United States
             Environmental Protection
              Office of Research and
              (8701 R)
October 1999
1999 Bioremediation
Research Program

                     Bioremediation Research Program Review 1999

In July 1995, the Biotechnology Research Subcommittee of the Committee on
Fundamental Science (now the Committee on Science) of the National Science and
Technology Council published a report, Biotechnology for the 21st Century: New
Horizons. The chapter on Environmental Biotechnology in the report stated, "the
successful development and application of bioremediation technologies depends on field-
based research to verify the efficacy of planned approaches under natural conditions."

In Fiscal Years 1996,1997, and 1998, four federal members of the Subcommittee - the
U.S. Environmental Protection Agency, the National Science Foundation, U.S.
Department of Energy, and the Office of Naval Research - joined together to initiate
research at the field level that would address some of the scientific gaps and augment our
understanding of basic chemical and physical principles as applied to the use of
bioremediation technologies  for the cleanup of hazardous wastes. The interagency
committee focused the three  solicitations for research grants on the fundamental issue of
the bioavailability of chemicals for bioremediation processes in complex mixtures under
field conditions. In the three years of the program, 29 grants were awarded for research
on various aspects of bioavailability.

Bioavailability, defined for the purpose of this program as the availability of
contaminants to an organism (including microorganisms, plants, and animals) that might
degrade or otherwise transform them, is one of the principal factors controlling
bioremediation processes. The future of this technology will depend on our ability to
facilitate the interaction between the organisms and the chemicals in their environment so
that they can be utilized as carbon sources or be otherwise co-metabolized and thus
degraded. The physical, chemical, and biological aspects of these interactions are the
subjects of the research grants that have been awarded.

This second program review is held in conjunction with the EPA-sponsored workshop on
Innovative Clean-up Approaches: Investments in Technology Development, Results and
Outlook for the Future held hi Bloomingdale, Illinois, November 2-4,1999.  Presenting
the results  obtained by grantees in this program to workshop attendees will facilitate
communication with the bioremediation community and provide useful information
directly to users.  In addition, the opportunity for feed-back from users to researchers will
be useful hi influencing future research directions. The results presented at the program
review will  also inform the participating agencies about research questions that need their
future support.

                            Bioremediation Research Program Review 1999

Forward	i

Agenda	vi

Interaction of Heavy Metal Sequestration and Production of Metal ION Ligands in Wheat Under Fe
Deficient, CD, and Soil Humic Treatments	1
    Teresa W-MFan, Fabienne Baraud, and Richard Higashi

The Influence of Nanoporosity in Soils from Contaminated Sites on Hydrocarbon Desorption
Kinetics and Bioavailability	2
    W. Braida, J.C. White, J.J. Pignatello, P.I. Ravikovitch, R. Russo and A. Neimark
Assessment of Biotic and Abiotic Processes Controlling the Fate of Chlorinated Solvents in
Mixed-Waste Under Iron- and Sulfate-Reducing Conditions Using Laboratory and In-Situ
Microcosms	4
    Kim F. Hayes, Peter Adriaens, andMichaelJ. Barcelona

Phytoremediation and Modeling of Land Contaminated by Hydrocarbons	6
    Clyde Munster, Malcom Drew, M. Yavuz Corapcioglu,

In-Situ Assessment of PCS Availability in Unsaturated Soils	8
    Richard G. Luthy and Sean W. McNamara

Population Biology of Bacteria Involved in Contaminant Bioremediation	10
    DM. Ward, E.A. Kern, G.M. Colores and W.P. Inskeep

The Influence of Soil Characteristics and Molecular Properties of Hydrophobic Contaminants on
Bioavailability in Aged Soils	12
    Michael H. Huesemann, Ph.D., Tom Hausmann, Tim Fortman and Ann Drum

Biogeochemical Interactions in Reactive Zero-Valent Iron Barriers	14
    Pedro J.J. Alvarez,  E. Sawvel, M. Wildman, B. Eberle, K. Gregory, G.F. Parkin, and
    J.L. Schnoor

Controls on Plant Bioavailability in Salt Marsh Environments Which Can be Manipulated for
Contaminated Sediment Remediation	17
    Richard F. Lee, Marc E. Frischer and Herbert L. Windom

Bioavailability of Aromatic Contaminants Bound to Solid and Aqueous Phase Natural Organic
Matter	19
    Jon Chorover, Patrick G. Hatcher, William D. Burgos

The Effects of Soil Organic Matter on Mineralization, Desorption, and Sequestration/
Transformation of Phenanthrene	21
    S.B. Soderstrom, A.D. Lueking, M. Johnson, M. Kim, W. Huang, W.J. Weber Jr.

                             Bioremediation Research Program Review 1999
Bioavailability of Toxicants as Reflected in the In-Situ Microbial Community Ecology and
Relationship to Defensible End-Points	23
    S.J. MacNaughton, J.R. Stephen, Y-J. Chang, Y-D. Gan, J. Bownas, D.C. White, K.R. Carman, R.
    Millward, M. Barcelona,

The Effects of Aging and Sorbent Decomposition on the Bioavailability of Toluene and Xylene in Solid
Waste	25
    Morton a. Barlaz, DetlefR. U. Knappe, Bingyan Wu, Matthew Pelton, and Caleb Taylor

Preliminary Investigation of U and Ni in Riparian and Wetland Sediments: Microbial Ecology
and the Potential of Apatite Amendments for Reducing Metal Availability and Toxicity	27
    P.M. Bertsch, P.J. Morris, A.G. Sawder, T.V. Khijniak, andM.T. Novak

Understanding Seasonal Variation of Bioavailability of Residual NAPL in the Vadose Zone	29
    Paranjpy, B. Bierwagen, S. Sirivithayapakorn, A.A. Keller and P.A. Holden

Biosurfactant Specificity and Influence on Microbial Degradation of Hydrocarbons by Microbial
Consortia in the Field	31
    Gina S. Shreve, William Finnerty

The Effects of Plants on the Bioavailability and Toxicity of Contaminants in Soil	33
    M.K. Banks, A.P. Schwab, J. Scott Smith

Bioavailability of PCBs in Surfactant-Washed Soils With and Without UV-Irradation	35
    John Sanseverino, Alice Layton, Betsy Gregory, James Easter, Fu-Min Menn,  T. Wayne
   Schultz, and Gary S. Sayler

Biogeochemical Factors Limiting Transformation of Co-Occuring Contaminants in Salt Marsh
Sediments	38
    MarcE. Frischer, Keith A. Maruya, Joel E. Kostka, and Herbert L. Windom

Enzymology of the Degradation of Organometallic Compounds	40
    Ann O. Summers, Keith Pitts, and Andreas Heltzel

Bioavailability and Biostabilization on Multicomponent Non-Aqueous Phase Liquids (NAPLs) in the
Subsurface	41
    Anu Ramaswami, Tissa Illangasekare, Angela Bielefeldt, Kendra Morrison, Timothy J.
    Donahue, Erich Vestal and Allison Riffel

Bioavailability of Organic Contaminants in Estuarine Sediments to Microbes and Benthic
Animals	43
    KarlRockne, Gary Taghon, Lily Young, David Kosson, Wenhsin Liang, Leslie Shor

Role of Metal Bioavailability in In-Situ Bioremediation of Metal and Organic Co-Contaminated
Sites	45
    Raina M. Maier and Ian L. Pepper

                            Bloremediatlon Research Program Review 1999
Wetland Plants' Roles in Uptake and Transport of Heavy Metals and Remediation	47
   Judith S. Weis andPeddrick Weis

Interaction Between Substrata Surface Chemistry, Conformation of Contaminant Upon
Adsorption and Availiability for Bacterial Degradation	49
    James D. Bryers

Bioluminescent Sensors for Measuring Pollutant Bioavailability and Validity of Environmentally
Acceptable Endpoints in Bioremediation	52
    Lisa Strong, Lawrence P. Wackett, and Michael J. Sadowsky

Influence of Soil Organic Matter Composition on Desorption and Biodegradation of Aromatic
Pollutants	53
    T.M. Young, KM. Scow, R.M. Higashi, T.W-MFan, E.Schwartz, L.F. SchultzN.  Watanabe
    and S. Loebmann

Genetic Expression During Biofilm Growth and Development	54
   R.S. Burlage, I. Foley, and R. Palmer

                        Biorcmedation Research Program Review 1999
                             NOVEMBER 3,4, & 5,1999

Wednesday. November 3.

10:00-10:30  Welcome and Introduction (Paul Bayer, DOE)

10:30       Clyde L. Munster, Malcolm Drew, M. Yavuz Corapcioglu,
            Texas A&M University.
             Phytoremediation and Modeling of Land Contaminated by Hydrocarbons

11:00       Richard F. Lee, Marc E. Frischer, Herbert L. Windom,
            Skidaway Institute of Oceanography.
            Controls on Plant Bioavailability in Salt Marsh Environments Which Can be
            Manipulated for Contaminated Sediment Remediation

11:30       Karl Rockne, Gary Taghon, Lily Young, David Kosson, Wenhsin Liang, Leslie
            Shor, Rutgers University.
            Bioavailability of Organic Contaminants in Estuarine Sediments to Microbes and
            Benthic Animals

11:30 to 1:15 Lunch

1:15        Marc E. Frischer, Keith A. Maruya, Joel E. Kostka, Herbert L. Windom,
            Skidaway Institute of Oceanography.
            Biogeochemical Factors Limiting Transformation of Co-Occurring Contaminants
            in Salt Marsh Sediments.

1:45        Peddrick Weis, Judith Weis, Rutgers Unversity.
            Wetland Plants' Roles in Uptake and Transport of Heavy Metals and Remediation

2:15 to 2:30  Break

2:30        John Sanseverino, Alice Layton, Betsy Gregory, James Easter, Fu-Min Menn,
            T. Wayne Schultz, Gary S. Sayler, University of Tennessee.
            Bioavailability of PCBs in Surfactant-Washed Soils With and Without UV-

3:00        Gina S. Shreve, William Finnerty, Wayne State University.
            Biosurfactant Specificity and Influence on Microbial Degradation of Hydrocarbons
            by Microbial Consortia in the Field.

3:30        Todd Sandrin, Raina M. Maier, Ivan L. Pepper, University of Arizona.
            Role of Metal Bioavailability in In Situ Bioremediation of Metal and Organic Co-
            Contaminated Sites

                        Bioremedation Research Program Review 1999
4:00        Teresa W-M. Fan, Fabienne Baraud, Richard Higashi, University of California,
            Interaction of Heavy Metal Sequestration and Production of Metal Ion Ligands in
            Wheat Under Fe Deficient, CD and Soil Humic Treatments

4:30        Anne O. Summers, Keith Pitts, Andreas Heltzel, University of Georgia.
            Enzymology of the Degradation of Organometallic Compounds

5:00        Speaker's corner Exhibits/Poster S essions
Thursday, November 4.1999

Recommend Session "A" on Metals - Unavailability for early birds

9:15-9:30  Comments by Linda Chrisey (ONR) '

9:30        Lisa Strong, Lawrence P. Wackett, Michael J. Sadowsky
            Bioluminescent Sensors for Measuring Pollutant Bioavailability and Validity of
            Environmentally Acceptable Endpoints in Bioremediation

10:00       Tom M.Young, K.M. Scow, R.M. Higashi, T.W-M. Fan, E.Schwartz, L.F. Schultz,
            N. Watanabe, S. Loebmann, University of California, Davis.
            Influence of Soil Organic Matter Composition on Desorption and Biodegradation
            of Aromatic Pollutants.

10:30       Morton A. Barlaz, Detlef R.U. Knappe, Bingyan Wu, Matthew Pelton, Caleb
            Taylor, North Carolina State University.
            The Effects of Aging and Sorbent Decomposition on the Bioavailability of
            Toluene and Xylene in Solid Waste

11:00-12:00 Lunch

1:00        James D. Bryers, University of Connecticut.
            Interaction Between Substrata Surface Chemistry, Conformation of Contaminant
            Upon Adsorption and Availability of Bacterial Degradation

1:30        Michael H. Huesemann, Tom Hausmann, Tim Fortman, Ann Drum, Battelle
            Pacific Northwest Division, Marine Science Laboratory.
            The Influence of Soil Characteristics and Molecular Properties of Hydrophobic
            Contaminants on Bioavailability in Aged Soils

                        Bioremedation Research Program Review 1999
2:00        S.B. Soderstrom, A.D. Lueking, M. Johnson, M. Kim, W. Huang, W.J. Weber Jr.,
            University of Michigan.
            The Effects of Soil Organic Matter on Mineralization, Desorption, and
            Sequestration/Transformation of Phenanthrene

2:30-3:00    Break

3:00        Pedro Alvarez, E. Sawvel, M. Wildman, B. Eberle, K. Gregory, G.F. Parkin,
            J.L. Schnoor, University of Iowa.
            Biogeochemical Interactions in Reactive Zero-Valent Iron Barriers

3:30        Jon D. Chorover, Patrick G. Hatcher, William D. Burgos, Pennsylvania State
            Bioavailability of Aromatic Contaminants Bound to Solid and Aqueous Phase
            Natural Organic Matter

4:00        Sean W. McNamara, Richard G. Luthy, Carnegie Mellon University.
            Bioavailability and Biostabilization of PCBs in Soils

4:30        Mark Brusseau

5:00        Adjourn for the day
Friday 5.1999

8:30 - 9:30   Check Out

9:30         Ami Ramaswami, Tissa Illangasekare, Angela Bielefeldt, Kendra Morrison,
             Timothy J. Donahue, Eric Vestal, Allison Riffel, University of Colorado.
             Bioavailability and Biostabilization of Multicomponent Non-Aqueous Phase
             Liquids (NAPLs) in the Subsurface

10:00-10:15 Break

10:15        P. A. Holden, A. Paranjpye, B. Bierwagen, S. Sirivithayapakorn, A.A. Keller,
             University of California, Santa Barbara.
             Understanding Seasonal Variation of Bioavailability of Residual NAPL in the
             Vadose Zone

10:45        David M. Ward, E.A. Kern, G.M. Colores, W.P. Inskeep, Montana State
             Population Biology of Bacteria Involved in Contaminant Bioremediation

11:15        Paul M. Bertsch, P. J. Morris, A.G. Sowder, T. V. Khijniak, University of Georgia.
             Microbial Ecology and the Potential of Apatite Amendments for Reducing Metal
             Availability and Toxicity

                        Bioremedation Research Program Review 1999
11:45-1:00 Lunch

1:00        A. P. Schwab, M.K. Banks, J. Scott Smith, Purdue University.
            The Effect of Plants on the Bioavailability and Toxicity of Contaminants in Soil

1:30        J.J. Pignatello, J.C. White, P.I. Ravikovitch, R. Russo, A. Neimark, Connecticut
            Agricultural Experiment Station.
            The Influence of Nanoporosity in Soils From Contaminated Sites on Hydrocarbon
            Desorption Kinetics and Bioavailability

2:00        Sara J. MacNaughton, J.R. Stephen, YJ. Chang, Y.D. Gan, J. Bownas,
            K.R. Carman, R. Millward, M. Barcelona, D.C. White, University of Tennessee.
            Bioavailibility of Toxicants as Reflected in the In-Situ Microbial Community
            Ecology and Relationship to Defensible End-Points

2:30        Peter Adriaens, Kim F. Hayes, Michael J. Barcelona, University of Michigan.
            Assessment of Biotic and Abiotic Processes Controlling the Fate of Chlorinated
            Solvents in Mixed-Waste Under Iron and Sulfate-Reducing Conditions Using
            Laboratory and In-Situ Microcosms

3:00 - 3:30  Wrap-up R.E. Menzer, (EPA)

                            Bioremediation Research Program Review 1999
  Interaction of Heavy  Metal Sequestration and  Production of Metal ION
  Ligands in Wheat Under FE Deficient, CD, and Soil Humic Treatments

                          Teresa W-M Fan, Fabienne Baraud, and Richard Higashi
                                   University of California, Davis, CA

Root exudation of metal ion ligands (MIL) such as phytosiderophoresis vital to nutritional acquisition of Fe and Zn,
and may also be important to metal contaminant mobilization by plants.  We have developed a broad  chemical
analysis of unfractionated exudates and plant tissue extracts for  MIL by  multidimensional NMR  and  GC-MS.
Organic anions including amino and organic acids and mugineic acid (MA)  phytosiderophores were identified and
quantified.  SH-rich peptides were also analyzed using  fluorescent tag and SDS-PAGE. The MIL profile differed
among plants and genotypes. Cd treatments of wheat caused a large reduction in the exudation of 2'-deoxymugineic
acid  (2'-DMA) and other MIL and yet a  substantial  increase  in Fe, Zn,  Cu, and Ni sequestration into roots,
regardless of the Fe sufficient or deficient status. This suggests a higher efficiency of MIL or a different mechanism
in facilitating transition metal (TM) uptake in Cd-contaminated rhizosphere.  A large increase in the production of
tissue SH-rich peptide and other MIL in Cd-treated wheat tissues may be related to the intracellular immobilization
of excess Cd and transition metal ions.

In addition, we investigated the interaction of soil humic substance (HS, an important extant rhizosphere ligand) and
Cd on metal ion uptake by wheat. The Cd treatment resulted in a large increase of metal ion content of Fe, Ni, Cu,
Zn, and Mn in roots but not in shoots (except for Zn). Zn translocation to shoots was enhanced by the Cd treatment.
The co-treatment of soil HS induced a biomass increase  and  a  higher exudation of 2'-DMA,  acetate, lactate,
glycinebetaine, Ala, and g-aminobutyrate. This was, in turn correlated with  a higher sequestration of Cd, Mn, Zn,
and Ni into roots. The HS alone treatment also stimulated biomass production but a significant decrease in the
exudation of major ligands 2'-DMA, malate, and acetate. The latter was related to the decreased content of Fe and
Zn in roots and that of Mn, Fe, Zn, Cu, and Ni in shoots.  It is likely that  the exudation of some of all of these
components were involved in the metal ion mobilization.

The concentrations of several MIL  (malate, citrate, lactate, Asn, and glyceraldehyde-3-phosphate) in wheat roots
were positively correlated with the root content of Fe, Ni, Cu, Mn, Zn, and Cd in the HS/Cd treatment series. It is
likely that these compounds were involved in the intracellular chelation of excess metal ions, in addition to that by
SH-rich peptides.
                                            Page 1 of54

                             Bioremediation Research Program Review 1999
    The  Influence of Na no porosity in Soils from Contaminated Sites on
               Hydrocarbon  Desorption  Kinetics and  Bioavailability

     W. Braida, J.C. White, J.J.Pignatello, The Connecticut Agricultural Experiment Station, New Haven; and
                  P. I. Ravikovitch, R. Russo, and A. Neimark, TRI/Princeton, Princeton, NJ

Biodegradation of contaminants in soil and sediment is often rate limited by contaminant mass transfer from remote
to more accessible microregions of soil particles. An  understanding of the mechanisms responsible for desorption
resistance is the subject of this research. We hypothesize that desorption resistance is due to hindered diffusion in
nanopores of molecular scale (0.2-2 nm) existing within  the interstices of soil organic  matter (SOM) and mineral
aggregates. The objectives of the work are to develop methodology to determine soil nanoporosity based on CO2
adsorption linked with theoretical models, and then to assess the relationship between  nanoporosity  and physical-
chemical and biological availability parameters for aromatic hydrocarbons.  In addition,  we suggest it is possible to
alter SOM nanoporosity by changing its glassy/rubbery properties via certain treatments or addition of co-solutes.

Phenanthrene sorption rates were measured  for soils spanning a wide  range  of organic  carbon (OC) content
(Wurtsmith AFB 1AB, 0.18 %; Mount Pleasant Silty  Loam,  4.45%; Pahokee Peat soil,  an IHSS reference sample,
43.9%). Characteristic diffusion times (D/L2, where D is the effective diffusion coefficient and L is the diffusion
path length) were estimated for 'infinite  bath' conditions (i.e., constant phenanthrene  concentration) by fining a
radial diffusion model to sorbed concentration data estimated from experimental Freundlich isotherms measured at
different times in batch experiments. The value of D/L2 (Figure 1) is seen to depend on phenanthrene  concentration
and on soil (Braida and Pignatello, submitted for publication).  The concentration dependence is a consequence of
the thermodynamic nonlinearity of sorption and may be related to the glassy character of the SOM.

Additional  work has focussed on competitive effects among  PAHs.  The existence  of competition  supports a
heterogenous 'polymer'  model of soil organic matter and is  relevant to contaminated sites where  mixtures are
commonplace. Work in the first year (White et al., Environ. Toxicol. Chem., 18:1728-32) showed that competitive
displacement of phenanthrene by pyrene increased the bioavailability  of phenanthrene.  Current research sought to
determine  whether competition was expressed thermodynamically or kinetically,  or both.   Pyrene significantly
reduced the sorption of phenanthrene and increased the  linearity of its isotherm.  Moreover, normalized rates of
phenanthrene  desorption  at  constant pyrene  concentration  increased  significantly with  increasing pyrene
concentrations (e.g., Fig 2).  This effect was observed  in two soils of widely different OC contents, and even at low
and equimolar phenanthrene and pyrene (closed circles). Competition may be due to the  plugging of pores in glassy
organic matter by the co-solute.

