 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 should 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.
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References
1. S. Tewari et. al.; Major pharmaceutical residues in wastewater treatment plants and receiving waters in Bangkok, Thailand, and associated ecological risks; Chemosphere, Volume 91, Issue 5, April 2013, Pages 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 ,visited 30.09.2020 3. https://www.usgs.gov/special-topic/water-science-school/science/wastewater-treatment-water-use?qt-science_center_objects=0#qt-science_center_objects , visited 30.09.2020 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.; Losick, R.; Kolter, R. 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 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 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 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 Zdarta 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