Team:TU Darmstadt/Project/Biofilm
Requirements of the Biofilm
In order to implement a Bacillus subtilis biofilm to render micropollutants in wastewater less toxic,
certain requirements have to be fulfilled. First of all,
the enzymes we are using have to be exported into the extracellular matrix to protect our bacteria from the toxicity of the substances.
We also have to avoid those enzymes from getting washed away by the water to make sure they can convert the micropollutants.
Furthermore, our biofilm as a whole has to absorb the substances to enable their enzymatic degradation.
Since we have to prevent genetically modified organism (GMO) release from the biofilm into the environment our biofilm needs to resist mechanical
pressure to which it is exposed in wastewater treatment plants.
Biofilm Engineering
Displaying our Degradation Enzymes in the Biofilm Matrix
The Major Biofilm Matrix Component (TasA)
The Bacillus subtilis biofilm matrix consists mainly of exopolysaccharides (EPS) and proteins. One protein essential for the structure and formation of a biofilm is the major biofilm matrix component (TasA)[1]. It polymerizes into long amyloid-like fibres which are attached to the cell wall by the TasA anchoring protein (TapA) outside of the cell[2]. The knockout of tasA leads to a mutant that forms a weak biofilm of decreased thickness[3]. B. subtilis secretes proteins in order to enable intercellular linkages and communication and therefore uses the Sec-dependent signal recognition particle (Sec-SRP) pathway. For proteins to be secreted via the SPR pathway, it is necessary that the proteins possess a 27 amino acids long N-terminal signal peptide, which is cleaved off during the membrane translocation. Following this scheme, TasA also possesses this N-terminal signaling peptide[4,5]. The Sec-SRP pathway consists of four main steps:- The signal peptide is recognized by a ribonucleoprotein complex, the signal recognition particle (SRP).
- SRP targets the Sec translocase which transports the protein through the membrane.
- During membrane translocation the signal peptide is cleaved off by the SipW peptidase leading to the release of the protein.
- Chaperones mediate the correct folding of the protein outside the cell[5].
A Fusion Protein of TasA with our Degradation Enzymes
We want to immobilize our degradation enzymes in the biofilm matrix, due to the improved stability and improved enzyme activity of immobilized
enzymes[6]. Furthermore, within the biofilm matrix the degradation targets are better accessible for enzymatic degradation than within the
cytoplasm. Therefore, we decided to generate fusion proteins with our degradation enzymes and the
protein TasA, following previous work by Huang et al.[7] (Figure 1).
They showed that fusion proteins consisting of TasA and a second protein domain
(e.g. mCherry, SpyTag) are successfully exported into the biofilm matrix (Figure 2). Even a fusion protein of TasA with the larger enzyme MHETase
(63.1 kDa) was localized in the biofilm matrix of B. subtilis. Based on the research of Huang et al., we planned the fusion protein of
superfolder green fluorescent protein (sfGFP) with TasA as a proof of concept. The sequence of the sfGFP gene is codon optimized for B. subtilis
and fused to the 3' end of the tasA gene with a gene fragment encoding a glycine-serine-rich linker (ARGGGGSGGGGS). The display of the
TasA-sfGFP fusion protein in the biofilm matrix can be verified using fluorescence microscopy. If TasA-sfGFP
is successfully expressed in the biofilm matrix, we hypothesize that analogously designed TasA fusion proteins with our targeted degradation
enzymes CotA, CueO and EreB will likely succeed.
Integration of the TasA Fusion Proteins in the B. subtilis Genome
We want to ensure that only TasA fusion proteins, but no endogenous TasA, are secreted into the matrix in order to increase the number of immobilized degradation enzymes in the biofilm matrix. Therefore, we use a B. subtilis TasA knockout strain supplied by the group of Prof. Stülke (Georg-August-Universität Göttingen)[8]. Then, as a proof of concept, the TasA fusion protein will be introduced in B. subtilis via plasmids (see Huang et al.). In the next step we want to integrate the TasA fusion proteins into the genome of our B. subtilis strain.
For further information on the workflow and analysis of our concept, please refer to our text on Engineering Success.
Improvement of Biofilm Formation
The matrix protein TasA is encoded in the tapA-sipW-tasA operon which is regulated by the repressing transcription factor SinR[1]. SinR also regulates exopolysaccharide synthesis by controlling the expression of the epsA-O operon[9].