We have developed and verified a new experimental protocol and molecular level theoretical models for assessing
nanoporosity in soils. The micropore size distributions are calculated from comparison of the experimental gas
adsorption isotherms and the theoretical isotherms in model pores predicted by means of density functional  theory
(DFT) and grand canonical  Monte Carlo (GCMC) simulations.  The DFT model, theoretical  foundations of which
were formulated in our earlier work, has  been modified  by taking into account specifics of CO2 interaction  with
carbonaceous matrixes. A new set of  intermolecular parameters for CO2/solid  potentials was defined and verified
against literature data. The MC simulation model has been developed and  is being verified against reference
samples. Preliminary results demonstrate consistency of the results of DFT and MC models.

We have designed and fabricated a new electric thermostat allowing us to maintain temperature in the adsorption
cell in the range -20° - +40 °C with a precision of 0.1 °C.  Isotherms at different temperatures allow us to estimate
the heat of sorption (see, for example, Figure 3).

The CO2 adsorption isotherms at 0°C and N2 adsorption isotherms at  77K on  20+ soils samples and reference
sorbents were measured and the pore size distributions were constructed. The CO2 adsorption-desorption isotherms
showed hysteresis that presumably indicates irreversibility of sorption in SOM nanopores. Hysteresis was especially
strong in samples having high OC content: peat and its purified humin and humic  acid fractions (Figure 4).  CO2
desorption was hindered until the vapor pressure was reduced tenfold. The adsorption isotherm on peat and humic
acid revealed a bend not observed in humin. This behavior probably indicates a phase transformation in the  humic
acid fraction upon CO2 sorption.  The hysteresis phenomenon requires further  study. We are currently developing
                                             Page 2 of 54

                              Bioremediation Research Program Review 1999
molecular models of sorption hysteresis that can be tested against capillary condensation isotherms collected on
reference adsorbents such as mesoporous molecular sieves.

We hypothesize that the nanopores of 0.3 - 1  nm  revealed in CO2 sorption measurements are responsible for a
delayed desorption kinetics of organic contaminants sequestered in  pores of soil particles. The  results obtained
justify that CO2 is a suitable molecular probe to study structural and sorption-desorption properties of soil particles
containing SOM.
          1e-6 -
            00001   0001    001    0.1
              Aqueous Phenanthrene cone. (
                                                                             i r f  i    r
                                                                             1234    5
                2         4
                 Amount adsorbed,
Figure 1. (upper left). Characteristic diffusion time in three soils as a function of phenanthrene concentration.

Figure 2. (upper right). Effect of pyrene on desorption of phenanthrene aged for 42 days in Cheshire fine sandy
laom  using  a sequential dilution technique in which the supernatant in each cycle was replaced with water at
constant pyrene concentration. The mm\6al is the initial mass present on the solids.  Pyrene was added 14 days before
phenanthrene at: 0.0 (•), 114 (O), and 1010 (D) ug (g OC)-1.

Figure 3. (lower left). Isosteric heats of CO2 sorption on Pahokee peat derived from the adsorption branches of the
isotherms at three temperatures using Clausius-Clapeyron equation.

Figure 4. (lower right).  CO2 adsorption-desorption isotherms at 0 °C on  Pahokee peat soil (93% SOM) and its
humin and humic acid fractions. Extreme  low-pressure hysteresis between sorption and desorption  isotherms is
observed. This suggests that sorption in the organic domain (SOM) is responsible for contaminant retention by soils.
                                               Page 3 of54

                             Bioremediation Research Program Review 1999
EPA Grant: R82-5958

    Assessment of Biotic and Abiotic  Processes Controlling the Fate of
 Chlorinated Solvents in Mixed-Waste Under Iron- and Sulfate-Reducin
              Conditions Using Laboratory and In-Situ Microcosms

                          Kim F. Hayes, Peter Adriaens, and Michael J. Barcelona
                               The University of Michigan, Ann Arbor, MI

The major objective of this  research is  to evaluate the relative importance of biotic and  abiotic reductive
dechlorination processes under iron- and sulfate-reducing conditions in both simple and mixed-waste systems. In
this project period, we performed abiotic reductive dechlorination using  synthetic Fe(II)  solids  as well as purel
biological studies  aimed at quantifying reductive dechlorination  in the absence of solids under sulfate and iron
reducing conditions. A comparison of rates and reaction products provides one means by which biotic and abiotic
transformation  might be distinguished.  We obtained a quantitative description of the kinetics of the abiotic
transformation of trichloroethylene (TCE) and tetrachloroethylene (PCE) b  FeS. Rates of hexachloroethane (HCA)
reductive dechlorination by FeS in the presence of metals and oxyacids were also measured to simulate the impacts
of mixed-waste composition.  In our  biological studies, the kinetics of  cell mediated carbon tetrachloride (CT)
dechlorination were characterized for the iron reducing bacteria Geobacter metallireducens.  Two sulfate reducing
bacteria, Desulforeducens autotrophica and Desulfovibrio vulgaris subsp. vulgaris have been  cultured during this
project  period  and  are  currently being  used to  generate FeS for  reductive dechlorination in sulfidogenic

Abiotic experiments were performed to determine the rates and products of transformation of TCE and PCE b  FeS.
The principal reaction product for TCE transformation by FeS was acetylene, while cw-l,2-dichloroethylene (cis-
DCE) and vinyl chloride (VC) were minor products. Data were interpreted assuming parallel transformation of TCE
to these two products, with VC most  likely forming from slow hydrogenolysis of cis-DCE.  The solution to the
differential equations describing such a reaction scheme indicated that TCE was transformed to  acetylene 11.8 ± 1.1
times faster than to cis-DCE. Detection of acetylene as the principal TCE reductive dechlorination product contrasts
with the sequential hydrogenolysis commonly observed in  the transformation of TCE in microbiological systems,
which can result in the accumulation of significant quantities of the harmful intermediates ds-DCE and VC. Similar
results were obtained for the transformation of PCE b  FeS. In addition, the impact of the metals Ag(I) Cu(II), and
Cr(III) and oxyacids chromate, arsenite, and selenite on HCA reductive dechlorination by FeS was investigated. T
date, the results indicate the softer metal acids Ag(I), Cu(II) tend to increase reductive rates while hard acid Cr(III)
and the oxyacids tend to reduce the rate.  We hypothesize that the former  interact with the Fermi level of FeS(s), a
known conductor,  while the later reduce reactivity by undergoing redox reactions with FeS(s). These studies reveal
that metals and oxyacids present in mixed wastes may impact reductive dechlorination.

Biological experiments have  been conducted using cell suspensions of  the dissimilative iron  reducing bacteria
Geobacter metallireducens    At carbon  tetrachloride  (CT) concentrations  less than 100  uM, cell mediated
dechlorination rates were found to follow first order kinetics.  The only volatile product detected was chloroform
(CF) which accounted for approximately 15% of the transformed CT. Approximately 85% of transformed C  was
recovered as either a cell bound product (68%) or as a  non-volatile aqueous product presumed to be cell lysate
(17%).  By contrast in magnetite mediated dechlorination, we have observed a similar amount of CF formation
(-20-30%) but also a minor amount of methane (-4-5%).  This suggests  methane may be a positive indicator for
Fe3O4 mediated CT dechlorination in systems containing both magnetite and iron reducing bacteria.

Extending the biological investigations to sulfidogenic environments, two pure cultures of sulfate-reducing bacteria,
Desulforeducens autotrophica and Desulfovibrio vulgaris subsp. vulgaris, were grown in liquid media under a range
of environmental  conditions to determine  the  effect of solution chemistry on bacterial  activity and iron solids
production. Preliminary results show that D. vulgaris, a freshwater species, is the more robust strain for generating
biologically-modified iron solids.

In the future, abiotic reductive dechlorination by biogenically produced FeS will be investigated.  A more extensive
evaluation of abiotic transformations of HCA, PCE and TCE by magnetite as a function of solution conditions will
also be conducted, as well as reductive dechlorination by FeS and magnetite in the presence of  metal cations and
oxyacids.  Future biological work will include examination of the impact  of temperature on biological and abiotic
                                             Page 4 of 54

                                Bioremediation Research Program Review 1999
reductive dechlorination.   We also anticipate  setting up  several in-situ  microcosm (ISMs) this Fall at the FT2
training  site at the Wurtsmith Air Force Base in Oscoda, MI.  Once the ISMs  are  in  place,  sulfate reducing
conditions will be stimulated and HCA will be injected and reductive dechlorination activity monitored for up to 120

Publications and Presentations

Jeong, H. and K. F. Hayes (1999), "Impact of Transition Metals and Oxyacids on Reductive Dechlorination of HCA by Iron
Sulfide,"  22nd Midwest Environmental Chemistry Workshop, Michigan Technological University, Houghton, Michigan, October
1-3, 1999.

Butler, E. C.  and K. F. Hayes (1999), "Kinetics of the Transformation of Trichloroethylene and Tetrachloroethylene by Iron
Sulfide,"Environ. Sci. Technol., 33, 2021-2027.

Adriaens, P.,  McCormick, M.L., Butler,  E.G., and  Hayes, K.F. (1999), "Biotic  and Abiotic Dechlorination of Alkyl Halides
under Iron and Sulfate Reducing Conditions," Swiss Federal Institute for Environmental Science and Technology,  Dubendorf,

McCormick, M.L., and P. Adriaens (1999), "Biotic and Abiotic Reductive Transformation of Chlorinated Solvents in Iron
Reducing Sediments," Fourth Annual EPA STAR Graduate Fellowship Conference, July  17-20, Arlington, VA.

McCormick,  M.L.  and P. Adriaens (1999), "Reductive  Transformation of Carbon Tetrachloride by the Dissimilative Iron
Reducing Bacteria Geobacter metallireducens," 99th General meeting of the American Society for Microbiology, May 30-June 3,
Chicago,  IL.

Butler, E. C. and K. F. Hayes (1999), "Correlation Analysis in the Reductive Transformation of Halogenated  Organic
Compounds by Iron Sulfide," 217th American Chemical Society National Meeting, March 21-25, 1999, Anaheim, California.

Butler, E. C. and K. F.  Hayes (1998), "Transformation  of Trichloroethylene and Tetrachloroethylene by Iron Sulfide" 21st
Midwest  Environmental Chemistry Workshop, University of Michigan, Ann Arbor, Michigan, October 16-18, 1998.

Danielsen, K. M.  and K. F. Hayes (1998),  "Abiotic Reductive Dechlorination of HCA  by Reduced  Iron" 21st Midwest
Environmental Chemistry Workshop, University of Michigan, Ann Arbor, Michigan, October 16-18, 1998.

McCormick, M. L., H. S. Kim, and P. Adriaens (1998), "Reductive Transformation of Chlorinated Organic compounds by
Biologically Generated Magnetite"  21st Midwest Environmental Chemistry Workshop, University  of Michigan, Ann Arbor,
Michigan, October  16-18,1998.

McCormick, M.L., H.S.  Kim,  E.J.  Bouwer,  and P. Adriaens  (1998),  "Abiotic transformation  of chlorinated solvents as a
consequence  of microbial  iron  reduction:  An investigation  of  the  role of  biogenic magnetite in   mediating reductive
dechlorination," Proceedings of the  30th  Mid-Atlantic Industrial and Hazardous Waste Conference, p.  339-349,  Technomic
Publishing, Lancaster, PA.

McCormick, M.L., and P. Adriaens (1998), "Tetrachloroethylene Transformation in an Iron Reducing Enrichment Culture," 98*
General meeting of the American Society for Microbiology, May 17-21, Atlanta, GA.
                                                  Page 5 of 54

                              Bioremediation Research Program Review 1999
           Phytoremediation and Modeling of Land  Contaminated by

    Clyde Munster, Associate Professor, Agricultural Engineering Dept, Malcolm Drew, Professor, Horticultural
       Sciences Dept.,  M. Yavuz Corapcioglu, A. P. and Florence Wiley Professor, Civil Engineering Dept.,
                                         Texas A&M University.

Plants may assist in the remediation  of recalcitrant chemicals at contaminated sites by various processes. The
possible  use  of deep-rooted plants  for phytoremediation of soil contamination has been offered as  a potential
alternative for waste management, particularly for in-situ remediation of large volumes  of contaminated soils. In
addition, a phytoremediation computer model  has been recently  developed for predicting the fate of  recalcitrant
hydrocarbons in soil. However, the model requires extensive testing with field data for validation and  calibration.
Therefore, the objective of this research was to undertake the principles, as well as the practices, of dealing with the
persistence of recalcitrant contaminants in the soil and  their simultaneous removal by plants, as well as the
collection of field data for model testing.

For this  research project, a  warm season grass (Johnsongrass) and a cool season grass (Canada wild-rye) were
chosen to evaluate the effectiveness of phytoremediation of soil contaminated with  a recalcitrant mix  of a
polybrominated biphenyl (PBB, 2,2',5,5'-tetrabromobiphenyl), a polycyclic aromatic hydrocarbon (PAH), chrysene,
and 2,4,6-trinitrotoluene  (TNT).  Two types  of lysimeters were developed  for field-testing. Twelve metal box
lysimeters, 1.5 m x 1.5 m x 0.75 m (length,  width, height) and 72  poly vinyl chloride (PVC) column lysimeters, 0.1
m diameter x 1.5 m in height were fabricated.  A translucent, corrugated PVC panel roof that provided shelter from
the rain  covered both sets of  lysimeters. A leachate collection system  was installed in  each lysimeter to obtain
leachate  for chemical analysis. The  box and column lysimeters were  placed  in the  ground to maintain an in-situ
temperature gradient throughout the  soil profile. The lysimeters were filled with virgin Weswood silt loam soil (23
% sand, 47, % silt, 30 % clay, 0.8 % organic carbon, and 7.9 pH). That was mixed with chrysene, TNT, and PBB to
a target concentration of 10  mg of each contaminant per kg of soil. As  the soil was added to the lysimeters,  time
domain reflectance  (TDR) probes, for soil moisture determination, were placed at depths of 0.125,  0.375, and
0.625m.  The soil was packed to a bulk density of 1400 kg m"3 to match field values.

Chemical losses during the initial 360 days of this experiment were similar for both box and column lysimeters, at
all depths, and between vegetated and unvegetated soils (Figure 1). The largest and most rapid loss in soil-chemical
concentration was TNT, which decreased to < 10 ug kg"1 after 360 days. Contaminant detection in plant herbage and
leachate has been insignificant. Enumeration of soil microorganisms reveals a robust population in both the bulk soil
and root rhizosphere,  but no significant differences. Field data are  being used to calibrate and validate the
phytoremediation computer model.  Simulations with TNT demonstrate that  the model  can predict decreases in
contaminant concentration as observed from actual field conditions (Figure 2).  Simulation for PAH and PBB are
currently being evaluated. Additional model  parameters  are being examined  and samples will continue to  be
collected and analyzed during a two-year period. The validated and calibrated  computer model may provide insight
into the selection and optimization of phytoremediation at contaminated sites.

Publications  and Presentations
Colville, C. J.,  R. L. Rhykerd, C. L. Munster, M. C. Drew and M. Y. Corapcioglu. 1999. Phytoremediation of soils
contaminated by TNT, PBBs and PAHs. The 1999 ASAE Annual International Meeting, Toronto, Canada, July 18-21, 1999.
ASAE Paper No. 992183. St. Joseph, MI, ASAE.
Corapcioglu, Y.M., C.L. Munster, M.C. Drew, R.L. Rhykerd, K. Sung, and Y.Y. Chang.  1999. Phytoremediation and modeling
of land contaminated by hydrocarbons. Proceedings, 5th International In  Situ On-Site  Bioremediation Symposium. San Diego,
Corapcioglu, M.Y., K. Sung, R.L. Rhykerd, C.L. Munster, and M.C. Drew. 1999.  Validation of a phytoremediation computer
model. Proceedings, Phytoremediation Technical Seminar. Calgary, Canada.
Corapcioglu, Yavuz  M., Robert  L. Rhykerd,  Clyde L.  Munster, Malcolm C. Drew, and Kijune Sung. 1998.  Modeling
phytoremediation of land contaminated  by hydrocarbons. Proceedings, The Fifth International Conference on remediation of
Chlorinated Compounds. Monterey, CA.
Rhykerd*, R. L., M. T. Hallmark* and C. L. Munster. 1998. A field facility for phytoremediation research. The 1998
ASAE  Annual International  Meeting, Orlando,  FL, July 11-16,  1998.  ASAE  Paper  No.  985026. St. Joseph,  MI,
                                               Page 6 of 54

                             Bioremediation Research Program Review 1999
                                        120      180     240
                                           Day of experiment
Figure 1. Soil concentrations of TNT, PBB, and PAH from column lysimeters under Johnsongrass, Canada wild-
rye, a Johnsongrass/Canadian wild-rye rotation or fallow.
                           0.0 1
> day 30 simulation
 day 90 simulation
 day 30 field data
 day 90 field data
60       90
 Depth (cm)
Figure 2. Loss of TNT from a contaminated Weswood soil determined by immunoassay analysis of field soil
(symbols) and predicted by computer model simulations  (lines). Computer simulations were based upon soil
parameters from the field  study. Analyses  and  simulations were made 30  and 90  days  after the soil  was
contaminated with 10 mg kg"1 TNT.
                                             Page 7 of 54

                              Bioremediation Research Program Review 1999
EPA Grant: R82-5365

          In-Situ Assessment of PCB Availability in Unsaturated Soils

                                 Richard G. Luthy and Sean W. McNamara
                Department of Civil and Environmental Engineering, Carnegie Mellon University

The objective of the current study is to develop understanding of the relationship between bioavailability and
biostabilization of polychlorinated biphenyls (PCBs) in aged soils that have undergone active land biotreatment, but
which still show a residual concentration.  At the study's conception we recognized a deficiency in field sampling
techniques that could assess the bioavailable fraction of PCBs, and other hydrophobic organic compounds (HOCs),
in impacted soils. Here, the bioavailable fraction is considered that present in the aqueous phase - i.e., available for
active or passive diffusion across a microbial membrane. Consequently, the research direction under this assistance
grant has been focused primarily on the  development  of field sampling devices and procedure  to measure the
mobility and availability of PCBs in unsaturated soils.

The new sampling device consists of a cylindrical porous stainless steel interface, a sorbent packing media (granular
activated carbon), and a fiberglass wick. The interface details are shown in Figure 1. The device can be installed in
field soils by direct-push or a soil auger pilot-hole, thus minimizing installation  requirements.  The device uses the
capillarity of a fiberglass wick and elevation potential to provide the necessary driving force to slowly sample water
from  unsaturated soils, thereby eliminating  the need for an external power source.  Water is drawn through the
cylindrical interface, into and along the wick, and is collected to quantify the amount of water sampled (maintains a
water balance). The annular space of the cylinder is packed with a sorbent media, so water that passes through the
interface must pass through the sorbent media before entering the wick.  Target constituents are  retained on the
sorbent  media for chemical analysis (allows for a chemical mass balance with  minimal losses) at the end of the
sampling interval (weeks or months). In this way, the new sampling device is designed to provide an integrated_/?eW
estimate of the time-averaged mass and volumetric flux rates of HOCs in the soil.  A patent application has been
submitted for the new device and sampling technique.

The new sampling  device has  been  optimized for its hydraulic performance when installed  in a  horizontal
configuration (Figure 2).  This configuration would  be  applicable for  shallow  soils in lined land  treatment units,
biopiles, confined disposal facilities, etc. (McNamara, 1999).  Laboratory and field evidence indicate that the device
can withstand  extended periods of drought and recover  hydraulically  when water becomes available  in  the
surrounding soil pore-water.  A hydraulic model has been conceived to predict the sampler's performance in soil
under a range of moisture conditions.

The device is currently being tested for  its chemical  capture  potential  in PCB-impacted soils.   Mass balance
calculations based on the analysis of the sampler's sorbent material and concurrent leaching studies should provide
insight into the sampler's ability to accurately predict the flux of PCBs in the subsurface. A new pressurized-solvent
extraction procedure has been developed to recover low-level PCBs from the sampler's sorbent material - granular
activated carbon. The new procedure allows for  higher recoveries and smaller RSDs than is possible with the
typical extraction procedures  for soil  (i.e.,  Soxhlet or sonication).  Similar procedures for additional HOCs (e.g.
PAHs and pesticides) are currently being evaluated.

The presentation will overview the hydraulic performance of the device in lab and field trials,  discuss work on the
chemical evaluation of the sampling device, and present preliminary information  on the field work scheduled for the
Spring of 2000 in PCB-impacted soils.
                                              Page 8 of 54

                               Bioremediation Research Program Review 1999
porous stainless steel cylinder
  pipe or tubing
                   sorbent material

15-cm porous sampling section
                                                end view
                                                of sampler
                                                               media fill hole
                                                                and conical tip
Figure 1.  Cross-section and plan view of new sampling device design
Figure 2.  Horizontal installation of sampling device in shallow soil.
Publications and Presentations
McNamara, S. W.  (1999). "Hydraulic Evaluation of an In Situ  Sampling Device for  Measuring  Hydrophobic Chemical
Availability in Unsaturated Soils," M.S. Thesis, Carnegie Mellon University, Pittsburgh.

