We expect to implement our project in the sewage treatment plants that face the problems of the requirement of regularly replacing old, overgrown biofilm with a new one. The requirement of regularly replacing biofilm can be expensive and time-wasting. However, with our inhibitors that can effectively prevent phage infection of Pseudomonas, we can reduce overgrown Pseudomonas used in the biofilm for waste-processing by adding bacteriophage specific to Pseudomonas into the overgrown biofilm and then adding our inhibitor to prevent too much damage from phage infection. In our proposed implementation, our drug acts as a switch to shut off excess phage infection when the quantity of biofilm is reduced to a proper level. In this way, we expect the drug that disrupts the binding between Aca1 and acr-promoter can successfully control the level of phage infection to an expected extent.

Still, there are some challenges we will face when we are going to apply our project in the real world, that is, the biofilm used in sewage treatment system actually comprise multiples bacteria instead of just Pseudomonas, and reducing simply one kind of important bacterium may affect the efficiency of the biofilm in sewage treatment. After looking deeper into biofilm in the waste-processing system, we find that in industrial sewage treatment, the biofilm contains a complex microbial community, including Acinetobacter, Aeromonas, Pseudomonas, and many other microbes¹. But we find that the way we found effective inhibitors to protect Pseudomonas from excessive phage infection is viable in finding molecules responsible for other microbes in the complex community of biofilm.

With multiple biofilm community characterization approaches², we expect the implementation in ways that we can add a complex community of phages to reduce the overgrown microbe community in the biofilm for sewage treatment, and when the state of biofilm meets our expectation, we can add mixture molecules to effectively protect the majority of the microbes in the biofilm from unexpected damages caused by phages. In this way, we can replace the expensive and time-wasting regularly replacement of biofilm with simply regularly applying phages and a mixture of molecules into the biofilm.

Pseudomonas shows great potential in biosynthesis, for example, Pseudomonas denitrificans can be used for vitamin B12 biosynthesis³. Also, Pseudomonas plays an important role in biodegradation, for instance, Pseudomonads putida plays a crucial role in the biodegradation process of polystyrene⁴. In the industry of biosynthesis and biodegradation, microbes are always cultured on a large scale. Under such conditions, phage infection can be a common and fatal problem. As our inhibitor can effectively protect Pseudomonas from phage infection, we propose that we can implement a strategy for Pseudomonas protection in the industry with massive cultivation of Pseudomonas, which is to add effective phage inhibitors during the cultivation.

However, there are still some challenges in such an application because the addition of molecules can affect the purity of the biosynthesis product and there can also be unexpected reactions between the phage inhibitor and chemical reagents in the industrial system. Therefore, before the implementation of effective phage inhibitors, we strongly recommend the users carry out a preliminary experiment to screen for proper phage inhibitors that do not disrupt the normal biosynthesis or biodegradation process. The screening experiment can be designed with the two-plasmid system that we developed in our project.

1. Kämpfer, P. et al. Characterization of bacterial communities from activated sludge: Culture-dependent numerical identification versus in situ identification using group- and genus-specific rRNA-targeted oligonucleotide probes. Microbial Ecology 32, 101-121, doi:10.1007/BF00185883 (1996).
   
2. Sehar, S. & Naz, I. Role of the Biofilms in Wastewater Treatment. in Microbial Biofilms-Importance and Applications (eds. Dhanasekaran, D. & Thajuddin, N.) (IntechOpen, 2016). doi: 10.5772/63499
3. Martens, .J., Barg, .H., Warren, .M. et al. Microbial production of vitamin B12. Appl Microbiol Biotechnol 58, 275–285 (2002). doi:10.1007/s00253-001-0902-7
4. Savoldelli, J., Tomback, D. & Savoldelli, H. Breaking down polystyrene through the application of a two-step thermal degradation and bacterial method to produce usable byproducts. Waste Management 60, 123-126, doi:1016/j.wasman.2016.04.017 (2017).