Final Report: Interactive Roles of Microbial and Spartina Populations in Mercury Methylation Processes in Bioremediation of Contaminated Sediments in Salt-Marsh Systems

EPA Grant Number: R825513C025
Subproject: this is subproject number 025 , established and managed by the Center Director under grant R825513
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: HSRC (1989) - South and Southwest HSRC
Center Director: Reible, Danny D.
Title: Interactive Roles of Microbial and Spartina Populations in Mercury Methylation Processes in Bioremediation of Contaminated Sediments in Salt-Marsh Systems
Investigators: Saunders, F. Michael , Frischer, Marc E , King, J. , Kostka, J.
Institution: Georgia Institute of Technology
EPA Project Officer: Hahn, Intaek
Project Period: January 1, 1998 through January 1, 2001
Project Amount: Refer to main center abstract for funding details.
RFA: Hazardous Substance Research Centers - HSRC (1989) RFA Text |  Recipients Lists
Research Category: Hazardous Substance Research Centers , Land and Waste Management


The goal of the project is remediation of mercury-contaminated sediments is being examined to establish the coupled roles arid effects of Spartina, microbial activity and sulfate reducing bacteria (SRB) on mercury methylation in saltmarsh sediments. The fundamental conceptual model (see Figure 1) is that the incidence of mercury methylation is biochemically driven by sulfate-reducing bacteria (SRB) in the top layers of sediments. Furthermore in Spartina systems the production of methyl mercury is potentially lowered in the root zones or altered by biologically mediated demethylation of methylmercury in marsh sediments by the microbial community. Mercury toxicity and availability in the biological food chain is driven by methylmercury and its production is controlled by the net rate of microbial formation and removal within sediments.

The growth of Spartina and the associated injection of root-zone organic exudates and molecular oxygen into microbially active sediments influence sulfate reduction, mercury methylation and demethylation processes. Hence the bioavailability and ultimately the risk associated with mercury contamination in saltmarsh sediments is related to presence and growth of Spartina. Thus, understanding the relationships among sulfate reducing bacteria (SRB), demethylating processes, Spartina and sediment-based mercury will be useful for assessing sediment-quality and developing and assessing in-situ remediation strategies and methods for risk assessment.

Summary/Accomplishments (Outputs/Outcomes):

The research plan for the current growth season (March '00 - February '01 include an enhanced focus on methylation and demethylation processes in sediments and pursuit of the effects of Spartina on the stabilization of mercury-contaminated sediments. The earlier research (year 01) focused on assessment of the biogeochemical properties of sediments has been reduced to focus on critical parameters directly linked to mercury availability and sulfate reducing populations. Earlier work in this area over the initial 18 months of the project clearly indicated the full equilibration of the sediments and the relevance of the current research to sediments systems in natural saltmarshes.

As described in our initial work plan, mercury demethylation rates will be determined in sediments by incubation with methylmercury using g-level injections of methylmercury in 1-cm, sediment-core lifts and in homogenized slurries from sediment cores using an incubation time of 12 hr in both systems. Demethylation rates will be determined by the loss of methylmercury after incubation. Methylmercury will be determined by distillation, derivatization, chromatographic separation and thermal decomposition using a Tekron 2500 CVAFS (Smith, 1993), as with.all current measurements. Recent improvements in the isolation of methylmercury from sediment and water involve distillation into a Teflon collection vessel with detection limits for methylmercury using this method of 2 ng/L for porewaters and 0.01 g/kg in sediments in SkIO laboratories.

These methods will provide potential demethylation rates because of the artificially high methylmercury concentrations involved in this experimental procedure. Therefore, in addition to these measurements, we are coordinating a multi-lab approach to make mercury demethylation measurements using radiotracer approaches using radioactive mercury and C-14 labeled methylmercury. These methods are projected to provide the most sensitive and accurate means to estimate demethylation rates. Technical and scientific details and radiotracer expense associated with conducting such tracer studies call for collaborative efforts with others who are conducting these measurements and are interested in their application to controlled mesocosm systems. We are currently organizing a joint exercise to make mercury demethylation rate measurements in a variety of mercury-contaminated sediment types using both multiple radiotracer and conventional methods. This exercise will provide for calibration of radiotracer methods with other methods and will provide a context for interpreting data generated in this study. In addition, these studies will provide a set of high-quality demethylation rate measurements that would otherwise not be available. Currently we are organizing these studies with Drs. Eric Roden (University of Alabama), Ron Oremland (USGS), and Mark Hines (University of Alaska) and anticipate this to be an integral component of the year 03 plan.

