H21A-0990 INVITED 0800h
Characterizing Spatial Variability of Microbial Processes: In-situ Determination of Biodegradation Activity
Characterizing the occurrence, magnitudes, and rates of biodegradation at the field scale has become of great interest, especially with respect to evaluating the feasibility of applying in-situ bioremediation and monitored natural attenuation for hazardous waste site remediation. Potential spatial variability of microbial activity, which may be influenced by a multitude of factors, complicates site characterization. Biotracer tests are one of several methods that have been used to characterize the biodegradation potential for field-scale systems. In this study, field experiments were conducted at two sites to evaluate the utility of the biotracer method for characterizing the spatial variability of biodegradation activity. The first site is a chlorinated-solvent contaminated regional aquifer in Arizona, and the second site is a mixed-waste contaminated surficial aquifer in Utah. Mass recovery of the biotracer decreased approximately linearly with increasing residence time for the chlorinated-solvent site. Similar behavior was observed at the mixed-waste site, except in the region adjacent to the injection zone, where percent recoveries were much lower. First-order biodegradation rate coefficients obtained from model calibration of the tracer data varied between 0.2 and 0.5 1/d for the chlorinated-solvent site. For the mixed-waste site, the values varied between 0.1 and 0.6 1/d down-gradient of the injection wells, and between 0.7 and 2.6 1/d near the injection wells. Considering the large range over which biodegradation rate coefficients can vary, the rate coefficient exhibited relatively minimal spatial variability (factor of 2.5) for the chlorinated-solvent site. Conversely, the spatial variability of the rate coefficient was an order of magnitude greater for the mixed-waste site. These differences in variability are consistent with conditions associated with the respective sites. For example, the greater microbial activity observed in the vicinity of the injection wells for the mixed-waste site is consistent with the biomass distribution determined from analysis of core samples, which shows larger bacterial cell densities for the region near the injection wells. These results illustrate the utility of biotracer tests for in-situ characterization of microbial activity (e.g., biodegradation potential), including evaluation of potential spatial variability.
H21A-0991 INVITED 0800h
Methanogenic and Methanotrophic Heterogeneity at a Crude Oil Spill Site
A study of the fate of crude oil in the subsurface at a 1979-spill site near Bemidji, Minnesota, shows the effect of subsurface heterogeneity on oil and methane gas degradation. Separate-phase oil is present in the surficial glacial outwash sediments at residual concentrations in the vadose zone and as an elongated oil body floating on the 6-8-m-deep water table. Gas and water concentrations and microbial data show that methanogenic conditions prevail in the area with separate-phase oil. Within the separate-phase oil, substantial degradation of the {\it n}-alkane fraction of the oil has occurred under methanogenic conditions. There is extreme spatial variability in the degradation rates such that the {\it n}-alkanes are mostly degraded in the upgradient oil-body limb, but in the downgradient limb {\it n}-alkane concentrations are comparable to oil archived from the original spill. Most Probable Number (MPN) microbial data show that numbers of methanogens are $\sim$10 times greater in the more degraded limb. These differences appear to be related to groundwater recharge. Data from two vertical arrays of moisture probes show that in 2002 the more degraded oil limb received over twice the recharge as the less degraded limb. Typically, samples located near the top of the 1-m-thick floating oil body, are more degraded than those located 10-30 cm lower. Consistent with the degradation state, numbers of methanogens and fermenters are generally greater near the top of the oil body compared to the lower locations. The lateral and vertical variation in degradation state and microbial data together suggest that a nutrient supplied from land surface enhances microbial growth. The resulting heterogeneity of microbial populations leads to greater degradation rates in some locations. There is also evidence of extreme heterogeneity in microbial activity between land surface and the oil body floating on the water table. A narrow methanotrophic horizon exists in the vadose zone where both oxygen from the land surface and $\sim$20 % (by volume) methane from methanogenic oil degradation are present. MPN data for methanotrophs show similar patterns to those for aerobes indicating that these are the same population. The largest methanotrophic activity (as indicated by aerobe MPN data) occurs just above a silt layer that restricts upward migration of methane and downward migration of oxygen. Changes in physical properties between this silt layer and an overlying unit of coarse sand result in greater than a 1000-fold increase in aerobe numbers over a vertical distance of less than 10 cm.
