Date of Award

5-2005

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

David C. White

Committee Members

Susan Pfiffner, Pam Small, Mark Radosevich

Abstract

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.

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