McNamara, S.  W.. and Luthy, R. G. (1999). "In Situ Measurement of PCB Availability in Unsaturated Soils." In Conference
Proceedings: The Fifth International In Situ and On-Site Bioremediation Symposium, San Diego, CA, 241-6.
                                                 Page 9 of 54

                             Bioremediation Research Program Review 1999
 Population  Biology of Bacteria Involved  in Contaminant Bioremediation

   D. M. Ward, E.A. Kem, G.M. Colores and W.P. Inskeep. Departments of Land Resources and Environmental
   Sciences and Microbiology, and the Center for Biofilm Engineering, Montana State University, Bozeman, MT

Molecular approaches allow us to investigate in detail the microbial populations that can potentially be involved in
bioremediation of contaminants and to assess which populations are likely to be most important under given sets of
environmental conditions. We have used denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene
segments, and sequencing of the resultant DGGE bands, combined with cultivation and molecular characterization
of isolates, to assay microbial populations that might occupy hypothesized specialized niches in contaminant
microenvironments. During the past year we have focused on three areas of research related to this theme.  Progress
in each area is summarized separately below.

Adaptations of Phenanthrene-Degrading Bacteria along a Bioavailability Gradient.  In previous work we
demonstrated that enrichment of soil bacteria on phenanthrene presorbed to an acrylate resin, BioBead SM7,
resulted in recovery of mycobacteria, whereas enrichments on a less strongly sorptive organic solid, the polystyrene
resin Amberlite IRC-50, or on sand or without a solid phase, led to recovery of Burkholderia sp. These
mycobacteria exhibited 5- to 7.5-fold greater relative rates of mineralization of SM7-associated phenanthrene than
Burkholderia from control enrichments, suggesting that the former may be adapted to low-bioavailability
microenvironments. Examination of near full-length 16S rRNA sequence data of isolates suggests that closely
related Burkholderia strains (differing by only 1.5 to 3.5%) that exhibit different distributions in Amberlite, sand and
control enrichments may also be adapted to high and moderately reduced bioavailability settings.  A current
objective is to examine the relative  fitness of these mycobacteria and Burkholderia isolates in competition with each
other across the bioavailability gradient.

Succession of Bacteria Associated With Chemical Changes During Biodegradation of Natural and Synthetic
Contaminant Mixtures. Contaminant spills often result in the introduction of mixtures of compounds to the natural
environment.  Using crude oil and simpler hydrocarbon mixtures as models, we  seek to understand relationships
between natural contaminant mixtures and niche diversity of contaminant-degrading microorganisms. Batch
enrichments inoculated with soil were used to study microbial population changes associated with the changing
composition of weathered crude oil and a simple mixture of normal- and isoprenoid-aliphatic hydrocarbons that are
among the most predominant oil components. A similar approach was used to determine populations that were
enriched during degradation of pure components of this mixture.  Parallel samples were taken for gas
chromatography/mass spectrometry analysis of column-separated aliphatic and aromatic compounds and for DGGE.
DGGE band profiles for crude oil and the synthetic mixture displayed identical successional patterns, documenting
that different bacterial populations appear to be associated with degradation of n- and isoprenoid-alkanes. Band
sequences suggest that an Acinetobacter population is likely to be responsible for n-alkane biodegradation, whereas
a Rhodococcus population may responsible for isoprenoid-alkane degradation. Liquid enrichments on pure
compounds yielded the same bands and band seqeunces for n- and isoprenoid-alkanes. respectively. However,
direct isolation from soil on solidified medium containing n- or isoprenoid-alkanes permitted recovery of only the
isoprenoid-degrading Rhodococcus population.  Since Rhodococcus isolates matching  the DGGE band associated
with branched hydrocarbon metabolism were obtained on plates containing either n- or isoprenoid alkanes. our
results suggest that the fitness of the Rhodococcus population to degrade n-alkanes seems lower than that of the
Acinetobacter population under our enrichment conditions.

Effects of Hydrocarbon and Surfactant  Amendments on Soil Microbial Community Composition.  We have
also  continued research in  more realistic soil environments studying changes  in microbial populations associated
with surfactant  (Witconol SN70, a nonionic alcohol ethoxylate) amendments  to hydrocarbon contaminated  soils.
Effects of surfactant and hydrocarbon amendment on catabolism were studied by monitoring conversion of 14C-
labeled  hydrocarbons  to  14CO2, while  effects  on populations were monitored  using DGGE  and cultivation.
Additions of 15 mg hexadecane gm"1 soil with or without 0.25 mg phenanthrene gm"1  soil caused an increase in the
intensity of one DGGE band whose sequence indicated  it was contributed by  a  Nocardia species.  Plating on
solidified media with added  hexadecane failed to recover an isolate matching this sequence.  However, we did
isolate a hexadecane-utilizing Rhodococcus sp. with 100% sequence identity to a band of lesser intensity observed in
treatments where hexadecane mineralization was observed. Amendment with Witconol below the CMC (2 mg gm"
soil) did not affect hydrocarbon metabolism or microbial populations with the exception of increased intensity of the
Rhodococcus sp. band.  Additional amendment of Witconol  at nearly  the  CMC'  (10 mg gm"1  soil) delayed
                                             Page 10 of 54

                             Bioremediation Research Program Review 1999
hexadecane mineralization and completely inhibited phenanthrene mineralization; addition above the CMC' (40 mg
gm"1 soil) inhibited all hydrocarbon mineralization.  At near and above CMC' there was a decline in the band
intensity of the Nocardia population and a rise in intensity of two DGGE bands that were contributed by relatives of
Pseudomonas putida and Alcaligenes xylosoxidans. Isolates matching these bands were able  to degrade  both
Witconol and hexadecane. Addition of high levels of Witconol thus appears to have caused a shift in species with
the ability to degrade hydrocarbons;  the basis for inhibition of hydrocarbon metabolism above CMC' remains
                                              Page 11 of 54

                             Bioremediation Research Program Review 1999
      The Influence of Soil Characteristics and Molecular Properties of
          Hydrophobic Contaminants on Bioavailability in Aged Soils

                 Michael H. Huesemann, Ph.D., Tom Hausmann, Tim Fortman, and Ann Drum
                Battelle Pacific Northwest Division, Marine Sciences Laboratory, Sequim, WA

It is the objective of the currently conducted research to provide answers to the following key questions: 1) How do
the properties  of soil  solids and the molecular structure  of hydrophobic model pollutants  such  as petroleum
hydrocarbons affect bioavailability in aged soils? 2) How does aging affect bioavailability?  3) What are the factors
that determine  whether bioremediation is reaction-rate or mass-transfer rate limited? 4) How does soil toxicity as
measured by solid-phase MicrotoxTM change during bioremediation treatment?  5) What mechanisms control the
leaching of soluble hydrocarbons  from  the aged, non-bioremediated soils?   6)  What  is  the distribution of
hydrocarbons on external particle surfaces before and after bioremediation?

In an effort to address these questions, eight model solids/soils (2 quartz sands, 2 silica gels, 2 clays, peat,  Richland
topsoil) were spiked with crude oil (5-10% wt) and aged for more than 2 years in the laboratory. These 8  aged soil
solids plus one weathered NAVY field soil, one freshly spiked Richland topsoil (not aged), and non-porous glass
beads spiked with crude oil or solvent extract  from the NAVY field soil have been subjected to bioremediation
treatment in well-mixed, aerated slurries in the laboratory (i.e., a total of 12 treatments) for more than 1.5 years.

Slurry samples were periodically  analyzed  for parent  and alkylated PAHs  and biomarkers (e.g., hopane) to
determine  the bioremediation kinetics of selected hydrocarbons. Abiotic release rates of PAHs from the soil slurries
were measured using XAD-2 resin as a sorbent. The changes in soil toxicity during bioremediation were periodically
measured using the solid-phase MictrotoxTM assay. Finally, continuous flow column experiments were performed
to evaluate the  leaching behavior of soluble hydrocarbons (BTEX) in the different aged, non-bioremediated model

Preliminary findings indicate that:

•   for most PAHs, the rate and extent of biodegradation is NOT significantly affected by soil properties, including
    organic matter content.

•   after ca. 1.5 years of slurry bioremediation, only a few  PAHs such  as  benzo(e)pyrene, perylene, and C4
    chrysenes  have not been  completely biodegraded. The extent of biodegradation varies with soil type. The
    reasons for this variability are not clear.

•   the effects of aging appear to be more pronounced with increasing molecular weight of the respective PAHs.
    However, in terms  of total PAHs, the effects of aging were not very significant.

•   PAHs with similar Kow's (e.g., IM-phenanthrene and pyrene) have similar desorption/dissolution  rates but
    different biodegradation rates indicating that
•   differences in biodegradation kinetics are due to microbial factors.

•   based  on modeling results, leaching of BTEX from all soils with the exception of montmorrilonite clay is NOT
    affected by soil properties or aging. Instead, it appears that equilibrium dissolution of BTEX from the  crude oil
    phase  controls the leaching kinetics.

•   soil toxicity as measured by MicrotoxTM decreases with increasing bioremediation time.

•   the conservative biomarker C30 17",21$ (H) -hopane disappeared after more than 6  months of bioremediation
    in two different slurry bioreactors. Current research is underway to detect potential biotransformation products.

•   based  on data from fluorescence photography, the interior of aged soil particles may contain significant levels
    of PAHs (NAPLs in mineral pore space?).
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                              Bioremediation Research Program Review 1999
Based on the above findings, it appears that aging and soil properties do not significantly affect the rate and extent of
biodegradation in soils contaminated with crude oils at levels commonly found in the environment (i.e., ca. 5% or
50,000 mg/kg). It is likely that at these concentrations, most hydrocarbons are present in a NAPL-phase in which
they are readily bioavailable for microbial degradation.

Publications and Presentations

Huesemann,  M.H.,  T. Hausmann, T.  Fortman, and  T.  Gilfoil, "Changes  in  Soil  Toxicity and Unavailability  During
Bioremediation of Aged Petroleum Contaminated Soils", Society of Environmental Toxicology and Chemistry  19th  Annual
Meeting, Charlotte, NC, November 1998
                                               Page 13 of 54

                             Bioremediation Research Program Review 1999
     Biogeochemical Interactions in Reactive Zero-Valent Iron Barriers

         Pedro J.J. Alvarez, E. Sawvel, M. Wildman, B. Eberle, K. Gregory, G.F. Parkin, and J.L. Schnoor
              The University of Iowa, Dept. of Civil & Environmental Engineering, Iowa City, Iowa

Permeable reactive barriers are receiving a great deal of attention as an innovative, cost-effective technology for in-
situ cleanup of groundwater contamination.  Fe(0) barriers promote favorable conditions  for treating mixtures of
redox-sensitive pollutants.  In addition  to direct contaminant reduction by  Fe(0), such in situ treatment systems
could induce anaerobic biodegradation through the depletion of O2 and the production of water-derived H2 during
anaerobic Fe(0) corrosion.
                                  Fe° + 2H2O -> Fe*2 + 2OIT + H2    [ 1 ]
Batch and column experiments performed to date suggest (1) that an integrated microbial-Fe(O) system holds great
promise for treating recalcitrant, redox-sensitive contaminants  (e.g., RDX) and chlorinated solvent-heavy metal
mixtures, (2) that indigenous microorganisms can colonize the  Fe(0) surface, and (3) that the Fe(0) surface area
concentration is an important design variable to optimize microbial activity and prevent  inhibitory effects when
multiple contaminants compete for sites on the Fe(0) surface.

RDX was rapidly reduced in aquifer microcosms amended with Fe(0) and  in flow-through columns packed with
steel wool. Adding anaerobic mixed  cultures enhanced the rate and extent  of RDX  degradation. Apparently, H2-
consuming bacteria exploited Fe(0)  corrosion as  a metabolic niche participated in  the further degradation of
heterocyclic intermediates produced by the reaction of RDX with Fe(0).   Reductive treatment of RDX with Fe(0)
also  reduced its toxicity to microorganisms and enhanced its subsequent  biodegradability under either aerobic or
anaerobic conditions. Therefore, a combined or sequential Fe(0)-biological treatment approach might  improve
treatment efficiency.

Contaminant interactions were studied in small reactors containing Fe(0) and various combinations of CC14, Cr(VI),
and NO3". The preferential degradation order for contaminants in these abiotic reactors was: Cr(VI) > CC14 > NO3".
Results show that at low Fe(0) surface area concentrations (11  m2/L) significant competitive effects  are observed
(Figure 1).  Yet, no inhibition was observed at high concentrations (1140 m2/L).  We hypothesize that inhibition was
due to competition for a limited number of reactive surface sites at a low Fe(0) dose.

The  effect  of  Fe(0) surface  area concentration on microbial activity was studied using  a mixed culture of H2-
consuming sulfate reducers. Fe(0) did not react with  sulfate within the  time  frame of the experiment. Yet,  H2
production  during Fe(0) corrosion stimulates microbial sulfate reduction.  Thus, this experimental system isolated
the effect of Fe(0) surface area concentration on microbial activity. Increasing the Fe(0) dose initially stimulated
sulfate reduction, possibly due to a higher production of H2. Nevertheless,  high Fe(0)  doses had an inhibitory effect
due to  a corrosion-induced increase in pH beyond the optimum  range of the bacteria (equation [1]).  An optimum
Fe(0) surface area concentration occurred at 570 m2/L for this combined microbial-Fe(O) treatment system (Figure
2). This optimum, however, is probably system specific and depends on the buffering capacity of the system.

Scanning electron  microscopy of samples from an Fe(0)  barrier that is treating a chlorinated solvents  plume in
Kansas City showed microbial colonization of the Fe(0) surface (Figure 3). Samples  from an Fe(0) barrier treating
an uranium plume  at Fry Canyon, CO, were also analyzed for the presence  of bacteria.  Fluorescent in  situ
hybridization with 16S rRNA probes showed that more eubacteria cells  were present within the barrier than in
upgradient  or downgradient aquifer samples. Interestingly, Archea cells (e.g., methanogens) were only detected in
Fe(0)  barrier  samples.  Apparently,  indigenous  microorganisms  colonize Fe(0) barriers to  exploit   cathodic
depolarization  and bioremediation as metabolic niches.

In conclusion, the performance of Fe(0) barriers might be enhanced by the concurrent  or subsequent participation of
indigenous microorganisms that increase the rate and extent of contaminant degradation.  Nevertheless, the effect of
such biogeochemical interactions on the long-term performance and permeability  of Fe(0) barriers remains to be
                                              Page 14 of 54

                                 Bioremediation Research Program Review 1999
              High Fe(0) dose (1140 nf/L)
                                                    Low Fe(0) dose (11 m2/L)
                               -.   CCI4 alone
                               o   CCI4+Cr(VI)
                               O   CCI. +Cr(VI) +NO,







                                                                   200 -
                                                                           CCI4 alone
                                                                           CCI4 + Cr(VI)
                                                                                                100   120   140   160
                        Time (Hours)                                                 Time (Hours)
   Figure 1. CCI4 Degradation in the presence of other contaminants with different Fe(0) doses.
   These results suggest that contaminants  will compete  for reactive sites on the Fe(0) surface. Such competitive
   interactions should not  significantly affect specific degradation rates when the available Fe(0) surface area  is
   relatively high (left panel). However, some contaminants will degrade slower when present in mixtures (relative to
   when present alone) when the Fe(0) surface area concentration is low (right panel). These observations illustrate the
   importance of the Fe(0) surface area concentration as a design variable

                                0.4 ^
                           5    0.3 H
     0.2 -
     0.1 -
                                                                                  - 9
                                                                                  - 8
                                                                                  - 7
                                     0      1000     2000     3000     4000     5000

                                       Fe(0) surface area concentration, m2/ L

   Figure 2. Effect of Fe(0) surface area concentration on sulfate reduction activity.
   A maximum rate occurred when the Fe(0) surface area concentration was 570 m2 I"1, which resulted in circum
   neutral pH. Apparently, an Fe(0) dose lower than 570 m2/L resulted in limited H2 production via Fe(0) corrosion,
   and hence a limiting supply of electron donor to respire sulfate. While an Fe(0) dose higher than 570 m2 I"1 produced
   much more H2, this had an inhibitory effect due to a corrosion-induced increase in pH beyond the optimum range of
   the bacteria.
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                            Bioremediation Research Program Review 1999
Figure 3. SEM Picture of Fe(0) Sample from a Barrier Treating a Chlorinated Solvent Plume.
Picture shows colonization of the iron surface by rod-shaped microorganisms, about 2 urn long.
                                            Page 16 of 54

                            Bioremediation Research Program Review 1999
Controls  on Plant Bioavailability in Salt Marsh  Environments Which Can
           be Manipulated for Contaminated Sediment Remediation

                         Richard F. Lee, Marc E. Frischer and Herbert L. Windom
                         Skidaway Institute of Oceanography, Savannah, GA 31411

The sediments  used in this study came from a former chloro-alkali  plant (LCP site) near Brunswick,GA. Marsh
sediments at this site showed a concentration gradient of total mercury (100 to 1 ppm) and PCBs (8000 to 2 ppm)
going from the high tide marsh(nearest the plant) to the low tide marsh. The roots and stem/leaves of two marsh
plants (Spartina alterniflora  and Juncus roemaricus), sediments from different  depths,  sediment  pore water,
microbial mats and benthic invertebrates from the site were analyzed  for PCBs, total mercury and methyl mercury.
The original PCB mixture (Arochlor 1268) used at the site have been modified over time with increasing amounts of
3-,4- and 5-ringed congeners due to reductive dechlorination  which is carried out by microbial systems in the
rhizosphere layer  of the  sediment.  The  subrhizosphere layer had  low concentrations of total mercury, methyl
mercury and PCBs.

Sediments from the most contaminated  stations were transferred  into mesocosms at the Bioremediation  and
Environmental  Mesocosm (BERM) facility located at the Skidaway Institute of Oceanography. The mesocosms are
one meter by three meters deep.  Water levels are tidally simulated using ambient estuarine water of intermediate
salinity (ca. 20 ppt) Four mesocosms are being used: (1) reference sediments without marsh plants; (2) reference
sediments with marsh plants;  (3) contaminated sediments without marsh plants; (4) contaminated sediments with
marsh plants.   Several observations  suggest that the  mesocosms with transferred sediments support  the same
estuarine ecosystem found at the LCP site.  For example, sediment porewater ion distribution and sulfate reduction
rate depth profiles resemble those found at the  LCP site.  In addition, Spartina has gone through  two growth
seasons, microbial mats are established, and the mesocosms are colonized  by fiddler crabs (Uca  sp.) periwinkle
snails (Littorina  sp. ) and small fish.  The identification of microbial communities in the contaminated sediments
from the LCP sites and in transferred sediments from this site are being done using 16S rRNA probes to determine
the important microbial groups in the sediments and rhizosphere community.

For the past 1.5 years we have followed changes in the  concentrations of total mercury, methyl mercury and PCB
congener profiles in the sediments in the BERM mesocosms.  For example the PCB 206 congener decreased from
48 to 0.6   g/g in the surface (0-3cm) sediments and from  98 to 5.8   g/g in the deeper (9-12cm) non-vegetated
sediments.  In the vegetated sediments PCB congener concentrations were much higher in the sediments after 500
days compared with the non-vegetated sediments.  Changes in total  mercury in sediment cores over the 500 day
period are shown in Table 1. Mercury concentrations decreased in all  depths sampled.  The nonvegetated sediments
showed a marked decreased in total mercury in the surface sediments (112 to 0.1   g/g) but little decrease in the
subsurface (3-6cm). In contrast, the vegetated sediments showed a marked decrease in total mercury concentrations
in the 3  to 6 cm depth.  One possible explanation may be that microbial mats, which were very abundant in the
nonvegetated sediments, formed volatile mercury.  Earlier studies showed  high production of methyl mercury by
microbial mats exposed to mercury contaminated sediments.  Methyl mercury can be  acted on by methyl mercury
lyase to form mercuric ions which can then be reduced by mercuric reductase to elemental mercury which volatilizes
from the sediment.  Thus, microbial mats may be making mercury more available to the action of bacterial lyases
and reductases.  The decrease in total mercury in the rhizosphere of the vegetated sediment suggests the importance
of the plants in  combination with rhizosphere microbes in volatilizing  mercury.

To pursue the possible importance of plants as an avenue for the volatilization of mercury from the sediments we are
measuring elemental mercury in the air above the BERM mesocosms  using a plexiglass enclosure to allow periodic
air samples from vegetated and non-vegetated mesocosms. An outlet and inlet to the enclosure allow for periodic air
sampling. Volatile mercury is collected on gold-coated quartz sand. The concentrations of elemental mercury in the
ambient air of the BERM facility ranges from 2 to 4ng/m .

Studies on the  effects of added  nitrogen to the  mesocosms on attenuation of PCBs and mercury are presently
                                            Page 17 of54

                              Bioremediation Research Program Review 1999
Table 1: Changes in Total Mercury in BERM Sediments Due to Natural Attenuation

  I. Sediments from contaminated BERM mesocosm B vegetated
                            Depth (cm)      Total Mercury ( g /g sediment)
                                            300 days          500 days
                            0-3               95               6.1
                            3-6               22               4.6
                            6-9               14               5.4
                            9-12              10               3.3

  II. Sediments from contaminated BERM mesocosm B nonvegetated
                            0-3             112                0.1
                            3-6              24               22.2
                            6-9               16                8.0
                            9-12              10                0.6
Publications based on support by NSF DEB-9706317	

Kannan,K., K.A. Maruya and S. Tanabe. 1997. Distribution and characterization of polychlorinated biphenyl congeners in soil
and sediments from a superfund site contaminated with Arochlor 1268. Environ. Sci. Technol. 31:1483-1488.

