Revision as of 15:45, 11 October 2020

Requirements of the biofilm Displaying our degradation enzymes in the biofilm matrix Improvement of Biofilm Formation Obviation of Sporulation final Bacillus Subtilis

Requirements of the biofilm

Biofilm engineering

Displaying our degradation enzymes in the biofilm matrix

Improvement of Biofilm Formation

Obviation of Sporulation

Bacillus subtilis is able to form endospores (Verlinkung Text über B. subtilis). During B. subtilis biofilm maturation, cells can sporulate and leave the biofilm ^[8] which could cause an escape of our genetically modified organisms into the environment. Since endospores are physiologically inactive, they do not express enzymes and thus do not contribute to micropollutant degradation. For these reasons, we aim to prevent any sporulation in the biofilm population.
The sigma factor F (σF) plays a critical role in the sporulation of B. subtilis by controlling several required genes ^[9] (Abbildung). The absence of σF renders B. subtilis unable to sporulate ^[10] , which is why we want to knockout the σF gene in the genome of our B. subtilis.

final Bacillus Subtilis

Testing

Flowchamber

AFM

Assay small molecule sorption into the biofilm

We want to produce our pollutant-degrading enzymes fused to one of the B. subtilis biofilm-forming proteins, the major protein component (TasA). This way it will be displayed in the matrix of the biofilm. We need to make sure that the substances are able to enter the biofilm to be converted by our displayed enzymes. Here we focused on the sorption of diclofenac because it poses the biggest issue in wastewater treatment plants. Torresi et al. recently established an assay to measure the uptake of small molecules into biofilms of various thickness on which our assay is based on^[1].

We grow the biofilm directly on carriers used in wastewater treatment to make the experiment as realistic as possible. After the biofilm is formed on the carriers, we test the diclofenac uptake. Therefore, we incubate the carriers with different concentrations of diclofenac and take samples of both the solution and the biofilm at certain time points. The biofilm sample is resuspended in water, centrifuged and washed repeatedly. After that, the cells are lysed via sonification and the suspension is centrifuged again to clear the lysate. The supernatants of this step and the samples of the diclofenac solutions are quantified via UV after HPLC separation. If diclofenac is absorbed by the biofilm at the assayed concentrations, we will do the same with concentrations that can be found in wastewater in Germany and then analyze the taken samples via LC-MS because it is more sensitive than HPLC with UV detection^[2].

Importantly, plastics has shown adsorption of hydrophobic substances^[3]. On that account, we perform the same assay with an empty carrier in diclofenac solution to see potential adsorption to the carrier itself.

Evaluation

We are confident that the biofilm will grow on these carriers, because the material is the same as used in the flowchamber where it grew (Verlinkung auf Flowchamber results). Furthermore, the carrier material was recommended by Prof. Dr. Lackner and these carriers are a common method in moving bed biofilm reactors^[4]. If the biofilm does not grow, we will try and use a different more porous carrier such as pumice stone or ceramics. Biofilm growth on the carrier is detectable by a mucous layer on the floating body which is visible by eye.
Diclofenac is a hydrophobic molecule which is positively charged at pH 7.4^[5]. The B. subtilis biofilm matrix is negatively charged and hydrophobic as well so diclofenac should be able to be absorbed into the biofilm^[6]. This test will be successful if the HPLC analysis shows a decrease of diclofenac even at low concentrations over time so we can test at real-life concentrations. The paper we based our assay on also tested diclofenac biofilm sorption but could not see a significant decrease of diclofenac in their bioreactor^[1]. As they used activated sludge and not a B. subtilis biofilm, we cannot directly compare results. Other reasons why this assay might not work could be wrong charge of the matrix or the size of the pores in the matrix. If this situation would occur, we could try to change the pH of the solution to consequently change the charge of the molecule of the matrix, e.g. the extracellular polymer poly-γ-glutamate. However, this approach would likely be not applicable in WWTP due to the high volume and later drain into the environment. Rather, we could change the conditions in which the biofilm is formed because that can affect the biofilm matrix and it also could be done in our proposed implementation^[7]. Furthermore, we would search for other solutions to make the biofilm matrix more receptive to diclofenac. That could be tried by overexpressing genes responsible for resorptive extracellular polymer substance synthesis^[6].
Here we exemplary focused on diclofenac, but there are further substances whose uptake into the biofilm we could test since they are relevant for this project (Verlinkung auf EreB).

References

1.Torresi, E.; Polesel, F.; Bester, K. Diffusion and Sorption of Organic Micropollutants in Biofilms with Varying Thicknesses. Water Res. 2017, 123, 388–400 Doi:10.1016/j.watres.2017.06.027 2.Abdel-Hamid, M. E. Comparative LC-MS and HPLC Analyses of Selected Antiepileptics and Beta-Blocking Drugs. Farmaco 2000, 55 (2), 136–145 Doi:10.1016/S0014-827X(00)00006-9 3.Zhang, H.; Pap, S.; Taggart, M. A. A Review of the Potential Utilisation of Plastic Waste as Adsorbent for Removal of Hazardous Priority Contaminants from Aqueous Environments. Environmental Pollution. Elsevier Ltd March 1, 2020, p 113698 Doi:10.1016/j.envpol.2019.113698 4.Andersson, S., Nilsson, M., Dalhammar, G. (2008). Assessment of carrier materials for biofilm formation and denitrification. Vatten, 64, 201–207. Retrieved from http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-10154 5. National Center for Biotechnology Information (2020). PubChem Compound Summary for CID 3033, Diclofenac. Retrieved October 4, 2020 from https://pubchem.ncbi.nlm.nih.gov/compound/Diclofenac 6.Marvasi, M.; Visscher, P. T.; Casillas Martinez, L. Exopolymeric Substances (EPS) from Bacillus Subtilis : Polymers and Genes Encoding Their Synthesis. FEMS Microbiol. Lett. 2010, 313 (1), 1–9 Doi:10.1111/j.1574-6968.2010.02085.x 7.Shukla, A.; Mehta, K.; Parmar, J. Depicting the Exemplary Knowledge of Microbial Exopolysaccharides in a Nutshell. European Polymer Journal. Elsevier Ltd October 1, 2019, pp 298–310 Doi:10.1016/j.eurpolymj.2019.07.044 8. Kolter et al. (2013) Sticking together: building a biofilm the Bacillus subtilis way. Nat Rev Microbiol. 11(3): 157-168 9. Errington, J. Regulation of endospore formation in Bacillus subtilis. Nat Rev Microbiol 1, 117–126 (2003). 10. Overkamp W, Kuipers OP. Transcriptional Profile of Bacillus subtilis sigF-Mutant during Vegetative Growth. PLoS One. 2015;10(10):e0141553. Published 2015 Oct 27. doi:10.1371/journal.pone.0141553

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