Microbial Incorporation of 13 C-Labeled Acetate at the Field Scale: Detection of Microbes Responsible for Reduction of U(VI)
A field-scale acetate amendment experiment was performed in a contaminated aquifer at Old Rifle, CO to stimulate in situ microbial reduction of U(VI) in groundwater. To evaluate the microorganisms responsible for microbial uranium reduction during the experiment, 13C-labeled acetate was introduced into well bores via bio-traps containing porous activated carbon beads (Bio-Sep). Incorporation of the 13C from labeled acetate into cellular DNA and phospholipid fatty acid (PLFA) biomarkers was analyzed in parallel with geochemical parameters. An enrichment of active sigma-proteobacteria was demonstrated in downgradient monitoring wells: Geobacter dominated in wells closer to the acetate injection gallery, while various sulfate reducers were prominent in different downgradient wells. These results were consistent with the geochemical evidence of Fe(III), U(VI), and SO(4)2- reduction. PLFA profiling of bio-traps suspended in the monitoring wells also showed the incorporation of 13C into bacterial cellular lipids. Community composition of downgradient monitoring wells based on quinone and PLFA profiling was in general agreement with the 13C-DNA result. The direct application of 13C label to biosystems, coupled with DNA and PLFA analysis,
Available from: Richard Mitchener
- "Since the beads are sterile and free of biomarkers when deployed, incorporation of 13 C into PLFA provides proof of in situ biodegradation of a target compound by indigenous organisms under aquifer conditions. Identification of the specific PLFA that becomes labeled can also potentially provide insight into the types of bacteria that are both metabolically active in an aquifer and involved in processing carbon from the labeled compound (Chang et al., 2005; White & Ringelberg, 1998). Bacteria containing labeled biomarkers may be primary degraders or organisms consuming labeled "
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ABSTRACT: The effect of phenol concentration on phenol biodegradation at an industrial site in the south of Wales, United Kingdom, was investigated using standard Bio-Sep® Bio-Traps® and Bio-Traps® coupled with stable isotope probing (SIP). Unlike many 13C-amendments used in SIP studies (such as hydrocarbons) that physically and reversibly adsorb to the activated carbon component of the Bio-Sep® beads, phenol is known to irreversibly chemisorb to activated carbon. Bio-Traps® were deployed for 32 days in nine site groundwater monitoring wells representing a wide range of phenol concentrations. Bio-Traps® amended with 13C-phenol were deployed together with non-amended Bio-Traps® in three wells.
Quantitative polymerase chain reaction (qPCR) analysis of Bio-Traps® post-deployment indicated an inhibitory effect of increasing phenol concentration on both total eubacteria and aerobic phenol-utilizing bacteria as represented by the concentration of phenol hydroxylase gene. Despite the chemisorption of phenol to the Bio-Sep® beads, activated carbon stable isotope analysis showed incorporation of 13C into biomass and dissolved inorganic carbon (DIC) in each SIP Bio-Trap® indicating that chemisorbed amendments are bioavailable. However, there was a clear effect of phenol concentration on 13C incorporation in both biomass and DIC confirming phenol inhibition. These results suggest that physical reductions of the phenol concentrations in some areas of the plume will be required before biodegradation of phenol can proceed at a reasonable rate.
- "Since the beads are sterile when deployed, incorporation of 13 C into PLFA provides proof of in situ biodegradation of a target compound by indigenous organisms under actual aquifer conditions. Identification of specific, labeled phospholipid fatty acids provides insight into the types of bacteria that are metabolically active in the aquifer and involved in processing carbon from the labeled compound (Chang et al. 2005; Busch-Harris et al. 2008). SIP studies can be performed for any compound that microorganisms use as a carbon source, including benzene, toluene, p-xylene, chlorobenzene, naphthalene , MTBE, and TBA (Geyer et al. 2005; Busch-Harris et al. 2006, 2008; Fiorenza et al. 2009). "
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ABSTRACT: A microbial survey of hydrocarbon-impacted groundwater and vadose zone at a Midwestern refinery employed molecular biological tools to elucidate the microbial processes involved in bioremediation occurring in the subsurface. qPCR analysis of bio-traps incubated in groundwater indicated that a large and diverse microbial community was present throughout the site and suggested that mechanisms of benzene, toluene, ethylbenzene, and xylene (BTEX) biodegradation included aerobic oxidation, sulfate reduction, methanogenesis, and possibly Fe+3 reduction. To assess the role of vadose zone microorganisms in hydrocarbon attenuation, RNA was extracted from soil core samples, and reverse transcriptase-qPCR (RT-qPCR) analysis indicated that microbial activity in the vadose zone generally increased with depth, likely supported by hydrocarbons and methane volatilizing from the groundwater. Stable isotope probing (SIP) with 13C6-benzene provided direct evidence of benzene biodegradation in six of the eight wells studied. The highest levels of 13C were detected in dissolved inorganic carbon (DIC) extracted from the two monitoring wells closest to the river. The influx of nutrients and oxygen coming from the river may help to maintain a robust population of hydrocarbon degraders in these wells. While qPCR analysis indicated that microorganisms with the genetic potential for hydrocarbon biodegradation were ubiquitous at the site, RT-qPCR and SIP results were used to refine the site conceptual model by identifying areas where that genetic potential was actively being expressed and locations where biodegradation was lagging.
Available from: Meriah Arias-thode
- "Inorganic amendments help to immobilize several toxic metals such as lead, zinc, and cadmium by forming stable compounds that render the metals no longer bioavailable   . Organic amendments, in contrast, may induce indigenous microbes capable of bio-reduction and/or of direct or indirect immobilization of toxic metals    . Apatite, an inorganic phosphate mineral, has been applied to facilitate the immobilization of several toxic metals including Cu, Zn and Pb   . "
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ABSTRACT: In situ amendments are a promising approach to enhance removal of metal contaminants from diverse environments including soil, groundwater and sediments. Apatite and chitin were selected and tested for copper, chromium, and zinc metal removal in marine sediment samples. Microbiological, molecular biological and chemical analyses were applied to investigate the role of these amendments in metal immobilization processes. Both apatite and chitin promoted micro-bial growth. These amendments induced corresponding bacterial groups including sulfide producers, iron reducers, and phosphate solubilizers; all that facilitated heavy metal immobilization and removal from marine sediments. Molecular biological approaches showed chitin greatly induced microbial population shifts in sediments and overlying water: chi-tin only, or chitin with apatite induced growth of bacterial groups such as Acidobacteria, Betaproteobacteria, Epsilon-proteobacteria, Firmicutes, Planctomycetes, Rhodospirillaceae, Spirochaetes, and Verrucomicrobia; whereas these bacteria were not present in the control. Community structures were also altered under treatments with increase of rela-tive abundance of Deltaproteobacteria and decrease of Actinobacteria, Alphaproteobacteria, and Nitrospirae. Many of these groups of bacteria have been shown to be involved in metal reduction and immobilization. Chemical analysis of pore-and overlying water also demonstrated metal immobilization primarily under chitin treatments. X-Ray absorp-tion spectroscopy (XAS) spectra showed more sorbed Zn occurred over time in both apatite and chitin treatments (from 9% -27%). The amendments improved zinc immobilization in marine sediments that led to significant changes in the mineralogy: easily mobile Zn hydroxide phase was converted to an immobile Zn phosphate (hopeite). In-situ amendment of apatite and chitin offers a great bioremediation potential for marine sediments contaminated with heavy metals.
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