Solid medium for the direct isolation of bacterial colonies growing with polycyclic aromatic hydrocarbons or 2, 4, 6-trinitrotoluene (TNT) as the sole carbon source

doi:10.21203/rs.3.rs-2701846/v1

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Solid medium for the direct isolation of bacterial colonies growing with polycyclic aromatic hydrocarbons or 2, 4, 6-trinitrotoluene (TNT) as the sole carbon source

https://doi.org/10.21203/rs.3.rs-2701846/v1

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published 26 Jun, 2023

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Isolation of hydrocarbon-degrading bacteria is a key step for the study of microbiological diversity, metabolic pathways and bioremediation, however current methods lack simplicity and versatility. We developed an easy method that allows the screening and isolation of bacterial colonies capable of degrading hydrocarbons, such as diesel or polycyclic aromatic hydrocarbons (PAHs), as well as the pollutant explosive, 2, 4, 6-trinitrotoluene (TNT). The method uses a two-layer solid medium, with a layer of M9 medium, with a second layer containing the carbon source deposited trough the evaporation of ethanol. Using this medium we grew hydrocarbon-degrading strains, using diesel, phenanthrene or anthracene as the sole carbon sources, as well as three TNT-degrading isolates. Using this medium we isolated PAHs-degrading bacterial colonies directly from diesel polluted soils. Analysis revealed that bacteria grown in medium using PAHs as carbon source maintain their morphological characteristics when compared to cells grown on traditional media with glucose.

Solid medium

Bacterial isolation

Hydrocarbon-degrading bacteria

Polycyclic aromatic hydrocarbons

6-trinitrotoluene

Environmental pollution by organic components of anthropogenic origin, such as diesel related hydrocarbons, is a widespread problem that affects all continents, even reaching remote environments as Antarctica (Aislabie et al., 1999; Mazzera et al., 1999). Diesel pollution is known to heavily impact the microbiome of soils, mainly by the action of one of its most persistent components (Adam et al., 2002), the toxic polycyclic aromatic hydrocarbons (PAHs) (Achten & Andersson, 2015; Szczybelski et al., 2019)(Agency for Toxic Substances and Disease, 1995). PAHs are known to reduce up to 80% of the endogenous species (Saul et al., 2005), while producing an enrichment of hydrocarbon-degrading bacteria (Gran-Scheuch et al., 2017). A similar effect is seen on soils polluted with 2,4,6-trinitrotoluene (TNT), a widely used explosive, found on soils near production plants, firing ranges, and detonation zones (Alothman et al., 2020; Bruns-Nagel et al., 1996; Williams et al., 2004). In spite of being an explosive, TNT can remain for long periods of time, leading to its accumulation on polluted soils (Lewis et al., 2004). Both, PAHs and TNT not only share their noxious effects over soil microbiome (Yang et al., 2021), but also share the benzene ring as their core structure in common (Wexler, 2005), sharing common biodegradation enzymatic pathways, such as the Protocatechuate Dioxygenases (Tsagogiannis et al., 2021; Xu et al., 2021).

The study of specific pollutant-degrading bacterial species to determine their degradation capacities and associated metabolic pathways requires the isolation of bacterial colonies. Currently, bacterial isolation of PAHs and TNT degrader-species is commonly done through bacterial enrichment in liquid media containing the pollutant of interest, for example motor oil (Uyar & Sağlam, 2021). This strategy has proven useful to our group for the isolation of PAH-degrading bacteria, such as Sphingobium xenophagum D43FB (Gran-Scheuch et al., 2017, p. 2) and TNT-degrading bacteria, such as the isolates TNT3, TNT11 and TNT19, of the Pseudomonas genus (Cabrera et al., 2020, 2022).

While useful, this strategy usually requires several steps over the span of weeks or even months (Sun et al., 2019). Furthermore, these enrichment-based methodologies require a final step for bacterial isolation, involving the growth of bacterial colonies on solid media, usually containing glucose or yeast extract as carbon source. In this final step, the selection pressure for pollutant-degrading bacteria is lost, which may lead to the over representation of bacteria better adapted to use the carbon source of the non-selective media rather than the hydrocarbons of interest.