Consequently, the genomic deletion of sinR improves the biofilm formation of B. subtilis, because involved genes are expressed without repressing effects by SinR (Figure 3)[10]. In addition, sinR knockout mutants have been found to show nonmotile phenotypes and thus are not able to disperse from the biofilm[11]. We are therefore planning the knockout of the sinR gene in our B. subtilis strain.
Consequently, the genomic deletion of sinR improves the biofilm formation of B. subtilis, because involved genes are expressed without repressing effects by SinR (Figure 3)[10]. In addition, sinR knockout mutants have been found to show nonmotile phenotypes and thus are not able to disperse from the biofilm[11]. We are therefore planning the knockout of the sinR gene in our B. subtilis strain.
Additionally, we hypothesize that the overexpression of tasA or respectively of the tapA-sipW-tasA operon could improve biofilm formation. To our knowledge no direct attempts are published. However, Lei et al. overexpressed the small protein Veg which led to a highly increased expression of tasA[12]. Although the authors are strongly suggesting that Veg negatively regulates the expression of sinR, we cannot assume that the overexpression of Veg will additionally improve stability after sinR knockout as Veg appears to mainly affect sinR expression and does not affect the biofilm stability via another mechanism. For investigating the mechanism of action, a comparison of a veg overexpressing strain with a veg overexpressing strain and simultaneous sinR knockout is necessary.
Overall, the approach of overexpressing veg sounds promising to gain a strain with high stability.
For comparison of the various strains we would use our flow chamber specifically designed for testing mechanical stability of biofilms.
Overall, the approach of overexpressing veg sounds promising to gain a strain with high stability.
For comparison of the various strains we would use our flow chamber specifically designed for testing mechanical stability of biofilms.
Obviation of Sporulation
Bacillus subtilis is able to form endospores.
During B. subtilis biofilm maturation, cells can sporulate and leave the biofilm
which could cause an escape of our genetically modified organisms into the environment[13].
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[14]. The absence of σF renders B. subtilis unable to sporulate (Figure 4), which is why we want to knockout the sigF gene in the genome of our B. subtilis[15].
The sigma factor F (σF) plays a critical role in the sporulation of B. subtilis by controlling several required genes[14]. The absence of σF renders B. subtilis unable to sporulate (Figure 4), which is why we want to knockout the sigF gene in the genome of our B. subtilis[15].
Testing
Flow Chamber
It is essential that our biofilm is stable enough to withstand the rough conditions that prevail in a wastewater treatment plant (WWTP), otherwise GMOs are likely to escape into the environment. Therefore, we sought for a method to measure the stability of biofilms. In order to simulate the most extreme conditions prevailing in WWTPs, we decided to design a custom-made flow chamber consisting of three parts which are stacked on top of each other.We printed the 3D models using a Creality Cr10V2 3D printer. The upper part contains two holes the for the influx and efflux water hoses. The bottom part is used to grow a biofilm on. The middle part is designed to direct the liquid to flow over the biofilm in a thin layer, simulating the most extreme conditions in a WWTP. Two clamps are used to compress the stacked parts and prevent leakage. We estimate that the roughness of the used printing material (ecoPLA filament by 3DJAKE) enables to form a biofilm on the carrier surface. The flow chamber is assembled as you can see in our video and our manual.
The goal of this experiment is analysing the amount of living cells which are washed off the biofilm at different flow velocities. We chose phosphate-buffered saline (PBS) as liquid since it has physiological osmolarity and ion concentrations. The test is conducted using a PBS reservoir of around 50 mL which is pumped circularly through the flow chamber by a peristaltic pump (Cole-Parmer Masterflex L/S Model 7523-60 Digital Peristaltic Pump with Easy-Load II Head).
After flowing PBS with a defined flow rate over the biofilm for 3 hours, the flow chamber is deconstructed, and the used PBS buffer is collected in a falcon tube and centrifuged at 6000 xg for at least 6 min. Subsequently, the supernatant is discarded and the pellet is resuspended in 2 mL lysogeny broth (LB) media. These first samples are later used to quantify the number of cells that have been washed out the biofilm. In addition, multiple areas of defined size are scrapped of the biofilm, each area separately resuspended in 2 mL LB media. These second samples are the remaining cells per mm2 to which we can compare the amount of washed-out living cells. As controls are essential, we simultaneously conduct the assay without a biofilm and with a tasA deletion strain of Bacillus subtilis which misses the major protein component of biofilm matrices and is thus assumed instable biofilm [3].
