Navy and Marine Corps aircraft operate around the world most frequently in hot, humid environments. In interior regions of the aircraft where sunlight/ultraviolet light (UV) cannot penetrate, and exacerbated by the heat and humidity, mold and mildew (fungal) spores may attach and proliferate leading to biofouling, biodegradation and ultimately microbiologically influenced corrosion (MIC). This type of growth-influenced corrosion is unsightly, produces objectionable odors, and ultimately can compromise aircraft structural integrity by breakdown of protective finish systems and production of corrosive by-products. In addition to heat, humidity, and the lack of natural deterrents (i.e., UV light), the presence of organic materials such as hydraulic fluid, corrosion preventative compounds, and organic coatings act as a feedstock for the microbes. Microbial digestion of the organic content produces metabolites such as organic acids, esterases, and lipases which are corrosive and destructive to protective coatings and structural metals. The researchers proposed a broad comprehensive study of the life cycle of the microbe in operational conditions to determine the factors that most closely influence coating degradation and microbial proliferation. With a greater understanding of the role of these and other environmental factors in degrading coatings, mitigation mechanisms can be devised which thwart the microbe exploitation of these conditions.

The activities needed to initiate microbe attack on organic coatings are summarized as: (1) spore attachment; (2) spore activation; and (3) microbe proliferation. During proliferation, microbes are hypothesized to be metabolizing organic coating constituents; furthermore, the researchers hypothesized that other conditions of the coating including its surface free energy, surface roughness, and anti-microbial additives play roles during these three stages of microbe activity where a greater understanding could contribute to alternative means of mitigation. Thus, they included various components of the operational environment that play a role in these activities. Regions of aircraft susceptible to microbe growth may contain primer or top-coat, environmental organic contaminants, corrosion preventative compounds, and known operational fluids such as hydraulic fluid. The goal of the study was to determine the role of each of the following in inhibiting microbial activity: (a) the corrosion inhibitor toxicity (by including chromated and non-chromated primers); (b) the surface roughness and energy of the coating by including four coatings with differing characteristics (two topcoats, two primers); and (c) the role of hydraulic fluid and corrosion preventative compounds in the microbe life cycle in this environment. They exposed five known microbes to temperature and humidity conditions typical in operational growth areas, and subsequently analyzed the compromised coating surfaces using multiple analytical techniques.

Environmental scanning electron microscopy clearly documented microbial growth patterns on the surface of test panels and the effectiveness of cleaning procedures used to remove microbes to permit the surfaces analyses. Significant amount of fungal growth was observed on the panels which were not purposely inoculated. As completely sterile conditions were not maintained throughout the 84-day experiment, some growth was expected on control surfaces, but not to the extent observed. Chromated epoxy primer was not as resistant to microbial propagation as expected compared to the non-chromated primer based on observations, however, resistant species will require DNA analysis for positive identification. Laser scanning microscopy documented comparatively smooth surfaces of the non-chrome primer which may be less likely to physically harbor spores from a geometric perspective relative to the chromate primer which had a rougher and more hydrophilic matte finish. Infrared spectral analyses showed that hydraulic fluid was consumed by the microbes. The lack of clear degradation in the coatings indicates the microbes are consuming either leachable material or applied fluids and not the polymer backbone of the coatings. Analysis of the extracts from all samples showed no peaks in the gel permeation chromatography indicating that molecular weights in the range of 2,500 to 936,000 were not detected for the exposure conditions in this project. The scanning Kelvin probe analysis differentiated both qualitatively and quantitatively amongst the fungi-inoculated, the climate controlled, and control panels, and therefore may provide a unique method to characterize microbiological growth effects on materials and coating systems. Various common cleaning solvents (e.g., isopropyl alcohol, acetone, ethanol, and methanol) used on the fungal hyphae/mycelia were shown to be relatively ineffective in removing the attached growths from the coating surfaces. NavClean was the most effective in removing growths from organic coatings surfaces. Combinations of cleaning procedures may be required to remove both fungal growths and organic films of various origin which are acting as additional nutrients for microbiological propagation.

Environmental scanning electron microscopy was most useful for defining the extent of coating areas affected by microbial growths, determining completeness of microbial removal after various cleaning/sanitizing procedures, and visually characterizing known species with high certainty; follow-on sampling and DNA analyses are still required for absolute species identification. Gas chromatography and mass spectrometry analysis of coating extracts showed new peaks indicating leaching or possible degradation; the exception where this technique did not detect leaching was for the climate controlled hydrophobic coating. Infrared spectroscopy was capable of detecting evidence of suspected polyurethane degradation based on reduction of peak areas of the Amide I and Amide II bonds after microbiological growth; this technique will be more effective with a smaller spot size. Infrared spectroscopy was also useful in documenting surface residues of contaminant films after ineffective cleaning procedures. Although the study was not able to ascertain completely, scanning Kelvin probe may effectively be used to differentiate both qualitatively and quantitatively among various fungi-attacked coating systems as compared to control coating conditions; likewise, it may provide a method to quantify fungal growth on other materials used across the Naval Aviation Enterprise. Further developments in characterizing what component of the fungal contamination results in changes in work function values of materials/coatings would refine this applied technique. Although the limit of the duration of exposure for this study prevented more definite conclusions regarding coating degradation, laser scanning microscopy could be a valuable quantitative tool for analyzing panels exposed for a longer duration or for more heavily degraded coatings obtained from systems in operational environments. Similarly, gel permeation chromatography is expected to provide more data when sufficiently large molecular degradation products are formed and extracted into analyte solutions.  Another potential benefit of gel permeation chromatography is that it could be effective in quantifying non-planar irregular shaped materials which have experienced growths.