Given its history of nuclear material processing, the Savannah River Site (SRS) was used to evaluate whether biofilms growing in water-cooling towers (WCTs) are effective passive collectors for environmental radionuclide surveillance. Uranium and plutonium analyses suggest WCT-sourced biofilms are efficient, indigenous, constantly running samplers that can be used for environmental monitoring, as their isotopic compositions are distinct from natural or atmospheric fallout and representative of SRS historical activities. The ubiquity of WCTs worldwide and demonstrated ability to detect nuclear material processing and constrain specific activities based on biofilm actinide isotopic compositions make WCT biofilms a promising means to improve monitoring.

Many microbial genes involved in degrading recalcitrant environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs) have been identified and characterized. However, all molecular mechanisms required for PAH utilization have not yet been elucidated. In this work, we demonstrate the proposed involvement of lasso peptides in the utilization of the PAH phenanthrene in Sphingomonas BPH. Transpositional mutagenesis of Sphingomonas BPH with the miniTn5 transposon yielded 3 phenanthrene utilization deficient mutants, #257, #1778, and #1782. In mutant #1782, Tn5 had inserted into the large subunit of the naph/bph dioxygenase gene. In mutant #1778, Tn5 had inserted into the B2 protease gene of a lasso peptide cluster. This finding is the first report on the role of lasso peptides in PAH utilization. Our studies also demonstrate that interruption of the lasso peptide cluster resulted in a significant increase in the amount of biosurfactant produced in the presence of glucose when compared to the wild-type strain. Collectively, these results suggest that the mechanisms Sphingomonas BPH utilizes to degrade phenanthrene are far more complex than previously understood and that the #1778 mutant may be a good candidate for bioremediation when glucose is applied as an amendment due to its higher biosurfactant production.

Biofouling and microbiologically influenced corrosion are major concerns to the long-term integrity of nuclear waste storage basins. The results of long-term microbial monitoring of nuclear storage at the Savannah River Site L-Basin nuclear material storage facility were examined to determine corrosion potential and biofouling. The densities of specific microbial species that can impact microbial corrosion and biofouling, including sulfate-reducing bacteria (SRB) and acid-producing (e.g., iron-oxidizing) bacteria (APB), were quantified by colony forming units (CFU). Several trends in microbial activity were observed during this time period. Overall microbial densities decreased slightly between 2000 and 2012. Metabolic diversity of microorganisms as measured by BIOLOG™ also decreased dramatically during this time. Aerobic microbial densities decreased from 2001 to 2010. Aerobes were most prevalent at 3.23 average CFU/mL and APB densities were lower at 0.80 average CFU/mL. From 2017 to present, iron-reducing bacteria were most prevalent at 3.41 average CFU/mL and APB were least prevalent at 0.44 average CFU/mL. All bacterial densities decreased except that of low-nutrient bacteria, which averaged 2.90 CFU/mL, and SRB averaged 0.19 CFU/mL. No distinct correlations between microbial concentrations, temperature, and either water chemistry parameters (pH and conductivity) or radiological conditions (19–30 disintegrations per minute (DPM) beta-gamma/mL; the equivalent to 0.32–0.50 Bq per mL) were determined. The occurrence of microbial biofilm formation on storage cannisters created biofouling issues in recent years. Although cleaning the biofouled surfaces provided temporary relief, the biofouling returned. Based on these results and physical observations of coupons, there has been minimal evidence of corrosion to date.

Phytoremediation, using plants for soil, sediment, or water contaminant clean-up, is an established technology dependent on plant health. Tritium (3H), a radioactive isotope of hydrogen that is generally found in the environment as tritiated water (HTO), is a low-level beta emitter with a half-life of 12.32 years. Chlorophyll fluorescence (CF) for monitoring risk assessment of tritium to plant health was conducted at the Tritium Irri-gation Facility (TIF) located on the US Department of Energy's Savannah River Site (SRS) near Aiken, SC. Two fluorometers were evaluated in conjunction with phytoremediation at the 25 -acre TIF where tritiated groundwater is being spray-irrigated on a mixed coniferous/deciduous forested watershed as a means of reducing tritium release to a nearby stream that serves as a tributary to the Savannah River. Tritium activity in irrigated water averaged 104 + 42 pCi mL-1 during the 2003 project. Fluorescence parameters measured by the two fluorometers were well correlated with each other (p < 0.0001). Tritium in water respired from oak leaves ranged up to 1845.13 pCi ml-1 and 2138.22 pCi ml-1 in pine needles. Trees in both the test and control sites were approximately 15 years old. Here we demonstrated that fluorescence parameters provide an effective way to estimate the impact of HTO on plant health in a noninvasive, extremely rapid, and cost-effective manner. In the current study applying fluorometry, plants within the TIF phytoremediation site exposed to the site tritiated water were not significantly impacted by the tritium phytoremediation based on CF parameters as compared to the control, a nascent non-irrigated site.