Quantifying the number of cells and cells within a biofilm matrix in our samples is not trivial. Although we found a method based on flow cytometry [16], we sought for an alternative as many laboratories worldwide do not have access to flow cytometry. As biofilms fluctuate in their density and thickness and thus in their cell number, quantifying the ratio and not the total numbers of washed-out living cells appears to be a reasonable approach. We therefore theoretically developed a microplate reader assay to roughly quantify the amount of washed-out living cells which yet has to be tested in practice. Both samples, of the remaining biofilm and the washed-out cells (see previous paragraph), are transferred into a microtiter plate and the samples are serial diluted in LB media. After a few hours of initial growth, a growth curve of the cells in the plate is recorded by measuring the optical density at 600 nm (OD600) of each well. Comparing optical densities of the serial diluted samples of the biofilm and samples, we are able to estimate the ratio of cells that have been washed out compared to all cells in the biofilm.
We had a short time frame of two weeks in the lab to establish biofilm growth and the assay, but were not able to grow a B. subtilis biofilm following a method based on MSgg media [17]. However, for illustration of the assay setup, we used red dye to prepare serial dilutions on microtiter plates. The plate represents samples after a few hours of growth under suitable conditions. The blue rectangle marks the resuspended cells of the biofilm in 2 mL LB media. Each dilution step in our example was performed 1:1.5 for the sake of a visible serial dilution over multiple steps. Assuming that the Xth (e.g. 7th) dilution of the biofilm sample corresponds to the optical density of the flow sample, we can calculate the ratio of cells washed out by using the following formulas.
Formula 1.
Formula 2.
In case of our example with a dilution factor of 1.5 and 7 dilution steps, 5.53 % of all living cells were washed out. The corresponding dilutions are outlined with a yellow rectangle.
Atomic Force Microscopy
An Atomic Force Microscopy (AFM) is a probe microscope with an up to 1000-fold higher resolution than common microscopes[18,19].
By measuring the surface, it will give precise information about the topology of our biofilm. A typical AFM setup is shown in Figure 8. The imaging process works with the offset of the laser pointing at the cantilver’s back, as well as the mechanical force bending the cantilever back.
AFM is capable of different measuring modes, that are favourable in varying conditions. For a biofilm’s soft surface, the non-contact mode is preferable since it reduces the friction of the cantilever’s tip on the surface[20].
Using our 3D printed flow chamber experiments in combination with AFM data analysis, we can determine the stability of our biofilm. For more details see Experiments.
AFM is capable of different measuring modes, that are favourable in varying conditions. For a biofilm’s soft surface, the non-contact mode is preferable since it reduces the friction of the cantilever’s tip on the surface[20].
Using our 3D printed flow chamber experiments in combination with AFM data analysis, we can determine the stability of our biofilm. For more details see Experiments.
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[21]. The following assay has been developed theoretically and could not be tested in the lab.
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 (Figure 9). 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[22]. For more details see Experiments.
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 (Figure 9). 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[22]. For more details see Experiments.
Importantly, plastics has shown adsorption of hydrophobic substances[23]. 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 flow chamber where it grew. Furthermore, the carrier material was recommended by Prof. Dr. Lackner and these carriers are a common method in moving bed biofilm reactors[24]. 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[25]. The B. subtilis biofilm matrix is negatively charged and hydrophobic as well so diclofenac should be able to be absorbed into the biofilm[9]. 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[21]. 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[26]. 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[9].
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 .
Diclofenac is a hydrophobic molecule which is positively charged at pH 7.4[25]. The B. subtilis biofilm matrix is negatively charged and hydrophobic as well so diclofenac should be able to be absorbed into the biofilm[9]. 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[21]. 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[26]. 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[9].
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 .
References
[1] Branda, S.; Chu, F.; Kearns, D. (2006): A major protein component of the Bacillus subtilis biofilm matrix. In: Molecular microbiology 59 (4), S. 1229–1238, DOI 10.1111/j.1365-2958.2005.05020.x.
[2] Romero, D.; Aguilar, C.; Losick, R. (2010): Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. In: Proceedings of the National Academy of Sciences of the United States of America 107 (5), S. 2230–2234,DOI 10.1073/pnas.0910560107x.
[3] Dogsa, Iztok; Brloznik, Mojca; Stopar, David; Mandic-Mulec, Ines (2013): Exopolymer diversity and the role of levan in Bacillus subtilis biofilms. In: PloS one 8 (4), DOI 10.1371/journal.pone.0062044.
