Metabolism and Biodegradation of Spacecraft Cleaning Reagents by Strains of Spacecraft-Associated Acinetobacter

Abstract

Spacecraft assembly facilities are oligotrophic and low-humidity environments, which are routinely cleaned using alcohol wipes for benchtops and spacecraft materials, and alkaline detergents for floors. Despite these cleaning protocols, spacecraft assembly facilities possess a persistent, diverse, dynamic, and low abundant core microbiome, where the Acinetobacter are among the dominant members of the community. In this report, we show that several spacecraft-associated Acinetobacter metabolize or biodegrade the spacecraft cleaning reagents of ethanol (ethyl alcohol), 2-propanol (isopropyl alcohol), and Kleenol 30 (floor detergent) under ultraminimal conditions. Using cultivation and stable isotope labeling studies, we show that ethanol is a sole carbon source when cultivating in 0.2 × M9 minimal medium containing 26 μM Fe(NH₄)₂(SO₄)₂. Although cultures expectedly did not grow solely on 2-propanol, cultivations on mixtures of ethanol and 2-propanol exhibited enhanced plate counts at mole ratios of ≤0.50. In support, enzymology experiments on cellular extracts were consistent with oxidation of ethanol and 2-propanol by a membrane-bound alcohol dehydrogenase. In the presence of Kleenol 30, untargeted metabolite profiling on ultraminimal cultures of Acinetobacter radioresistens 50v1 indicated (1) biodegradation of Kleenol 30 into products including ethylene glycols, (2) the potential metabolism of decanoate (formed during incubation of Kleenol 30 in 0.2 × M9), and (3) decreases in the abundances of several hydroxy- and ketoacids in the extracellular metabolome. In ultraminimal medium (when using ethanol as a sole carbon source), A. radioresistens 50v1 also exhibits a remarkable survival against hydrogen peroxide (∼1.5-log loss, ∼10⁸ colony forming units (cfu)/mL, 10 mM H₂O₂), indicating a considerable tolerance toward oxidative stress under nutrient-restricted conditions. Together, these results suggest that the spacecraft cleaning reagents may (1) serve as nutrient sources under oligotrophic conditions and (2) sustain extremotolerances against the oxidative stresses associated with low-humidity environments. In perspective, this study provides a plausible biochemical rationale to the observed microbial ecology dynamics of spacecraft-associated environments.

Keywords: Acinetobacter; Bioburden; Cleaning; Extreme survival; Metabolism; Planetary protection; Spacecraft.

FIG. 1.

(A) Growth rates and (B) mid-log phase plate counts for differing strains of spacecraft-associated Acinetobacter (50v1, 2P01AA, 2P08AA, 2P07AA, 2P07PB, and 2P07PC) cultivated in 0.2 × M9 (32°C) containing 26 μM Fe²⁺ and 16 mM (0.1% v/v) or 160 mM (1.0% v/v) ethanol; the respective growth curves are provided in Supplementary Figure S1 (X demarks no measurable growth, n = 6–7, errors bars in (A) represent the standard error of regression and in (B) the standard deviation).

FIG. 2.

(A) Growth rates of Acinetobacter radioresistens 50v1 and A. radioresistens 43998^T cultivated (32°C) in 0.2 × M9 and 26 μM Fe²⁺ containing 2–650 mM ethanol (n = 2–6, errors bars represent the standard error of regression); (B) mid-log phase plate counts of A. radioresistens 50v1 obtained from cultivations (32°C) in 0.2 × M9 and 26 μM Fe²⁺ containing 200 mM ethanol (mole ratio 0), 170 mM ethanol, and 30 mM 2-propanol (mole ratio 0.15), 100 mM ethanol and 100 mM 2-propanol (mole ratio 0.50), 30 mM ethanol and 170 mM 2-propanol (mole ratio 0.85), and 200 mM 2-propanol (mole ratio 1.0) (*demarks statistical significance of p < 0.05, X demarks no growth, n = 5–11, and error bars represent the standard deviation); and (C) growth rates of A. radioresistens 50v1 cultivated (32°C) in 0.2 × M9 and 26 μM Fe²⁺ containing 200 mM ethanol (mole ratio 0; triangles) or 170 mM ethanol and 30 mM 2-propanol (mole ratio 0.15; squares).

FIG. 3.

Mass spectra for (A) oleic acid (18:1^Δ9) and (B) trehalose [α-d-glucopyranosyl-(1 ↔ 1)-α-d-glucopyranose] extracted from cultures of A. radioresistens 50v1 cultivated (32°C) in 0.2 × M9 and 26 μM Fe²⁺ containing 16 mM [1,2-¹³C₂] ethanol; inset structures represent the underivatized compound, rounded corner boxes highlight the molecular ion (arrow) or fragment ion, and the representative total ion chromatogram is provided in Supplementary Figures S2A and S2B.

FIG. 4.

Michaelis–Menten kinetics and nonlinear least-squares regressions (fitted line) for (A) ethanol and (B) 2-propanol catalysis using suspended membrane fractions of A. radioresistens 50v1 (cultivated in 0.2 × M9, 26 μM Fe²⁺, and 16 mM ethanol at 32°C), and (C) comparisons of the maximum specific activities (pkat/mg) for ethanol and 2-propanol (error bars represent the standard deviation, n = 7–9 for ethanol and n = 3–7 for 2-propanol).

FIG. 5.

Biodegradation and impacts of 0.1% and 1.0% v/v Kleenol 30 on cultures of A. radioresistens 50v1 (0.2 × M9, 26 μM Fe²⁺, 16 mM ethanol, 32°C), where (A) the relative abundances of the degradation products for Kleenol 30 (K30) are compared with those in the control samples of (1) K30 incubated in 0.2 × M9 (1%K30) and (2) the 50v1 strain grown in the absence of K30 (50v1 0.2 × M9); (B) impacts on the extracellular metabolites, as displayed by the logarithm of the ratio of abundances measured in the presence of K30 (0.1% and 1.0%) and absence of K30 (50v1 0.2 × M9, control sample), where negative values indicated a decrease in abundance compared with the control, and positive values indicated an increase in abundance compared with the control (for these experiments, biological replicates referred to samples cultivated from plates prepared from glycerol stocks, and technical replicates referred to samples cultivated from separate colonies from the same plate; all metabolites were detected in three biological replicates [n = 3], with at least one technical replicate per condition, except for citrate [n = 1 with three technical replicates] and suberic acid [n = 1 with two technical replicates]; error bars represent (A) the standard deviation and (B) propagated error).