Maruya,K.A. and R. F. Lee.  1998. Aroclor 1268 and toxaphene in fish from a southeastern U.S. estuary. Environ. Sci. Technol.
32: 1069-1075.

Lee.R.  and Y. Oshima. 1998. Effects of selected pesticides, metals and organometallics on development of blue crab (Callinectes
sapidus) embryos. Mar. Environ. Res. 46:479-482.

Maruya,K.A. and  R.F. Lee 1998.  Biota-sediment  accumulation and trophic transfer factors for  extremely hydrophobic
polychlorinated biphenyls. Environ. Toxicol. Chemc. 17: 2463-2469.

King,J.K., F. M. Saunders, R. F. Lee and R.A. Jahnke. 1999. Coupling mercury methylation rates to sulfate
reduction rates in marine sediments. Environ. Toxicol. Chem. 18: 1362-1369.

Lee,R.F., S.A. Steinert, K. Nakayama and Y. Oshima. 1999. Use of DNA strand damage (comet assay) and embryo development
effects  to assess contaminant exposure by blue crab (Callinectes sapidus) embryos.  IN:  Environmental Toxicology and Risk
Assessment: 8* Volume, ASTM STP 1364 (D. Henshel.M.C. Black and M.C. Harrass, eds.), American Society for Testing and
Materials, West Conshohocken, PA (in press).
                                               Page 18 of 54

                             Bioremediation Research Program Review 1999
 Bioavailability of Aromatic Contaminants Bound to Solid and Aqueous
                                Phase Natural Organic Matter

Jon Chorover, Department of Agronomy, Patrick G. Hatcher, Department of Chemistry, Newman and Wolfrom Lab,
                  William D. Burgos, Department of Civil Engineering, Penn State University

The overall goals of this project are (a) to examine the effects of solution chemistry on interaction of contrasting
polycyclic aromatic compounds (PACs) with dissolved and mineral-sorbed humic acid (HA) and (b) to determine
the consequences for PAC bioavailability to microbial  degraders.  Naphthalene, naphthol and quinoline were
selected to represent neutral, acidic and basic PACs, respectively. PAC-HA sorption was studied using equilibrium
dialysis, fluorescence spectroscopy  and nuclear  magnetic resonance (NMR) spectroscopy.  The PACs exhibit
different affinities for humic acid as a function of solution chemistry, as can be seen  from Figure 1 (Karthikeyan and
Chorover, in  prep.).  These differences can be attributed to functional group chemistry.  The neutral compound
(naphthalene) shows relatively  low  but positive sorption to HA, and the extent was unaffected by pH or ionic
strength [both are known to affect the conformation of humic acid] (Fig. la).  Hydroxylation of the C-l carbon, as
occurs during naphthalene biodegradation in the presence of monooxygenases, produces 1-naphthol.  Sorption to
HA of this weakly acidic PAC exhibits strong time, pH and  ionic strength dependencies that were found to result
from (a) weak initial complexation of 1-naphthol by HA and (b) oxidative (abiotic) transformation of 1-naphthol
[slow reaction] resulting in the formation of strongly bound  secondary products detectable by HPLC/MS analyses
(Karthikeyan  and Chorover, in review). Data on 1-naphthol  sorption after 7 d equilibration time are shown in Fig.

Quinoline is an N-heterocyclic compound that becomes protonated (cationic) below pH 4.9. Sorption to humic acid
was  dominantly via cation exchange and was highly dependent  on protonation and competition with H+ and
background cation.  Biodegradation experiments (Pisutpaisal et al., in prep.)  were conducted under conditions of
maximum sorption, as dictated by dialysis and fluorescence data.  Degradation by isolated pure cultures of PAC
degraders and consortia were compared using biomass additions corresponding to a known degrader activity
(Tuntoolavest and Burgos, in prep.). Figure 2 provides a partial summary of degradation extent as a  function of
PAC and HA concentration.  Napthalene degradation was slightly reduced in the presence of HA (65 mg  L"1 as
DOC, Fig. 2a) because of positive - but low - napthalene  affinity for HA.   Quinoline degradation was slightly
reduced (Fig. 2c), despite the fact that it forms only weak  complexes with HA via cation exchange (Chorover et al.,
1999). The effect of humic sorption on 1-naphthol biodegradation was dependent on the concentration ratio of PAC
to HA.  In the absence of HA, a decrease in biodegradation resulted from the production of toxic intermediates
(naphthoquinones).  Since HA was shown to be an effective sequestering  agent for naphthoquinones (Karthikeyan
and Chorover, in review), the presence of this sorbent in  solution nullified the toxic effect (Fig. 2b). These results
underscore the importance of considering the reactivity of intermediate compounds in bioremediation of organic

Publications  and Presentations	^^	

Burgos, W. D., C.M. Munson, and C.J. Duffy. 1999. Phenanthrene Adsorption-Desorption Hysteresis in Soil described Using
Discrete-Interval Equilibrium Models. Water Resources Research, 35:2043-2051

Burgos, W.D.,  D.F. Berry, A. Bhandari, and J.T. Novak.  1999. Impact of Soil-Chemical Interactions on the Bioavailability of
Naphthalene and 1-Naphthol. Water Research (in press).

Chorover, J., K. G. Karthikeyan, R. Saunders, and P. G.  Hatcher. 1998. Sorption of 1-naphthol to humic acid. In Proceedings of
the World Congress of Soil Science, Montpellier, France. 5p. (on CD/ROM).

Chorover, J., M. K. Amistadi, W. D. Burgos, and P. G. Hatcher. 1999.  Quinoline sorption on kaolinite-humic acid complexes.
SoilSci. Soc. Am. J. 63 (4).

Karthikeyan, K. G., J. Chorover, J. M. Bortiarynski, and P. G. Hatcher. 1999.  Interaction of 1-naphthol and  its oxidation
products with aluminum hydroxide. Environ. Sci. Technol. (in press, November 15  issue).

Karthikeyan, K. G., and J. Chorover. Abiotic transformation of 1-naphthol affects sorption to humic acid. Environ. Sci. Technol.
(in review)
                                              Page 19 of 54

Bioremediation Research Program Review 1999
were sup
o 8"
1 20-
° 15-
? 1°~
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van, K. G.. and J. Chorover. Complexation of acidic, basic and neutral aromatic compounds with humic acid. Ir
al, N., W.D. Burgos, M. Tuntoolavest, J. Chorover, and R.F. Unz. Biodegradation of 1-Naphthol in the Presence o
d Humic Acid. In preparation for Environmental Engineering Science.
vest, M., and W. D. Burgos. Measuring Organic Contaminant Degradation Activity of Mixed and Pure Cultures. Ir
on for Applied and Environmental Microbiology.
3 M.S. (Soil Science, Environmental Engineering, and Chemistry) and one Ph.D. thesis (Environmental Engineering
ported by this grant.

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              Page 20 of 54

                             Bioremediation Research Program Review 1999
   The Effects of Soil Organic Matter on Mineralization, Desorption, and
                  Sequestration/Transformation of Phenanthrene

   S.B. Soderstrom, A.D. Lucking, M. Johnson, M. Kim, W. Huang, W.J. Weber Jr., Environmental Engineering,
                                  University of Michigan, Ann Arbor, MI

Sorption, bioavailability,  and sequestration  are  interrelated  phenomena affecting the transport and  ultimate
environmental fate of organic contaminants in subsurface systems.  An important, yet poorly defined, condition that
influences these phenomena is the  physicochemical character of the sorbent, particularly that of its associated
natural organic matter.  Previous studies in our laboratories have shown that soil/sediment organic matter (SOM) can
be modeled by two separate domains.   Older soils and sediments that have undergone  significant diagenetic
alteration typically have "hard-carbon" dominated-SOMs that  are physically condensed and chemically  reduced.
These soils  exhibit more nonlinear, slower, and only partially reversible sorption of hydrophobic organic compounds
(HOCs) and have greater organic-carbon-normalized sorption capacities for such contaminants.   Conversely,
younger soils that have undergone little or no diagenetic alteration  have  "soft  carbon" dominated-SOMs that are
more physically amorphous and chemically oxidized, and typically exhibit nearly linear, faster, and reversible HOC
sorption, and lower organic-carbon-normalized sorption capacities.

By using three  geosorbents that exhibit different degrees of diagenetic alteration, the desorption rate, mineralization
extent, and  degree of sequestration/transformation were shown to vary with the degree of diagenesis. The  chemical
nature  and  relative degree of diagenetic alteration of the  organic matter associated with each geosorbent were
characterized using solid state 13C-NMR spectrometry.   50°C water column  flow-through extraction of the sorbed
phenanthrene results in a desorption profile similar to that using an infinite-sink method within a shorter time period.
This suggests that 50 °C water extraction can be used as  an assay to predict long-term desorption.

Mineralization  profiles in aqueous suspensions, using sorbed I4C- phenanthrene as a representative HOC, illustrated
that initial degradation rates were much faster for the younger  geosorbents (Michigan peat and Chelsea soil) when
compared to the older geosorbent (Lachine shale). For the younger geosorbents, mineralization slowed  after the
initial degradation period; mineralization in the older geosorbent-water system  continued at a nearly constant rate
after the initial degradation period. Abiotic desorption  experiments using the infinite-sink method provided similar
trends suggesting that desorption is the rate-limiting step in the system.  The mineralization and desorption profiles
are shown in Figures 1 and 2. After completion of the  mineralization experiments, both combustion and methanol
Soxhlet extraction were used to recover 14C-organics from the geosorbents.

The amount of extractable 14C-organic material varied with the  degree of diagenetic alteration of the soil: relatively
small amounts  of 14C-organics were  extractable from the younger geosorbents while virtually all l4C-organics were
extractable  from the older geosorbents.  The extraction results imply that biological activity alters the SOM and
changes the nature of sequestration/transformation, and that this alteration is  more pronounced for geosorbents that
are younger, more chemically oxidized, and more biologically active.
                                             Page 21 of 54

                             Bioremediation Research Program Review 1999
Figure 1. Mineralization profile for three geosorbents
Figure 2.  Desorption profile for three geosorbents
                                             Page 22 of 54

                             Bioremediation Research Program Review 1999
       Bioavailability of Toxicants as Reflected in the in-situ Microbial
       Community Ecology and Relationship to  Defensible End-Points

  S. J. MacNaughton, J.R. Stephen, Y-J. Chang, Y-D. Gan, J. Bownas, D.C. White: University of Tennessee, TN.,
K.R. Carman, R. Millward: Department of Biological Sciences, Louisiana State University,  M. Barcelona: National
     Center for Integrated Bioremediation Research and Development, Department of Civil and Environmental
   Engineering, University of Michigan, and D.C. White, Environmental Sciences Division, Oak Ridge National

Pollution of the subsurface with bioavailable toxins has been shown to induce marked shifts in the viable biomass,
community structure and nutritional/physiological status of the microbial community.  Combined lipid biomarker
analysis and PCR-DGGE of the bacterial 16S rDNA shows the impact of pollutants on the in-situ soil microbiota.
Recovery of communities from perturbation may provide quantitative definition of defensible endpoints for natural
and attenuated bioremediation.

Two ongoing projects: First, a constructed plume at the  Michigan  Integrated Remediation Laboratory (MIRTL),
shows the impact of MTBE (methyl-t-butyl-ether) and  BTEX on the microbiota within the plume. To simulate the
impact of fuel constituents that were not present in this experiment, a reducing barrier was placed ahead of the test
lane to stimulate suboxic  redox conditions within the  aquifer  prior to the injection of the MTBE,  BTEX and an
aqueous tracer solution. An oxidizing barrier utilizing ORC (Regenesis Bioremediation Products, Inc.) was placed
within the test lane to evaluate its potential for use in  the in-situ treatment of MTBE.  Samples were taken at bi-
monthly intervals from transects located along the constructed plume, with the ORC located between Transects at
145' and 155' from the injection well. Herein we present lipid biomarker and PCR-DGGE 16S rDNA data from the
first 6 months of this ongoing study. The presence of the reducing barrier resulted in the expected shift in the redox
potential to  a  suboxic conditions immediately down  gradient, and resulted in an increased relative  abundance
Flavobacteria, Pseudomonas and Clostridia. Independent of sampling time, higher biomass levels were detected in
samples obtained from closer to the center of the plume.  The shift in biomass/community structure  generally took
the form of increased biomass compared to background samples and as yet unexposed areas  of the plume, as well as
an increase in the proportion of PLFA indicative of Gram-negative bacteria, anaerobic biomass and sulfate reducing
bacteria. The presence of the ORC resulted in an increase in viable biomass with substantially more PLFA indicative
of Gram negative biomass at Transect 155. Phylogenetic characterization of the dominant bacteria in the plume
region has consisted of the excision and sequence analysis of 125  PCR-DGGE 16S rDNA fragments.  The  great
majority of these are related to proteobacteria (a-, P-  and y-subgroups; including methanotrophs, caulobacters and
pseudomonads),  Flavobacteriaceae,  and  the   Geobacteraceae   (Gram-),   and  Gram+   bacteria  of  the
Bacillus/Staphylococcus group and Clostridiaceae.

The second project concerns the impact of co-contaminants (toxic metal and diesel-fuel) on the microbiota of coastal
salt marshes. Previous studies have focused on both these contaminants separately, but essentially nothing is known
about the microbial response to co-contamination.  Herein we present the results of the first of two preliminary
microcosm studies. This study was designed to determine the concentration of toxic metals required to elicit a
response from  the benthic community.  Salt marsh sediments were  contaminated with known concentrations of a
mixture of Navy-relevant toxic metals including Cu,  Cr, Cd,  Pb and Hg.  Metal concentrations were chosen to
simulate the range of concentrations typical of San Diego Harbor (SDH), with samples analyzed on days 0, 12 and
30 following contamination. Metal addition had no significant impact (/*>0.05) on the total biomass content (PLFA)
of the sediments, however, at the higher metal concentrations (x 1, and x 10 SDH levels) changes were detected in
the community structure.  Principal components analysis of the PLFA profiles showed clustering of the samples
from days 12 and 30 of the x 10 SDH metal concentration samples. The PLFA with the greatest influence on the
clustering were indicative of certain eukaryote biomass, increased  metabolic stress and anaerobic biomass. The
PLFA indicative of bacterial response to metabolic stress were shown to increase in samples from day 12 and 30
from both the x 1 and x 10 SDH metal concentrations.  PCR-DGGE detected changes in the bacterial population
structure of samples from day 30 at both xlO SDH and, too a lesser extent, at xl SDH. The primers used also detect
chloroplast rDNA, and indicated that the increased eukaryotic biomass was in part due to the growth of the oxygenic
phototrophic eukaryote Amphora  delicatissima or a related species.  Subsequent microcosms  will use the  data
obtained regarding metal  concentrations to examine the impact  of the metal/diesel on the microbiota and
bioavailability  of the co-contaminants.  Diesel and metals will be used  at known-effect concentrations and at
elevated but sub toxic concentrations.
                                             Page 23 of 54

                             Bioremediation Research Program Review 1999
Publications and Presentations
Quantitative PCR-DGGE analysis of bacterial communities in natural samples. 1999. American Society for Microbiology, 99th
General Meeting, Chicago, IL

J., HART, W.E., & WHITE. D.C. (1999) Evaluating the diversity of soil microbial communities using molecular techniques and
established ecological indicators. American Society for Microbiology Conference on Microbial Diversity, Chicago, IL

CORRONDO, M.J.T (1999). Microbial typing for management of remediation in contaminated soils. African International
Environmental Protection Symposium (AIEPS-99). ISBN 0-620-23945-X)

MACNAUGHTON, S.J., STEPHEN, J.R., VENOSA, A.D., CHANG, Y-J, DAVIS, G.A. and WHITE, D.C. (1999). Microbial
population changes during bioremediation of an experimental oil spill. Applied and Environmental Microbiology. 65 (8):3566-

MACNAUGHTON, S.J. & STEPHEN, J.R. (1999). A combined phospholipid and 16S rDNA PCR-DGGE analysis
to study bioremediative microbial communities in situ. In press, Molecular Environmental Microbiology: Protocols
and Applications (P. Rochelle, Ed). Horizon Scientific Press
                                            Page 24 of 54

                              Bioremediation Research Program Review 1999
           The  Effects of Aging and  Sorbent  Decompostion  on the
              Bioavailability  of  Toluene and Xylene in Solid Waste

             Morton A. Barlaz, Detlef R.U. Knappe, Bingyan Wu, Matthew Pelton, and Caleb Taylor
                                      North Carolina State University

Approximately 25% of the sites on the National Priority List of Superfund are municipal landfills that accepted
hazardous waste. Unlined landfills usually result in groundwater contamination, and priority pollutants such as
alkylbenzenes are typically present. Ultimately, the  EPA must develop strategies to manage these sites in a manner
that is both cost-effective and protective of the environment. To select cost-effective risk management alternatives,
better information on factors controlling the fate of contaminants in landfills is required.

In this research, we focus on the importance of sorption/desorption, contaminant  aging, leachate composition,
sorbent decomposition, and humification on the bioavailability and fate of organic contaminants in municipal solid
waste (MSW). Results from prior studies suggest  that sorbed contaminants  are less bioavailable than dissolved
contaminants, and  that aged contaminants are less bioavailable than freshly sorbed contaminants. However, most
studies focusing on bioavailability  have been conducted in soils that had lower organic carbon contents than MSW
and in aqueous phases  that had lower organic carbon concentrations than landfill leachate.

The sorption  capacity  of l4C-labeled toluene and o-xylene on major organic MSW components [polyvinyl-chloride
(PVC), high density polyethylene  (HOPE), newsprint, office paper, and model food and yard waste (rabbit  food)]
was determined. Batch isotherm data were collected in phosphate-buffered organic-free water and in  acidogenic
leachate. Flame-sealed glass ampules were used to  minimize volatilization losses, and sodium azide was added to
the liquid phase  to prevent aerobic sorbate degradation. Acidogenic leachate  was prepared by recirculating water
through fresh residential municipal waste. Generation of methanogenic leachate from decomposed refuse is ongoing.
In addition, anaerobically degraded office paper and newsprint have been prepared, and generation of anaerobically
degraded rabbit food is ongoing.

Initial tests were conducted to determine the time required to reach short-term sorption equilibria.  Short-term
sorption equilibria for toluene on  HOPE,  newsprint, office paper, and  rabbit  food were reached within 2 days in
organic-free water and acidogenic  leachate while 20 days were required with PVC. Sorption kinetics of toluene on
PVC  exhibited a rapid initial rate of toluene uptake followed by a slower phase. A slower approach to equilibrium
for the glassy polymer PVC was expected given that diffusion in glassy polymers is orders of magnitude slower than
in rubbery polymers such as HOPE.

The Freundlich model (q  = Kf CN) was  employed to describe toluene  isotherm data. Table  1  summarizes the
parameters Kf and  N with 95%  confidence limits that describe single-solute toluene  sorption on each material. In
addition, Table 1 depicts the correlation  coefficients (R2), the number of data points for  each isotherm (n), and the
studied equilibrium liquid-phase concentration ranges  (C). A comparison of the KF values in Table 1 shows that
PVC exhibited the largest sorptive  capacity for toluene. In contrast, the sorption capacity  of office paper for toluene
was about 100 times smaller than  that of PVC. The Freundlich N values for the studied materials were generally
close  to 1, indicating that partitioning dominated toluene uptake. The largest deviations from linearity were observed
for office paper and PVC.

Table 2 summarizes the Freundlich isotherm  parameters describing toluene sorption from acidogenic leachate. A
comparison of Freundlich KF  values in Tables 1 and 2 shows that the single-solute toluene sorption capacities of
HOPE, rabbit food, and newsprint were statistically similar to those obtained in acidogenic  leachate. In contrast, the
sorptive capacity of office paper for toluene from acidogenic leachate was approximately  20% of that from organic-
free water. Given that the single-solute toluene isotherm on office paper was not quite  linear (N=0.87), it  is possible
that components in acidogenic leachate competed with toluene for adsorption sites on office paper. The single-solute
toluene sorption capacity of PVC was also greater than that determined in acidogenic leachate; however, it is unclear
at this point whether we attained true equilibrium with PVC. Longer-term isotherm tests with PVC are ongoing.

Table 3 summarizes single-solute  isotherm parameters describing o-xylene sorption on  HOPE and rabbit food. A
comparison of KF values  in Tables  1 and 3  indicates that the sorption capacity of the tested MSW components for o-

                             Bioremediation Research Program Review 1999
xylene was about 2 to 3 times larger than that for toluene, a result that is consistent with the greater hydrophobicity
of o-xylene.