[4] Stöver, A.; Driks, A. (1999): Secretion, Localization, and Antibacterial Activity of TasA, a Bacillus subtilis Spore-Associated Protein. In: Journal of Bacteriology 181 (5), S. 1664–1672.
[5] Ling, L.; Zi, R.; Wei, F. (2007): Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. In: Biotechnology advances 25 (1), S. 1–12 DOI 10.1016/j.biotechadv.2006.08.002.
[6] Brady, D., Jordaan, J. Advances in enzyme immobilisation. Biotechnol Lett 31, 1639 (2009),DOI 10.1007/s10529-009-0076-4.
[7] Huang, J., Liu, S., Zhang, C. et al. Programmable and printable Bacillus subtilis biofilms as engineered living materials. Nat Chem Biol 15, 34–41 (2019), DOI 10.1038/s41589-018-0169-2.
[8] Gerwig J, Kiley TB, Gunka K, Stanley-Wall N, Stülke J. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis. Microbiology (Reading). 2014 Apr;160(Pt 4):682-691, DOI 10.1099/mic.0.074971-0.
[9] Marvasi, M; Visscher, P; Casillas Martinez, L (2010): Exopolymeric substances (EPS) from Bacillus subtilis: polymers and genes encoding their synthesis. In: FEMS microbiology letters 313 (1), S. 1–9, DOI 10.1111/j.1574-6968.2010.02085.x.
[10] Diethmaier, C; Pietack, N; Gunka, Ket al. (2011): A novel factor controlling bistability in Bacillus subtilis: the YmdB protein affects flagellin expression and biofilm formation. In: Journal of Bacteriology 193 (21), S. 5997–6007, DOI 10.1128/JB.05360-11.
[11] Bartolini, M.; Cogliati, S.; Vileta, D. Regulation of Biofilm Aging and Dispersal in Bacillus subtilis by the Alternative Sigma Factor SigB. 2018, DOI 10.1128/JB.00473-18.
[12] Ying L; Taku O; Naotake O; Shu I; Functional Analysis of the Protein Veg, Which Stimulates Biofilm Formation in Bacillus subtilis. In: Journal of Bacteriology, 2013, 193,8: 1697–1705, DOI 10.1128/JB.02201-12.
[13] Kolter et al. (2013) Sticking together: building a biofilm the Bacillus subtilis way. Nat Rev Microbiol. 11(3): 157-168
[14] Errington, J. Regulation of endospore formation in Bacillus subtilis. Nat Rev Microbiol 1, 117–126 (2003), DOI 10.1038/nrmicro750.
[15] Overkamp W, Kuipers OP. Transcriptional Profile of Bacillus subtilis sigF-Mutant during Vegetative Growth. PLoS One. 2015;10(10):e0141553. Published 2015 Oct 27
[16] Kerstens et al., A flow cytometric approach to quantify biofilms, Folia Microbiologica, 2015, DOI 10.1007/s12223-015-0400-4
[17] Branda et al., Fruiting body formation of Bacillus subtilis, Proceedings of the National Academy of Sciences of the United States of America, 2001, 98, 20: 11621-11626, doi: 10.1073/pnas.191384198
[18] Huang, Qiaoyun; Wu, Huayong; Cai, Peng; Fein, Jeremy B.; Chen, Wenli (2015): Atomic force microscopy measurements of bacterial adhesion and biofilm formation onto clay-sized particles. In: Scientific reports 5, S. 16857,DOI 10.1038/srep16857.
[19] Ramazan Asmatulu and Waseem S. Khan, Synthesis and Applications of Electrospun Nanofibers Micro and Nano Technologie
2019, Pages 257-281, DOI 10.1016/B978-0-12-813914-1.00013-4.
[20] Sean A. James, Lydia C. Powell and Chris J. Wright. Atomic Force Microscopy of Biofilms—Imaging, Interactions, and Mechanics, DOI 10.5772/63312.
[21] 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.
[22] 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.
[23] 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
[24] Andersson, S., Nilsson, M., Dalhammar, G. (2008). Assessment of
carrier materials for biofilm formation and denitrification. Vatten,
64, 201–207. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-10154 (accessed on Oct 3, 2020)
[25] National Center for Biotechnology Information (2020). PubChem Compound Summary for CID 3033, Diclofenac. https://pubchem.ncbi.nlm.nih.gov/compound/Diclofenac (accessed on October 4, 2020)
[26] 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.