ENHANCED RESISTANCE OF BIOFILMS

Biofilm growth confers greater than usual resistance to a diversity of environmental extremes,1 and microbial functional redundancy in biofilms might also confer resilience to environmental extremes.2 Future research can address the extent to which organisms within communities or biofilms may exhibit increased resistance to the high temperatures used for terminal bioload reduction. Although the exterior of a spacecraft that has been assembled in a clean room is unlikely to harbor communities within biofilms, the protected interior of spacecraft might contain microenvironments in which organisms are in contact and behave as biofilms.

Protected microenvironments within spacecraft have to be characterized, and their microbial ecology has to be assessed. Moreover, research is needed to determine whether biofilm growth of organisms associated with spacecraft microhabitats can influence their resistance to heat treatment and other environmental extremes encountered on journeys to icy bodies.

Recommendation: Research should be undertaken to characterize the protected microenvironments within spacecraft and to assess their microbial ecology.

Recommendation: Research should be undertaken to determine the extent to which biofilms might increase microbial resistance to heat treatment and other environmental extremes encountered on journeys to icy bodies.

IMAGING METHODOLOGY TO DETERMINE BIOLOAD

The long-standing NASA standard protocol used to assess microbial contamination on spacecraft during assembly, test, and launch operations uses a Petri-plate-based culturable assay method to determine the number of cultivable aerobic bacterial endospores present on surfaces of interest. This assay takes 72 hours to complete, which can be extremely challenging and costly in a time-constrained hardware assembly environment. Because it relies on swab or wipe sampling, the assay method cannot be used directly for parts that cannot be touched or that are sensitive to the water matrix used for sampling. New techniques for obtaining real-time accurate assessments of microbial burden on flight hardware could provide a significant improvement over the current culture method.

The ideal solution would be a non-invasive, non-destructive technique that can be used to scan a spacecraft’s surfaces and identify living microbes through detection of morphologies of cells that are alive, as indicated by the presence of ribosomal RNA transcripts. There are several techniques that might be stepping stones toward the goal of detecting individual microbes on a spacecraft: e.g., Raman and fourier transform infrared spectroscopy. However, some of these techniques, for example, lack a scanning capability or cannot distinguish between living and dead or metabolically inactive cells.

One particularly promising new technique that might be applicable to the assessment of the bioload on a spacecraft is deep-ultraviolet (224 to 250 nm range) imaging.3,4 The advantage of using short-wavelength ultraviolet radiation is that most minerals and solid surfaces are non-fluorescent at this wavelength, whereas strong fluorescence is seen from the amino acids tryptophan, phenylalanine, and tyrosine, so that any organism containing proteins with these amino acids will be detectable by autofluorescence (i.e., without the addition of fluorescent stains or dyes). The identification of single cells through fluorescence scanning at low magnification can also provide a quantitative measurement of bioload. Real-time analyses of positive targets at higher magnification can enable identification of cells’ morphological properties, including the ability to differentiate bacterial spores from vegetative cells.

The deep-ultraviolet imaging method should be applicable to the assessment of bioload on the surfaces of spacecraft. As shown in the adjacent images (of Figure 6.1), bacteria “hiding” in the matrices of well-scrubbed surfaces of stainless steel are easily seen using deep-ultraviolet fluorescence imaging. In addition, because of their high tyrosine content (and probably other chemical differences), the spores have a fluorescent signal that differs from the signal of vegetative cells, making the approach valuable for direct determination of bacterial spores on



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