Table 1. Single-solute Freundlich isotherm parameters for toluene sorption on MSW components. The 95%
confidence intervals for Kf and N are shown in parentheses
Rabbit food
Office paper
804.1 (751.8,860.0)
66.9 (59.9, 74.8)
28.2 (27.0, 29.4)
16.6(15.6, 17.8)
1.01 (0.99, 1.03)
1.00(0.99, 1.01)
0.96 (0.94, 0.98)
0.87 (0.82, 0.92)
C (ug/L)
Table 2. Freundlich isotherm parameters for toluene sorption on MSW components from acidogenic leachate. The
95% confidence intervals for Kf and N are shown in parentheses
Rabbit food
Office paper
487.4 (452.9, 528.7)
57.8 (50.0, 66.8)
26.1 (25.1, 27.1)
15.2(13.2, 17.4)
0.98(0.96, 1.00)
1.04(1.01, 1.07)
1.02(1.01, 1.03)
0.97(0.93, 1.00)
1.02(0.99, 1.05)
C (ug/L)
Table 3. Single-solute Freundlich isotherm parameters for o-xylene sorption on MSW components. The 95%
confidence intervals for Kf and N are shown in parentheses
Rabbit food
1.02(1.01, 1.04)
1.01 (1.00, 1.03)
C (ug/L)
                                             Page 26 of 54

                             Bioremediation Research Program Review 1999
        Preliminary Investigation of U and  Ni  in Riparian and Wetland
 Sediments: Microbial Ecology and the Potential of Apatite Amendments
                     for Reducing Metal Availability and Toxicity

                P. M. Bertsch1, P. J. Morris2'3, A. G. Sowder1, T. V. Khijniak2, and M. T. Novak3
 'Savannah River Ecology Laboratory, The University of Georgia, 2Department of Microbiology and Immunology, Medical University of South
                 Carolina, 3Marine Biomedicine and Environmental Sciences, Medical University of South Carolina

The direct discharge of metallurgical process effluents to the environment on the Department of Energy's Savannah
River Site (SRS)  has led to extensive contamination of the vadose zone and groundwater with trichloroethylene
(TCE) and tetrachloroethylene (PCE) and stream  sediments and  wetlands with  uranium (U)  and  nickel (Ni).
Riparian and wetland  systems  are recognized for their natural  attenuation capacity  due to high organic  matter
content, diverse microbial populations, and range of geochemical  conditions conducive to the  biodegradation of
organics and the biotransformation of metals. However, metal toxicity is a potential obstacle for natural attentuation
of organic plumes in critical SRS riparian and wetland areas,  where U and Ni sediment concentrations can exceed
 1000 mg'kg"1. It is critical, therefore, to understand the effect  of metals on in situ biodegradation of organics and to
design strategies for eliminating metal toxicity in support of intrinsic and enhanced biodegradation. In situ chemical
stabilization of metals using apatite and other phosphate media offers a low-cost and minimally invasive alternative
to traditional metal remediation methods such as excavation.  The efficacy of hydroxyapatite (HA) for stabilizing a
large assortment of metals, metalloids, and radionuclides has been demonstrated in a number of studies.1'2'3  In our
initial  work,  we have found HA amendments to be effective  for application rates as  low as 1% by weight.3
Accordingly, research was initiated to:

•   Determine the  chemical  speciation of U and Ni  in wetland sediments and  establish  linkages between
    contaminant speciation and availability, bioavailability,  and microbial toxicity.

•   Assess the impact of U and Ni on native microbial communities, especially with respect to their capacity for
    degrading TCE in riparian and wetland sediments on the SRS.

•   Evaluate the effectiveness of in situ apatite treatments  for reducing U and Ni availability and toxicity and for
    facilitating TCE degradation.

Denaturing gradient gel electrophoresis (DGGE) profiles of  sediment DNA amplified using a universal bacterial
primer indicate no significant differences in bacterial diversity  between contaminated and uncontaminated sites.
However, overall diversity  is not the only important indicator of metal stress.  High U and Ni levels may adversely
impact specific groups of microorganisms (e.g. methanotrophs, sulfate reducers, nitrifiers, and ammonia oxidizers),
and it is this diversity of metabolic activity in systems that supports natural attenuation capacity.  Ongoing research
using selective bacterial primers focuses on the effect of U and  Ni sediment contamination on specific functional
groups of bacteria. Microorganisms isolated from highly contaminated sediments  (pH 4.55; 2140 mg-kg'1 U; 581
mg'kg"' Ni) displayed no enhanced U resistance but did exhibit unusually high Ni tolerance with respect to cultures
isolated from unimpacted sediments (Figure 1). In preliminary assessments of metal availability (extractions in 10
mM CaCl2 solutions), dissolved U and Ni concentrations  on the order of 0.1 and 10 mg-L"1,  respectively, were
observed for the most contaminated sediments.  These results explain the dissimilar responses of sediment bacteria
to U and Ni.  While total U sediment concentrations are over  3.5 times greater than Ni, Ni appears to be 100 times
more available. Consequently, the adaptive response of sediment bacteria to Ni and not U is a reflection of metal
 lability and bioavailability in their environment. A metal specific toxicity assay, MetPLATE™, indicated metal
toxicity for dissolved concentrations of 3 mg-L"1 Ni (EC50 =10-20 mg-L'1) and 10 mg-L"1 U (EC50 > 50 mg-L'1),
which is consistent with the expression of an adaptive response of sediment bacteria for Ni only  as observed U
concentrations fall well below toxic levels.  Apatite amendments (5% w/w) were able to reduce metal availability by
 one order of magnitude, and therefore, appear to be effective  for maintaining both U  and Ni below  dissolved
 concentrations of concern.

 Preliminary results from sediment extractions and microbial metal  resistance studies indicate that Ni is of greater
 concern than U  in  terms  of environmental availability and toxicity to native microorganisms.  Consequently,
 minimizing ecological and human risk and maximizing natural attenuation capacity in the Tims Branch system
 require careful consideration of U and Ni speciation and environmental availability.
                                              Page 27 of 54

                              Bioremediation Research Program Review 1999
 1. Ma, Q.Y., Traina, S.J., Logan, T.J., Ryan, J.A.: Environ. Sci. Technol. 27:1803-1810(1993).
 2. Chen, X., Wright, J. V., Conca, J. L., Peurrung, L. M.: Water Air Soil Pollut. 98, 57-78 (1997).
 3. Arey J. S, Seaman, J. C., Bertsch, P. M.: Environ. Sci. Technol. 33, 337-342 (1999).
CFU/g sediment x 106
0000 -» -»
ContaminatecT(SP5-~ZT) njncontaminaYe^ (BGT^ZT)
1 A
(a) BpH5
Va/ DpH6
Qpn /


. J" JTi
« 1.2
^~ 1
"1 06
-5> 04
O 0.2


fhl -BpH5:

x ' DPH6
mnU 7

0 10 100 1000 5000 o 10 100 1000 5000
Cone, of U (mg-kg'1) Cone, of U (mg-kg'1)

Contaminated (SP5-Z1)
§ 08
"g 0.6
§ 0.4
O 0.2
(c) BPH5
»w' OPH6
jgjpri /

' i— H

r _ iTl
>dimentx 106
3) 0 -J. -A
j> bo -» k) itk
§ 0.4
O 02
Uncontaminated (BG1-Z1)


frl\ 1PH5

mnU 7

LJr1 ' '

1 n
0 10 100 1000 5000 0 10 100 1000 5000
Cone, of N\ (mg-kg"1) Cone, of Ni (mg-kg'1)

Figure 1. Culturing of sediment bacteria on U and Ni loaded media indicates the metal tolerance of organisms, (a)
Cultures from a contaminated sediment (SP5-Z1) exhibited the same tolerance for U as those (b) from an
uncontaminated sediment (BG1-Z1). In contrast, (c) organisms from the same contaminated sediment displayed a
much greater resistance to Ni than those (d) from the reference sediment.  (SP5-Z1: pH 4.55; 2140 mg-kg'1 U; 581
mg'kg''Ni. BGl-Zl:pH4.97;2mg'kg-'U;4mg-kg-|Ni.)

Publications Associated with Project

Arey, J. S., Seaman, J. C.,  Bertsch, P. M. Immobilization of Uranium in Contaminated Sediments by Hydroxylapatite Addition.
Environmental Science & Technology. 33, 337-342 (1999).

Sowder, A. G., Khijniak, T. V., Novak, M.  T., Morris, P. J., Bertsch, P. M. Impact of U and Ni on the Microbial Ecology of
Aged-Contaminated Sediments and the Potential of Apatite Amendments for Reducing Metal  Availability  and Toxicity.
Radiochimica Acta. (Submitted).
                                              Page 28 of 54

                             Bioremediation Research Program Review 1999
 Understanding Seasonal Variation  of Bioavailability of Residual NAPL in
                                         the Vadose Zone

                 A. Paranjpye, B. Bierwagen, S. Sirivithayapakorn, A.A. Keller and P.A. Holden
                                University of California, Santa Barbara, CA

Natural attenuation of non-aqueous phase liquid (NAPL) hydrocarbon pollutants proceeds in the vadose zone at
unknown  rates and as  a function of uncertain mechanisms.  While we  know that mass transfer and microbial
physiology both play a role in bioattenuation of pollutants in-situ, we don't know the relative role of these processes
or the operative environmental factors. Here, we are investigating how bioavailability and natural bioattenuation of
NAPL hydrocarbon pollutants are affected by natural cycles of wetting (precipitation) and evaporative drying. We
are performing this work at three scales: the pore or microscale, the core or mesoscale, and the field or macroscale.
The specific objectives are to:

•   determine the effects of  wetting  and drying cycles on NAPL distribution in pore  spaces for  specific
    environmentally-significant pollutants with differing physicochemical properties;

•   determine the mass transfer and biological responses to sequential wetting and drying;

•   determine the influence of the  microbial  exopolymeric substances (EPS) on  spreading  and partitioning
    characteristics of selected pollutants;

•   relate the temporal patterns of microbial  growth and biodegradation to  spatial  locations  of biodegrading
    microbes and residual NAPL;

•   relate the effects of wetting and drying cycles to field-scale observations of natural attenuation; and

•   mathematically describe bioavailability as a function of drying and wetting cycles.

We are conducting pore scale studies using glass micromodels  operated under unsaturated conditions and visualized
with fluorescent and visible light microscopy. Using micromodels, our abiotic goals are to determine spreading and
distribution characteristics of study compounds (toluene and hexadecane) under conditions of water-wet and mixed
wettability surfaces. Thus far, we have studied the imbibition of NAPLs into a dry matrix in the micromodel, or the
drainage of a water-filled matrix using NAPLs.  We have also recorded the dissolution and volatilization processes,
to determine the rate of mass transfer at the pore scale.  We are next determining the spatial response of NAPLs to
changes in moisture under abiotic conditions.

In separate micromodel studies, we are studying bacterial colonization of micromodel surfaces when  either non-
toxic and  pollutant substrates are provided for growth.  We have developed a method for applying Pseudomonas
aeruginosa to the model, and have acquired images of the distribution of fluorescently-stained inocula.  Through a
tri-parental mating procedure, we have developed a mutant of our P. aeruginosa strain that overproduces alginate,
the primary constituent of EPS for this organism.  Our current work is aimed at confirming expression  of alginate
overproduction in liquid and in sand culture. We will use the wild-type and mutant strains  in the micromodel to
study spreading and distribution of pollutants with microbial biofilms to determine the influence of the EPS matrix
on mass transfer and bioavailability in response to sequential wetting and drying events. Water potential will be
varied  across  a range that represents wet to very dry conditions (0.0  MPa to 1.5  MPa)  by equilibrating  the
micromodel with a polyethylene glycol solution  (PEG-8000).  We are  in progress with engineering a mutant of our
P. aeruginosa strain that produces green fluorescent protein (GFP) in response to hexadecane and our plan is to use
this organism to estimate the location of biodegradation activity within the micromodel.

Core studies using silica sand  as the substratum for growth and mass transfer will be conducted with the same
pollutants, water potential controls and biodegradation measurements used in micromodel studies.  Thus far,  we
have begun development of prototype reactors for the core experiments. NAPL saturation distribution in cores as a
function of wetting and drying will be determined using X-ray imaging. Currently we are developing the best
conditions for X-ray imaging.
                                              Page 29 of 54

                              Bioremediation Research Program Review 1999
Results from the pore and core scale experiments will be coupled to develop a mechanistic explanation of seasonal
variation in bioavailability. Field test data from well-monitored sites of intrinsic or managed bioremediation will be
used to evaluate the effect that drying and wetting cycles have on biodegradation rates. The results from these three
scales  will be modeled to provide an empirical  and a mechanistic interpretation of the functional relationship
between bioavailability and soil moisture. Overall, we expect to gain an understanding of the physicochemical and
biological mechanisms that determine bioavailability under realistic climatic conditions.  With an understanding of
bioavailability / biodegradation seasonality, it is increasingly feasible to predict the efficacy of natural  biological
attenuation and the necessity of engineered or managed remediation for specific vadose zone pollutants.
                                              Page 30 of 54

                             Bioremediation Research Program Review 1999
    Biosurfactant Specificity and Influence on  Microbial Degradation of
                 Hydrocarbons by  Microbial Consortia in the Field

 Gina S. Shreve, Department of Chemical Engineering, Wayne State University, Detroit, MI and William Finnerty,
                                       Dynazyme Inc., Athens, GA

A  comprehensive research program involving  basic and applied field investigations is defined to establish the
efficacy of various  classes of biosurfactants in the  remediation of soils contaminated with mixed hydrocarbon
wastes.   The  proposed research objectives are (1) to determine the basis  for the hydrocarbon specificity  of
biosurfactants  in terms of micelle  size, micelle dielectric constant, and targeting of minimal interfacial tension
values for mixed micelle solutions  to mixed wastes; (2) elucidation of the influence of pollutant mixtures on the
effectiveness of pure and mixed biosurfactant micelles upon solubilization of hydrocarbons; (3) the assessment of
hydrocarbon solubilization on the microbial degradation of target pollutants; and (4) field studies to determine the
role and influence of biosurfactants in the remediation of polluted target sites.

The  research objectives  addressed to date  include: (1)  examining the basis of the hydrocarbon specificity  of
biosurfactants based on structural and chemical properties of the biosurfactants, and, (2) determining the influence
of contaminant mixtures on the effectiveness of pure and mixed micelle preparations of microbial biosurfactants for
solubilization of specific  classes of hydrocarbons.  The properties of three biosurfactants are currently  under
investigation for a  number of contaminants that  are present  or  structurally  similar to  those present in the
contaminated soils  from the field  site.  Two biosurfactants under investigation,  Dyna 270 and Dyna 200, are
provided by Dynazyme Incorporated. A mixture of rhamnolipids Rl & R3 is also being used in these studies. Dyna
270 and the rhamnolipids have been characterized with respect to their solubilization, interfacial tension, and micelle
structural properties against target hydrocarbons from various hydrocarbon classes, including: straight chain alkanes,
branched alkanes, polyaromatic hydrocarbons, monoaromatic hydrocarbons, and a light weight petroleum mixture.
Various other physical-chemical properties such as the pH optima, the  most effective alkane  carbon number
(EACN) were also determined. The pH optimas are 5.0 for Dyna 270, 11.0 for Dyna 200 and 6.8 for rhamnolipid.
The EACN for Dyna 270 is 6, the EACN for Dyna 200 is 5, and the EACN determined for rhamnolipid is dodecane.
Data on the structural characterization of Dyna 270, the biosurfactant solubilization of target hydrocarbons, and the
physical-chemical characterization of the biosurfactant micelles are contained in the Annual Progress report and will
be presented.

Purification procedures have been developed and mass spectrometry has been performed to determine the structure
of the previously uncharacterized biosurfactant Dyna 270.  The molecule is currently identified as a mannosyl ester
fatty acyl compound using carbohydrate  chemistry  and subsequent TLC analysis.  The remaining linkages and
functional groups are being determined currently through LC-MS analysis at the University of Georgia Center for
Complex Carbohydrates.

Rhamnolipid and the mannosyl ester 3-hydroxydecanoic  acid biosurfactant (Dyna 270) have been evaluated with
respect  to their  physical properties and their ability to  solubilize  several  classes of hydrocarbons  (Table 1  of
Technical Report/Summary).  Both biosurfactants were similar  in demonstrating the ability to solubilize alkane
hydrocarbons, branched alkane hydrocarbons and monoaromatic hydrocarbons in order of decreasing effectiveness.
Their effectiveness  is currently under  investigation for  polyaromatic and chlorinated hydrocarbons as well as
mixtures of the target hydcarbons of each  structural class.  While the surfactant show similar packing properties at
the interface as represented by similar area per surfactant  monomer head group, Dyna 270 consistantly reduces the
interfacial tension to a greater extent.  The critical micelle concentration determined for Dyna 270 is also much
lower than that of the rhamnolipid Rl and  R3 mixture. This results in a much higher measured solubilization of the
target hydrocarbon on a milligram of hydrocarbon solubilized per milligram of biosurfactant basis.  Hydrocarbon
mixture results are currently under examination to determine if these observed trends continue for complex mixtures
of hydrocarbons. Data on the structural  characterization of Dyna 270, the biosurfactant solubilization of target
hydrocarbons, and the physical-chemical characterization of the biosurfactant micelles will be presented.

The  direct solubilization of various structural  classes of  hydrocarbons was measured by equilibrium  partitioning
experiments followed by extraction of the aqueous phase and gas chromatography analysis for the  hydrocarbon
species.  These results were graphed for each hydrocarbon species and biosurfactant mixture and the slope  of the
line was calculated for each target contaminant and is reported in Table 1 as the solubilization in units of moles of
                                              Page 31 of 54

                              Bioremediation Research Program Review 1999
target hydrocarbon per mole of rhamnolipid and moles of target hydrocarbon per microliter of Dyna 270 in buffer
solution.  Both Dyna 270 and rhamnolipid solubilized linear alkane hydrocarbons effectively.  Rhamnolipid also
solubilized branched alkanes and to a lesser extent monoaromatic hydrocarbons. Rhamnolipid solubilization of the
PAH's (napthalene and phenanthrene) appear low, therefore, these experiments are being repeated. Dyna 270 is also
being reexamined to determine it's solubilization of the branched hydrocarbons and PAHs.

Mixtures of these same hydrocarbons are now  being examined to determine if these trends also occur for the
solubilization of these same hydrocarbon species from mixed organic phases or whether the solubilization of a target
contaminant from a matrix presented by complex mixtures of contaminants differs from the results observed with
pure hydrocarbons.

Interfacial  properties of  the  biosurfactants, Dyna  270  and rhamnolipid,  were investigated  at  25  C  in  a
buffer/hydrocarbon solution (for Dyna  270)  or in a hydrocarbon/water system for rhanmnolipid against specific
hydrocarbons from various structural classes of hydrcarbons.  Other parameters of importance that were obtained
from these experiments are the critical micelle concentration (CMC) in the hydrocarbon/biosurfactant system and
the interfacial tension at the CMC.

Dyna 270 is more effective in lowering the  interfacial tension against all hydrocarbons except hexadecane.  For
rhamnolipid, lower interfacial tension appears to correlate somewhat with the solubilization trends observed.

Soil cores from the LNAPL plum near FT-2 at the former Wurtsmith AFB have been obtained and column studies
are currently being set up to examine the properties of each biosurfactant for microscopic displacement of the target
hydrocarbons from  the  soil matrix.  We intend to  model the results using a  combined transport/equilibrium
partitioning model.   Batch solution  phase experiments will be conducted to examine the biodegradability  of the
micellar phase  hydrocarbon and the applicability  of an organic/aqueous  solution phase biodegradation  model
(Sekelsky and Shreve, 1999) to describe this. The microbial consortia  showing optimal growth and degradation of
the target hydrocarbons will then be applied to  the column experiments.  Mathematical models will  be used to
describe the effect of biosurfactnat mediated microbial degradation of sorbed contaminant prior to the beginning of
the field studies at the former Wurtsmith AFB.
                                              Page 32 of 54

                             Bioremediation Research Program Review 1999
                                  CONTAMINANTS IN SOIL

 M.K. Banks, Civil Engineering, Purdue University, A.P. Schwab, Agronomy, Purdue University, and J. Scott Smith,
                                 Animal Sciences, Kansas State University

The  objectives  of  the  research  are  to:  (1)  evaluate the  impact  of plants  on  contaminant  toxicity  and
bioremediation/phytoremediation efficiency, (2) investigate the fate of contaminants  in plant/soil systems, and (3)
determine the impact of plants on leaching of contaminants.

Remediation of Petroleum Contaminated Soil: Port Hueneme, California
In 1997, we established a site at the Port Hueneme Naval Station near Oxnard, California in cooperation with the
Naval Facilities Engineering Service Center.  The soil was contaminated with bunker oil with an average total
petroleum hydrocarbon (TPH) contamination concentration of 3,000 mg/kg.  A small phytoremediation cell was
constructed (30 feet  by 60 feet by 3.5 feet deep) and filled with the contaminated soil.  Vegetative treatments were
imposed in a randomized complete block design with four replications and treatments of unvegetated, a mix of
native grass and legume species, and a typical mixture of plant species used in roadside revegetation. An automatic
irrigation system  keeps the plots moist, and fertilizer N and P are added quarterly. The soil is sampled quarterly and
monitored for changes in TPH, petroleum degrading bacteria, and
toxicity   as determined  by  lettuce  germination,  earthworm
response, and Microtox0.
The  standard TPH determination reveals that the vegetated and
unvegetated treatments are preceding at the same, slow rates (see
figure at right).  The lack of difference between the vegetated and
unvegetated treatments  in due  in  part  to  the  fact  that the
contaminant residues have been heavily aged.  The TPH as
determined by standard protocols is only a small fraction of the
total extractable hydrocarbons; the largest fraction  consitsts of
weathered, polar organic materials that are beginning to resemble
soil humus and are very slowly degraded. The combination of
low  TPH  levels, low polyaromatic hydrocarbon concentrations,
and the weathering stage of the residues suggests that the soils
probably represent a very small environmental threat.
                                                                            TPH Content
                                                                    As Impacted by Vegetative Treatments


I5 1500
Ql 1000
I —
(S invegei.


