Bioremediation of Hexanoic Acid and Phenanthrene in Oil Sands Tailings by the Microbial Consortium BioTiger™.

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.

Amplicon Sequencing Reveals Microbiological Signatures in Spent Nuclear Fuel Storage Basins.

Water quality is an important determinant for the structural integrity of alloy cladded fuels and assemblies during long-term wet storage. Detailed characterization of a water filled storage basin for spent nuclear reactor fuel was performed following the formation and proliferation of an amorphous white flocculent. White precipitant was sampled throughout the storage basin for chemical and spectroscopic characterization, and environmental DNA was extracted for 454 pyrosequencing of bacterial 16S rRNA gene diversity. Accordingly, spectroscopic analyses indicated the precipitant to be primarily amorphous to crystalline aluminum (oxy) hydroxides with minor associated elemental components including Fe, Si, Ti, and U. High levels of organic carbon were co-localized with the precipitant relative to bulk dissolved organic concentrations. Bacterial densities were highly variable between sampling locations and with depth within the water filled storage basin; cell numbers ranged from 4 × 103to 4 × 104 cells/mL. Bacterial diversity that was physically associated with the aluminum (oxy) hydroxide complexes exceeded an estimated 4,000 OTUs/amplicon library (3% cutoff) and the majority of sequences were aligned to the families Burkholderiaceae (23%), Nitrospiraceae (23%), Hyphomicrobiaceae (17%), and Comamonadaceae (6%). We surmise that episodic changes in the physical and chemical properties of the basin contribute to the polymerization of aluminum (oxy) hydroxides, which in turn can chemisorb nutrients, carbon ligands and bacterial cells from the surrounding bulk aqueous phase. As such, these precipitants should establish favorable microhabitats for bacterial colonization and growth. Comparative analyses of 16S rRNA gene amplicon libraries across a selection of natural and engineered aquatic ecosystems were performed and microbial community and taxonomic signatures unique to the spent nuclear fuel (SNF) storage basin environment were revealed. These insights could spur the development of tractable bio-indicators that are specific of and diagnostic for water quality at discrete locations and finer scales of resolution, marking an important contribution for improved water quality and management of SNF storage facilities.

Environmental impact of ongoing sources of metal contamination on remediated sediments.

A challenge to all remedial approaches for contaminated sediments is the continued influx of contaminants from uncontrolled sources following remediation. We investigated the effects of ongoing contamination in mesocosms employing sediments remediated by different types of active and passive caps and in-situ treatment. Our hypothesis was that the sequestering agents used in active caps and in situ treatment will bind elements (arsenic, chromium, cadmium, cobalt, copper, nickel, lead, selenium, and zinc) from ongoing sources thereby reducing their bioavailability and protecting underlying remediated sediments from recontamination. Most element concentrations in surface water remained significantly lower in mesocosms with apatite and mixed amendment caps than in mesocosms with passive caps (sand), uncapped sediment, and spike solution throughout the 2520h experiment. Element concentrations were significantly higher in Lumbriculus variegatus from untreated sediment than in Lumbriculus from most active caps. Pearson correlations between element concentrations in Lumbriculus and metal concentrations in the top 2.5cm of sediment or cap measured by diffusive gradient in thin films (DGT) sediment probes were generally strong (as high as 0.98) and significant (p<0.05) for almost all tested elements. Metal concentrations in both Lumbriculus and sediment/cap were lowest in apatite, mixed amendment, and activated carbon treatments. These findings show that some active caps can protect remediated sediments by reducing the bioavailable pool of metals/metalloids in ongoing sources of contamination.

Discovery of Bioactive Metabolites in Biofuel Microalgae That Offer Protection against Predatory Bacteria.

Microalgae could become an important resource for addressing increasing global demand for food, energy, and commodities while helping to reduce atmospheric greenhouse gasses. Even though Chlorophytes are generally regarded safe for human consumption, there is still much we do not understand about the metabolic and biochemical potential of microscopic algae. The aim of this study was to evaluate biofuel candidate strains of Chlorella and Scenedesmus for the potential to produce bioactive metabolites when grown under nutrient depletion regimes intended to stimulate production of triacylglycerides. Strain specific combinations of macro- and micro-nutrient restricted growth media did stimulate neutral lipid accumulation by microalgal cultures. However, cultures that were restricted for iron consistently and reliably tested positive for cytotoxicity by in vivo bioassays. The addition of iron back to these cultures resulted in the disappearance of the bioactive components by LC/MS fingerprinting and loss of cytotoxicity by in vivo bioassay. Incomplete NMR characterization of the most abundant cytotoxic fractions suggested that small molecular weight peptides and glycosides could be responsible for Chlorella cytotoxicity. Experiments were conducted to determine if the bioactive metabolites induced by Fe-limitation in Chlorella sp. cultures would elicit protection against Vampirovibrio chlorellavorus, an obligate predator of Chlorella. Introduction of V. chlorellavorus resulted in a 72% decrease in algal biomass in the experimental controls after 7 days. Conversely, only slight losses of algal biomass were measured for the iron limited Chlorella cultures (0-9%). This study demonstrates a causal linkage between iron bioavailability and bioactive metabolite production in strains of Chlorella and Scenedesmus. Further study of this phenomenon could contribute to the development of new strategies to extend algal production cycles in open, outdoor systems while ensuring the protection of biomass from predatory losses.