http://wwwmn.cr.usgs.gov/bemidji/
H21A-0992 INVITED 0800h
Use of Microarray-based Genomic Technologies for Assessing Microbial Community Composition and Dynamics in Contaminated Groundwater
To effectively monitor microbial populations involved in various important processes, a 50-mer-based oligonucleotide microarray was developed based on known genes and pathways involved in: biodegradation, metal resistance and reduction, denitrification, nitrification, nitrogen fixation, methane oxidation, methanogenesis, carbon polymer decomposition, and sulfate reduction. This array contains approx 2000 unique and group-specific probes with $<$85% similarity to their non-target sequences. Based on artificial probes, our results showed that at hybridization conditions of 50$^{o}$C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having $<$88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. Detection limits were about 5-10ng genomic DNA in the absence of background DNA, and 50-100ng ($\sim$1.3 -A10$^{7}$ cells) in the presence background DNA. Strong linear relationships between signal intensity and target DNA and RNA concentration were observed (r$^{2}$ = 0.95-0.99). Real-time PCR analysis of 12 representative genes was consistent with microarray-based quantification (r$^{2}$ = 0.95). Also novel approaches were developed and used to increase microarray detection sensitivity of both DNA and mRNA. Application of these array-based technologies to analyze microbial communities in contaminated groundwaters from the US Department of Energy's Natural and Accelerated Bioremediation Research Program (NABIR) Field Research Center at Oak Ridge, TN, demonstrated that it is feasible to biostimulate the indigenous microbial populations for contaminant remediation but the process could be very complicated due to highly spatial heterogeneous microbial distributions. Based on these results, more comprehensive functional gene arrays containing $\sim$27,000 probes from the genes important for biogeochemical cycling of C, N, S, P, metal resistance and contaminant degradation have been designed and constructed. This is the most comprehensive array available so far for environmental studies and will also be useful for biogeochemistry studies in general.
H21A-0993 0800h
Microbial Heterogeneity in the Subsurface and its Effects on DNAPL Interfacial Properties
Remediation of dense non-aqueous phase liquid (DNAPL) is affected by the heterogeneous spatial distribution of the DNAPL in the subsurface. This distribution is contingent upon porous media properties such as grain size, mineral composition as well as hydrodynamic and capillary forces on the DNAPL. Processes resulting in reduced interfacial tension (IFT) and, thus, capillary forces, may affect DNAPL distribution in the subsurface. The goal of this research is to determine the effect of heterogeneous microbial distributions on the DNAPL interfacial properties at A-14 Outfall Area of the DOE Savannah River Site (SRS). Soil cores were collected from both PCE contaminated areas and background locations near the A-14 outfall. Cores were characterized for microbial concentrations, volatile compounds, and grain size and mineral content. Anaerobic enrichment cultures were grown to assess the effects of microbial activity on the IFT. Results show that bacterial distributions vary significantly with depth but are well correlated with the PCE and TCE concentration in the contaminated area. In the non-contaminated area, bacterial concentrations also vary significantly with depth and are at least 1-2 orders of magnitude lower than concentrations in the DNAPL contaminated borings. These results indicate that the bacterial distribution is predominantly affected by the presence of DNAPL and secondarily by the pore space and soil moisture. Anaerobic enrichments cultures generated from the site reduced PCE/water IFT as much as 50% when the culture was stressed with oxygen or elevated PCE concentrations. The decrease in IFT was predominantly attributed to bacterial adhesion at the PCE/water interface. Anaerobic enrichment cultures using background cores have been developed to determine whether this observed effect on IFT is unique to anaerobic PCE degrading organisms and what bacterial concentrations at the DNAPL interface are required to observe significant IFT reductions. Results of these experiments will be presented at the conference.