An additional objective for this period will be to investigate the influence of Spartina on sulfate reducing bacteria (SRB) populations in contaminated marsh sediments. The influence of Spartina on methylation (see Figure 2) indicates that the resident SRB population could be influenced such that lower levels of methylmercury are produced in these sediments. Our previous investigations have demonstrated that the primary source of bioavailable methylmercury in mercury contaminated marine sediments is derived from the activity of SRB. Furthermore, our recent HSRC funded studies have shown that genetically distinct sulfate-reducing bacteria (SRB) methylate mercury with various incidence rates, relative to sulfate reduction activity (King et al., 2000). In particular, the Desulfobacterium group of the SRB appears to be the mercury methylators with the highest incidence rate for methylation in marsh sediments. Desulfobacterium methylated mercury at an incidence rate that was 92-fold higher than the lowest mercury methylating group Desulfobulbus that was studied (Table 1). Based on these studies, it was concluded that the composition of SRB populations in mercury contaminated sediments is a primary parameter responsible for controlling the biological availability of mercury in contaminated marine sediments.

Table 1. Incidence of Mercury Methylation in Various Sulfate Reducing Bacteria

SRB Taxon MMR/SRR (pmole [Hg]/mole[S04]) Relative rate (normal to Desulfobulbus)
Desulfobulbus 1.40 ± 0.4 1
Desulfovibrio 6.83 ± 1.0 4.9
Desulfobacter 20.44 ± 4.7 14.6
Desulfococcus 22.93 ± 0.65 16.4
Desulfobacterium 128.62 ± 15.0 91.9

Through our HSRC sponsored activities we have utilized these data to develop a kinetic model that can be used to predict the formation rate of methyl mercury in contaminated sediments. This model incorporates measurements of sulfate reduction activity, mercury concentration, and the structure of SRB populations. Initial studies suggest that this model significantly improve our ability to predict mercury methylation activity in contaminated marine sediments.

As a component of our studies during this phase of the project, we will pursue and utilize these findings (and methods developed during the course of these studies) to explore the influence of Spartina on SRB populations and activity. In addition, we will determine the species composition of Desulfobacterium species present in the BERM mesocosm systems. This information will be used to refine existing models and to extend our understanding of how complex biogeochemical and plant processes interact to influence mercury cycling in contaminated salt marsh systems. Specifically, our objectives are twofold. First, we will determine the structure of SRB communities in sediment cores from pristine and contaminated mesocosm systems. These studies will be conducted in the BERM mesocosms over a full annual cycle to capture the influence of plant growth on SRB populations. These studies will utilize SRB group-specific 16S rRNA targeted oligonucleotide probes and methods utilized in our previous studies (Frischer et al., 2000). This information will be used as model input so that methylation rate can be estimated in the contaminated marsh sediments. Second, we will determine the species composition (richness) of Desulfobacterium species in each of the three mesocosms. In these studies 16S rDNA targeted oligonucleotide PCR primers specific for the Desulfobacterium group of SRB will be utilized to amplify, clone, and sequence representative Desulfobacterium species from LCP marsh sediments in the presence or absence of Spartina.

In summary, the results of these studies will add to our continued understanding of the interactions between plant processes and mercury cycling, particularly with respect to methylmercury formation and cycling. These insights will then form the basis for improved risk assessment and the development of innovative in situ bioremediation strategies for mercury contaminated marine sediments. Understanding controlling processes for methylmercury production in sediments is the critical question being addressed to understand the processes, which control mercury endpoints in evaluating risk and designing and evaluating in-situ bioremediation technologies. Our research is directed at a fundamental assessment of methylation/demethylation processes and their application to sediments contaminated with hazardous wastes.

The project is being conducted at the Bioremediation Environmental Research Mesocosm facility (BERM) at the Skidaway Institute of Oceanography (SkIO). Three mesocosms are in their second season of operation with sediment placement and equilibration having been completed. Sediments in the three mesocosms were from the contaminated tidal saltmarsh at the LCP site (EPA IV Superfund site in Brunswick GA) and an uncontaminated Skidaway saltmarsh. One contaminated saltmarsh mesocosm and an uncontaminated saltmarsh mesocosm contain Spartina, while the remaining contaminated saltmarsh mesocosm contains LCP sediment without Spartina.