As an attempt to solve this issue, a method using solid growth medium with oil powder was developed for the screening of oil degrading bacteria (Olga et al., 2008). The protocol involves mixing oil and silica powder, using diethyl ether as solvent, and adding an enzymatic indicator for dehydrogenase activity. Although this method allows the screening and isolation of cultivable oil degrading bacteria from soil samples, it requires several steps to add the carbon source, and due to the opacity of the added oil powder, an enzymatic indicator must be incorporated to help discerning the isolated colonies. This method constituted a significant improvement from bacterial enrichment; however, it was not described for the addition of individual hydrocarbons, which along with the mentioned need for an indicator, has hindered its widespread use. Other alternative has been recently reported in which acetone was used for solubilization of pyrene for incorporation in solid media (Zeng et al., 2019). Though this method was used to screen for hydrocarbon degrading bacteria from already obtained isolates, the addition of less soluble hydrocarbons into melted agar does not warrant an even distribution or their availability. Furthermore, incorporation in solid medium of pollutants with low water solubility, like diesel or TNT, and its implementation as an isolation medium is still understudied.

Considering the necessity of an easy and rapid alternative for the isolation of hydrocarbon degrading bacteria in solid media, and the limitations of the methods currently published, we developed a simple medium that allows the growth and isolation of organic pollutant degrading bacteria from soil samples. The method developed here was tested for incorporation of diesel as carbon source in solid medium, as well as individual components such as PAHs, and TNT, characterized by low water solubility (Agency for Toxic Substances and Disease, 1995), that does not allow for their direct addition or even distribution into a solid medium or soft agar medium. This new method will facilitate the isolation and characterization of bacteria capable of degrading common and highly persistent soil pollutants, contributing to the understanding of the degradation pathways present on different bacteria and their behavior when exposed to organic pollutants, such as diesel, PAHs, and TNT.

Reagents and solutions: Na₂HPO₄, KH₂PO₄, NaCl, NH₄Cl, MgSO₄ and CaCl₂ salts, as well as Phenanthrene was purchased from Merck Supelco (USA), LB media components were purchased from Becton, Dickinson and Company (USA), except for agar and NaCl, which were purchased from Winkler (Santiago, Chile). TNT was obtained from the US Army. Diesel, phenanthrene and TNT were solubilized in HPLC grade ethanol, obtained from J.T. Baker, under constant stirring overnight at ambient temperature.

Solid medium preparation and inoculation: The solid growth medium developed is composed by a layer of agarised M9 minimal medium with no carbon source (composition: 42.2 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.56 mM NaCl, 18.7 mM NH₄Cl, 1 mM MgSO₄, and 0.1 mM CaCl₂), followed by a layer of a filtration-sterilized diesel solution, dissolved in absolute ethanol. This solution was allowed to evaporate under a laminar flow hood to form a homogeneous layer of diesel on top of the medium. For preparation of media with phenanthrene, anthracene or TNT as the sole carbon source, the same method was performed. Concentrations of the respective carbon sources were calculated as a mean of mass over surface area. For diesel and phenanthrene plates, concentrations of 0.08 to 0.16 mg/cm² were used. For anthracene plates, concentrations of 0.12 to 0.16 mg/cm² were used. For TNT plates, a concentration of 4 µg/cm² was used. As a negative control, a solid M9 minimal medium with no carbon source was used. For inoculation of hydrocarbon-containing plates, S. xenophagum D43FB (Gran-Scheuch et al., 2017)d silesiensis species were used. For TNT-containing plates, the Pseudomonas species termed TNT3, TNT11 and TNT19 (Cabrera et al., 2020) were used. As a negative control, Escherichia coli BW25113 was used. For inoculation on solid media, a single colony of each species was grown in lysogeny broth (LB) (1% tryptone, 0,5% yeast extract, and 1% NaCl) at 28°C until overnight density. This culture was diluted a hundred-fold on fresh broth and incubated at 28°C with constant agitation (160 RPM) until stationary phase was reached (OD₆₀₀ ~ 0.6), measured on a Synergy H1 microplate reader (BioTeK, USA). To ensure that no carbon source from the inoculum was transferred to the solid media, the inoculums were centrifuged at 6000 g and washed with M9 medium with no carbon source and then resuspended in fresh M9 medium with no carbon source. A 5 µl droplet of washed bacteria was deposited on the plates. After the droplets were dried, the plates were incubated for five days at 28° C.

Microscopy analysis: Bacterial species were inoculated on solid media as described above. After incubation, 5 colonies were picked and resuspended on 200 µl of 50 mM potassium phosphate pH 7.4 buffer. An aliquot of 5 µl was mounted on a glass slide for observation on a MF606 microscope (BW OPTICS), equipped with a 100X oil immersion objective (N.A.: 1.25). As control, bacteria grown on a plate with 0.2% (w/v) of glucose as carbon source were analyzed.