• Fescue

S//A CL,
1: .




                                                                                   9    12
                                                                           Months of Growth
Lettuce germination was restricted  only in soil collected at the
time of seedbed establishment; germination at all other times was statistically equal to 100% (data not shown). The
initial, low germination was likely because of the high salinity of the soil, which was reduced by management of the
application of irrigation water. The Microtoxc results were quite similar.
Several species of the Pseudomonas bacteria are capable of degrading PAHs and other petroleum hydrocarbons.
Therefore, we quantified the populations of these micro-organisms by plate counts.  As expected, there were no
                                            treatment differences  at the time of seeding, but the vegetated
                                            plots had signifi-cantly higher Pseudomonas spp by the  end  of
                                            only three months, and the  trend continued through at least  15
                                            months of plant growth (see figure at left).
             Pseudomonas spp.
t  10'

I  103
LL  1Q2

"o  10'
                            ^3 Unveget.
                   3     9     15
               Months of Growth
                                            The US EPA standard protocol for earthworm toxicity is based
                                            upon mortality, and these soils did not induce mortality regardless
                                            of the time of sampling or treatment. This result is consistent with
                                            lettuce  germination and Microtox0,  but  appears to  be an
                                            insensitive measure of overall toxicity.   Therefore, a modified
                                            procedure  was used in which worms  were added to the soil,
                                            incubated for two weeks, and  change in mass quantified.  This
                                            approach proved to be much more sensitive to  sampling time and
                                             Page 3 3 of 54

                               Bioremediation Research Program Review 1999
treatment.  In the figure to the right, 0% growth would mean that the
worms had the same mass before and after incubation.  Growth of
100% would indicate mass gain equal that of worms incubated in an
uncontaminated, control soil.  Worms in soils from the vegetative
treatments  had  significantly higher  mass  gain   than  those  in
unvegetated soils. Thus, even though the soils were not toxic, the
presence of plants enhanced the overall quality of the soil and clearly
improved the ability of the worms to thrive.

The  Port Hueneme study is scheduled to be completed in January,
2000 at which time we have final measures of TPH, specific PAHs,
and toxicity determinations.
The Impact of Aging and Phytoremediation on Contaminant Toxicity
                                                                              Earthworm Evaluation
                                                                   9   60
                                                                            E3 unvegel.
                                                                            • Fsscue
                                                                            m Cal,'.
                                                                                0      12      15
                                                                                 Months cf Growth
In an on-going study, soils have been contaminated with petroleum products (diesel fuel, motor oil, and selected
PAHs).  The soils are allowed to age for 0, 3, 6, 12, and 18 months followed by  12 months of phytoremediation.
Degradation of contaminants and toxicity will be monitored as a function of time and remediation treatments. We
also are engaged in a study of the impact of the rhizosphere on the mineralization of benzo[a]pyrene in which plants
are grown in chambers  that isolate the roots from shoots and allow us to evaluate CO2(g) evolution from soil and
aerial portions of the system.

Submitted Publications
Kulakow, P.A., A.P. Schwab, and M.K. Banks. Screening plant species for growth on  weathered sediments
contaminated with petroleum hydrocarbons. J. Phytoremediation (submitted)

Published Abstracts

Banks, M.K., A.P.  Schwab, J.S. Smith, P. Kulakow, and B. Liu.  1999. Effect of plants on bioavailability and toxicity of soil
contaminants. The Fifth International Symposium on In-Situ and On-Site Bioremediation, April 19-22, San Diego

Schwab, A.P., M.K.  Banks, and S. Lewis. 1999. Phytoremediation of Petroleum-Contaminated Soils: Greenhouse and Field
Studies. International Business Conferences, Toronto, Canada, June, 1999

Schwab, A.P., M.K. Banks, and J.S. Smith. 1999. Field assessment at Port Hueneme Naval Facility. West  Coast Contaminated
Soils Conference, Oxnard California. March, 1999

Banks, M. K., Schwab, A. P., Smith, J. S., Kulakow, P., and K. Miller.  1998. "Evaluation of Bioavailability in the Field for
Petroleum Contaminated Soil," 14th Annual Conference on Contaminated Soils, Amherst, MA,  1998

Kulakow,  P., A.P.  Schwab,  M.K. Banks, S.  Lewis, K.  Rathbone, and  J.  Keller. 1998.  Phytoremediation of petroleum
contaminated soils: laboratory and greenhouse studies. 90th Annual Meeting of the American  Society of Agronomy, Baltimore,

Schwab, A.P., M.K.  Banks. P. Kulakow,  K. Rathbone, and S. Lewis.  1998.  Phytoremediation of contaminated soils: Field
studies. 90th Annual Meeting of the American Society of Agronomy, Baltimore, MD

Banks, M. K., A. P. Schwab, and J.  S. Smith. 1998. Bioavailabiiity of Petroleum Contaminants in Vegetated Soil. Annual West
Coast Conference on Contaminated Soils and Groundwater, Oxnard, CA
                                               Page 34 of 54

                             Bioremediation Research Program Review 1999
DOE contract No: DE-FG02-97ER62350

   Bioavailability of PCBs in Surfactant-Washed  Soils With and Without

   John Sanseverino, Alice Layton, Betsy Gregory, James Easter, Fu-Min Menn, T. Wayne Schultz, and Gary S.
        Sayler. Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee.

A major factor in developing PCB bioremediation technology and gauging endpoints of effective  treatment is
overcoming the relative insolubility of PCB and its sorptive properties, hence low bioavailability in many soils and
sediments.  The problem of poor bioavailability (defined as availability of substrate to the microorganism) has been
overcome by soil washing with nonionic surfactants and feeding the surfactant/PCB mixture to 2 previously
developed  genetically-engineered  organisms,  Pseudomonas  putida  IPL5::TnPCB  and  Ralstonia  eutrophus
B30P4::TnPCB. These organisms degrade surfactant and constitutively express a biphenyl operon for degradation
of lessor chlorinated PCB congeners.

Bioavailability (defined now as availability to human and ecological receptors) and measuring associated toxicity is
fundamental to risk assessment and defining environmentally acceptable endpoints.  After treatment, PCBs removed
and remaining on contaminated soil should not pose an unacceptable risk to human health and the environment.  The
long-range goal of this project is to develop an alternative, low-cost treatment process for the  in  situ/ex situ
remediation of PCBs.  This goal is being accomplished by (i) increasing PCB  bioavailability by surfactant washing,
(ii) increasing the extent of PCB biodegradation by photolytic dechlorination of highly chlorinated congeners, and
(iii) testing the surfactant/PCB solutions and the washed soil for environmental estrogens and toxicity.

A PCB-contaminated soil (designated PR3-1; 1,100 mg Aroclor 1248/kg soil  and 90 mg Aroclor 1260/kg soil) was
washed with  2% polyoxyethylene 10 lauryl ether (POL(IO)).   Approximately 86% of soxhlet-extractable PCBs
were removed after 3 wash cycles.  Surfactant/PCB solutions were divided with half subjected  to biodegradation and
the other half subjected to photolysis followed by biodegradation. In all cases, approximately  95% of the surfactant
was  degraded in 2-3 days.  The combination of photolysis  and biodegradation significantly enhanced the total
removal of PCBs (approximately 80%; Fig. 1) than biodegradation alone (approximately 59%; Fig. 1). The purpose
of photolysis was to dechlorinate  the heavily chlorinated congeners economically and shift the congener profile
towards the more biodegradable congeners.  Either method used alone is not practical for 2 reasons:  (1) Photolysis
as a means to completely dechlorinate PCBs is too expensive, and (2) biodegradation is not effective with the highly
chlorinated congeners.  The combination of both methods provides an efficient, economical means to treat PCB-
contaminated soil.

Three molecular-based assays are being utilized to assess bioavailability of PCBs and by-products:  a bacterial
bioluminescent reporter assay, the  presence of environmental  estrogens using a yeast-based human estrogen assay,
and the presence of potentially toxic aromatic compounds using a yeast-based human aryl  hydrocarbon receptor
(AHR) assay.  The bioluminescence assay detects biphenyl and mono-chlorinated biphenyls.  These  compounds
were present in pure Aroclor  1242 and photolyzed surfactant/PCB solutions (Table 1). Nonphotolyzed solutions
were not reactive  in this  assay implying that photolysis produced some biphenyl and monodichlorinated by-

Nonphotolyzed  surfactant/PCB solutions were reactive with the human estrogen receptor  (Table 1)  indicating that
environmental estrogens were present.  However, tests  with pure aroclors  did not react with this assay further
confirming that unmodified PCBs are not estrogenic. Photolyzed solutions however, were  not estrogenic indicating
that photolysis had the dual effect of dechlorinating highly chlorinated congeners as well as preferentially degrading
potentially estrogenic compounds.  Hydroxylated PCBs have been previously  identified as environmental estrogens.
Work is on-going to determine if bacterial metabolism can generate hydroxylated congeners that may  act as
environmental estrogens and if they were present in the soil.

The AHR binds aromatic compounds and enhances transcription of cytochrome  P450 genes.  Cytochrome P450
oxidizes these aromatic compounds which  may result in serious health effects. A yeast-based reporter system has
been developed with a human AHR enabling testing of the bioavailability of PCBs in soil.  At  this time, the assay is
being validated for pure PCBs. Future work will  test the washed soil directly to determine if soil washing reduced
                                             Page 35 of 54

                             Bioremediation Research Program Review 1999
the bioavailability (to human receptors) of remaining contaminants and environmental estrogens.  This data will be
used to develop environmentally acceptable endpoints for remediation of this soil.
iuu -
1 80 -
^ 60-
I 40-
g 20-

' -\ > ',,x

S *' -i &•*
• .->-"'
•* i*CM

r t>- '
- ; c 

Biodegradation Photolysis Biodegradation
Alone Alone +
                                                Treatment Conditions
Figure 1.  Percent PCB removal from surfactant/PCB micelles undergoing treatment by biodegradation, photolysis,
and biodegradation combined with photolysis
Table 2.  Summary of bioluminescence, estrogenic activity and aryl hydrocarbon receptor assays
Aroclor 1242
Aroclor 1248
Aroclor 1260
POL (10)
Nonphotolyzed Sol'n
Photolyzed Sol'n

hER Activity

AHR Activity
Not tested
Not tested
Not tested
                                             Page 36 of 54

                                Bioremediation Research Program Review 1999
Publications and Presentations
LaTorre, K.A., Z. Shi, M.M. Ghosh, and A.C. Layton.  1998. Aerobic biodegradation of PCBs in photolyzed and non-photolyzed
surfactant solutions. In Designing and Applying Treatment Technologies: Remediation of Chlorinated Recalcitrant Compounds.
Vol. 16:225-230

Layton, A.C., M.  Muccini,  M.M. Ghosh, and G.S. Sayler.  1998.  Construction of a bioluminescent reporter strain to detect
polychlorinated biphenyls. Applied and Environmental Microbiology. 64:5023-5026

Layton, A.C., B. Gregory, T.W. Schultz, and G.S. Sayler. 1999.  Validation of genetically engineered bioluminescent surfactant
resistant bacteria as toxicity assessment tools.  Ecotoxicology and Environmental Safety. 43:222-228

Muccini, M., A.C. Layton, G.S. Sayler and T.W. Schultz. 1999.  Aquatic toxicities of halogenated benzoic acids to Tetrahymena
pyriformis.  Bulletin of Environmental Contaminants and Toxicology. 62:616-622

Sanseverino J., A. C. Layton, B. Gregory, T.W. Schultz, and G.S.  Sayler. Estrogenic Activity of PCBs in Surfactant-Washed
Soils. The 99th Annual Meeting of the American Society for Microbiology.  Chicago, II. May 31-June3, 1999

Sanseverino J., F-M. Menn, B. Gregory, E. Sampsel, Z. Shi, M. Ghosh, T.W. Schultz, and G.S. Sayler. 1999. Bioavailability,
estrogenicity, and toxicity of surfactant-PCB mixtures after biodegradation and photolysis.  In Bioremediation ofNitroaromatic
and Haloaromatic Compounds. (B.C. Alleman and A. Leeson, eds.)  5(7):155-160

Sampsel E., M. Ghosh, Z. Shi, J. Sanseverino, F-M. Menn, M.M. Wong, B. Gregory, and G.S. Sayler.  1999. Desorption and
destruction of PCBs in micellar media. In Bioremediation ofNitroaromatic and Haloaromatic Compounds. (B.C. Alleman and
A. Leeson, eds.) 5(7): 161-166

Schultz, T.W., D.H. Kraut, G.S.  Sayler, and A.C. Layton.   1998.  Estrogenicity of selected biphenyls  evaluated using a
recombinant yeast assay.  Environmental Toxicology and Chemistry. 17(9):1727-1729

Shi, Z., K.A. LaTorre, M.M. Ghosh,  A.C. Layton, S.H. Luna, L.  Bowles, and G.S. Sayler.  1998.  Biodegradation of UV-
irradiated polychlorinated biphenyls in  surfactant micelles. Water Science Technology. 37(7):25-32

Shi, Z., M.M. Ghosh, and M.E. Sigman.  1998. Surfactant-enhanced photolysis of polychlorinated congeners.  Water Research

Shi, Z,, F.-M. Menn, J. Sanseverino, and G.S. Sayler.  Extraction of PCBs from nonionic surfactant media using a reverse-phase
absorbent (tCi8) as the emulsion breaker.  In preparation. To be submitted to Environmental Toxicology and Chemistry
                                                   Page 37 of 54

                             Bioremediation Research Program Review 1999
     Biogeochemical  Factors  Limiting Transformation  of Co-Occurring
                         Contaminants in Salt Marsh Sediments

                  Marc E. Frischer, Keith A. Maruya, Joel E. Kostka, and Herbert L. Windom.
                                 Skidaway Institute of Oceanography, GA

A common difficulty associated with devising bioremediation strategies for many contaminated sites is the co-
occurrence of several classes  of toxic chemicals.  In the case where mixed contaminants are present, the likelihood
of interactions between candidate transformation pathways is high and greatly complicates remediation processes.
Because these pathways are believed to involve complex microbial consortia, this program seeks to investigate the
linkages among biogeochemical parameters, microbial activity, microbial diversity and  community structure, as they
apply to the biotransformation of co-occurring contaminants. It is hypothesized that a fundamental understanding of
these linkages will lead to the development of enhanced in situ remediation strategies.  To address this thesis we
have assembled a multi-disciplinary research team with expertise in biogeochemistry, microbial ecology, molecular
biology, and environmental organic chemistry/engineering to focus on saltmarsh sediments in which co-occurring
polychlorinated biphenyls  (PCBs-principally as Aroclor 1268) and mercury  dominate  a complex assemblage of
contaminants.  Studies are focusing on a saltmarsh Superfund site ("LCP") in southeastern coastal Georgia (USA)
that has been active since the  1920's.

Surprisingly, during the first project year observations made at the LCP site suggested that PCB dechlorination are
likely occurring under sulfidogenic conditions. Therefore, one of the primary objectives  of the current phase of the
project has been to study the linkage between sulfate reduction and PCB dechlorination. Using native contaminated
sediment from the LCP site, controlled microcosm studies indicated that  PCB dechlorination activity could  be
induced under both methanogenic and sulfidogenic conditions in the  presence of high mercury concentrations.
Microcosms that were primed with 2,3,4,5,6-pentachlorobiphenyl (a "meta" primer) exhibited the greatest decrease
in Aroclor 1268 (18%  by mass after 3 months). Furthermore, the distribution of PCB dechlorination products
differed under sulfidogenic vs. methanogenic conditions suggesting that different microbial dechlorination activities
were inducible.  In experiments supported by our AASERT augmentation  project, temperature and/or pH were
found to influence the  extent and rate of primer dechlorination in  sulfate-amended  sediment slurry incubations,
suggesting that specific dechlorination activities can be induced under varying environmental conditions.

To  identify potentially important  microbial consortia involved  in contaminant transformation, studies were
undertaken to correlate the structure of microbial communities associated with pristine  and contaminated sediments.
Characterization of microbial community structure was determined using several molecular approaches including
direct sequencing of the 16S rRNA gene, cell blot hybridization with suites of phylogenetic broad group-specific
16S rRNA targeted oligonucleotide probes,  and Amplified Ribosomal DNA  Restriction Analysis (ARDRA).
Characterization of 16S rDNA clone collections by ARDRA and sequence analysis suggests that microbial diversity
is at. least as  high in contaminated sediments as those found  in other  environments. Although sulfate reducing
bacteria (SRB) accounted for the  majority of activity and probe hybridization signal, respectively, the delta
proteobacteria did not numerically dominate the microbial consortium as determined by sequence analysis of clones
obtained from pristine or contaminated sites.  Within the SRB populations, the abundance of Desulfobacterium was
positively correlated to the presence of contaminants.  These studies suggest the role of sulfate reducing populations
of bacteria and  indicate the importance of diverse microbial assemblages for the tolerance and transformation of
complex contaminant mixtures in saltmarsh sediments.


Reyes, N.S., M.E. Frischer, And P. A. Sobecky. (In Press). Molecular characterization of marine microbial communities from
PCB and Hg-contaminated saltmarsh sediments. FEMS Microbial Ecology


Maruya, K.A.,  R.F. Lee,  M.E. Frischer, J.E. Kostka, and H.L.  Windom.   1998. Bioavailability and bioremediation  of co-
occurring PCBs and mercury in saltmarsh sediments. Society of Environmental Toxicology and Chemistry 19* Annual Meeting.
Charlotte, NC.  November 15-19,1998
                                             Page 38 of 54

                                Bioremediation Research Program Review 1999
Maruya,  K., R.  Lee, M. Frischer, J. Kostka  and H. Windom "Bioavailability and bioremediation of saltmarsh sediments
contaminated with Aroclor 1268", First International Conference on Remediation of Chlorinated and Recalcitrant Compounds,
Monterey, CA, May 18-21, 1998

Maruya, K.A., R.F. Lee, M.E. Frischer, J.E. Kostka, H.L. Windom, J.K. King, and P.M. Saunders. 1999 Biotransformation of co-
occurring PCBs  and mercury by sulfate reducing bacteria in estuarine sediments.   Society of Enviromental Toxicology and
chemistry 20th Annual Meeting. Philadelphia, PA.  November 14-18,1999

Kostka, J.E., L.B. Cowden, J. King, and M.E. Frischer. 1999. Microbial biogeochemistry of saltmarsh sediments co-contaminated
with mercury and PCBs. American Society for Microbiology General Meeting, 1999, Chicago, IL

J.K. King, M.E. Frischer, J.E. Kostka, and F.M. Saunders.  Identification of sulfate-reducing, phylogenetic groups most capable
of methylating mercury in pure culture and organic acid amended sediment slurries. American Society for Microbiology General
Meeting, 1999, Chicago,  IL

Frischer,  M.E., J.D. Danforth, L.B. Cowden, and J.E. Kostka. 1999. Characterization of microbial assemblages associated with
saltmarsh sediments co-contaminated with mercury and PCB. American  Society for Microbiology General  Meeting, 1999,
Chicago, IL

N.S. Reyes, M.E. Frischer, And  P.A. Sobecky. 1999. Molecular characterization of marine microbial communities from PCB
and Hg-contaminated saltmarsh sediments. American Society for Microbiology General Meeting, 1999, Chicago, IL
                                                  Page 39 of 54

                             Bioremediation Research Program Review 1999
       Enzymology of the  Degradation  of Organometallic Compounds

                            Anne 0. Summers, Keith Pitts, and Andreas Heltzel,
                         Dept. of Microbiology, Univ. of Georgia, Athens GA 30602

For decades the form  of mercury of greatest public  health concern has been  the neurotoxic organomercurial
monomethylmercury (MMM). This simple but potent compound is biomagnified in the  food chain and, apart from
accidental exposure to now-outlawed MMM fungicides, the most common route of exposure is through freshwater
fish and  seafood.   While precise routes of accumulation in fish are not well-defined, the  origin of the  initial
methylation process has been increasingly well detailed in freshwater settings in just the last 5 years.

Industrially produced organotin compounds are also of considerable environmental concern. They have been used
for decades as marine antifouling agents, agricultural fungicides, and plasticizers  on the assumption that they are
relatively benign to higher organisms. However, recent work makes it clear that compounds such as relatively stable
tributyltin (TBT) exert potent estrogenic effects.

Early work on our project established that another enzymatic process, Hg(0) oxidation by catalase, takes place in
bacteria at rates sufficiently high to constitute a potentially important source of methylatable  Hg(II) in nature (1).
Our present work focusses on a bacterial enzyme capable of effecting the direct degradation of both organomercury
and organotin compounds, the organomercurial lyase, product of the plasmid-encoded merB gene.  MerB catalyzes
protonolysis of a large variety of organomercurials with the alkyl mercurials being poorer substrates than the aryl
mercurials. In collaboration with Genetics Dept. colleague, Rich Meagher, we have recently shown that this novel
enzyme  in  combination  with  another  bacterial  enzyme, mercuric  reductase,   can  confer resistance  to
organomercurials in plants (2).  Interestingly, MerB  also  effects degradation of certain organotin compounds
including  tetravinyltin, triethylvinyltin, tetramethyltin, and trimethyltin fluoride, although various other organotin
compounds including the widely used anti-fouling agent TBT oxide are not substrates for the existing enzyme.

The remaining goals of our work are: (1) to determine the 3 dimensional solution NMR structure of the  MerB
enzyme and (2) to  derive variants of MerB  optimized for degradation  of the most  environmentally important
organometals, MMM and  TBT.

