Effects of road salt on microbial communities: Halophiles as biomarkers of road salt pollution

Abstract

Increased use of salting to de-ice roadways, especially in urban areas, is leading to elevated salinity levels in soil as well as surface- and ground water. This salt pollution may cause long-term ecological changes to soil and aquatic microbial communities. In this study, we examined the impact on microbial communities in soils exposed to urban road salt runoff using both culturing and 16S amplicon sequencing. Both methods showed an increase in halophilic Bacteria and Archaea in samples from road salt-exposed areas and suggested that halophiles are becoming persistent members of microbial communities in urban, road salt-impacted soils. Since salt is a pollutant that can accumulate in soils over time, it is critical to begin assessing its impact on the environment immediately. Toward this goal, we have developed a facile semi-quantitative assay utilizing halophilic microbes as biomarkers to evaluate on-going salt pollution of soils.

Fig 1. Trends of snow accumulation and salt usage for de-icing roadways.

Average snow accumulation in Central Park, New York, and salt usage for de-icing for the USA are shown using a 20-year sliding window starting in the winter season of 1940/41. The sliding window was chosen to minimize annual fluctuations which would otherwise mask the long-term trend. Snow accumulation data were obtained from the National Weather Service [13], and salt usage data from the U. S. Geological Survey [14].

Fig 2. Sampling locations and relative salinity measurements.

Sample sites are located in the Jones Falls watershed (MD 8-digit watershed code: 02130904), a sub-watershed of the Patapsco River watershed (MD 6-digit watershed code: 021309). The control un-impacted site (with an osmolality of 48 mOsm kg^-1) is located in an arboretum on a ridge-top away from any road. The seasonally impacted site (with an osmolality of 51 mOsm kg^-1) is located in a park receiving runoff from streets in a residential neighborhood, and the continuously impacted site (with an osmolality of 603 mOsm kg^-1) is located adjacent to a year-round salt storage and distribution facility. The location of the Jones Falls, the main contributor to the Inner Harbor of Baltimore, Maryland, USA, and one of its tributaries, the Stony Run are also indicated. Maps were created using Generic Mapping Tools v. 6.0.0 [44] and GIMP v. 2.8.22. Map data were obtained from Geofabrik GMBH (download.geofabrik.de). Map data OpenStreetMap contributors are licensed under the Open Data Commons Open Database License (ODbL; https://opendatacommons.org/licenses/odbl/1.0/index.html) by the OpenStreetMap Foundation, and the Creative Commons Attribution-ShareAlike 2.0 License (CC BY-SA; https://creativecommons.org/licenses/by-sa/2.0/legalcode).

Fig 3. Spot test for a semi-quantitative assessment of halophiles.

Twenty μl of serially diluted soil suspensions were spotted in triplicate onto HB agar plates supplemented with 2 M NaCl and incubated for 48 hours at 37°C. Note that the brown to light brown spots seen at the lower dilutions (10⁰ to 10⁻¹) are residual insoluble materials from the soil suspensions. Colonies appear as cream-colored dots. Un-impacted soil shows no growth on the high salt agar, while seasonally and continuously impacted sites show halophile growth. Continuously impacted sites show the highest growth levels. Summer season shows the least amount of growth across all samples.

Fig 4. Number of colony forming units of halophiles at the different sites.

Soils collected in November 2014, February 2015, and June 2015 were resuspended in HB broth supplemented with 2 M NaCl, serial dilutions plated onto HB agar plates supplemented with 2 M NaCl, and plates incubated for 48 hours at 37°C. Colonies were counted and colony forming units normalized to 1 cm³ of soil. The seasonal salt impacted site (blue) harbors significantly higher numbers of halophiles of at least one order of magnitude compared to the un-impacted site (green) at all seasons. Continuous salt exposure enhances the differences as the site that is continuously impacted (red) has at least one order of magnitude more halophiles than the seasonally impacted site (green). The y-axis of the plot is a logarithmic scale. Standard deviations are indicated by error bars.

Fig 5. Relative abundance of representative halophiles in soil samples collected in 2014 and 2015.

Amplicon sequencing of the 16S rRNA gene identified 3 prokaryotic groups comprised of predominately halophilic members that were present at the sites. The abundance of these groups was significantly higher at the continuously impacted site (adjusted P-values = 8.21 x 10⁻¹⁰ and 2.96 x 10⁻⁹, respectively; 2-sided Fisher’s exact test with Holm-Bonferroni correction). Note that Nanohaloarchaea were not found at the continuously impacted site.

Fig 6. Relative abundance of Cyanobacteria in soil samples collected in 2014 and 2015.

Based on amplicon sequencing of the 16S rRNA gene Cyanobacteria abundance was significantly higher at the seasonally and continuously impacted sites (adjusted P-values = 2.32 x 10⁻¹⁵ and 1.45 x 10⁻¹⁶, respectively; 2-sided Fisher’s exact test with Holm-Bonferroni correction).