Progress Report

CICEET Progress Report for the period 9/01/02 through 3/01/03

Project Title: Hydrogen-Enhanced Dechlorination In Contaminated Coastal Sediments
Principal Investigator(s): Peter Adriaens, Cyndee L. Gruden and John Hull

Accomplishments
Scheduled Tasks:
Complete Objective 2: Screening of hydrogen-enhanced dechlorination activity in sediment-eluted microorganisms, under optimal hydrogen fluxes and maximum hydrogenase activity.

Complete Objective 3: Assessment of hydrogen fluxes in capped and uncapped sediment column configurations.

Commence Objective 4: Monitoring of hydrogen-stimulated degradation reactions and contaminant mobilization in capped and uncapped columns.

Progress on Tasks
Progress was made on the scheduled tasks. Objective 3 was completed. Work on Objectives 2 (Screening of hydrogen-enhanced dechlorination activity in mixed sediment systems, under optimal hydrogen fluxes and maximum hydrogenase activity) and 4 (Monitoring of hydrogen-stimulated degradation reactions and contaminant mobilization in capped and uncapped sediments) is still ongoing.

Difficulties Encountered
We have had some difficulty locating and acquiring standard sediments for completion of Objective 2.

Preliminary Data
Objective 2: Screening of hydrogen-enhanced dechlorination activity in sediment-eluted microorganisms, under optimal hydrogen fluxes and maximum hydrogenase activity.
The addition of hydrogen to sediment-eluted microorganisms resulted in a statistically significant decrease in HCB over time. Initial degradation rates (t = 9 hours) were higher in sediment-eluted microorganisms exposed to hydrogen fluxes above the activity threshold (6.02 + 1.10 ng/cell/d) than those exposed to lower hydrogen fluxes (3.55 + 0.63 ng/cell/d) (data not shown). However, after 5 days of incubation, the sediment-eluted microorganisms exposed to lower fluxes of hydrogen resulted in increased degradation (See Figure 1, Hexachlorobenzene Degradation: 5-day exposure in resting cells). Several of the samples appear to contain a potential dechlorination daughter product (pentachlorobenzene). This compound was identified but not quantified.

Terminal restriction fragment length polymorphism (T-RFLP) was carried out on sediment-eluted microorganisms before and after hydrogen addition to document any changes in microbial community composition resulting from various treatments. FCM data reported earlier indicated a shift in community composition for samples containing 25nM H2 (See Figure 2). A flow cytometer was used to generate a density plot of green fluorescence (FL1) as a function of internal complexity (SSC) at (a) 0 nM H2 and (b) 25 nM H2. Community DNA was extracted and the phylogenetic marker 16S rRNA was used for microbial community analysis.

T-RFLP of sediment microorganisms in response to varying hydrogen amendments indicated a change in the community at both the lower and higher levels of hydrogen addition indicated by the appearance of a novel "peak" or rRNA fragment length in both instances (See Figure 3, Peak height as a function of DNA fragment length (in base pairs) is shown for (A) no hydrogen addition, (B) 2nM hydrogen addition, and (C) >25 nM hydrogen addition. The restriction enzyme used was MSPI. The primers selected were 8F and 1392R, which are used for bacteria only when analyzing 16S rRNA). Novel peaks occurred at 142 bp fragment length for low hydrogen addition and at 537 and 601 bp fragment length for high hydrogen addition (See Figure 3). These results suggest that selection may be occurring in sediment-eluted microbial populations exposed to hydrogen concentrations. An attempt to identify community members is currently underway using the Ribosomal Database Project website (www.rdp.cme.msu.edu).

This work is currently being repeated with sediments from San Diego Bay and from Pearl Harbor. Preliminary results indicate similar hydrogen thresholds for microorganisms eluted from these sediments. Degradation studies are also being carried out this quarter to determine if increases in CTC activity correlate with an increase in rate and extent of dechlorination for these additional sediment samples.

Objective 3: Assessment of hydrogen fluxes in capped and uncapped sediment column configurations.
The aim of this objective was to determine hydrogen diffusivity in sediments in order to establish a zone of influence for eventual field application of this technology. Preliminary research presented much difficulty in working with hydrogen gas over long periods of time. Discussion with colleagues at other institutions regarding this issue prompted a thorough literature review. An exhaustive literature search failed to identify any theoretical models specifically describing hydrogen gas diffusion in saturated porous media, such as sediments. However, accurate models describing gaseous hydrogen diffusion through unsaturated porous media were plentiful. Models developed for unsaturated porous media have been extended in a few instances to account for hydrogen gas diffusion in a fully saturated porous media. Two approaches are used to estimate permeability in porous, unsaturated soils; the Kozeny-Carmen approach, based upon path-length, tortuosity, and shape factors (Millington and Quirk, 1960) and the Childs and Collis-George "cut- and random-rejoin-type" approach, based upon a series parallel arrangement of pores within a porous material (Millington and Quirk, 1964). The work of Millington and Quirk and, later, Millington and Shearer (1971) has been extended by Collin and Rasmuson to account for gaseous hydrogen diffusion in saturated pores (Collin and Rasmuson, 1988). Only one measurement of gaseous hydrogen diffusivity in sediments was found in the literature, based upon in-situ measurement of gaseous hydrogen concentrations in sediments located in a fjord in British Columbia, Skan Bay, Alaska, and from the Mexican coastline (Novelli et al., 1987). This value was reported to be 1.3 X 10-5cm2 s-1. An investigation into models for calculating hydrogen diffusivity in sediments and sediment column experimentation were combined to provide a gaseous hydrogen diffusivity estimate.