Oily sludge is a residue from the petroleum industry composed of a mixture of sand, water, metals, and high content of hydrocarbons (HCs). The heavy oily sludge used in this study originated from Colombian crude oil with high density and low American Petroleum Institute (API) gravity. The residual waste from heavy oil processing was subject to thermal and centrifugal extraction, resulting in heavy oily sludge with very high density and viscosity. Biodegradation of the total petroleum hydrocarbons (TPH) was tested in microcosms using several bioremediation approaches, including: biostimulation with bulking agents and nutrients, the surfactant Tween 80, and bioaugmentation. Select HC degrading bacteria were isolated based on their ability to grow and produce clear zones on different HCs. Degradation of TPH in the microcosms was monitored gravimetrically and with gas chromatography (GC). The TPH removal in all treatments ranged between 2 and 67%, regardless of the addition of microbial consortiums, amendments, or surfactants within the tested parameters. The results of this study demonstrated that bioremediation of heavy oily sludge presents greater challenges to achieve regulatory requirements. Additional physicochemical treatments analysis to remediate this recalcitrant material may be required to achieve a desirable degradation rate.

Infectious diseases and nosocomial infections may play a significant role in healthcare issues associated with biomedical materials and devices. Many current polymer materials employed are inadequate for resisting microbial growth. The increase in microbial antibiotic resistance is also a factor in problematic biomedical implants. In this work, the difficulty in diagnosing biomedical device-related infections is reviewed and how this leads to an increase in microbial antibiotic resistance. A conceptualization of device-related infection pathogenesis and current and future treatments is made. Within this conceptualization, we focus specifically on biofilm formation and the role of host immune and antimicrobial therapies. Using this framework, we describe how current and developing preventative strategies target infectious disease. In light of the significant increase in antimicrobial resistance, we also emphasize the need for parallel development of improved treatment strategies. We also review potential production methods for manufacturing specific nanostructured materials with antimicrobial functionality for implantable devices. Specific examples of both preventative and novel treatments and how they align with the improved care with biomedical devices are described.

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.

Glass, a near infinitely recyclable material, can be upcycled to create new products such as foamed glass ceramics, which are essentially a synthetic pumice-like material. This material has been demonstrated to sustain preserved biofilms which have application in various fields based on the deployability of the product and the preserved microbes. Foamed glass ceramics have increased surface area compared to typical soda-lime glass cullet. This material has been explored for variety of applications including the growth, storage and transport of biofilms and microbial colonies which can be preserved and deployed later. Here, we demonstrate the ability for microbial cultures including BioTiger™, Escherichia coli K-12, Bacillus thuringiensis , and two environmental eukaryotic cells to colonize the upcycled glass products, undergo preservation, and regrow after 84 days of storage. The growth of preserved samples is correlated to the time spent incubating prior to preservation. These results demonstrate the applicability of this novel glass-biofilm combination in which various preserved microorganisms are able to be rapidly grown after storage on an upcycled glass product.

Cupriavidus basilensis SRS was isolated from stream sediments from the Savannah River Site in South Carolina. Here, we report the draft genome sequence and annotation of Cupriavidus basilensis SRS. The genome contains 8,918,236 bp and 7,916 predicted protein-coding genes, with a total G+C content of 65.2%.

The SRNL L-Basin corrosion surveillance and microbial monitoring programs provide early detection and characterization of corrosion attack to the fuel and storage system materials resulting from prolonged exposure to the L-Basin water environment and of changes to and impact of the diverse microbial population, respectively. The early detection of corrosion allows for adjustment of the water quality, engineering management, and fuel storage configurations to mitigate excessive corrosion attack. While microbial influenced corrosion in L-Basin has not been detected, tracking and understanding the effect of microbial populations on the stored fuel and basin water will aid in identifying remedial measures to mitigate any detrimental impact. This report reviews the microbial monitoring activities since initial characterizations in the mid-1990s.

