In Situ Biostimulation of Uranium Reducing Microorganisms at the Old Rifle UMTRA Site

Bioremediation is a promising strategy for cleaning up heavy metal and radionuclide contamination. Nutrient or electron donor amendment is an increasingly accepted practice used to stimulate the growth of microorganisms capable of immobilizing dissolved uranium in situ, but there is scant understanding of the systematic effects of nutrient addition on indigenous microbial populations or the progress of the bioremediation. Successful implementation of metal and radionuclide bioremediation in heterogeneous environments requires an understanding of the complex microbial and geochemical interactions that influence the redox speciation and mobility of toxic metals. The major challenge in microbial ecology and biogeochemistry is to connect observed biogeochemical processes to the microbial populations responsible for carrying them out. This thesis thus investigated the effects of electron donor addition to indigenous microbial populations actively involved in uranium bioremediation. Stable Isotope Probing (SIP) technique for environmental application was developed and established. A microcosm study was designed in parallel to a field biostimulation test at the Old Rifle, UMTRA site. In the microcosm study that simulated Rifle in situ biostimulation of uranium reducing organisms, the microbial community dynamics were analyzed quantitatively and qualitatively using Phospholipid Fatty-acid Analysis (PLFA) and Denaturing Gradient Gel Electrophoresis (DGGE) analysis combined with SIP, which was modified to accommodate low biomass environmental samples. The microcosms consisted of sediment and groundwater from the Rifle, Colorado UMTRA site and activated carbon bead microbial traps (Biosep beads). 13C labeled acetate amended and non-amended microcosms were compared. Lipid analyses showed a significant biomass increase with acetate amendment, specifically monounsaturated PLFA. The data also demonstrated a community shift in acetate-amended microcosms, mirroring the observation of DGGE analysis. The bacterial community in non-amended microcosms showed notable differences from those amended with acetate. β-proteobacterial sequences dominated the non-amended community. Furthermore, 13C DNA analysis indicated that acetate treatment encouraged the growth of Gram-negative microorganisms such as Pseudomonas, Geobacter, and sulfate reducing bacteria (SRB). PLFA extracted from beads and sediment also showed uptake of the 13C-acetate, mainly in 14:0, 16:1ω7c, 16:1 ω5c, 16:0, cy17:0 and 18:1 ω7c, supporting the DNA results. Geobacter and SRB sequences were not detected until day 20, while Pseudomonas sequences were prevalent by day 5 and continued to be one of the dominant sequences retrieved. The dominance of Geobacter was much more pronounced in bead samples than in sediments. GC-IRMS analysis also demonstrated the 13C enrichment in fatty acids of i15:0, i17:0, 17:0 and 18:0 extracted from beads samples, which might be indicators of Geobacter, SRB or Gram+.The SIP technique enabled an evaluation of the taxonomic and metabolic diversity of key groups of microbes actively involved in biostimulation. The microbial monitoring in microcosms can elucidate the bacterial populations responsible for uranium reduction and may indicate that SIP using 13C-acetate added to microbial traps can provide important data on ecosystem function in the field. At Rifle, Colorado, a field-scale acetate amendment experiment was performed to stimulate in situ microbial reduction of U (VI) in groundwater. Geochemical measurements indicated reduction of iron, uranium, and sulfate, which were stimulated by acetate injection. The PCR-DGGE analysis of 16S rRNA genes revealed 15 major lineages in the bacterial domain, enriched during biostimulation. A temporal (T1, T2, and T3, T4) and spatial (B-02, M-03, M-08 and M-13) distribution of the bacterial community structure was demonstrated. The background well showed no significant community shift throughout the experiment, and was dominated by β-proteobacteria with no Geobacteraceae detected. The down-gradient monitoring wells, on the other hand, shared similar community structure with background wells before acetate injection, but exhibited significant enrichment of Geobacter and Desulfuromonas sequences during the injection. This enrichment disappeared after the injection of electron donor ceased and was replaced by sequences originating from organisms of Sulfuricurvum, SRB within δ- proteobacteria, and gram positives closely related to either Desulfotomaculum, or Clostridium. Consistent with the reducing activities determined by geochemical analysis, well M-13, furthest away from the injection gallery, appeared to be less similar with other down gradient monitoring wells in community composition. PLFA analysis indicated a similar trend in community shift and displayed an increase in monounsaturated PLFAs (indicative of Gram-negative bacteria), as well as terminally branched saturated LFAs (indicative of anaerobic sulfate reducing bacteria) relative to the background well. The data presented demonstrates the effects of biostimulation and bioreduction by addition of acetate, and lead to the conclusion that Geobacteraceae was initially responsible for enzymatic uranium reduction, but had no role afterwards. The sulfate reducers played an important role in reducing uranium and also maintaining the low concentration of uranium at the Old Rifle site. Nitrate reducers such as Sulfuricurvum bacteria may also had important part in maintaining the stability of reduced uranium by removing the subsurface nitrate. To evaluate the microorganisms responsible for uranium microbial reduction during the field experiment, Biosep beads baited with 13C labeled acetate were deployed into well boreholes and sampled when groundwater chemistry indicated metal and or sulfate reduction. Incorporation of the 13C into cellular DNA and PLFA biomarkers was examined. The 13C labeled DNA fraction demonstrated an enrichment of Geobacteraceae sequences in down gradient monitoring wells. Geobacter sequences dominated in wells approximately 3.7 meters from the injection gallery. Further down gradient, sequences belonging to Desulfuromonas increased. Pseudomonas sequence was also found to be stimulated. PLFA profiling of activated carbon beads suspended in the monitoring wells showed the incorporation of 13C into the bacterial cellular lipids, particularly the 16:1ω7c. A comparison among groundwater, sediment, and biotraps was performed, which indicated that the biotraps captivated the key populations of both groundwater and sediment but are probably more representative of the groundwater. The research presented in this thesis demonstrates the importance of metal reduction and sulfate reduction in stimulated uranium immobilization, also expands our knowledge of quantitatively important iron and sulfate reducing bacteria in uranium contaminated subsurface environment. The direct introduction of 13C labeled substrates into ecosystems, coupled with DNA and PLFA analyses, which combine detailed taxonomic description with a quantitative measure of metabolic diversity allowed detection and definition of the metabolically active subset of the microbial community. This study provides an effective technique and experimental model to identify particular microbial populations involved in a desired process. Future research may explore whether the sediment or groundwater has even greater diversity of uranium reducing populations than those we have identified. More focused study on sulfate reducers are needed to shed light on their involvement in uranium reduction, either biotic or abiotic, or both