Gamma radiation interacts with melanin to alter its oxidation-reduction potential and results in electric current production.

The presence of melanin pigments in organisms is implicated in radioprotection and in some cases, enhanced growth in the presence of high levels of ionizing radiation. An understanding of this phenomenon will be useful in the design of radioprotective materials. However, the protective mechanism of microbial melanin in ionizing radiation fields has not yet been elucidated. Here we demonstrate through the electrochemical techniques of chronoamperometry, chronopotentiometry and cyclic voltammetry that microbial melanin is continuously oxidized in the presence of gamma radiation. Our findings establish that ionizing radiation interacts with melanin to alter its oxidation-reduction potential. Sustained oxidation resulted in electric current production and was most pronounced in the presence of a reductant, which extended the redox cycling capacity of melanin. This work is the first to establish that gamma radiation alters the oxidation-reduction behavior of melanin, resulting in electric current production. The significance of the work is that it provides the first step in understanding the initial interactions between melanin and ionizing radiation taking place and offers some insight for production of biomimetic radioprotective materials.

Proteomic and physiological responses of Kineococcus radiotolerans to copper.

Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II) concentrations (0-1.5 mM) in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II) concentration in the growth medium (R(2)=0.7). Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration.

Stimulatory and inhibitory effects of organohalides on the dehalogenating activities of PCB-dechlorinating bacterium o-17.

Bacterium o-17, a microorganism capable of the ortho dechlorination of 2,3,5,6-polychlorinated biphenyl (PCB), is a member of a sediment-free, nonmethanogenic mixed culture. The culture was examined for the ability to dechlorinate 26 PCB congeners, 12 chlorobenzenes (CBZs), and 6 chlorinated ethenes (CEs). Eight of the PCBs and 4 of the CBZs were dechlorinated including single-flanked ortho PCB chlorines, but double-flanked chlorines of PCBs and CBZs were preferentially dechlorinated. The dechlorination of three of the PCBs (2,3,4,5,6-, 2,3,4,6-, and 2,3,5,6-PCB), three of the CBZs (hexa-, penta-, and 1,2,3-CBZ), and PCE could be sustained for three or more sequential transfers of the bacterial community. Two PCBs (2,3,4- and 2,3,5-PCB), two CBZs (1,2,3,5- and 1,2,4,5-CBZ), and trichloroethene were dechlorinated only when a more extensively chlorinated parent compound was present. Aroclor 1260 and 2,4,6-PCB, not dechlorinated by the culture, inhibited the dechlorination of 2,3,5,6-PCB. Within the culture only bacterium o-17 was linked to dechlorination by PCR-DGGE analysis, confirming that this dehalogenating species was the catalyst for the dechlorination of the compounds tested. The microorganism is capable of dechlorinating several different congeners of PCBs, CBZs, and CEs, and it remains a rare example of an ortho-PCB dechlorinator. However, its limited ability to dechlorinate more extensively chlorinated congeners and Aroclor plus the inhibitory effects of some PCB congeners upon the bacterium is consistent with the observed infrequency of this reaction in the environment. An assessment of bioremediation potential of this microorganism in situ will require a greater understanding of the synergistic, cometabolic and competitive interactions of PCB dechlorinating microbial communities.

Reductive dechlorination of tetrachloroethene to trans-dichloroethene and cis-dichloroethene by PCB-dechlorinating bacterium DF-1.

Polychlorinated biphenyls (PCBs) and chlorinated ethenes (CEs) are known to pollute sediment, soil, and groundwater. The anaerobic dechlorination of these compounds is an integral part of their biodegradation in polluted environments. We report for the first time the dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) by bacterium DF-1. This PCB and chlorobenzene dechlorinating bacterium dechlorinated PCE to TCE, which was then converted into trans-1,2-dichloroethene (trans-DCE) and cis-1,2-dichloroethene (cis-DCE). The ratio of trans-DCE to cis-DCE produced by the culture had a range of 1.2-1.7. Bacterium DF-1 has been enriched in co-culture with a desulfovibrio-like microorganism. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the 16S rRNA genes of the co-culture demonstrated that DF-1 was enriched during the dechlorination of PCE, PCB, and chlorobenzene. DF-1 was not detected in the absence of PCE dechlorination and the desulfovibrio-like organism, isolated in pure culture, did not dechlorinate PCE. This is the first identification of a microorganism capable of producing high amounts of trans-DCE from PCE and indicates that microorganisms such as DF-1 are a possible biological source of trans-DCE in the environment.

Short-term administration of (-)-epigallocatechin gallate reduces hepatic steatosis and protects against warm hepatic ischemia/reperfusion injury in steatotic mice.