At the U.S. Department of Energy’s Savannah River Site (SRS) in Aiken, SC, cooling tower water is routinely monitored for Legionella pneumophila concentrations using a direct fluorescent antibody (DFA) technique. Historically, 25–30 operating SRS cooling towers have varying concentrations of Legionella in all seasons of the year, with patterns that are unpredictable. Legionellosis, or Legionnaires’ disease (LD), is a pneumonia caused by Legionella bacteria that thrive both in man-made water distribution systems and natural surface waters including lakes, streams, and wet soil. Legionnaires’ disease is typically contracted by inhaling L. pneumophila , most often in aerosolized mists that contain the bacteria. At the SRS, L. pneumophila is typically found in cooling towers ranging from non-detectable up to 10 8 cells/L in cooling tower water systems. Extreme weather conditions contributed to elevations in L. pneumophila to 10 7 –10 8 cells/L in SRS cooling tower water systems in July–August 2017. L. pneumophila concentrations in Cooling Tower 785-A/2A located in SRS A-Area, stayed in the 10 8 cells/L range despite biocide addition. During this time, other SRS cooling towers did not demonstrate this L. pneumophila increase. No significant difference was observed in the mean L. pneumophila mean concentrations for the towers ( p < 0.05). There was a significant variance observed in the 285-2A/A Tower L. pneumophila results ( p < 0.05). Looking to see if we could find “effects” led to model development by analyzing 13 months of water chemistry and microbial data for the main factors influencing the L. pneumophila concentrations in five cooling towers for this year. It indicated chlorine and dissolved oxygen had a significant impact ( p < 0.0002) on cooling tower 785A/2A. Thus, while the variation in the log count data for the A-area tower is statistically greater than that of the other four towers, the average of the log count data for the A-Area tower was in line with that of the other towers. It was also observed that the location of 785A/2A and basin resulted in more debris entering the system during storm events. Our results suggest that future analyses should evaluate the impact of environmental conditions and cooling tower design on L. pneumophila water concentrations and human health.

Polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) are toxic contaminants of environmental concern found in process water and mature fine tailings, or tailings, from the oil sands industry. BioTiger™, a patented microbial consortium of twelve natural environmental isolates, was found to cometabolically biodegrade the NA hexanoic acid and the PAH phenanthrene in the presence of tailings. Hexanoamide was found to be produced and consumed during cometabolism of hexanoic acid. Mechanistic analysis demonstrated three of the BioTiger™ strains generated biosurfactants with the bacterial adhesion to hydrocarbons assay, seven with the methylene blue active substances assay, and nine with a hemolysis assay. Serial transfers of the BioTiger™ consortium demonstrated the stability of hexanoic acid degradation over several generations. The results demonstrate that BioTiger™ cometabolically biodegrades combinations of phenanthrene and hexanoic acid in tailings. This work reveals the potential for in situ bioremediation of tailings with this natural microbial consortium.

Three potential causes of the strip effluent (SE) coalescer (SEC) failure were investigated: inorganic fouling, organic fouling, and biological fouling. From the analysis of the SEC and leachate samples from the SEC, there is no evidence that the fouling is caused by inorganic precipitates, solids, or salts. The analysis of the SEC shows the presence of organic material such as sec-butylphenol and Modifier (1-(2,2,3,3-Tetrafluoropropoxy)-3-(4-secbutylphenoxy)-2- propanol). The source of the sec-butylphenol would be from the decomposition of the Modifier in the solvent. The analysis of the SEC shows the presence of amide bonds that are likely from bacteria and other biological material. In addition, samples from the Strip Feed Tank (StFT) and the boric acid delivery tanker showed the presence of bacteria and other biological material. The most likely cause of the SEC fouling is biofouling by bacteria and other organisms that form a biofilm on the SEC fibers. The biofilm would sorb organic material in the feed to the SEC, and not readily release this material. The bacteria would use this trapped organic material to grow the biofilm, which would decrease the porosity of the SEC. The biofilm would thereby impede SEC operation through biofouling.

Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soil resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. These results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications.