Soil-isolation of hydrocarbon-degrading bacteria: Diesel-polluted (DPS) and not polluted (NPS) soil samples with similar mineralogical characteristics were collected near the Chilean Antarctic station Escudero, during the 55th Chilean Antarctic Expedition (ECA-2018) organized by the Chilean Antarctic Institute (INACH). Two grams of each soil sample were suspended in sterile water (20% w/v) and incubated at 28°C with agitation (160 RPM) overnight. Then, the soil substrate was removed from the inoculum using sterile nitrocellulose filter paper. The inoculums were washed three times as previously described and 100 µl were evenly spread over one half of solid media plates, using diesel, phenanthrene, anthracene or glucose as carbon sources, with the concentrations described above. Plates were incubated at 28°C for 5 days.

Selective solid media for hydrocarbon degrading bacteria

To incorporate the different pollutants of an agar plate, these were first solubilized using ethanol, as it allowed the solubilization of the pollutants and could be easily removed via evaporation. This solution was deposited on top of an agar plate of M9 minimal medium without carbon source (See “Methods").

To assess the required amount of Diesel to allow the growth of hydrocarbon degrading bacteria, several concentrations of diesel were evaluated for the hydrocarbon layer on top of the agar. As expected, colonies of the hydrocarbon degrading strain S. xenophagum D43FB were observed when adding 0.08 mg/cm² of Diesel, while the E. coli control did not grow (Fig. 1A). Further testing showed that diesel content up to 0.16 mg/cm² in the solid media allowed the growth of S. xenophagum D43FB, while E. coli failed to grow in all concentrations (Fig. 1B). Considering the difference in optimal growth temperature of E. coli, the same plating and incubation process was repeated at 37°C and no growth of E. coli was observed in any plate containing hydrocarbons as the sole carbon source (not shown).

The method was used to prepare solid media with phenanthrene and anthracene as the sole carbon source. We tested various amounts of both PAHs to find the range allowing the growth of S. xenophagum D43FB. Phenanthrene content between 0.08 mg/cm² (Fig. 1C) and 0.16 mg/cm² (Fig. 1D) allowed the growth of S. xenophagum. This range of concentrations formed a uniform layer of phenanthrene over the agar. In the case of anthracene, concentrations between 0.12 mg/cm² (Fig. 1E) and 0.16 mg/cm² (Fig. 1F) allowed the growth of S. xenophagum D43FB, while E. coli failed to grow at all concentrations tested. Anthracene did not leave an even layer after the solvent evaporated, however it did not seem to affect the growth of bacterial colonies, although the borders of the colonies was affected. An opaque spot was observed on the agar surface when E. coli was plated directly from its source media. This opaque spot was not observed when E. coli was washed three times before plating (Fig. 1A-F) or when no carbon source was added to the M9 agar (Fig. 1H). Overall, washing S. xenophagum D43FB had no effect over the growth on the solid media. On the same plates, we tested a P. silesiensis strain that we recently isolated from a diesel polluted site in Antarctica (unpublished work). The P. silesiensis isolate tested grew on the solid media in the presence of diesel, phenanthrene and anthracene at all tested concentrations. Both the Pseudomonas and Sphingobium isolates could grow at all tested concentrations, although, the higher concentrations tested decreased the size of bacterial colonies (not shown), indicating that growth can be controlled by changing the pollutant concentration. No colonies of neither Pseudomonas nor Sphingobium were observed on plates with no carbon source (Fig. 1H).

Selective solid media for TNT degrading bacteria

We tested this technique with the less common pollutant TNT, based on its similar polarity with the PAHs previously tested due to the presence of the benzene ring. We used a plate containing 4 µg/ cm² of TNT as the sole carbon source and we plated following the same inoculation method already described. All strains were washed three times to ensure no carbon source from the liquid media was transferred to the TNT containing plate. The plated strains were Pseudomonas spp. TNT3, TNT11, and TNT19, described as TNT degraders (Cabrera et al., 2020). The three strains were able to grow using TNT as the sole carbon source, whereas the negative control E. coli failed to grow (Fig. 1G). The same inability to grow using TNT as carbon source was observed with the PAH degraders P. silesiensis and S. xenophagum, despite having dioxygenases capable of degrading the benzene ring on PAHs (Gran-Scheuch et al., 2017). As expected, none of the TNT degraders grew on the plate with no carbon source (Fig. 1H).

