Hexavalent chromium bioreduction and chemical precipitation of sulphate as a treatment of site-specific fly ash leachates.

Most of the power generation globally is by coal-fired power plants resulting in large stockpiles of fly ash. The trace elements associated with the ash particles are subjected to the leaching effects of precipitation which may lead to the subsequent contamination of surface and groundwater systems. In this study, we successfully demonstrate an efficient and sustainable dual treatment remediation strategy for the removal of high levels of Cr6+ and SO42- introduced by fly ash leachate generated by a power station situation in Mpumalanga, South Africa. The treatment consisted of a primary fixed-bed bioreactor kept at a reduction potential for Cr6+ reduction. Metagenome sequencing clearly indicated a diverse bacterial community containing various bacteria, predominantly of the phylum Proteobacteria which includes numerous species known for their ability to detoxify metals such as Cr6+. This was followed by a secondary BaCO3/dispersed alkaline substrate column for SO42- removal. The combination of these two systems resulted in the removal of 99% Cr6+ and 90% SO42-. This is the first effective demonstration of an integrated system combining a biological and chemical strategy for the remediation of multi-contaminants present in fly ash leachate in South Africa.

Bioremediation in Fractured Rock: 2. Mobilization of Chloroethene Compounds from the Rock Matrix.

A mass balance is formulated to evaluate the mobilization of chlorinated ethene compounds (CE) from the rock matrix of a fractured mudstone aquifer under pre- and postbioremediation conditions. The analysis relies on a sparse number of monitoring locations and is constrained by a detailed description of the groundwater flow regime. Groundwater flow modeling developed under the site characterization identified groundwater fluxes to formulate the CE mass balance in the rock volume exposed to the injected remediation amendments. Differences in the CE fluxes into and out of the rock volume identify the total CE mobilized from diffusion, desorption, and nonaqueous phase liquid dissolution under pre- and postinjection conditions. The initial CE mass in the rock matrix prior to remediation is estimated using analyses of CE in rock core. The CE mass mobilized per year under preinjection conditions is small relative to the total CE mass in the rock, indicating that current pump-and-treat and natural attenuation conditions are likely to require hundreds of years to achieve groundwater concentrations that meet regulatory guidelines. The postinjection CE mobilization rate increased by approximately an order of magnitude over the 5 years of monitoring after the amendment injection. This rate is likely to decrease and additional remediation applications over several decades would still be needed to reduce CE mass in the rock matrix to levels where groundwater concentrations in fractures achieve regulatory standards.

Bioremediation in Fractured Rock: 1. Modeling to Inform Design, Monitoring, and Expectations.

Field characterization of a trichloroethene (TCE) source area in fractured mudstones produced a detailed understanding of the geology, contaminant distribution in fractures and the rock matrix, and hydraulic and transport properties. Groundwater flow and chemical transport modeling that synthesized the field characterization information proved critical for designing bioremediation of the source area. The planned bioremediation involved injecting emulsified vegetable oil and bacteria to enhance the naturally occurring biodegradation of TCE. The flow and transport modeling showed that injection will spread amendments widely over a zone of lower-permeability fractures, with long residence times expected because of small velocities after injection and sorption of emulsified vegetable oil onto solids. Amendments transported out of this zone will be diluted by groundwater flux from other areas, limiting bioremediation effectiveness downgradient. At nearby pumping wells, further dilution is expected to make bioremediation effects undetectable in the pumped water. The results emphasize that in fracture-dominated flow regimes, the extent of injected amendments cannot be conceptualized using simple homogeneous models of groundwater flow commonly adopted to design injections in unconsolidated porous media (e.g., radial diverging or dipole flow regimes). Instead, it is important to synthesize site characterization information using a groundwater flow model that includes discrete features representing high- and low-permeability fractures. This type of model accounts for the highly heterogeneous hydraulic conductivity and groundwater fluxes in fractured-rock aquifers, and facilitates designing injection strategies that target specific volumes of the aquifer and maximize the distribution of amendments over these volumes.

Seasonal and Diel Variability in Dissolved DNA and in Microbial Biomass and Activity in a Subtropical Estuary.