H21A-0994 0800h
Bacterial Community Structure Response to Petroleum Concentration in Groundwater
This study characterized the bacterial community present in groundwater samples from the Guadalupe Dunes Restoration Project on the central California coast. The purpose of the study was to determine the changes in bacterial community structure and function in response to variations in the concentration of dissolved phase total petroleum hydrocarbons (TPH) in groundwater plumes at the site. For the purpose of this study groundwater samples were collected at varying distance from TPH source zones in 10 different plumes. All samples were analyzed for ammonia, phosphate, TPH, methane, oxygen, carbon dioxide, nitrate, sulfate, and dissolved iron levels. Chemical analysis revealed that the groundwater chemistry varied between plumes and on a well-to-well basis within a plume. Principle component analyses (PCA) demonstrated that TPH degradation related parameters explained 28% of the variation in the groundwater chemistry. In addition to the physical and chemical analyses, four liters of each groundwater sample were filtered and bacterial DNA was isolated to determine the relationship between groundwater chemistry and bacterial community structure and function. Specific Polymerase Chain Reaction (PCR) primers were used to characterize populations of Eubacteria, and Archaea, as well as function genes for sulfate reducing, methanotrophic, and methanogenic bacteria. Terminal Restriction Fragment (TRF) Length Polymorphisms (or T-RFLP) were used to analyze community structure. Eubacterial and Archaeal groundwater communities were separated into distinct clusters which did not clearly reflect changes in groundwater chemical parameters unless individual plumes were analyzed separately. However, specific Eubacterial and Archaeal TRF peaks did correspond to known petroleum degrading organisms and methanogenic bacteria, respectively. Only one sample produced a positive result for the sulfite reductase gene (dsrAB), indicating that sulfate reduction may not be a dominant process at the site. While the particulate methane monooxygenase gene (pmoA) was not detected in all samples, the specific type of pmoA gene present in a sample correlated directly to methane concentration, indicating that different types of methanotrophic bacteria are affected by changes in groundwater chemistry. In contrast to the methanotroph data, the presence of different types of the methyl coenzyme M reductase gene (mcrA) specific for methanogens did not correlate to physical and chemical groundwater parameters.
H21A-0995 0800h
In Situ Subsurface Cometabolic Transformation of Chlorinated Solvent Mixtures by Native and Bioaugmented Butane Utilizing Microorganisms
An aquifer test zone at Moffett Federal Airfield, CA, was used to assess the cometabolic transformation of chlorinated solvent mixtures by native and bioaugmented butane-utilizing microbes. Two parallel, but hydraulically separate, well legs were controlled by separate injection/ extraction systems and were fed pulses of butane and oxygen and were continually fed chlorinated aliphatic hydrocarbons \(CAHs\). Groundwater monitoring wells were located 1.0, 2.2, and 4.0 m from the respective injection wells and were semi-continuously analyzed for butane, CAHs, pH, anions, and dissolved oxygen. One well leg was bioaugmented with approximately 5 g dry weight of a butane-utilizing enrichment culture known to cometabolically transform 1,1-dichloroethene \(DCE\), 1,1-dichloroethane \(DCA\), and 1,1,1-trichloroethane \(TCA\). Periodically, samples were taken from the groundwater monitoring wells, or from two fully-penetrating wells placed 0.5 m and 1.5 m from the injection wells, for molecular microbial analyses including terminal restriction fragment length polymorphism \(T-RFLP\), real time quantitative PCR. Bioaugmentation stimulated butane uptake and CAH transformation with the greatest removal efficiencies observed within the first week, followed by declining CAH treatment efficiency over a period of days to months. DCE was at least partially transformed in both the indigenous and bioaugmented well legs. An injection concentration of 175 mg/L DCE was readily transformed in the bioaugmented well leg with a removal efficiency of greater than 90% while only 25% removal occurred in the indigenous well leg. Neither DCA nor TCA was transformed in the indigenous well leg, while both DCA and TCA transformation were obtained in the bioaugmented well leg. However, less than 80% TCA transformation efficiency was obtained and it was difficult to maintain TCA transformation over time. A clear and repeatable microbial community transition from dominance of organisms with a T-RFL of 277 bp, when restricted with MnlI, to an organisms with a T-RFL of 126 bp occurred upon bioaugmentation and stimulation of the aquifer test zone with butane and oxygen. Microbial community structure varied between the two parallel well legs while reasonably similar communities were observed along the flow path of the bioaugmented well leg. A Rhodococcus sp. present in the bioaugmentation culture was quantified in samples taken from the field using real time PCR. The organism was successfully transported at least 2 m through the subsurface, but was found to be present in insufficient quantity relative to the total microbial community to appear in T-RFLP analyses.