Based on our year-to-date quarterly measurements in 10-cm, sediment cores, the mercury levels of the sediments are as indicated in Table 2. The LCP sediments contain 13.3 mg/kg of total mercury and are generally representative of the extensive saltmarsh area associated with the LCP site, while the uncontaminated Skidaway (Priests Landing site) sediments contain 51 mg/kg of total mercury. Whole-sediment methylmercury levels are 3.62 and 0.27 g/kg, respectively for the LCP and Skidaway sediments.

Table 2. Mercury content of LCP and Skidaway (Priests Landing) saltmarsh sediments for period of June '99 to March '00.

  Sediment Porewater
  Total Hg Methyl Hg Total Hg
Priests Landing 0.051 mg/kg 0.27 g/kg 227 ng/L
LCP Site 13.3 mg/kg 3.62 g/kg 220 ng/L

Of critical relevance are the porewater levels of total mercury species in the sediments. The total mercury in porewater samples, inclusive of data for 10-cm cores and parallel in-situ sipper samples, in the mesocosm systems is 227 ng/L and 220 ng/L for the Skidaway and LCP sediments, respectively. These results are indicative of the potential similar bioavailability of mercury for methylation in the two sediments. In that porewater concentrations of mercury are virtually equal in both contaminated and uncontaminated sediments, containing total mercury at levels that are different by a factor of over 200 (i.e., 260:1), indicates the controlling link for methylmercury production may be related to factors controlling and influencing biological activity.

The current data for the systems are those through March '00 of the current growth season. The results to date for porewater methylmercury concentrations are presented in Figure 2 and indicate the correlation of porewater methylmercury levels with sulfate reduction rates (SRR). The indicated SRR values are 10 cm-integrated averages for intact core analyses for the sediments and porewater methylmercury levels are averaged values for 10-cm cores and parallel in situ sipper analyses. The SRRs indicate activities ranging from 100 to 500 nmole/cm3-day over the season and reflect in part, the temperature variations for the period.

Sediments with Spartina contain background levels of methylmercury in the range of 1-1 lng/L and indicate no significant correlation with SRR. This is the outcome for both of the uncontaminated and contaminated sediments with Spartina. For the contaminated LCP sediment without Spartina, there is a positive correlation indicated for porewater methylmercury levels and SRRs. In Spartina- free systems, sulfate reduction is therefore paralleled by the elevation of porewater methylmercury in the sediment. This correlation is as expected through the stimulation of sulfate reducing bacteria found previously in related HSRC/S&SW research. The concentration values for porewater methylmercury are well above the background levels for vegetated Spartina mesocosms.

The lack of stimulation of methylmercury formation in the contaminated mesocosm with Spartina and its close correlation with the pristine sediment are projected to be related to (i) a low level of methyl ation in both Spartina sediments due to the phylogenetic composition of the microbial communities and (ii) the enhancement of demethylation of in situ methylmercury in parallel with production. The equal levels of total available mercury in the sediment porewaters indicate a similar potential for methylation in the three sediment systems, independent of "contamination" status. The assessment of the positive impact of Spartina growth on remediation of mercury-contaminated sediments is being pursued in the current growing season. Furthermore, an enhanced understanding of the microbial role in the methylation and associated demethylation of mercury is planned.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 4 publications 2 publications in selected types All 2 journal articles
Other center views: All 392 publications 154 publications in selected types All 106 journal articles
Type Citation Sub Project Document Sources
Journal Article Frischer ME, Danforth JM, Healy MAN, Saunders FM. Whole-cell versus total RNA extraction for analysis of microbial community structure with 16S rRNA-targeted oligonucleotide probes in salt marsh sediments. Applied and Environmental Microbiology 2000;66(7):3037-3043 R825513C025 (Final)
not available
Journal Article King JK, Kostka JE, Frischer ME, Saunders FM. Sulfate-reducing bacteria methylate mercury at variable rates in pure culture and in marine sediments. Applied and Environmental Microbiology 2000;66(6):2430-2437. R825513C025 (Final)
not available