In respect of the first sub-goal, we have demonstrated that merB is a cytosolic enzyme, over-expressed the protein,
devised a facile spectrophotometric assay, and  optimized its purification (3). The protein is a monomer of 24 kDa,
well  within the range of  solution structure determination by highfield NMR. In  collaboration with Biochemistry
Dept. colleagues Jim Omichinski and Pascale Legault we have determined a 1-H, 15-N HSQC spectrum in which
nearly 200 of the protein's 220 amide protons are well-resolved.  On the basis of these  very promising results we
have obtained supplemental  funding from DOE and the University  of Georgia for determination of the  entire
solution structure of MerB, and anticipate completion of the structure determination during 2000.

In respect of the second  sub-goal of the project, we have  used high efficiency PCR and mutator strain random
mutagenesis of merB to derive several dozen variants (detected by various screening and  selection strategies) which
have lost  or gained the ability to  metabolize organometal  compounds. DNA sequence determination currently
underway will, for the first time, reveal residues  involved in  the novel functions of this  enzyme.   Detailed
biochemical and biophysical characterization of key mutant  proteins will follow during 2000.  In addition to being
incorporated into living bacterial  cells  for remediative purposes, with enhanced ability to degrade MMM or TBT
could be used alone on a solid support or introduced into plants or animals (including insects) for contained or field

(1). Smith, CT, Pitts, KE, McGravey, JM, and Summers, A.O. (1998) Bacterial oxidation of mercury metal vapor, Hg(0). Appl.
Environ. Microbiol. 64:1328-1332

(2). Bizily, S. P., Rugh, C.L., Summers, A.O., and Meagher, R.B. (1999). Phytoremediation of methylmercury pollution: merB
expression in Arabidopsis thaliana confers resistance to organomercurials. Proc. Nat. Acad. Sci. 96:6808-6813

(3). Pitts, KP, Heltzel, A., Zeng, Q., and Summers, A.O. (1999) The organomercurial lyase (MerB) is a cytosolic enzyme. MS in
preparation for J. Bacteriol
                                              Page 40 of 54

                             Bioremediation Research Program Review 1999
EPA Grant: R82-5961

   Bioavailability and Biostabilization  of Multicomponent Non-Aqueous
                      Phase Liquids (NAPLs) in the Subsurface

        Anu Ramaswami, Tissa Illangasekare, Angela Bielefeldt, Kendra Morrison, Timothy J. Donahue,
                                      Eric Vestal & Allison Riffel
          University of Colorado, Denver; Colorado School of Mines; University of Colorado, Boulder

The objective of this project is to understand key factors that control the bioavailability and biostabilization of high
molecular weight organic contaminants (PAHs and PCBs) sequestered within multi-component DNAPLs entrapped
in heterogeneous soil systems. The main hypothesis of this project is that slow dissolution of contaminants released
from DNAPL pools entrapped in the subsurface, when combined with low-level microbial activity in the vicinity of
the  DNAPL source  region, can result  in stabilization of contamination with diminished plume formation and
associated risk reduction.

The project consists of four phases:

Phase 1: Biostabilization Screening  Tests and Biokinetic Studies. Bench-scale biostabilization screening tests are
developed to examine the potential for biostabilization of various DNAPLs. The  protocols are tested with DNAPL
coal tar (composed of a mix of PAHs), and an Aroclor DNAPL (composed of a mix of PCBs). Phase 1 research also
examines the biodegradation kinetics of individual PAH and PCBs in the absence of mass transfer constraints.

Phase 2: Mass Transfer and Bioavailability Tests measure  the equilibrium partitioning and kinetics of release of
multiple organic compounds from DNAPL in two  abiotic  tank systems: a small-scale, bench-top system and a
larger, pilot-scale system. The goal is to determine key factors that control the availability of organic contaminants
released from DNAPL pools, at two different spatial scales.

Phase 3: Biostabilization Tests examine the combined effect of contaminant dissolution from  DNAPLs and
microbial degradation on stabilization and risk reduction at two spatial scales.

Phase 4: Modeling and Scale-up of Laboratory Data: The goal is to develop mathematical models and engineering
protocols that would enable scale-up of biostabilization processes (mass transfer and biokinetics) from the bench-
scale to the tank-scale, ultimately to the field.

The work to-date in Years 1 and 2 has resulted in completion of Phase 1 activities, and steady progress in Phase 2
and Phase 4 research. As part of Phase  1 research, the biostabilization potential of coal tar and Aroclor  1242 has
been examined,  and the biodegradation kinetics  of bi-phenyl,  styrene,  naphthalene, methyl-naphthalene and
phenanthrene have been studied and modeled. As part of Phase 2 work, bench-scale and tank-scale systems have
been designed and tested to  examine the kinetics of release  of contaminants from DNAPL pools at different water
flow rates. Diagnostic tests with octanol and coal tar revealed problems in localizing and immobilizing the DNAPL
pool, due to which a smaller pilot-scale  tank was designed  and  constructed with glass and metal walls. A bench-
scale cell has also been  developed and tested with solid naphthalene. Both systems are currently being operated to
assess mass transfer from DNAPL pools at the  small-scale  and  the intermediate-scale. We are evaluating current
methods used to model NAPL pool dissolution as they may place  limitations on modeling dissolution under biomass
at the pool-water interface. A new modeling methodology that uses a local dispersivity that gets modified due to
biomass growth is proposed and tested in our ongoing research.  As part  of Phase 4  work, a  finite  element
groundwater  contamination modeling  software  package,  SUTRA,  is  under modification to incorporate
biostabilization  phenomena. These modifications involve the incorporation of multiple  species in the  transport
equations, incorporating bio-kinetics and bio-growth, modeling of oxygen supply,  modification of hydraulic
conductivity field due to biomass growth  and biomass transport.

Results from Phase 1 research are presented in this  section.  This work has resulted in successful completion of an
M.S thesis  and an M.S. report by two students, Ms. Allison Riffel (University of Colorado, Boulder) and Ms.
Kendra Morrison (University of Colorado, Denver) who examined biodegradation of PCBs and PAHs, respectively.
The  Monod model  was found to  describe the biodegradation kinetics  of biphenyl, naphthalene and methyl
naphthalene, with the following average estimated parameter values: biphenyl: Ks=22 mg/L, k= 0.3 mg biphenyl/
mg VSS-hr, Y=l g VSS/g biphenyl;  naphthalene: Ks=0.5 mg/L, k= 1.4 mg naphVmg VSS-hr,  Y=0.83; m-
                                             Page41 of 54

                               Bioremediation Research Program Review 1999
naphthalene: Ks=0.006 mg/L, k=4.3 mg m-naph./mg VSS-hr, Y=0.83. Styrene exhibited toxicity to the microbes at
a concentration > -15 mg/L. Phenanthrene exhibited first order kinetics with a degradation rate constant of 1.8 L/mg
VSS-hr. The biostabilization screening tests indicated potential for biostabilization of both coal  tar and Aroclor.
Unlike coal  tar, which demonstrated stabilization of aggregate  aqueous  phase  toxicity,  DNAPL  composition
(depletion of more soluble PAHs), as well as microbial counts after a 100 day period, Aroclor studies revealed
stabilization  of two parameters:  DNAPL  composition (depletion of higher solubility congeners) and microbial
counts. Aqueous phase PCB concentrations were not significantly different from controls and aggregate aqueous-
phase toxicity is currently being re-evaluated.

Publications and Presentations:

Vestal, E.; Illangasekare, I.; Ramaswami, A. Modeling of Net Interphase Mass Exchange in  NAPL-Water Systems Undergoing
Biodegradation at the Spill-Site  Scale,  Paper presented at the National Conference  of the American Geophysical Union,
December, 1998

Vestal, E.; Illangasekare, I.; Bielefeldt, A.; Ramaswami, A. Modeling of Net Interphase Mass  Exchange in NAPL-Water Systems
Undergoing Biodegradation, Paper presented at the 1999 EPA  Hazardous Substance Research Center  Conference, Kansas City,
April, 1999

Morrison, K.;  Ramaswami, A.; Riffel, A.; Bielefeldt Biodegradation and Biostabilization of PAHs in  Coal Tar-Water Systems,
Poster presented at the 1999 EPA Hazardous Substance Research Center Conference, Kansas City, April, 1999

Riffel, A.; Bielefeldt, A.; Morrison, K.; Ramaswami, A; Vestal, E.; Illangasekare, I. Biostabilization of PCBs, Poster presented at
the Regional Conference  of the American Waste Management Association, Denver, CO, March, 1999.  Received Best Poster

Morrison, K.; Ramaswami, A.; Riffel, A.; Bielefeldt, A. Biodegradation Kinetics and Biostabilization of PAHs in Coal Tar-Water
Systems Poster presented at the Regional Conference of the American Waste Management Association, Denver, CO, March,

Riffel, A. M.S. Thesis, Biostabilization of Polychlorinated Biphenyls, University of Colorado, Boulder,  July, 1999.
Morrison, K. M.S. Report, Biodegradation and Biostabilization of PAHs in Coal Tar-Water Systems, University of Colorado,
Denver, August 1999

Morrison, K.; Johansen,  P.; Ramaswami, A.  The  Potential for Biostabilization of Coal Tar in the Aqueous Environment,
nresenteH at the 4-th International Svmnniinm of Snhciirfare Mirrnhinlnov  American Srvietv nf Mir.mhinlnoicte  Anoiict  1QQQ
                                                Page 42 of 54

                             Bioremediation Research Program Review 1999
     Bioavailability of Organic Contaminants  in Estuarine Sediments to
                               Microbes and Benthic Animals

              Karl Rockne, Gary Taghon, Lily Young, David Kosson, Wenhsin Liang, Leslie Shor
                                           Rutgers University

The  goal of this project is to determine the bioavailability of sediment-associated contaminants to microbes and
benthic animals. Most often  bioavailability refers to the fraction of a contaminant that may enter  biological
processes. Alternatively, bioavailability can refer to the flux of contaminants. When defined as a flux, bioavailability
will  be a function of the local environmental conditions, including contaminant interactions with the solid matrix,
concentration gradients, pH, redox potential, and solution composition.  If, under a given set of conditions,  the
resulting flux is below the minimum flux required by the organism for uptake or utilization, then the contaminant
would not be  bioavailable. Understanding and quantifying the relationship between the physical and chemical
characteristics of sediments and fluxes of contaminants to microbial and animal communities is essential for prudent
risk-based decision making.

Our approach has focused on characterizing contaminant partitioning, biodegradation, and uptake behavior in field-
aged estuarine  sediments. The test sediments come from two locations in the heavily industrialized New York/New
Jersey Harbor.  Piles Creek is a tidal creek located within an industrial park consisting of petroleum refineries and oil
storage tanks. Newtown Creek is a small channel off the East River in New York City with a long  history of
contamination  from  industrial and municipal waste sources. Sediments  from both  locations contain a complex
assortment of  organic and inorganic contaminants. Our emphasis is  on the  polycyclic aromatic  hydrocarbons
(PAHs). The physical and chemical properties of these sediments have been extensively characterized,  and many
differences in these properties which may affect the bioavailability of contaminants have been observed. Newtown
Creek sediment is very fine-grained, with 94% (by mass) silt and clay, while Piles Creek sediment is relatively
coarser at 55% silt and clay. The sediments have been fractionated on the basis of particle size and particle  density.
Large (>500 Fm), low-density (<1.9 g/cm3) particles from Piles Creek have the highest PAH concentrations,
approaching 3000 ppm. PAH concentrations in Newtown Creek sediment are lower, peaking at 800 ppm in the 125-
300 Fm sized low-density fraction. For both sediments, the majority of the total PAHs are found in the low-density
fraction, although this fraction makes up only a minor part of the total sediment mass (4% for Piles Creek,  15% for
Newtown Creek). When  PAH concentrations are normalized to the  carbon  content of the  size-  and  density-
fractionated particles,  both sediments show deviations from the predictions of equilibrium partitioning theory,
especially for Piles Creek sediment.

Characterization of the physical structure of the sediments demonstrated that Newtown creek sediment  had much
greater macropore (pores > 50 nm) and mesopore (pores 2-50 nm) volume and surface area. This resulted in much
higher PAH concentrations (normalized to surface area) in Piles Creek sediment and over twice the pore volume
(and three'times the pore surface area) of pores accessible to bacteria (>0.5  Fm) in Newtown Creek sediment.

We investigated the  abiotic desorption flux of field-aged  PAHs (PAHs already present in the sediment) from  the
various sediment fractions to determine whether the flux rates  were independent of size- or density-fraction.  We
found substantial differences in desorption kinetic behavior between the sediments. For example, the desorption rate
of benzo[a]pyrene was an order of magnitude higher in the low density fraction than in the high density fraction, and
had a very different flux curve.

A bacterium isolated from Piles Creek sediment has been partially characterized and used in biodegradation rate
studies. The strain, designated PC01, is weakly motile, Gram positive, and capable of growing on phenanthrene and
pyrene.  The degradation of phenanthrene by PC01 varies depending on sediment type. For freshly spiked Piles
Creek sediment, phenanthrene degradation  rate is 67  Fm/h/mg protein, whereas the rate on  Newtown Creek
sediment is much lower, at 25 Fm/h/mg protein. PC01 can degrade pyrene and phenanthrene simultaneously,  but
pyrene degradation proceeds  more rapidly in the presence of phenanthrene. Degradation of freshly added pyrene
also  proceeds faster  in Piles Creek sediment than in Newtown  Creek sediment.  These biodegradation rates were
similar  to initial PAH desorption flux  measurements in the  field-aged  sediments,  suggesting that initial
biodegradation rate is biologically-limited. However, the rapid decrease in desorption flux after approximately 24
hours observed in the abiotic desorption experiments suggests that biodegradation will rapidly  become desorption
rate-limited. Degradation by PC01 of field-aged pyrene shows a different pattern from the freshly spiked sediment
                                             Page 43 of 54

                               Bioremediation Research Program Review 1999
experiments, with biodegradation rates approximately six times faster in Newtown Creek sediment.  This difference
may be due to the greater pore volume and pore surface area of Newtown Creek sediment, resulting in increased
surficial contact area for bacteria.  Not surprisingly, the biodegradation rates were much lower than those observed
with the freshly spiked sediment, demonstrating the effect of field aging on biodegradation rates.

Our data demonstrate the effect sediment physical and chemical structure has on sequestration, biodegradation, and
abiotic  desorption  behavior. These results led us to hypothesize that sediment detritivory (sediment feeding by
benthic animals), which can change the physical and chemical structure of sediment, will have a substantial effect on
partitioning behavior. To test this hypothesis,  we first quantified the changes in physical structure  caused by
sediment detritivory using Capitella sp. I as our model polychaete.  We characterized the pore structure in our test
sediments before and after feeding, and found substantial differences in particle size selectivity. In addition, in both
sediments (and a non-contaminated control)  there were substantial  changes in the  macropore structure caused by
feeding. This suggests that PAH transport processes affected by macropore structure may be affected significantly
by detritivory.

Publications and Presentations

Rockne, K.J., G.L. Taghon, L.Y. Young, D.S. Kosson.  1999. Sequestration of PAHs in size- and density fractionated estuarine
sediments. In Situ and On Site Bioremediation, Fifth International Symposium, San Diego

Rockne, K. J., Young, L. Y., Kosson, D. S., Taghon, G. L. 1999. Correlation of PAHs with organic carbon, surface area, and soot
in organic-rich estuarine sediments. Society of Environmental Toxicology and Chemistry, Philadelphia

Rockne, K. J., Young, L. Y., Kosson, D. S., Taghon, G. L. 1999 Metal content versus particle size and organic matter content in
estuarine sediments:  factors affecting potential uptake  by  detritivores. Society of Environmental Toxicology and Chemistry,

Rockne, K. J., Shor, L. M., Young,  L. Y., Kosson, D. S., Taghon, G. L.  1999 PAH sequestration and desorption kinetics in
density-fractionated estuarine sediment. Society of Environmental Toxicology and Chemistry, Philadelphia

Shor, L.M., Rockne, K.J., Taghon, G.L., Young, L.Y., Kosson, D.S. 1999. Effect of particle size on sequestration and release of
PAHs in field-aged estuarine sediment. Society of Environmental Toxicology and Chemistry, Philadelphia

Young,  L.Y., So, C.M., Zhang, X., Phelps, C.D., Sullivan, E.R.,  Palermo,  C.  1999. Novel mechanisms for anaerobic
biodegradation of PAH  and alkanes. Society of Environmental Toxicology and Chemistry, Philadelphia
                                                Page 44 of 54

                             Bioremediation Research Program Review 1999
     Role of Metal  Bioavailability in In-Situ Bioremediation of Metal and
                              Organic Co-Contaminated  Sites

                                    Raina M. Maier and Ian L. Pepper
                                    University of Arizona, Tucson, AZ

A  large proportion of hazardous waste sites are  co-contaminated with organics and  various metals.  Such  co-
contaminated sites are difficult to bioremediate due to the nature of the mixed contaminants.  Specifically,  the
presence of a co-contaminating metal imposes increased stress on indigenous populations already impacted by
organic contaminant stress. As a result, rates of biodegradation of organics may be reduced significantly in  the
presence of a co-contaminating metal.  The overall objective of this research is to investigate the effect of varying
metal bioavailability on microbial populations and biodegradation of organics to allow a better understanding of
how to optimize remediation of co-contaminated sites.

One of the first steps in understanding the response of indigenous microbial populations to metal stress is to examine
different mechanisms of metal resistance.  We have chosen cadmium as a model  metal for this study and have
obtained samples of cadmium-contaminated soil as well as similar uncontaminated soil for comparison.  Cadmium-
resistant isolates resistant were isolated,  discriminated by ERIC DNA fingerprinting, and identified at  the genus
level by metabolic fingerprinting (BIOLOG).  Isolates obtained include Arthrobacter,  Bacillus, Corynebacterium
and Pseudomonas spp.  Six isolates were studied further to determine the specific mechanism of metal resistance.
Three different mechanisms were observed; biosurfactant production (1 isolate), exopolysaccharide complexation of
metal (2 isolates), intracellular accumulation of cadmium (2 isolates), and an unidentified mechanism (1 isolate).
These results suggest that there is much greater diversity in cadmium-resistance mechanisms within environmental
isolates than was previously thought.  These isolates were then used  to evaluate the impact of different resistance
mechanisms in a co-contaminated soil system.  The  organic contaminant was 2,4-dichlorophenoxyacetic acid (2,4-
D) and the co-contaminating metal was cadmium.  Initial experiments were performed in pure culture using one of
the cadmium-resistant isolates obtained  as a part of this study and a cadmium-sensitive bacterium Alcaligenes
eutrophus JMP134 that carries the pJP4 plasmid for 2,4-D degradation.   While none of the  cadmium-resistant
isolates could  degrade 2,4-D,  four of the resistant isolates supported the degradation of 500 ug/ml 2,4-D  by  the
cadmium sensitive 2,4-D degrader, Alcaligenes eutrophus JMP134. Degradation occurred in the presence of up to
24 ug/ml cadmium in pure culture and up to 60 ug/g cadmium in amended soil microcosms. In a pilot intermediate-
scale  field study  conducted in five-gallon  soil bioreactors, one cadmium resistant  isolate, Pseudomonas HI,
enhanced 2,4-D degradation in the presence of 60 ug/g cadmium.

A second approach being evaluated for stimulation  of biodegradation within co-contaminated sites is to alter  the
bioavailability of the toxic metal through the addition of a  biosurfactant.   Previous work has shown  that a
rhamnolipid biosurfactant can complex metals such as cadmium, lead, and  zinc.   However, it was not known
whether this complexation affected metal bioavailability.  Therefore, a series of experiments were conducted to
investigate the effect of  rhamnolipid on  biodegradation in a model co-contaminated system  containing cadmium
and naphthalene.  In this system, cadmium was found to inhibit naphthalene degradation at 45 and 89 uM.  When
rhamnolipid was added at a 10-fold greater concentration than cadmium, complete protection against metal toxicity
was observed. Additions of equimolar concentrations of rhamnolipid reduced cadmium toxicity.  Rhamnolipid
added at a  10-fold smaller concentration than cadmium had no effect on cadmium toxicity. The degrader did  not
utilize rhamnolipid as a carbon source under the conditions of this study, suggesting  that the observed effect of
rhamnolipid on cadmium toxicity was protective rather than nutritive.  A series  of  soil experiments was then
performed to determine whether rhamnolipid could mitigate the toxic effect of cadmium on an indigenous soil
population  during the degradation of phenanthrene.  A Brazito sandy loam  with an indigenous population  of
phenanthrene degraders was tested.  Cadmium (866.2 mg/Kg) was  added to the  soil to achieve a bioavailable
cadmium concentration of 20  mg/L which inhibited phenanthrene (500 mg/kg) degradation for over two weeks.
Rhamnolipid was added at two concentrations, 100  and 1,000 mg/kg.  Results indicate that rhamnolipid added at
1,000 mg/kg protected against cadmium  toxicity while rhamnolipid  added  at  100  mg/kg had no effect.  Further
studies indicated that rhamnolipid is degraded by the indigenous soil population. This led us to try a strategy of
pulsed addition of rhamnolipid.  The results indicate that  at a concentration of 20 mg/L bioavailable  cadmium,
phenanthrene mineralization levels achieved in the absence of cadmium could be attained with an initial rhamnolipid
concentration of 1,000 mg/Kg  and a second pulse of that concentration at 336 hours. This strategy was tested on a
second soil, a  Gila loam, with similar results. These results suggest that rhamnolipid may be useful in enhancing
                                             Page 45 of 54

                                Bioremediation Research Program Review 1999
bioremediation of organic contaminants in sites that are co-contaminated with organics and metals and that a pulsed
application strategy may be necessary to maximal complete degradation.