H21A-0996 0800h
Using {\it nirS} gene expression as a proxy for denitrification rate
Quantification of microbial respiration rates in subsurface environments is complicated by difficult access to samples, impervious aquifer materials, and a lack of information about groundwater transport processes. Here, Quantitative Reverse Transcriptase Polymerase Chain Reaction (Q-RT-PCR) is investigated as a novel method of directly quantifying biological denitrification rates on a single sample using a single measurement. Benchtop batch reactors innoculated with the model organism {\it Paracoccus denitrificans} are sampled for denitrification rate as measured by mass balance, and these rates are then correlated with the abundance of expressed copies of the nitrite reductase gene {\it nirS}. Future work will examine the applicability of this technique to mixed cultures and environmental samples.
H21A-0997 0800h
Heterogeneity in Extracellular Polymeric Substances Production and its Effects on the Transport and Attachment of Trichloroethene Degrading Toluene Oxidizing Bacteria
The role of extracellular polymeric substances (EPS) heterogeneity in microbial transport and retention was investigated for pure cultures of trichloroethene (TCE) degrading toluene oxidizing bacteria. A wildtype and a mutant strain of {\it Ralstonia pickettii} were employed in sand column experiments, based upon their ability to metabolize alkyl aromatic hydrocarbons and cometabolize TCE by a toluene-3-monooxygenase (T3MO) enzyme system. These wildtype and mutant strains are identical, except that the TbuX protein is knocked out in the latter, resulting in less EPS synthesis. A pulse of $^{3}$H radiolabeled cell-EPS suspension was introduced to saturated packed sand columns under steady flow conditions. Biomass and EPS were quantified in the influent, effluent, and within the porous medium. Experimental results indicate that increased EPS enhanced bacterial retention and reduced detachment. While biomass deposition (mass/g sand) declined, EPS (mg/g sand) increased with travel distance along the column. EPS mass balance calculations suggest that EPS were synthesized by surface-associated bacteria within the column. The biomass-normalized EPS along the column ranged from values that were fairly consistent with those found under free-living conditions (near the column entrance) to five-fold increases (near the column outlet), suggesting that most EPS synthesis took place in microbes deposited near the column outlet. Flow cytometric results revealed no significant change in average cell size within the two day starvation period. However, cell influent was comprised of at least two subpopulations, as assessed through differences in fluorescence intensity. Additionally, a better prediction of the observed microbial deposition pattern was achieved with a model that incorporates a power-law distribution for the particle-collector deposition rate coefficients, consistent with a variation of attachment properties within the population. This observed microbial heterogeneity could also be attributed to differential EPS coatings on cell surfaces.