Supplemental Keywords:

methylmercury, mercury toxicity, and demethylation., RFA, Scientific Discipline, Waste, Water, Chemical Engineering, Contaminated Sediments, Environmental Chemistry, Analytical Chemistry, Hazardous Waste, Bioremediation, Ecology and Ecosystems, Environmental Engineering, Hazardous, Mercury, environmental technology, sediment treatment, hazardous waste management, hazardous waste treatment, risk assessment, decontamination of soil and water, soil and groundwater remediation, microbial degradation, biodegradation, decontamination of soil, risk management, contaminated sediment, slurry reactors, Spartina, chemical contaminants, microbes, contaminated soil, bioremediation of soils, contaminants in soil, remediation, methyl mercury, biotransformation, anaerobic biotransformation, waste mixtures, technology transfer, extraction of metals, contaminated soils, metal compounds

Progress and Final Reports:

Original Abstract
  • 1998
  • 1999

  • Main Center Abstract and Reports:

    R825513    HSRC (1989) - South and Southwest HSRC

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R825513C001 Sediment Resuspension and Contaminant Transport in an Estuary.
    R825513C002 Contaminant Transport Across Cohesive Sediment Interfaces.
    R825513C003 Mobilization and Fate of Inorganic Contaminant due to Resuspension of Cohesive Sediment.
    R825513C004 Source Identification, Transformation, and Transport Processes of N-, O- and S- Containing Organic Chemicals in Wetland and Upland Sediments.
    R825513C005 Mobility and Transport of Radium from Sediment and Waste Pits.
    R825513C006 Anaerobic Biodegradation of 2,4,6-Trinitrotoluene and Other Nitroaromatic Compounds by Clostridium Acetobutylicum.
    R825513C007 Investigation on the Fate and Biotransformation of Hexachlorobutadiene and Chlorobenzenes in a Sediment-Water Estuarine System
    R825513C008 An Investigation of Chemical Transport from Contaminated Sediments through Porous Containment Structures
    R825513C009 Evaluation of Placement and Effectiveness of Sediment Caps
    R825513C010 Coupled Biological and Physicochemical Bed-Sediment Processes
    R825513C011 Pollutant Fluxes to Aquatic Systems via Coupled Biological and Physicochemical Bed-Sediment Processes
    R825513C012 Controls on Metals Partitioning in Contaminated Sediments
    R825513C013 Phytoremediation of TNT Contaminated Soil and Groundwaters
    R825513C014 Sediment-Based Remediation of Hazardous Substances at a Contaminated Military Base
    R825513C015 Effect of Natural Dynamic Changes on Pollutant-Sediment Interaction
    R825513C016 Desorption of Nonpolar Organic Pollutants from Historically Contaminated Sediments and Dredged Materials
    R825513C017 Modeling Air Emissions of Organic Compounds from Contaminated Sediments and Dredged Materials title change in last year to "Long-term Release of Pollutants from Contaminated Sediment Dredged Material"
    R825513C018 Development of an Integrated Optic Interferometer for In-Situ Monitoring of Volatile Hydrocarbons
    R825513C019 Bioremediation of Contaminated Sediments and Dredged Material
    R825513C020 Bioremediation of Sediments Contaminated with Polyaromatic Hydrocarbons
    R825513C021 Role of Particles in Mobilizing Hazardous Chemicals in Urban Runoff
    R825513C022 Particle Transport and Deposit Morphology at the Sediment/Water Interface
    R825513C023 Uptake of Metal Ions from Aqueous Solutions by Sediments
    R825513C024 Bioavailability of Desorption Resistant Hydrocarbons in Sediment-Water Systems.
    R825513C025 Interactive Roles of Microbial and Spartina Populations in Mercury Methylation Processes in Bioremediation of Contaminated Sediments in Salt-Marsh Systems
    R825513C026 Evaluation of Physical-Chemical Methods for Rapid Assessment of the Bioavailability of Moderately Polar Compounds in Sediments
    R825513C027 Freshwater Bioturbators in Riverine Sediments as Enhancers of Contaminant Release
    R825513C028 Characterization of Laguna Madre Contaminated Sediments.
    R825513C029 The Role of Competitive Adsorption of Suspended Sediments in Determining Partitioning and Colloidal Stability.
    R825513C030 Remediation of TNT-Contaminated Soil by Cyanobacterial Mat.
    R825513C031 Experimental and Detailed Mathematical Modeling of Diffusion of Contaminants in Fluids
    R825513C033 Application of Biotechnology in Bioremediation of Contaminated Sediments
    R825513C034 Characterization of PAH's Degrading Bacteria in Coastal Sediments
    R825513C035 Dynamic Aspects of Metal Speciation in the Miami River Sediments in Relation to Particle Size Distribution of Chemical Heterogeneity