Assessment of biofilms in sewer systems and on building materials
PostDoc: Dr. Adrian Augustyniak 
Supervisor: Prof. Dr. Dietmar Stephan 
Background information of the objectives
Biofilm formation in sewer systems and on building materials can lead to the biodeterioration of the cementitious material. Even though the biofilm growth is a known phenomenon, its activity is still being studied (Flemming et al., 2016). Thousands of kilometres of sewer pipes are buried below the ground level in every city. These systems are in fact bioreactors in which a plethora of biogenic processes occur (Hvitved-Jacobsen et al., 2013). This activity is associated with certain interfaces. Biofilm-forming microorganisms are forming an interface with air, water, sediment, and pipe wall. Each of them may have different activity, which was not extensively studied to date (Hughes et al., 2013; Yuan et al., 2015). On the one hand, better understanding of the biofilm formation in sewer systems can be the base for new countermeasures against odour and corrosion (Jiang et al., 2013). On the other hand, studying the physiology, morphology, and diversity of microorganisms dwelling on pipes and buildings could be used to affect abovementioned interfaces and positively contribute to the sustainability of construction materials. Such studies would serve modern urbanization with sustainable solutions that have minimal impact on the climate change (Bertron, 2014). There is a necessity to develop methods that could be used in the monitoring of biogenic effects on building materials, e.g. using molecular (PCR-based) approach. In the sewers perspective, developing a molecular test could improve biofilm monitoring and provide data for modelling. Assessing the impact of microbiological acidic attack on building materials could be used to develop or efficiently improve protective layers that could be used to protect materials from biogenic activity. Many microorganisms, such as urease-producing strains have potential to be used in such application. Introducing these bacteria into biofilms could positively alter the biodiversity, physiological state and metabolic activity of microorganisms residing on building materials (Wu et al., 2018). Another interesting direction can be the evaluation whether biofilms associated with technical systems can harbour antibiotic resistance markers (Kollef et al., 2014; WHO, 2014). Such studies could answer the question whether the type of construction material can have an impact on the transfer of antibiotic resistance.
Six main objectives were established for the project:
- Studying the influence of free nitrous acid (FNA) dosage on biofilms in sewer systems
- Isolation and characterization of urease-producing bacteria, and their activity in increasing the chemical resistance of concrete
- Detecting antibiotic resistance markers in biofilms growing on building materials and technical systems
- Studying bacterial biodiversity on building materials and sewer systems with particular attention to harmful and potentially beneficial taxa
- Developing a methodology for studying microorganisms and biofilms on building materials
- Proposing biosafety measures for building materials
The above-mentioned objectives are based on the following hypotheses:
- Free nitrous acid can reduce the viability of biofilm-forming bacteria in sewers
- Urease-producing bacteria are abundant in sewer systems and can be used to protect cementitious materials from acidic attack
- Biofilms on building materials can harbour antibiotic resistance
- Recipe of cementitious material can affect the biodiversity of biofilm-forming microorganisms
- Standard methods need to be adjusted in order to be used on cementitious materials
- Current standards do not provide clear biosafety measures for the connection between biofilm-forming microorganisms and building materials
Studies are performed in combination of classic microbiological methods with modern techniques based on microscopy, flow cytometry, and molecular biology. Studies associated with biofilms growing in sewer systems are carried out in the pilot plant.
The main methods on which the project is based are:
- PCR-based methods, including quantitative and qualitative analyses by Real-time PCR
- Culture methods joined with biochemical testing
- Spectroscopic methods
- Use of microbiological standards (JIS and ISO), that will be modified for the building materials
- Modern microscopy, including fluorescence microscopy (Figure 1) and confocal laser scanning microscopy
- Figure 1. Microorganisms scraped from the sewer pipe treated with FNA visualised with Live/Dead staining in fluorescence microscopy; red – dead cells, green – viable cells
- © Augustyniak
The following working steps need to be done in order to verify the hypotheses:
- Using the pilot plant to perform a treatment with FNA. For this step, visualisation techniques and viability testing experiments will be required.
- Isolation and characterisation of culturable microorganisms gathered from samples taken from the pilot plant. Isolation will be performed with standard microbiological procedures. For the characterisation, molecular and biochemical tests will be used. Taxonomy of the strains will be based on sequence coding 16S rRNA. The collection of strains will be stored for further tests on building materials.