To confirm that the solid media described here does not negatively affect the morphology of the plated isolates, cells were analyzed through microscopy. S. xenophagum and P. silesiensis strains were plated on M9-glucose agar medium or M9 agar medium containing diesel or phenanthrene (0.08 mg/cm²) as carbon source and incubated for 48 hours at 28°C. For the TNT3, TNT11 and TNT19 strains, a solid medium with 4 µg/cm² of TNT was used, as well as M9- glucose agar.

Microscopy analysis showed that, both S. xenophagum and P. silesiensis maintain their morphology in all 3 media, independently of the carbon source (Fig. 2A). This suggests that the hydrocarbon-supplemented media keeps the cellular viability and allows to maintain a cellular morphology comparable to other traditional media (such as M9-glucose) which is necessary for morphology based bacterial identification. Due to the irregular crystallization of anthracene, microscopy analysis of cells was not possible (not shown). In the same way, when the different TNT-degrader species were grown with either TNT or glucose as carbon source, they kept rod shape, characteristic of Pseudomonas genus (Fig. 2B).

Isolation of hydrocarbon-degrading bacteria from diesel-polluted soil samples

Lastly, we tested the ability of the media to directly isolate hydrocarbon degrading bacteria from soil samples (Fig. 3). For this, we used soil samples from two sites with similar mineralogical characteristics and closely located near the Chilean Antarctic station Escudero; one site was polluted with diesel (DPS) and the other was not polluted (NPS). Two grams of each soil sample were suspended in sterile water (20% w/v) and incubated at 28°C with agitation (160 RPM) overnight. Then, the soil substrate was removed from the inoculum using sterile nitrocellulose filter paper. The inoculums were washed three times as previously described, and 100 µl were evenly spread over one half of a plate. The growth of bacterial colonies directly from soils was evaluated in plates containing different concentrations of diesel, phenanthrene or anthracene (Fig. 3).

When plates were inoculated with the polluted soil, bacterial colonies were observed in solid media containing diesel, anthracene or phenanthrene (Fig. 3, left side of plates). A higher number of bacterial colonies was present in the solid media containing diesel and phenanthrene. On the other hand, no bacterial colonies were visible after five days incubation when the unpolluted soil was used to inoculate the solid media (Fig. 3, right side of plates). Both soil samples, contaminated and non-contaminated, showed similar bacterial growth when plated over solid M9 medium with glucose (0,2% w/v) (Fig. 3G). This suggests that the soil sample from the polluted site contained a much higher number of bacteria capable of degrading hydrocarbons, which was not the case for the unpolluted soil sample. This is consistent with previous reports indicating that soils polluted with diesel contain a higher proportion of hydrocarbon-degrading bacteria (Bej et al., 2000; Gran-Scheuch et al., 2020). This result in addition to the lack of growth on the plate with no carbon source from either soil sample (Fig. 3H), indicates that the solid media developed here is capable of isolating bacteria capable of growing with hydrocarbons as the sole carbon source directly from diesel polluted soil samples.

In summary, we developed of a solid medium that allows the selective growth of bacteria capable of degrading organic pollutants, including diesel, PAHs, and TNT, despite their low solubility. This rapid, simple, and economic method maintains the selective pressure during the complete isolation process and does not require extra steps for effective isolation. Furthermore, this method keeps the bacterial morphology after growth, which allows further bacterial identification and characterization. This would allow to directly isolate bacterial colonies capable of degrading hydrocarbons in presence of other compounds, a topic of growing interest.

Funding

This study was financially supported by Fondecyt 1200870 (JMP-D).

Competing Interests

The authors declare that there are no conflicts of interest.

Author contributions

C.D-V: Conceptualization, Methodology, Validation, Investigation, Writing – original draft, Writing – review and editing, Visualization; F.V-I: Methodology, Validation, Writing – original draft, Writing – review and editing, Visualization; J.M.P-D: Conceptualization, Investigation, Resources, Writing – original draft, Writing – review and editing, Supervision, Project administration, Funding acquisition.

Acknowledgment

This work was supported by Erika Elcira Donoso López, and Fondecyt 1200870 (JMP-D). We would like to thank Dr. Sebastian Lagos Moraga for his suggestions that ultimately led to this work. In the loving memory of Dr. Claudio Vásquez Guzmán, an excellent friend, mentor, and scientist, but an even better human being. Thanks for all the adventures and for showing us the beauty of science and friendship.

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No competing interests reported.

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Solid medium for the direct isolation of bacterial colonies growing with polycyclic aromatic hydrocarbons or 2, 4, 6-trinitrotoluene (TNT) as the sole carbon source

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