Dissolved hydrogen concentrations in sediment columns have been investigated (See Figure 4, Experimental Apparatus for Measuring Hydrogen Diffusion in Sediments). Passaic River Estuary sediment (TS ~ 52% w/w; VS ~ %15 w/w), classified as very fine silt/clay using a standard particle size distribution, demonstrated a hydrogen diffusion rate in repeated experiments of approximately 5x10-5cm2/sec which is similar to diffusivity of hydrogen in water. Breakthrough was achieved in several column experiments, however, we were unable to capture a complete breakthrough curve due to eventual short-circuiting of hydrogen. The molecular size of hydrogen facilitates leaking and short-circuiting in column-based research. However, our results, similar to published literature, suggest that there may be difficulties in creating a sufficiently large zone of influence with passive diffusion due to the low solubility of hydrogen and the tortuosity of sediments. Although it has been determined in our work and in the literature that hydrogen is effective at stimulating dechlorination, difficulties with direct hydrogen addition resulting from its low aqueous solubility and molecular size was reported (Fang et al., 2002). The zone of influence will be studied in further detail in Objective 4 with CTC activity used as a surrogate for hydrogen concentration.

Collin M., Rasmuson, A., 1988, A comparison of gas diffusivity models for saturated porous media, Soil Science Journal of America, 52:1559-1565.

Fang, Y., Hozalski, R.M., Clapp, L.W., Novak, P.J. and Semmens, M.J., 2002, Passive dissolution of hydrogen gas into groundwater using hollow-fiber membranes, Water Research, 36: 3533-3542.

Millington, R., Quirk, J., 1960, Permeability of porous solids, Transactions of the Faraday Society, 57:1200-1207.

Millington, R., Shearer, R., 1971, Diffusion in aggregated porous media, Soil Science, 111, 372-378.

Millington, R., Quirk, J., 1964, Formation factor and permeability equations, Nature, 202:143-145.

Novelli, P., Scranton, M., Michener, R., 1987, Hydrogen distributions in marine sediments, Limnology and Oceanograpgy, 32:565-576.

Objective 4: Monitoring of hydrogen-stimulated degradation reactions and contaminant mobilization in capped and uncapped columns.
We are in the process of assembling columns for carrying out Objective 4. We expect this objective to be studied during Summer 2003. In this work, we are initially going to measure the microbial activity in relation to physical distance from the hydrogen source in an effort to determine the zone of influence for this hydrogen technology. Sediment subsamples will be processed, sediment bacteria will be eluted from subsamples, and CTC activity will be measured using a flow cytometer. Contaminant mobility will be measured with a Tenax trap located on the end of the column to account for ebullition of target contaminants during hydrogen stimulation.

Tasks and activities for the next reporting period

Tasks for the next reporting period
Complete Objective 2: Screening of hydrogen-enhanced dechlorination activity in sediment-eluted microorganisms, under optimal hydrogen fluxes and maximum hydrogenase activity.

Complete Objective 4: Monitoring of hydrogen-stimulated degradation reactions and contaminant mobilization in capped and uncapped columns.

Work plan to accomplish tasks
Complete Objective 2: Screening of hydrogen-enhanced dechlorination activity in mixed systems, under optimal hydrogen fluxes and maximum CTC activity.

A. Sediment Systems
Hydrogen-enhanced dechlorination activity of sediment-eluted microorganisms (three unique sediments) in standard sediments (purchased) spiked with model contaminant, under optimal hydrogen fluxes and maximum CTC activity.

Objective 2 Tasks in "Standard Sediment" Systems

  1. Elute bacteria from 3 types of sediments. For each sediment repeat tasks 2 through 6.
  2. Determine equilibrium time to reach steady state hydrogen concentration in mixed reactors.
  3. Determine incubation time to achieve maximum CTC activity.
  4. Establish hydrogen threshold above which a statistically significant increase in CTC activity is achieved.
  5. Evaluate shifts in microbial community in response to hydrogen fluxes using fluorescent microscope and FCM.
  6. Prepare resting cells above and below the hydrogen threshold and spiked with "standard sediments" to evaluate rate and extent of dechlorination.

Objective 2 Tasks Completion Date: 6/31/03

Complete Objective 4: Monitoring of hydrogen-stimulated degradation reactions and contaminant mobilization in capped and uncapped columns.

Objective 4 Tasks

  1. Acquire sediment apparatus and test.
  2. Amend sediments with the contaminant of choice.
  3. Prepare sterile controls.
  4. Carry out degradation experiment at achievable hydrogen fluxes for approximately 2 months.
  5. Dismantle columns and subsample sediments for analysis of contaminant of choice and microbial activity assessment.
Objective 4 Completion Date 08/30/03.

Concerns or difficulties
From our preliminary results regarding hydrogen diffusivity and the challenges associated with moving gases through sediments, it may be necessary to evaluate other hydrogen delivery mechanisms. Our final report will address this issue in further detail.

Expenditures
Expenditures were in the range anticipated for the work accomplished to date.

 

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