XTT assay for detection of bacterial metabolic activity in water-based polyester polyurethane

Abstract

Cellular metabolic activity can be detected by tetrazolium-based colorimetric assays, which rely on dehydrogenase enzymes from living cells to reduce tetrazolium compounds into colored formazan products. Although these methods have been used in different fields of microbiology, their application to the detection of bacteria with plastic-degrading activity has not been well documented. Here, we report a microplate-adapted method for the detection of bacteria metabolically active on the commercial polyester polyurethane (PU) Impranil®DLN using the tetrazolium salt 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT). Bacterial cells that are active on PU reduce XTT to a water-soluble orange dye, which can be quantitatively measured using a microplate reader. We used the Pseudomonas putida KT2440 strain as a study model. Its metabolic activity on Impranil detected by our novel method was further verified by Fourier-transform infrared spectroscopy (FTIR) analyses. Measurements of the absorbance of reduced XTT at 470 nm in microplate wells were not affected by the colloidal properties of Impranil or cell density. In summary, we provide here an easy and high-throughput method for screening bacteria active on PU that can be adapted to other plastic substrates.

Fig 1. Growth curves of bacteria in BM medium based on OD630.

A) P. putida KT2440 and B) E. coli BL21 were grown in BM medium with either 20 mM citrate or 20 mM citrate and 1 mg⋅mL^-1 Impranil for 25 hours at 30°C, 180 rpm in a microplate reader. Biotic controls correspond to bacterial cultures without any carbon source. The data are the mean of three independent experiments performed in duplicate. Error bars indicate the standard deviation (SD).

Fig 2. putida KT2440 grown in BM-citrate-Impranil tend to form aggregates (see C-3).

P. Photographs of microplate wells containing cultures of P. putida KT2440, and E. coli in BM medium added with either citrate or citrate-Impranil after 24 h of incubation at 30°C, 180 rpm. Differences in the color of cultures in wells are due to the presence of Impranil (compare 2-BM-citrate with 3-BM-citrate-Impranil), E. coli BL21 inocula, or the growth of P. putida KT2440 using citrate or citrate-Impranil as carbon sources.

Fig 3. Metabolically active P. putida KT2440 cells reduce the XTT salt to a soluble, orange-colored formazan.

Photographs of microplate wells containing cultures of P. putida KT2440 and E. coli grown in BM medium added with either citrate or citrate-Impranil and XTT after 25 hours of incubation. The intensity of the orange formazan is directly proportional to the number of active cells. E. coli BL21 negative control and abiotic controls did not show XTT reduction.

Fig 4. Kinetics of orange-colored formazan production based on OD470.

50 μL of XTT (2 mg⋅mL^-1) were added to the cultures in microplates of (A) P. putida KT2440 and (B) E. coli BL21 described in the legend of Fig 1. The data are the mean of the corrected absorbance (OD470-OD630) of three independent experiments performed in duplicate. Error bars; SD.

Fig 5. FTIR spectra of Impranil treated with P. putida KT2440 for 0 and 15 days of incubation.

Changes in functional groups are observed. The FTIR spectra show the average of three biological replicates for each sampling day averaged with Spectragryph software.