Metabolomic and cultivation insights into the tolerance of the spacecraft-associated Acinetobacter toward Kleenol 30, a cleanroom floor detergent

Abstract

Introduction: Stringent cleaning procedures during spacecraft assembly are critical to maintaining the integrity of life-detection missions. To ensure cleanliness, NASA spacecraft are assembled in cleanroom facilities, where floors are routinely cleansed with Kleenol 30 (K30), an alkaline detergent.

Methods: Through metabolomic and cultivation approaches, we show that cultures of spacecraft-associated Acinetobacter tolerate up to 1% v/v K30 and are fully inhibited at ≥2%; in comparison, NASA cleanrooms are cleansed with ~0.8-1.6% K30.

Results: For A. johnsonii 2P08AA (isolated from a cleanroom floor), cultivations with 0.1% v/v K30 yield (1) no changes in cell density at late-log phase, (2) modest decreases in growth rate (~17%), (3) negligible lag phase times, (4) limited changes in the intracellular metabolome, and (5) increases in extracellular sugar acids, monosaccharides, organic acids, and fatty acids. For A. radioresistens 50v1 (isolated from a spacecraft surface), cultivations yield (1) ~50% survivals, (2) no changes in growth rate, (3) ~70% decreases in the lag phase time, (4) differential changes in intracellular amino acids, compatible solutes, nucleotide-related metabolites, dicarboxylic acids, and saturated fatty acids, and (5) substantial yet differential impacts to extracellular sugar acids, monosaccharides, and organic acids.

Discussion: These combined results suggest that (1) K30 manifests strain-dependent impacts on the intracellular metabolomes, cultivation kinetics, and survivals, (2) K30 influences extracellular trace element acquisition in both strains, and (3) K30 is better tolerated by the floor-associated strain. Hence, this work lends support towards the hypothesis that repeated cleansing during spacecraft assembly serve as selective pressures that promote tolerances towards the cleaning conditions.

Keywords: Acinetobacter; cleanrooms; detergent; metabolomics; planetary protection; spacecraft; survival.

Figure 1

Impacts of 0–1.0% v/v Kleenol 30 (K30) on (A) the survival and (B–D) cultivation kinetics of Acinetobacter johnsonii 2P08AA (red) and Acinetobacter radioresistens 50v1 (blue). Survival (N/N^⊝) is expressed as the ratio of plate counts (cfu mL⁻¹) from cultures grown in the absence of K30 (N^⊝) and presence of K30 (N). Fitted regressions of growth curves yielded changes in (B) growth rates (h⁻¹), (C) lag time (h), and (D) maximum relative biomass (log (N/N₀)), which is assumed to represent a ratio of the total biomass (N) at stationary phase and the biomass at the start of the culture (N₀). Error bars represent the standard errors (n = 4–5, growth curves; n = 3–5, plate counts). Univariate tests are represented as asterisks (*) for t-tests with p < 0.05, hashtags (#) for t-tests with p ≥ 0.05, and asterisks (*) with an underlying line for one or two-way ANOVA with p < 0.05.

Figure 2

Measured and fitted growth curves for A. johnsonii 2P08AA (left panel) and A. radioresistens 50v1 (right panel) cultured in the presence of 0–1.0% v/v Kleenol 30 at 28°C in 0.2x M9 media containing 25 μM Fe²⁺ and 150 mM ethanol (the sole supplied carbon source). Optical density measurements (blue empty circles; right-hand y-axis) were converted to log(N/N₀) (red filled circles; left-hand y-axis) as described and fit by non-linear regression to yield the parameters of time in lag phase, growth rates, and maximum ratiometric and logarithmic change in biomass (log (N/N₀)) at stationary phase.

Figure 3

Maps showing the impacts of cultivation in 0.1% v/v K30 on the intracellular and extracellular metabolomes of A. johnsonii 2P08AA (A–C) and A. radioresistens 50v1 (B,D). Metabolites are represented as nodes and organized by structural and metabolic connections (MetaMapp and Cytoscape 3.9.1). Important changes in abundance (p < 0.05) are represented as red (increases), blue (decreases), and yellow (no change) nodes, where node sizes represent the degree of change. Relevant metabolites that show important (black text; p < 0.05) and significant changes (red text; p < 0.05; FDR ≤ 0.20), as listed in Tables 1, 2, are displayed using the associated identity numbers (ID#). Shaded circles represent biochemical classes (red text) that show significant changes (ChemRich); biochemical classes showing no change are listed for comparison purposes (black text).

Figure 4

Canonical correspondence analyses (CCA) that compare the impacts of cultivation in 0.1% v/v Kleenol 30 (K30) on (A) the intracellular metabolome of A. johnsonii 2P08AA, (B) the extracellular metabolome of A. johnsonii 2P08AA, (C) the intracellular metabolome of A. radioresistens 50v1, and (D) the extracellular metabolome of A. radioresistens 50v1 against the quantitative descriptors of growth rates at exponential phase (blue callout boxes, green vectors), plate counts at late-log phase (purple callout boxes, green vectors), maximum relative biomass at stationary phase (brown callout boxes, green vectors), and either the total concentrations of detergents (0.025% w/w; assumed) or chelators (0.02% w/w; assumed) in the cultivation media (black callout boxes, green vectors); dimension reduction of the triplicate metabolomic measures associated with 0% v/v K30 (red squares, pink circles, and red and pink diamonds) and 0.1% v/v K30 (black triangles) are provided, reduced terms are highlighted by the green and red circles and callout boxes, and metabolites are arrayed as text.