Green roof technology: investigation of crumb rubber effects on microbial growth
University of Delaware
Green roofs incorporate plant communities in the roof structure. The use of these structures is a growing practice worldwide, particularly in densely populated areas. In an attempt to find new methods for recycling crumb rubber, incorporation of this material into the growth medium of green roofs has become an attractive option. Though this approach decreases waste in landfills, there are concerns about the leaching of zinc, other heavy metals and nutrients into the environment. The present study analyzed the impact of leachate from crumb rubber on the growth of Salmonella enterica subsp. enterica serovar Typhimurium. The study showed shown that microbes can colonize crumb rubber and that zinc-tolerant strains of Salmonella can be obtained after sub-culturing in increasing levels of zinc. Gene expression of Salmonella zinc efflux and influx pumps were measured using qPCR, and the potential of increased resistance to antimicrobial agents due to increased zinc tolerance was determined. Crumb rubber extracts were obtained by incubation in synthetic rainwater at 28○C. The liquid was filtered from the crumb rubber after 1, 24, and 48 hours and microbes were exposed to these extracts. Results of growth studies involving S. Typhimurium and the 24-hour extract showed that crumb rubber contains compounds that were inhibitory to the bacterium, but when diluted at least 10-fold the extract could serve as a source of nutrients, resulting in up to about 15-fold increased colony forming units compared to growth in the synthetic rainwater. Salmonella were able to attach to crumb rubber as shown by the crumb rubber biofilm assays. Since crumb rubber is known to contain as much as 1% of its weight in zinc, S. Typhimurium were exposed to increasing amounts of zinc and strains with reduced susceptibility (SRS) capable of growing in the presence of 20 mM zinc or more were isolated. qPCR was used to quantify the expression of the major zinc influx and efflux pumps in cells exposed to 0 mM and 20 mM zinc. The SRS strain exhibited a seven-fold increase in the gene expression of efflux pump gene zntA and a 2.5–fold decrease in the gene expression of the influx pump gene znuA compared to the parent when exposed to 20 mM added zinc. In biofilm formation studies, the SRS strain was less able to attach to an abiotic surface, but was more motile than the parental strain as observed by motility assays on semisolid agar plates. The SRS strain did not show an increase in resistance to oxytetracycline, tylosin tartrate, fumagillin, DTAC, or triclosan. These data suggest that simultaneous addition of zinc and antimicrobial to cultures in TSB did not lead to cross-resistance.