Multiple Events of Allopolyploidy in the Evolution of the Racemose Lineages in Prunus (Rosaceae) Based on Integrated Evidence from Nuclear and Plastid Data.

Prunus is an economically important genus well-known for cherries, plums, almonds, and peaches. The genus can be divided into three major groups based on inflorescence structure and ploidy levels: (1) the diploid solitary-flower group (subg. Prunus, Amygdalus and Emplectocladus); (2) the diploid corymbose group (subg. Cerasus); and (3) the polyploid racemose group (subg. Padus, subg. Laurocerasus, and the Maddenia group). The plastid phylogeny suggests three major clades within Prunus: Prunus-Amygdalus-Emplectocladus, Cerasus, and Laurocerasus-Padus-Maddenia, while nuclear ITS trees resolve Laurocerasus-Padus-Maddenia as a paraphyletic group. In this study, we employed sequences of the nuclear loci At103, ITS and s6pdh to explore the origins and evolution of the racemose group. Two copies of the At103 gene were identified in Prunus. One copy is found in Prunus species with solitary and corymbose inflorescences as well as those with racemose inflorescences, while the second copy (II) is present only in taxa with racemose inflorescences. The copy I sequences suggest that all racemose species form a paraphyletic group composed of four clades, each of which is definable by morphology and geography. The tree from the combined At103 and ITS sequences and the tree based on the single gene s6pdh had similar general topologies to the tree based on the copy I sequences of At103, with the combined At103-ITS tree showing stronger support in most clades. The nuclear At103, ITS and s6pdh data in conjunction with the plastid data are consistent with the hypothesis that multiple independent allopolyploidy events contributed to the origins of the racemose group. A widespread species or lineage may have served as the maternal parent for multiple hybridizations involving several paternal lineages. This hypothesis of the complex evolutionary history of the racemose group in Prunus reflects a major step forward in our understanding of diversification of the genus and has important implications for the interpretation of its phylogeny, evolution, and classification

Evaluation of bile salt hydrolase inhibitor efficacy for modulating host bile profile and physiology using a chicken model system