Dissolved DNA and microbial biomass and activity parameters were measured over a 15-month period at three stations along a salinity gradient in Tampa Bay, Fla. Dissolved DNA showed seasonal variation, with minimal values in December and January and maximal values in summer months (July and August). This pattern of seasonal variation followed that of particulate DNA and water temperature and did not correlate with bacterioplankton (direct counts and [H]thymidine incorporation) or phytoplankton (chlorophyll a and CO(2) fixation) biomass and activity. Microautotrophic populations showed maxima in the spring and fall, whereas microheterotrophic activity was greatest in late summer (September). Both autotrophic and heterotrophic microbial activity was greatest at the high estuarine (low salinity) station and lowest at the mouth of the bay (high salinity station), irrespective of season. Dissolved DNA carbon and phosphorus constituted 0.11 +/- 0.05% of the dissolved organic carbon and 6.6 +/- 6.5% of the dissolved organic phosphorus, respectively. Strong diel periodicity was noted in dissolved DNA and in microbial activity in Bayboro Harbor during the dry season. A noon maximum in primary productivity was followed by an 8 p.m. maximum in heterotrophic activity and a midnight maximum in dissolved DNA. This diel periodicity was less pronounced in the wet season, when microbial parameters were strongly influenced by episodic inputs of freshwater. These results suggest that seasonal and diel production of dissolved DNA is driven by primary production, either through direct DNA release by phytoplankton, or more likely, through growth of bacterioplankton on phytoplankton exudates, followed by excretion and lysis.

A modular injection system, multilevel sampler, and manifold for tracer tests.

Ground water injection and sampling systems were developed for bacterial transport experiments in both homogenous and heterogeneous unconsolidated, surficial aquifers. Two types of injection systems, a large single tank and a dynamic mixing tank, were designed to deliver more than 800 L of amended ground water to the aquifer over 12 hours, without altering the ground water temperature, pH, Eh, or dissolved gas composition. Two types of multilevel samplers (MLSs) were designed and installed. Permanent MLSs performed well for the homogenous surficial aquifer, but their installation procedure promoted vertical mixing, which could obfuscate experimental data obtained from vertically stratified, heterogeneous aquifers. A novel, removable MLS was designed to fit in 2- and 4-inch wells. Expandable O-rings between each sampling port hydraulically isolated each port for sample collection when a nut was tightened at the land surface. A low-cost vacuum manifold system designed to work with both MLS designs used 50 mL centrifuge tubes to efficiently sample 12 MLS ports with one peristaltic pump head. The integrated system was developed and used during four field campaigns over a period of three years. During each campaign, more than 3000 ground water samples were collected in less than one week. This system should prove particularly useful for ground water tracer, injection, and push-pull experiments that require high-frequency and/or high-density sampling.

Lessons learned from bacterial transport research at the South Oyster Site.

This paper provides a review of bacterial transport experiments conducted by a multiinvestigator, multiinstitution, multidisciplinary team of researchers under the auspices of the U.S. Department of Energy (DOE). The experiments were conducted during the time period 1999-2001 at a field site near the town of Oyster, Virginia known as the South Oyster Site, and included four major experimental campaigns aimed at understanding and quantifying bacterial transport in the subsurface environment. Several key elements of the research are discussed here: (1) quantification of bacterial transport in physically, chemically, and biologically heterogeneous aquifers, (2) evaluation of the efficacy of conventional colloid filtration theory, (3) scale effects in bacterial transport, (4) development of new methods for microbial enumeration and screening for low adhesion strains, (5) application of novel hydrogeophysical techniques for aquifer characterization, and (6) experiences regarding management of a large field research effort. Lessons learned are summarized in each of these areas. The body of literature resulting from South Oyster Site research has been widely cited and continues to influence research into the controls exerted by aquifer heterogeneity on reactive transport (including microbial transport). It also served as a model (and provided valuable experience) for subsequent and ongoing highly-instrumented field research efforts conducted by DOE-sponsored investigators.

Relative dominance of physical versus chemical effects on the transport of adhesion-deficient bacteria in intact cores from South Oyster, Virginia.