Hepatic steatosis increases the extent of cellular injury incurred during ischemia/reperfusion (I/R) injury. (-)-Epigallocatechin gallate (EGCG), the major flavonoid component of green tea (camellia sinensis) is a potent antioxidant that inhibits fatty acid synthase (FAS) in vitro. We investigated the effects of EGCG on hepatic steatosis and markers of cellular damage at baseline and after I/R injury in ob/ob mice. Animals were pretreated with 85 mg/kg EGCG via intraperitoneal (ip) injection for 2 days or oral consumption in the drinking water for 5 days before 15 minutes of warm ischemia and 24 hours of reperfusion. After EGCG administration, total baseline hepatic fat content decreased from baseline. Palmitic acid and linoleic acid levels also were reduced substantially in all ECGC-treated animals before I/R. Alanine aminotransferase (ALT) levels decreased in all EGCG-treated animals compared with control animals after I/R. Histologic analysis demonstrated an average decrease of 65% necrosis after EGCG administration. EGCG administration also increased resting hepatic energy stores as determined by an increase in cellular adenosine triphosphate (ATP) with a concomitant decrease in uncoupling protein 2 (UCP2) before I/R. Finally, there was an increased level of glutathione (GSH) in the EGCG-treated mice compared with the vehicle-treated mice both at baseline and after I/R. In conclusion, taken together, this study demonstrates that treatment with ECGC by either oral or ip administration, significantly protects the liver after I/R, possibly by reducing hepatic fat content, increasing hepatic energy status, and functioning as an antioxidant.

Dechlorination of chlorobenzenes by a culture containing bacterium DF-1, a PCB dechlorinating microorganism.

Polychlorinated benzenes were reductively dechlorinated by an enrichment culture containing the polychlorinated biphenyl (PCB) dechlorinating bacterium DF-1. The culture dechlorinated hexachlorobenzene (hexa-CB) --> pentachlorobenzene (penta-CB) --> 1,2,3,5-tetrachlorobenzene (1,2,3,5-CB) --> 1,3,5-trichlorobenzene (1,3,5-CB) and did not dechlorinate other tetrachlorobenzenes or any trichlorobenzenes. This restricted series of reactions is the most predominant and frequently reported pathway for the dechlorination of hexa-CB and penta-CB by enrichment cultures inoculated with either freshwater or estuarine sediments. The culture did not dechlorinate hydroxylated and methoxylated polychlorinated benzenes or a hydroxylated PCB. Bacterium DF-1 was detected by PCR/DGGE analysis following dechlorination of penta-CB but was not detected when a chlorinated benzene (CB) was not dechlorinated; detection of other members in the communitywas unaffected by the presence or absence of CB dechlorination. This is the first report of a bacterium that reductively dechlorinates both PCBs and CBs and the first identification of an organism that can dechlorinate a CB with more than four chlorines.

Phase 1/2 placebo-controlled, double-blind, dose-escalating trial of myocardial vascular endothelial growth factor 2 gene transfer by catheter delivery in patients with chronic myocardial ischemia.

BACKGROUND: This phase 1/2 study investigated the safety of percutaneous catheter-based gene transfer of naked plasmid DNA encoding for vascular endothelial growth factor 2 (phVEGF2) to left ventricular (LV) myocardium in a prospective, randomized, double-blind, placebo-controlled, dose-escalating study of inoperable patients with class III or IV angina. METHODS AND RESULTS: A steerable deflectable 8F catheter with a 27-gauge needle at its distal tip was advanced percutaneously to the endocardial surface of the LV in 19 patients (age, 61+/-2 years) with chronic myocardial ischemia who were not candidates for conventional revascularization. Patients were randomized in a double-blind fashion to receive 6 injections (total volume, 6.0 mL) of placebo or phVEGF2 in doses of 200 microg (n=9), 800 microg (n=9), or 2000 microg (n=1) guided by LV electromechanical (NOGA) mapping with a gene-to-placebo ratio of 2:1. A total of 114 LV injections were delivered and caused no hemodynamic alterations, sustained ventricular arrhythmias, ECG evidence of infarction, or ventricular perforation. End-point analysis at 12 weeks disclosed a statistically significant improvement in Canadian Cardiovascular Society (CCS) angina class in phVEGF2-treated versus placebo-treated patients (-1.3 versus -0.1, P=0.04). Remaining efficacy end points--including change in exercise duration (91.8 versus 3.9 seconds), functional improvement by > or =2 CCS classes (9 of 12 versus 1 of 6), and Seattle Angina Questionnaire data--all showed strong trends favoring efficacy of phVEGF2 versus placebo treatment. CONCLUSIONS: This phase 1/2, double-blind, randomized trial provides preliminary data that support safety of phVEGF2 catheter-mediated myocardial gene transfer. The statistically significant reduction in anginal class and strong positive trends for remaining end points suggest that a larger phase 2/3 trial is warranted.