Fungi can survive on any polyurethane coating if oxygen and moisture are present resulting in the deterioration of protective coatings and, potentially, of the supporting weapon system structure. This is a systemic problem for sustaining our fleet and providing a safe environment to our military personnel. The objective of this project was to correlate the biodegradation of polyurethane coatings to the physiological and chemical real-time responses of fungi isolated and identified from coatings inside of active aircraft.

The research team performed experiments with two different fungal morphotypes using a total of 4 fungal species (Fungus 1, Fungus 2, Fungus 3, and Fungus 4) that were isolated from inside aircraft and were cultured/and screened against a colloidal polyester polyurethane (Impranil®DLN). These fungi were then challenged individually as biofilms on two polyesters (polyethylene succinate, polyethylene adipate), a polyurethane-based coating with polyester and polyether blocks (Irogran®; PS455-203), and ultimately with two polyether polyurethanes at two different relative humidity ranges (relative humidity: 70% and 100%). The two different humidity levels were used to evaluate the role of water in the degradation process. These polyester and polyether polyurethane coatings were spin-coated and cured on silicon, quartz, or zinc selenide transparent substrates and then challenged with the fungal strains listed above as air-dried biofilms. Polyurethane biodegradation products and fungal biodegradation mechanisms for all polymers were determined using spatially and/or temporally resolved spectroscopy techniques and imaging including Raman and IR microscopies. Single-cell microscopy, characterization of localized polymer degradation, and the relationship between degradation and the fungal response were determined using atomic force microscopy (AFM)-IR techniques (NanoIR™) and 3D confocal microscopy. The potential metabolic responses as the coatings were degraded and their reaction to the potential hydrolysis products were compared using carbon dioxide production over time from biofilms and a growth screen based on planktonic optical density changes.

The research team completed a comprehensive evaluation of two non-motile yeast strains (Fungus 1 and Fungus 2) during the degradation of all polymers and started to compare those results to a fungal mold, Fungus 3, which was capable of spreading over the polymer surface during the degradation process. The results confirm that the team has isolated and characterized three new fungal strains that are capable of degrading polyester polyurethane coatings. The project's success with identifying fungi capable of biodeterioration places the research team in the position to identify actual degradation pathways that are shared between non-motile yeasts and Basidiomycetes. Thus far, all fungi have hydrolyzed the soft polyester segments of these coatings and not the polyurethane nor polyether segments. Highlights include:

• Fungus 1 and Fungus 2 were isolated from the inside of aircraft and cleared Impranil®DLN containing agar plates
• Biofilms of both strains metabolized polyethylene succinate, polyethylene adipate, and the polyester component of Irogran® to different degrees without additional carbon sources
• Carbon dioxide production in the headspace above polymer degradation experiments confirmed that both fungal strains were metabolically active while degrading the polymers
• Fungus 1 and Fungus 2 grew on and degraded polyethylene adipate (PEA) coatings rapidly while only Fungus 1 grew and degraded polyethylene succinate (PES) coatings.
• Fungus 1 degraded Irogran® to a greater degree than Fungus 2 and infrared microscopy data confirmed that the soft polyester segment was hydrolyzed preferentially to the hard polyurethane segment
• Adipate was a viable carbon source for Fungus 1 and Fungus 2 biofilm growth on the polymer surface but not as a soluble carbon source for planktonic cultures.
• AFM-IR demonstrated non-homogenous Irogran® film degradation at the single cell level by Fungus 1 biofilms. Aggregation and transport of degradation resistant polymer components was also revealed.
• The two remaining fungi, Fungus 3 and Fungus 4, are capable of growing and degrading Irogran® but the mechanisms for degradation were not determined.

This project has identified two new non-motile yeast strains (Fungus 1 and Fungus 2) that are active degraders of polyurethane coatings from active aircraft. The team has isolated several polymer degrading organisms to date and have defined how moisture leads to the greatest difference in reactivity on all the coatings. Based on the data, both Fungus 1 and Fungus 2 are model organisms for studying and developing predictive biodegradation models for new alternative coating formulations and techniques. The activity of these yeasts indicates that they are direct degraders of the polyester coatings since they can both hydrolyze polyester based coatings and metabolize the hydrolysis products to CO₂; i.e. the polymer was considered a viable carbon source. Fungus 1 and Fungus 2 are ideal candidates for future studies as their attachment and degradation mechanisms in conjunction with their lack of motility results in predictable patterns of degradation (based on pitting by fungal generated secondary compounds) on any polymer surface. The results also indicated that moisture is critical to the rapid degradation of the coatings; no biodegradation was observed below a relative humidity of 70%.

Thwarting biofilm and coating degradation is not a new area of study; however, the research team believes that research and development in this area is still guided by legacy compounds and concepts where cell death is more important than control over the microorganism. The Department of Defense (DoD) cannot control when and if an organism will land on weapon systems, it can only respond and adapt to what happens as a result of the organism being on that surface. The more sensitive the solutions are to an organism’s natural behavior, the more likely that a universal solution to coating biodegradation can be discovered.