Bioremediation of copper in sediments from a constructed wetland ex situ with the novel bacterium Cupriavidus basilensis SRS

Abstract

The H-02 constructed wetland was designed to remove metals (primarily copper and zinc) to treat building process water and storm water runoff from multiple sources associated with the Tritium Facility at the DOE-Savannah River Site, Aiken, SC. The concentration of Cu and Zn in the sediments has increased over the lifetime of the wetland and is a concern. A bioremediation option was investigated at the laboratory scale utilizing a newly isolated bacterium of the copper metabolizing genus Cupriavidus isolated from Tim's Branch Creek, a second-order stream that eventually serves as a tributary to the Savannah River, contaminated with uranium and other metals including copper, nickel, and mercury. Cupriavidus basilensis SRS is a rod-shaped, gram-negative bacterium which has been shown to have predatory tendencies. The isolate displayed resistance to the antibiotics ofloxacin, tetracycline, ciprofloxacin, select fungi, as well as Cu²⁺ and Zn²⁺. Subsequent ribosomal sequencing demonstrated a 100% confidence for placement in the genus Cupriavidus and a 99.014% match to the C. basilensis type strain. When H-02 wetland samples were inoculated with Cupriavidus basilensis SRS samples showed significant (p < 0.05) decrease in Cu²⁺ concentrations and variability in Zn²⁺ concentrations. Over the 72-h incubation there were no significant changes in the inoculate densities (10⁶-10⁸ cells/ML) indicating Cupriavidus basilensis SRS resiliency in this environment. This research expands our understanding of the Cupriavidus genus and demonstrates the potential for Cupriavidus basilensis SRS to bioremediate sites impacted with heavy metals, most notably copper.

Figure 1

Images of relevant areas of the Savannah River Site. (A) GoogleMaps image of the H-02 wetland. Water exits the retention basin through a culvert where it flows to a splitter box that equally feeds into two separate treatment cells. These cells house the water in an artificial wetland environment. Image from Google Maps. (B) Map showing the Tim’s Branch Creek area where samples from which Cupriavidus basilensis SRS was originally isolated. Black arrows highlight likely sites of initial contamination. The box highlights H area and pink box highlights the constructed wetland area, shown in detail in (A). Map was created using ArcGIS Destop Release 10 (https://www.arcgis.com/index.html) software by Esri using the USGS National Boundaries Database.

Figure 2

Plates of Bacillus subtilis (A) and Exophiala-moniliae (B) were grown on plates of R2A agar, had filters (25 mm) overgrown with Cupriavidus basilensis SRS placed on them and were incubated. Clear zones developed around Cupriavidus basilensis SRS containing filter after incubation, and no zone developed around the blank control. The right white filter (B) contained no Cupriavidus basilensis SRS.

Figure 3

Phylogenetic tree of 16S rRNA relating Cupriavidus basilensis SRS to other closely aligned Cupriavidus spp, resulting from a near-full length nt sequence indicating a near 100% confidence that the Cupriavidus basilensis SRS belongs to the Cupriavidus genus.

Figure 4

Growth of Cupriavidus basilensis SRS with Cu and Co in liquid medium with Bioscreen for 23 h and 46 h for Cu and Co, respectively. Five different concentrations were monitored, 0, 0.1, 0.35, 0.75, and 1.5 mM. Growth was monitored via optical density at 600 nm. Average of 5 runs, error bars are smaller than symbols shown. The general trend shows a decease as metal concentrations increase; however, Co is significantly more inhibitory than Cu.

Figure 5

Microbial density of Cupriavidus basilensis SRS in various wetland test samples after inoculation. Cupriavidus acts as a control sample for comparison, and denotes the initial microbial concentration added to the wetland samples. The data is shown as mean values with the error bars representing the 95 percent confidence intervals around the average value. Samples measured in triplicate.

Figure 6

Changes in water chemistry after incubation with Cupriavidus basilensis SRS after three hours, depicted with boxplots. The pH of the water, sediment (sed) and organic layer (OL) sample treatments are shown in (A); Cu ion concentrations in the water, sediment (sed) and organic layer (OL) in (B); and Zn ion concentrations in the water, sediment (sed) and organic layer (OL) in (C). The data is shown as mean values with the error bars representing the 95 percent confidence intervals around the average value; samples measured in triplicate.

Figure 7

Model for microbially induced changes from pH 6 to 9 over three days for Cu⁺². Model reflects standard temperature and pressure.