Bacterial transport experiments were conducted using intact sediment cores collected from sites on the Delmarva Peninsula near South Oyster, VA, to delineate the relative importance of physical and chemical heterogeneity in controlling transport of an adhesion-deficient bacterial strain. Electron microscopy revealed that the sediments consisted of quartz and feldspar with a variable amount of clay and iron and aluminum hydroxide coatings on the grains. A nonmotile, gram-negative indigenous groundwater strain, designated as Comamonas sp. DA001, was injected into the cores along with a conservative tracer bromide (Br). DA001 cells were 1.2 x 0.6 microm in size with a hydrophilic surface and a slightly negative surface charge. Bacterial breakthrough preceded that of Br. This differential advection phenomenon can be accounted for by reduction of the effective porosity for the bacteria relative to Br. The distribution of cells remaining in the core as determined by scintillation counting and phosphor imaging techniques was variable, ranging from nearly uniform concentrations throughout the core to exponentially decreasing concentrations with distance from the point of injection. The fraction of bacterial retention in the core was positively correlated with the abundance of the metal hydroxides and negatively correlated with grain size. Because grain size was inversely correlated with the abundance of the metal hydroxide coatings, it was necessary to separate the effects of grain size and mineralogy. The fraction of the bacterial retention accounting for the effect of grain size, the collision efficiency, exhibited no correlation with the abundance of the metal hydroxides, indicating that the bacterial retention was primarily controlled by grain size. Reasons for the lack of influence of mineralogy on bacterial transport include (i) the slightly negatively charged bacterial surfaces; (ii) an insufficient heterogeneity of sediment surface properties; and (iii) the masking of the positive charge of the metal hydroxide surfaces by adsorbed organic carbon (up to 1180 ppm). This study demonstrates that the laboratory-based bacterial transport experiments are effective in delineating physical versus chemical controlling factors and provide an important link to field-based bacterial transport studies.

Simultaneous transport of two bacterial strains in intact cores from Oyster, Virginia: biological effects and numerical modeling.

The transport characteristics of two adhesion-deficient, indigenous groundwater strains, Comamonas sp. strain DA001 and Erwinia herbicola OYS2-A, were studied by using intact sediment cores (7 by 50 cm) from Oyster, Va. Both strains are gram-negative rods (1.10 by 0.56 and 1.56 by 0.46 microm, respectively) with strongly hydrophilic membranes and a slightly negative surface charge. The two strains exhibited markedly different behaviors when they were transported through granular porous sediment. To eliminate any effects of physical and chemical heterogeneity on bacterial transport and thus isolate the biological effect, the two strains were simultaneously injected into the same core. DA001 cells were metabolically labeled with (35)S and tagged with a vital fluorescent stain, while OYS2-A cells were metabolically labeled with (14)C. The fast decay of (35)S allowed deconvolution of the two isotopes (and therefore the two strains). Dramatic differences in the transport behaviors were observed. The breakthrough of DA001 and the breakthrough of OYS2-A both occurred before the breakthrough of a conservative tracer (termed differential advection), with effluent recoveries of 55 and 30%, respectively. The retained bacterial concentration of OYS2-A in the sediment was twofold higher than that of DA001. Among the cell properties analyzed, the statistically significant differences between the two strains were cell length and diameter. The shorter, larger-diameter DA001 cells displayed a higher effluent recovery than the longer, smaller-diameter OYS2-A cells. CXTFIT modeling results indicated that compared to the DA001 cells, the OYS2-A cells experienced lower pore velocity, higher porosity, a higher attachment rate, and a lower detachment rate. All these factors may contribute to the observed differences in transport.

Application of a vital fluorescent staining method for simultaneous, near-real-time concentration monitoring of two bacterial strains in an Atlantic coastal plain aquifer in Oyster, Virginia.

Two differentially labeled bacterial strains were monitored in near-real time during two field-scale bacterial transport experiments in a shallow aquifer in July 2000 and July 2001. Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107 were grown and labeled with the vital fluorescent stain TAMRA/SE (5 [and -6]-carboxytetramethylrhodamine, succinimidyl ester) or CFDA/SE (5 [and -6]-carboxyfluorescein diacetate, succinimidyl ester). Fluorescently labeled cells and a conservative bromide tracer were introduced into a suboxic superficial aquifer, followed by groundwater collection from down-gradient multilevel samplers. Cells were enumerated in the field by microplate spectrofluorometry, with confirmatory analyses for selected samples done in the laboratory by epifluorescence microscopy, flow cytometry, and ferrographic capture. There was general agreement in the results from all of the vital-stain-based enumeration methods, with differences ranging from <10% up to 40% for the analysis of identical samples between different tracking methods. Field analysis by microplate spectrofluorometry was robust and efficient, allowing thousands of samples to be analyzed in quadruplicate for both of the injected strains. The near-real-time data acquisition allowed adjustments to the predetermined sampling schedule to be made. The microplate spectrofluorometry data sets for the July 2000 and July 2001 experiments allowed the transport of the injected cells to be related to the site hydrogeology and injection conditions and enabled the assessment of differences in the transport of the two strains. This near-real-time method should prove effective for a number of microbial ecology applications.