BioTiger™ (BT) is a consortium of 12 bacteria designed for petroleum waste biodegradation. BT is currently being studied and could be considered for bioremediation of the Athabasca oil sands refineries in Canada and elsewhere. The run-off ponds from the petroleum extraction processes, called tailings ponds, are a mixture of polycyclic aromatic hydrocarbons, naphthenic acids, hydrocarbons, toxic chemicals like heavy metals, water, and sand. Due to environmental regulations the oil industry would like to separate and degrade the hazardous chemical species from the tailings ponds while recycling the water. It has been shown that BT at 30 C° is able to completely degrade 10 mM hexanoic acid (HA) co-metabolically with 0.2% yeast extract (w/v) in 48 hours when starting at 0.4 OD 600nm. After establishing this stable degradation capability, variations were tested to explore the wider parameters of BT activity in temperature, pH, intermediate degradation, co-metabolic dependence, and transfer stability. Due to the vast differences in temperature at various points in the refineries, a wide range of temperatures were assessed. The results indicate that BT retains the ability to degrade HA, a model surrogate for tailings pond contaminants, at temperatures ranging from 15°C to 35°C. Hexanamide (HAM) was shown to be an intermediate generated during the degradation of HA in an earlier work and HAM is completely degraded after 48 hours, indicating that HAM is not the final product of HA degradation. Various replacements for yeast extract were attempted. Glucose, a carbon source; casein amino acids, a protein source; additional ammonia, mimicking known media; and additional phosphate with Wolffe’s vitamins and minerals all showed no significant degradation of HA compared to control. Decreasing the yeast extract concentration (0.05%) demonstrated limited but significant degradation. Finally, serial inoculations of BT were performed to determine the stability of degradation over several generations. Overall, BT has shown to be moderately flexible for HA co-metabolic biodegradation.

Oil sands are a major source of oil, but their industrial processing generates tailings ponds that are an environmental hazard. The main concerns are mature fine tailings (MFT) composed of residual hydrocarbons, water, and fine clay. Tailings ponds include toxic contaminants such as heavy metals, and toxic organics including naphthenics. Naphthenic acids and polyaromatic hydrocarbons (PAHs) degrade very slowly and pose a long-term threat to surface and groundwater, as they can be transported in the MFT. Research into improved technologies that would enable densification and settling of the suspended particles is ongoing. In batch tests, BioTiger, a microbial consortium that can metabolize PAHs, demonstrated improved oil sands tailings settling from a Canadian tailings pond. Results also showed, depending on the timing of the measurements, lower suspended solids and turbidity. Elevated total organic carbon was observed in the first 48hours in the BioTiger-treated columns and then decreased in overlying water. Oil sands tailings mixed with BioTiger showed a two-fold reduction in suspended solids within 24hours as compared to abiotic controls. The tailings treated with BioTiger increased in microbial densities three orders of magnitude from 8.5 x 105 CFU/mL to 1.2 x 108 CFU/mL without any other carbon or energy source added, indicating metabolism of hydrocarbons and other available nutrients. Results demonstrated that bioaugmentation of BioTiger increased separation of organic carbon from particles in oil sands and enhanced settling with tailings with improved water quality. Journal style is for Abstract to be less than 200 words, and contain no citations to other sources; please edit as needed

The impact of phenolic-contaminated water on microbial community structure was assessed using Biolog microarrays techniques. The following Biolog plates were used: GEN III plates, new test panel for identification of both Gram-negative and Gram-positive bacteria, EcoPlates microarray for evaluation of functional diversity of microbial communities and phenotype microarrays (PMs) for characterization of the selected bacterial strains. Most of the isolated strains were identified as: Paenibacillus castaneae, Chryseobacterium indoltheticum, Pseudomonas fluorescens, Acinetobacter johnsonie, Mycobacterium flavescens, Ralstonia pickettii, Acinetobacter schindleri, Microbacterium maritypicum. The mean value of substrate richness (S) was high (30.67). Also, microbial activity in contaminated water evaluated by AWCD and AUC was high. The mean values of AWCD and AUC were 1.5 and 740.10, respectively. Instead, the mean values of Shannon-Weiner functional diversity index (H) and Shannon Evenness index were low, 1.46 and 0.978, respectively. The carbohydrates (Carb) and carboxylic and acetic acids (C & AA) were the most utilized carbon sources by the microbial communities of phenol-contaminated water. The proprieties of Pseudomonas fluorescens and Paenibacillus castaneae to oxidize 190 different substrates as sole carbon sources (PM1 and PM2), and the sensitivity to various toxic chemical compounds at 4 different concentrations (PM11, PM12 and PM13) were evaluated. Phenotypic microarrays used identified the differences between species. Both studied bacterial strains showed high ability to metabolize aminoacids as well as carbohydrates. Among carboxylic acids Pseudomonas fluorescens was able to use more of substrates as a sole of carbon in comparison with Paenibacterium castaneae.