- Isolating resistant microorganisms with the use of previously described preselection method (Augustyniak et al., 2018). The antibiotic resistance will be then studied in accordance to EUCAST guidelines. PCR-based methods will be used to determine the type of resistance.
- Testing various recipes in the pilot plant and analysing biofilms. This step will require the use of visualisation methods that were described above. For this stage, also new generation sequencing techniques are planned to be used. Based on 16S rDNA fragments, the changes in the taxonomy will be studied over the selected time period.
- Toxicity of building materials will be verified with the use of international standards (e.g. JIS Z 2801:2000). The standards should be adjusted so that they can provide repetitive results from tests on cementitious materials.
- Last hypothesis will be verified through the analysis of data generated in the studies described above. Morphology, physiology and metabolic activity of bacteria exposed to building materials will create a base to introduce ideas for biosafety measures in regard to building materials and their ecotoxicity.
Connections to interfaces and doctoral theses within UWI
The main connection within UWI is with the group dedicated to interfaces in sewer systems. Nevertheless, microbiological approach can address the problems indicated also in other projects. Moreover, a collaboration with the modelling and remote sensing postdoc is also planned in order to connect the modelling in technical systems with biological processes. Major collaborations include following projects:
- S1: corrosion and odour in sewers caused by biochemical processes of sulphurous compounds
- T2: corrosion in sewers caused by biochemical processes of sulphurous compounds
- H4: redox gradients in natural and technical systems: population structure and physiological properties
- W2: Scaling and connectivity assessment of critical source areas of diffuse pollution in urban catchments
Augustyniak, A., Grygorcewicz, B., Nawrotek, P., 2018. Isolation of multidrug resistant coliforms and their bacteriophages from swine slurry. Turkish J Vet Anim Sci 42, 319–325. doi:10.3906/vet-1710-102
Bertron, A., 2014. Understanding interactions between cementitious materials and microorganisms: a key to sustainable and safe concrete structures in various contexts. Mater Struct 47, 1787–1806. doi:10.1617/s11527-014-0433-1
Flemming, H.C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S.A., Kjelleberg, S., 2016. Biofilms: An emergent form of bacterial life. Nat Rev Microbiol 14, 563–575. doi:10.1038/nrmicro.2016.94
Hughes, P., Fairhurst, D., Sherrington, I., Renevier, N., Morton, L.H.G., Robery, P.C., Cunningham, L., 2013. Microscopic study into biodeterioration of marine concrete. Int Biodeterior Biodegrad 79, 14–19. doi:10.1016/j.ibiod.2013.01.007
Hvitved-Jacobsen, T., Vollertsen, J., Nielsen, A.H., 2013. Sewer Processes. Microbiological and Chemical Process Engineering of Sewer Networks, Second. ed. CRC Press, Boca Raton, Florida, USA.
Jiang, G., Keating, A., Corrie, S., O’halloran, K., Nguyen, L., Yuan, Z., 2013. Dosing free nitrous acid for sulfide control in sewers: Results of field trials in Australia. Water Res 47, 4331–4339. doi:10.1016/j.watres.2013.05.024
Kollef, M.H., Chastre, J., Fagon, J.-Y., François, B., Niederman, M.S., Rello, J., Torres, A., Vincent, J.-L., Wunderink, R.G., Go, K.W., Rehm, C., 2014. Global Prospective Epidemiologic and Surveillance Study of Ventilator-Associated Pneumonia due to Pseudomonas aeruginosa*. Crit Care Med 42, 2178–2187. doi:10.1097/CCM.0000000000000510
WHO, 2014. ANTIMICROBIAL RESISTANCE Global Report on Surveillance. World Health Organisation, Geneva, Switzerland.
Wu, L., Hu, C., Liu, W.V., 2018. The sustainability of concrete in sewer tunnel-A narrative review of acid corrosion in the city of Edmonton, Canada. Sustain 10. doi:10.3390/su10020517
Yuan, H., Dangla, P., Chatellier, P., Chaussadent, T., 2015. Degradation modeling of concrete submitted to biogenic acid attack. Cem Concr Res 70, 29–38. doi:10.1016/j.cemconres.2015.01.002