Investigating the Reactivity of Marine Bacteria with Copper and other Heavy Metals

Abstract

Marine bioinorganic chemistry is an emerging field that focuses on how inorganic elements impact biological functions within marine organisms. Exploration of heavy metals within marine environments is the connection that holds the world presented herein together. The work begins with an examination of Interspeed 640, a popular antifouling (AF) coating that utilizes cuprous oxide (Cu2O) as the primary biocide, to understand the rationale behind its declining efficacy. This effort was approached in two distinct ways: 1) by designing an assay to characterize the impact of environmental conditions on surface composition and copper (Cu) release to define the interface primary colonizing bacterial species interact with, and 2) by describing the bacterial communities involved in primary colonization to understand acquired Cu tolerance. First, a newly developed and highly tunable lab-scale assay was utilized to measure the impact of environmental conditions on surface composition and Cu release. This method was utilized to describe the behavior of Interspeed 640 in an inert environment (double-distilled, 18.2 MΩ water, (ddH2O)) and under conditions which may facilitate biofilm growth (tryptic soy broth, (TSB)). We utilized x-ray photoelectron spectroscopy (XPS) to characterize the coating surface and inductively coupled plasma optical emission spectroscopy (ICP-OES) to measure Cu release over a 14-day period. Under both immersion conditions, XPS analysis of the coating surface showed signs of hydrolysis. Coated substrates immersed in TSB demonstrated an increase in both nitrogen and copper as compared to those in water. Additionally, coated substrates immersed for 14 days in TSB released approximately 500% more Cu than those immersed in water. Next, polyvinyl chloride panels either left blank or coated with Interspeed 640 were deployed at three field sites to collect, isolate, and identify bacterial species involved in the early stages of biofouling. We have created a library of primary colonizing bacteria for all three field sites and, for one site, have completed a preliminary assessment of bacterial Cu tolerance capabilities using broth microdilution assays. These results indicate that Cu tolerance is not unique to species isolated from coated panels. Further investigations into the interplay between metal ions and marine bacteria continue in the following section where Marinobacter atlanticus is evaluated for potential applications in bioremediation. M. atlanticus can produce small amounts of electricity and this ability is likely dependent upon labile metal ion concentration and mineral cycling. We hypothesized that M. atlanticus may be a promising candidate for applications in heavy metal remediation or as a biosensor. We utilized broth microdilution assays to expose M. atlanticus to a concentration range of essential and non-essential metal ions. M. atlanticus demonstrated a profound ability to grow in the presence of metal concentrations that significantly exceed those found in native environments. In a metal detoxification assay, where the ability of M. atlanticus to remove metal ions from solution was assessed, we found that this bacterium is unable to significantly reduce metal concentrations. In one final shift of focus, we provide an examination of bioactive molecules isolated from evolutionarily resilient organisms via an investigation of the Clavanin family of antimicrobial peptides. Prior literature on these peptides indicates the possession of broad-spectrum antimicrobial activity. We focused on the antimicrobial of ClavA against fungal pathogens Candida albicans and Cryptococcus neoformans and the bacterium Escherichia coli strain BW25113; ClavC was tested against C. albiancs and E. coli. Overall, both peptides exhibited enhanced antimicrobial activity under acidic (pH 5.5) and, in most cases, ClavC was more potent than ClavA. When supplemental Cu or Zn were introduced, significant metal modulated activity increases were seen between ClavA and Cu at pH 5.5 against C. albicans and C. neoformans. Conversely, the combination of ClavC and Cu at pH 5.5 seemed to inhibit peptide activity.