H21A-0998 0800h
Single-Well Push-Pull Tests for Evaluating In Situ TCE, cis-DCE, and trans-DCE Cometabolism by Toluene-Utilizing Microorganisms
Single-well-push-pull tests were performed to assess the feasibility of in situ aerobic cometabolism of chlorinated aliphatic hydrocarbons (CAHs), such as trichloroethylene (TCE) and cis-1,2-dichloroethylene (cis-DCE), by toluene-grown microorganisms. The tests were performed in the saturated zone at Fort Lewis, Washington, which is contaminated with TCE and cis-DCE. The tests assessed the heterogeneity of the indigenous microorganisms towards toluene utilization and CAH transformation. The tests were conducted in two multi-level monitoring wells at two different depths. Transport characteristics of the dissolved solutes were compared using bromide as a conservative tracer. Toluene utilization was evaluated by observing repeated uptake under natural gradient flow conditions and during push-pull activity tests. For the push-pull activity tests, the injected solution was amended with the substrates of interest, and after injection was permitted to reside in the formation for 24 hours and then extracted. Toluene utilization was indicated by decreases in concentration, when normalized to bromide as a conservative tracer, dissolved oxygen uptake, and the production of ortho-cresol as an intermediate oxidation product. Isobutene added to the injected groundwater was transformed to isobutene oxide, indicating that microorganisms that express an ortho-monooxygenase were stimulated. Similar rates of toluene utilization, isobutene, cis-DCE, and trans-DCE transformation were achieved at the four different locations tested. Rate estimates obtained in the 24-hour activity tests were similar to those achieved in the 50-hour natural gradient tests. The results indicated that at the four locations tested, there was little difference in the rates of toluene utilization and cometabolic transformation.
H21A-0999 0800h
Modeling Subsurface Biogeochemical Cycling of Heavy Metals
Mining of precious metals since the late 1800's has left the Western U.S. sediments at many sites heavily enriched with toxic metals, including Cd, Cu, Pb, and Zn. Such sites include Lake Coeur d'Alene, the Yuba-Bear Watersheds and the Iron Mountain mining sites. Most of these metals are toxic, mutagenic and carcinogenic to biota, including humans, such that significant negative human/ecological impacts result from the metal-enriched dust, soil, and water. However, microorganisms indigenous to metal contaminated sediments are capable of catalyzing reactions that detoxify their environment, and thus constitute an important driving component in the biogeochemical cycles of these metals. It is vital to understand and quantify the biogeochemical reactions controlling the fate and transport of these metals to effectively model and predict changes in metal concentrations and microbial populations with respect to time and space. We present a preliminary microbial ecology model for simulating trace metal fate and transport in 1-D sediment systems, including Cu and Pb, and their effect on microbial consortia including multiple trophic groups. The model accounts for multiple electron acceptors, product inhibition, metal toxicity via an accounting of dose. Dose is expressed in a novel fashion as a structural variable in the population dynamics of the microbial consortia.
H21A-1000 0800h
Evaluation of the Factors that Control the Time-Dependent Inactivation Rate Coefficients of Bacteriophage MS2 and PRD1
Batch experiments were conducted under both static and dynamic conditions to study the effects of temperature and the presence of sand on the inactivation process of viruses. The male--specific RNA coliphage, MS2, and the {\sl Salmonella typhimurium} phage, PRD1, were used as model viruses for this study. Over 100 oven--baked borosilicate glass bottles with or without Monterey sand were filled with a low--ionic--strength phosphate buffered saline solution containing both bacteriophage and incubated at temperatures of 4$^\circ$, 15$^\circ$, or 25$^\circ$C. The results of the batch experiments indicate that the inactivation process can be represented by a pseudo first-order expression with time--dependent rate coefficients. A combination of high temperature and the presence of sand appears to produce the greatest disruption to the surrounding protein coat of MS2. However, for PRD1, the lower activation energies derived from Arrhenius plots indicate a weaker dependence of the inactivation rate on temperature. Furthermore, the presence of an air--liquid--solid interface in the dynamic batch experiment containing sand produces the greatest damage to specific viral components of PRD1 that are required for infection. These results indicate the use of thermodynamic parameters based on the pseudo first--order inactivation expression allows better prediction of the inactivation of viruses in the environment.