Gut microbial enzymes, bile salt hydrolases (BSHs) are the gateway enzymes for bile acid (BA) modification in the gut. This activity is a promising target for developing innovative non-antibiotic growth promoters to enhance animal production and health. Compelling evidence has shown that inhibition of BSH activity should enhance weight gain by altering the BA pool, host signalling and lipid metabolism. We recently identified a panel of promising BSH inhibitors. Here, we address the potential of them as alternative, effective, non-antibiotic feed additives, for commercial application, to promote animal growth using a chicken model. In this study, the in vivo efficacy of three BSH inhibitors (caffeic acid phenethylester, riboflavin, carnosic acid) were evaluated. 7-day old chicks (10 birds/group) were either untreated or they received one of the specific BSH inhibitors (25 mg/kg body weight) via oral gavage for 17 days. The chicks in treatment groups consistently displayed higher body weight gain than the untreated chicks. Metabolomic analysis demonstrated that BSH inhibitor treatment led to significant changes in both circulating and intestinal BA signatures in support of blunted intestinal BSH activity. Consistent with this finding, liver and intestinal tissue RNA-Seq analysis showed that carnosic acid treatment significantly altered expression of genes involved in lipid and bile acid metabolism. Taken together, this study validates microbial BSH activity inhibition as an alternative target and strategy to antibiotic treatment for animal growth promotion

Administration of a Decoction of Sucrose- and Polysaccharide-Rich Radix Astragali (Huang Qi) Ameliorated Insulin Resistance and Fatty Liver but Affected Beta-Cell Function in Type 2 Diabetic Rats

The current investigation attempted to confirm the beneficial actions of a chemically characterized Radix Astragali decoction (AM-W) against type 2 diabetic (T2D) Sprague-Dawley (SD) rats. Using a case/control design, after 2 months of treatment with AM-W (500 mg/kg, daily i.p.) in T2D rats therapeutic outcomes were compared. Sucrose and Astragalus polysaccharides (ASPs) were shown to exist in nearly equal proportions in AM-W. Body weight loss, an improvement in insulin sensitivity, and an attenuation of fatty liver after AM-W administration in T2D rats were evident. Surprisingly, blood sugar, beta-cell function, and glucose tolerance in T2D rats did not improve with AM-W treatment. Further investigation indicated the deleterious effects of the addition of sucrose (100 and 500 μg/mL) and APSs (500 μg/mL) on glucose-stimulated insulin secretion and viability, respectively. In conclusion, a proper administration dosage and a reduction in the sucrose content are keys to maximizing the merits of this herb

Bioequivalence Evaluation of Two Formulations of Celecoxib 200 mg Capsules in Healthy volunteers by using a validated LC/MS/MS method

The bioequivalence study to compare a new formulation of celecoxib to its reference formulation was designed as an open-label, randomized, single-dose, two-way crossover, comparative bioavailability study by using a validated LC/MS/MS method. In order to determine the plasma concentrations of celecoxib, a sensitive LC/MS/MS method was developed. The method was validated to possess adequate specificity, linearity, precision, accuracy and stability. The linearity of calibration curve was assessed between the concentration intervals (5–2000 ng/mL) with a correlation coefficient over 0.999. Regarding pharmacokinetic investigation, the mean celecoxib AUC0-t values from the test and reference drug formulations were 7360.44 ± 1714.14 h•ng/mL and 7267.48 ± 2077.68 h•ng/mL, respectively, and the corresponding AUC0-∞ values were 8197.45 ± 2040.31 h•ng/mL and 7905.54 ± 2286.12 h•ng/mL, respectively. The Cmax of the test and reference drugs was 705.30 ± 290.63 ng/mL and 703.86 ± 329.91 ng/mL, respectively, and the corresponding Tmax was 3.4 ± 1.6 h and 2.9 ± 1.4 h. Lastly, the T1/2 values of the test and reference drugs were 13.9 ± 7.9 h and 12.9 ± 7.7 h, respectively. The 90% confidence intervals for AUC0-t, AUC0-∞, and Cmax were 97.00-108.85, 98.01-112.09, and 93.20-116.13, respectively, satisfying the bioequivalence criteria of 80-125% range. In conclusion, these results demonstrated that the bioequivalence of two formulations of celecoxib was established successfully by utilizing present developed LC/MS/MS method