Al-Tahhan, R. and R.M. Maier.  1999.  Cell Surface Hydrophobicity of Pseudomonas Aeruginosa: Effects of Monorhamnolipid
and Substrate on Fatty Acid and Lipopolysaccharide Content. Appl. Environ. Microbiol., manuscript in review

Sandrin,  T.  R., A.M. Chech,  and R.M.  Maier.   1999.  Protective effect  of a  rhamnolipid  biosurfactant on naphthalene
biodegradation in the presence of cadmium. Appl. Environ. Microbiol., manuscript in review

Marlowe, E.M., R.M. Maier, and I.L. Pepper.  1999a. An RT-PCR assay for evaluating expression and divergence of the nahAc
genotype within Pseudomonas spp. Appl. Environ. Microbiol., manuscript in review

Roane, T.M. and I.L. Pepper.  1999.  Diversity of cadmium resistance  mechanisms in six bacterial isolates.  Appl.  Environ.
Microbiol., manuscript in review

Roane, T.M., K.L.  Josephson,  and I.L.  Pepper.  1999.   Microbial cadmium  detoxification  allows  remediation of a co-
contaminated soil. Environ. Microbiol., manuscript in review

Maslin, P. and R.M. Maier. 1999. Biosurfactant-induced protection against cadmium toxicity during phenanthrene degradation
in soil. Environ. Sci. Technol., manuscript  in preparation

Marlowe, E.M., I.L. Pepper, and R.M. Maier.  1999b. Application of a RT-PCR assay to monitor the influence of bioavailability
on the gene expression patterns of the  catabolic nahAc gene during phenanthrene biodegradation.  Appl. Environ. Microbiol.,
manuscript in preparation

Marlowe, E.M.  An evaluation of bacterial gene expression during the biodegradation of organic  contaminants.  Ph.D.
Dissertation, Defended 2/18/99

Al-Tahhan, R. A.  Cell surface hydrophobicity of Pseudomonas aeruginosa: effects  of monorhamnolipid and substrate on fatty
acid and lipopolysaccharide content. Ph.D.  Dissertation, Defended 6/19/98

Roane, T.M.  Bioaugmentation with metal-resistant microorganisms in the remediation of metal and organic contaminated soils.
Ph.D. Dissertation, Defended 4/22/99

Maslin, P. Biosurfactant-induced protection against cadmium toxicity during phenanthrene degradation in soil.  M.S. Thesis, To
be defended 11/99
                                                  Page 46 of 54

                             Bioremediation Research Program Review 1999
                 Wetland Plants'  Roles in Uptake and  Transport of
                               Heavy Metals and Remediation

                    Judith S. Weis and Peddrick Weis, Rutgers University, Newark, NJ, and
                             UMDNJ - New Jersey Medical School, Newark, NJ

Wetlands can be used to mitigate pollution runoff and to bioremediate contaminated soils. However, wetland plants
can subsequently act as sources, as well as sinks of contaminants in an ecosystem.  We are testing the hypothesis
that the saltmarsh cordgrass, Spartina alterniflora, while yielding a more productive marsh by putting more detritus
into its ecosystem than the reed Phragmites australis, also transports more metals from the sediment into the water
column and into higher trophic levels than does Phragmites. In our first year, we  have examined the nutritive value
of the detritus, contaminant flux and uptake/trophic transfer of metals, both in situ and in plants and detritus brought
into the laboratory from natural and restored Spartina marshes and from Phragmites marshes.

The two species of plants, both raised in a greenhouse and studied in the field (the metal-contaminated Hackensack
Meadowlands (HM)), were analyzed for metals in leaf tissue and for excretion of metals.  Utilizing natural and
restored Spartina and natural Phragmites, all from HM, the nutritive value of the detritus was evaluated by raising
juvenile fiddler crabs and grass shrimp on the  three sources of detritus and comparing their growth, mortality and
regenerative ability.  Parallel studies were performed with the three types of detritus from a relatively clean salt
marsh for comparison. The crabs and shrimp were analyzed for metal uptake, focusing on Hg, Cu, Zn, Cr and Pb.

Our hypothesis that Spartina would be a more nutritious diet than Phragmites, but at the same time a more effective
source of transfer of toxicants to fiddler crab and grass shrimp consumers via detritus,  was not proven.  These
animals grew, regenerated and took up metals similarly from the several diets, despite substantial differences in
metal levels among the several detritus diets (Table 1).  Our hypothesis that Spartina would export more metals into
the water column via excretion  through salt glands was proven. Spartina was found to excrete significantly more of
all metals through leaf tissue than Phragmites under both field and laboratory conditions (Figure 1). Spartina was
also found to accumulate significantly more Cr and Pb in leaves than Phragmites.  Therefore, Spartina both removes
more of certain metals from sediments and excretes more of all analyzed metals into the water column.

Experiments are currently under way to determine which plant species breaks down sooner as detritus and releases
its metal burden into the environment.  The results of the experiments completed thus far suggest that Phragmites,
rather than Spartina, may be the more appropriate species to plant in a wetland designated for bioremediation.

Table 1. Metal contents (ug/g  + SD) in detritus. HM = Hackensack Meadowlands, AC = Accabonac Harbor, NS =
natural Spartina, RS = restored Spartina, P = Phragmites. For each metal, groups with the same superscript are not
significantly different from one  another.
Site Plant
* not certified
104 + 8.5"
107 +9.4"
23.4 + 3.6c
25.1 + 1.9bc
28.2 +14.71*
4.02 + 0.31
3.7 + 0.4

124+ 2.6"
34.8 + 9.2 b
54.8 + 7.7 ^
67.4 + 3.3b
62.5 + 8.1"
17.7 + 0.6
17.9 + 0.4

43.5 + 5.9b
108 + 24.5"
16.2 + 4.8"
17.2 + 5.2"
0.97 + 0.02
1.0 (n.c.)*

4.7 + 0.4C
9.8 + 2.3"
9.2 + 2.4""
4.7 + 0.4C
5.3 + 0.2**
5.3 + 0.7*
0.42 + 0.02
0.87 + 0.03

0.30 + 0.37"
2.22 + 1.13"
1.63 + 1.27"
0.09 + 0.04"
0.04 + 0.03"
0.04 + 0.02"
2.05 + 0.12
1.99 + 0.10

                                             Page 47 of 54

                             Bioremediation Research Program Review 1999

I- 10

                   ^        JL  *

                                                  -— us
                                                  g ••»
                                                                                  3 SPARTINA
                                                                                  i PHRAGMTES
                                                                                      1 Spartina
                                                                                      i Phragmites
                                                                                I	v//r///A
Figure 1. Metal excretion by Spartina and Phragmites leaves under field and lab conditions over a 48-hour period.
Differences between field and lab probably relate to differences in evapo-transpiration rates.
                                             Page 48 of 54

                             Bioremediation Research Program Review 1999
NSF Grant: DEB 9817719

    Interaction  Between Substrata Surface Chemistry, Conformation of
Contaminant Upon Adsorption  and Availability for Bacterial Degradation

                                            James D. Bryers
                 Department of Civil & Environmental Engineering, University of Connecticut

We hypothesize that the bioavailability of organic  contaminants adsorbed to subsurface materials is determined by
the contaminants' conformation upon adsorption and that this conformation is affected by the chemical properties of
the solid support and the contaminant.  Concomitant adsorption of mixed organics or metals will also affect the
molecule's conformation upon a surface.  Research will  quantify the surface chemistry effects on adsorption
conformation and subsequent availability of adsorbed contaminants for bacterial degradation.

We have experimentally determined the bacterial degradation kinetics of three bacterial species, each individually
degrading a specific type of hazardous waste organics. The three organic contaminants  considered are a PAH
(naphthalene-NPH), an s-Triazine (cyanuric acid-CN), and a chlorinated solvent (trichloroethylene; TCE). The
respective bacterial species degrading these organics are Pseudomonas aeruginosa 19SJ (naphthalene) , Klebsiella
pneumonia 99 (cyanuric acid), and Burkholdia cepacia  17616-pTOMSlc (TCE). Suspended batch and continuous
culture aerobic degradation studies have been carried out on the three contaminant:bacterial species combinations to
develop a data base for degradation rates and bacterial growth kinetic constants.  Currently underway are similar
experiments to evaluate degradation rates of mixtures of organics by combinations of defined mixed cultures of all

Bacterial adhesion rates, biofilm formation kinetics, and the rates of extracellular polysaccharide synthesis were
determined under flowing fluid conditions (to minimize mass transfer limitations) for each species.  Growth limiting
substrate in these control experiments was glucose.  Silicon  1,1 crystal, pre-treated with an oxygen beam to affect a
smooth surface (surface topography characterized by atomic force spectroscopy), served as the substratum. Rates of
bacterial cell adhesion and biofilm formation were measured  non-invasively  using fluorescent  microscopy and
digital image analysis and verified  from  destructive  samples of the surface, followed by conventional cell

We have employed self assembled monolayers (SAMs) of akyl-silanes  as molecular "tethers", to  bind desired
functional groups to the substrata; thus manipulating the spatial chemistry of the substratum while maintaining its
topography constant (Figure 1). Oxygen beam pre-treated silicon 1,1  crystal substrata (topographic distortions < ±
10 nm), were coated with a self-assembly  monolayer of vinyl terminated  alkyl silane .  The alkyl silane binds
covalently to the surface at the oxygen terminal end, creating a structured layer of molecules that presents vinyl end
groups to the environment.  Using a spatial template, a pattern of desired chemical functional groups  can be
fabricated across a substratum (Figure 2). In the  first series of fabrications, the resultant  SAMs were left alone,
affecting a layer of vinyl end groups exposed to the environment. Time-of-Flight Secondary Ion Mass Spectroscopy
(TOF SIMS), was used to confirm both the  spatial location and chemical composition of each substratum pre-
treatment layer  (Figure 3).  AFM was used to quantify the apparent  "topography" of the  chemically modified

Currently, we are refining these techniques to sulfonate the vinyl groups, thus  providing a reactive group for the
deposition of goethite (FeOOH) from a supersaturated  ferric salts solution. The resulting surface will be a thin,
nonporous, iron oxide film, providing a surface that is an analog to a soil mineral.  In  Year three, a series of these
SAM-coated substrata will be exposed to single organic contaminants (NPH, CN or TCE) or various combinations
of the three organics and the spatial content and conformation of organics determined using a combination of AFM
(Figure 4 A&B), XPS, and TOF-SIMS. Each contaminant-coated substratum above will be exposed to suspended
cultures of the appropriate degrading bacterial species.  Rates of bacterial cell adhesion and biofilm formation will
be measured non-invasively using fluorescent microscopy  and digital image analysis.  Degradation of adsorbed
contaminants will be qualitatively monitored using fluorescent reporter genes under the control of the contaminant
degradation gene promoter. Rates of contaminant degradation and adherent cell growth will also be determined from
invasive sampling and destructive analysis of the substratum  surface.
                                             Page 49 of 54

                            Bioremediation Research Program Review 1999
                 8 min.

24 min.
48 min.
                72 min.
                                       ..i^ >w*  ;
82 min.
92 min.
Figure 1. Evolution of a alkyl silane self-assembling monolayer as seen via atomic force microscopy.

                   Deplete Exposed Photoresist
                        5. Alkytsilane
                        6. Deplete Photoresist
                                                               7. Aminosilane
                                                      H3C  CH3
                                                         X  /
                                                         o  o

                                     O O  O
                                     I   I  I
Figure 2. Schematic of template process to affect surfaces of known spatial chemistry.
                                           Page 50 of 54

                             Bioremediation Research Program Review 1999
Figure 3. ToF-SIMS analysis of patterned silicone substratum chemistry.  Total negative ion map (top-L); location
of the CN" fragment related to the amino-silane (top-R); location of Si" related to methyl-silane (lower-L); location
      "  related to amino-silane.
Figure 4. A. AFM images of naphthalene adsorbed to silicone 1,1 crystal with a methyl-silane SAM.
B. Naphthalene on a hydroxyl-silane SAM (on the right).
                                                               U.S. EPA Headquarters Library
                                                                       Mail code 3201
                                                               1200 Pennsylvania Avenue NW
                                                                   Washington  DC  20460
                                            Page 51 of 54

                            Bioremediation Research Program Review 1999
   Bioluminescent Sensors for Measuring Pollutant Bioavailability and
   Validity of Environmentally Acceptable Endpoints  in Bioremediation

       Lisa Strong, Lawrence P. Wackett, and Michael J. Sadowsky, University of Minnesota, St. Paul, MN

Research is being conducted to construct and characterize a panel of whole-cell based bioluminescent sensors for
determination of biological availability of the major classes of pollutants.  The biosensors are useful to determine
changes in bioavailability of the organic pollutants. The biosensors are constructed by cloning specific regulatory
regions, derived from bacterial  DNA conferring the  ability to  catabolize xenobiotic compounds, upstream of a
promoterless luxCDABE operon (encoding for bacterial luciferase) in plasmid pUCD615. Cells exposed to certain
pollutant classes respond by emittting light.  Light emission is readily quantitated  by scintillation  counting. A
camR-o/p:lux CD ABE  plasmid was constructed and expressed in Escherichia coli [strain HMS174 (designated
OS30)]. This strain responded to a variety of monocyclic, bicyclic and tricyclic alkanes.  It was a surprise, but also
very useful, that the strain  emits light in the presence of highly  chlorinated aliphatic  compounds such as
pentachloroethane, hexachloroethane and 1,1,1,2-tetrachloropropane.  Another  strain constructed, E. coli (ipbR
o/p::luxCDABE ),  improves on a previously constructed strain strain OS25.  Although  OS25 responds well to
aromatic hydrocarbons such as BTEX and to chlorinated solvents such as TCE and perchloroethylene (PCE), light
emission from OS25 gradually became constitutive due to an unstable insertion sequence element present in the
cloned region of the ipb gene region. The destabilizing insertion sequence element has now been removed. We are
investigating the potential to detect pesticides by biosensors. Atrazine is being used as a model herbicide, building
on our extensive knowledge of microbial atrazine degradation.  The approach  is to use cells which constitutively
express lux.   Upon exposure to chemicals that inhibit photosystem II electron transfer reactions, bioluminescence
will be inhibited.  We have supplied our colleagues at Minnesota with a mer-lux  strain for sensitively sensing
mercury and they have developed immobilized containing E. coli cells in patches within a 30 mm latex polymer film
(1). The copolymer patches could be stored at -20°C for at least 3 months with minimal loss of activity. This offers
a practical method for mercury detection in the field with only inexpensive equipment brought on site.


(1).  Lyngberg, OK, DJ Stemke,  JL  Schottel and MC  Flickinger. 1999. A single-use luciferase-based mercury
biosensor using Escherichia coli HB101 immobilized in a latex copolymer film J.  Indus. Microbiol. & Biotech.
                                            Page 52 of 54

                             Bioremediation Research Program Review 1999
      Influence of Soil Organic Matter Composition on Desorption and
                         Biodegradation of Aromatic Pollutants

                          T.M. Young*, K.M. Scow+R.M. Higashi", T.W-M. Fan+,
                        E. Schwartz*, L.F. Schultz+, N. Watanabe* and S. Loebmann*

                             Department of Civil & Environmental Engineering
                              Department of Land, Air and Water Resources
                                       "Crocker Nuclear Laboratory
                                    University of California, Davis, CA

The availability of sorbed organic chemicals to organisms appears to be controlled in many cases by desorption rates
at the grain scale. Adequate risk assessment or bioremediation system  design will therefore depend on a thorough
understanding of the desorption process, particularly the role of sorbent structure and contact time in determining
desorption rates. Previous efforts to understand how soil organic matter (SOM) composition affects both desorption
and biodegradation rates have  relied upon elemental analysis  (carbon, hydrogen, nitrogen, oxygen) of SOM for
characterizing the sorbing medium.  The goal of this research is to elucidate linkages between the rate and extent of
desorption and biodegradation with SOM composition determined by pyrolysis GC-MS, diffuse reflectance Fourier
transform infrared (DRIFT)  microscopy, and solution-phase I3C-NMR.  The study design  includes  determining  (i)
biodegradation  behavior and active  microbial communities, (ii) SOM  composition,  (iii) sorption-desorption
characteristics, and (iv) supercritical CO2 extractability of aromatic organic pollutants in soils with different types
and amounts of organic matter.

Pyrolysis-gas chromatography/mass spectrometry was combined with standard degradative chemical methods and
DRIFT to characterize nine soils varying in organic matter content, litter input, and degree of humification. Thirty-
five pyrolysis fragments were quantified and grouped into pools based on the structures they most likely originated
from in soil.  Structural hypotheses  based on these  pyrolysis pools were consistent with  findings  by degradative
chemical methods and DRIFT.

Linear correlations were determined between structural properties, including pyrolysis pools, degradative chemical
parameters, and ratios of DRIFT peak heights,  and functional properties, including Freundlich parameters  from
phenanthrene aqueous and supercritical CO2 sorption and desorption experiments, thermal  desorption experiments,
and phenanthrene microbial mineralization  studies.  Results suggest that the organic  carbon-normalized Freundlich
coefficient is negatively correlated with polar features in SOM and positively correlated with hydrophobic features.
Conversely, measurements of non-ideal sorption behavior, including sorption-desorption hysteresis and slow uptake,
as well  as decreased microbial mineralization appear to be correlated with hydrophobic features in soils.  This
supports the hypothesis that hydrophobic regions in SOM, particularly aromatic structures, sequester hydrophobic
contaminants contributing to non-ideal behavior. Correlations with isotherm nonlinearity were less definitive.


Schultz. L.F., T.M. Young and R.M. Higashi, "Sorption-Desorption Behavior  of Phenanthrene Elucidated  by
Pyrolysis GCMS Studies of Soil Organic Matter," Environmental Toxicology and Chemistry, 1999, 18: 1710-1719

Schwartz E. and K.M. Scow, "Using Biodegradation Kinetics  to Measure Availability of Aged Phenanthrene to
Bacteria Inoculated into Soil, " Environmental Toxicology and Chemistry, 1999, 18: 1742-1746
                                             Page 53 of 54

                              Bioremediation Research Program Review 1999
         Genetic Expression During Biofilm Growth And Development

                                   R.S. Burlage1,1. Foley2 and R. Palmer3.
                          1. Environmental Sciences Division, Oak Ridge National Laboratory, TN.
                             2. Department of Microbiology, Stanford University, Stanford, CA.
                         3. National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD

Despite the acknowledged importance of biofilms,  little is known about gene expression once bacterial cells are
attached to a surface. This is partly because there are relatively few cells present, and partly because true biofilms
are mixed cultures, making it difficult to study any single species.

We have used bioreporters of specific  contaminants to determine  when genes are active  in  biofilms. These
bioreporter  strains  produce bioluminescence  when active, and  sensitive photodetecting equipment  is  able  to
visualize the response.  In addition,  we have  used the confocal  microscope to determine the three-dimensional
structure and development of the biofilms under investigation. The convergence of these techniques has revealed
unexpected  patterns of gene expression for genes involved in metal reduction and for biodegradation  of toluene.
These results have been  conformed  using  reverse  transcriptase-polymerase chain reaction and with in situ
hybridization to specific gene probes, demonstrating that the system does not generate artefacts.

It was found that biofilms aged 48 hours showed the most gene activity, although the presence of casamino acids
severely reduced gene expression. This was unexpected, since there was no immediate reason for this level  of
control and the effect had not been observed in bulk phase cultures. However, this phenomenon points up the
differences in gene expression between attached and free bacteria. Experiments with flowcells demonstrated that
gene expression could be greatly influenced by medium components that were not classical inducers of the system.

Subsequent experiments showed  that precursors of homoserine lactones were able to  affect gene expression,
suggesting that a quorum sensing effect is important for gene  expression in  attached  microcolonies. This was
directly observed using  the confocal microscope, and showed that the cells of a  microcolony (less than 100  cells)
were influenced to different degrees by a change in medium composition. This suggests that some cells are sentinels
of environmental changes, and influence the gene expression of other cells in the colony. Whether this represents a
true differentiation of cells is open to debate.


R.S. Burlage and I. Foley. Gene expression in biofilm-associated Pseudomonas strains is influenced by medium constituents, (in

Khang, Y. and R. Burlage. 1998. In-situ monitoring of biofilm formations of Escherichia coli and Pseudomonas putida by use of
Lux and GFP reporters. Biotechnol. Bioprocess Eng. 3: 6-10

Khang, Y., Z.K. Yang, and R.S.  Burlage. 1997. Measurement of iron-dependence of pupA promoter activity by a pup-lux
bioreporter. J. Microbiol. Biotechnol. 7:352-355

Burlage, R. Visualizing gene expression  in-situ. Proceedings, SPIE conference on  Environmental Monitoring and Remediation
Technologies, Boston, MA. October 1998

Palmer, R.J., B. Applegate, R. Burlage, G. Sayler, and D.C.  White. Heterogeneity of gene expression and activity in bacterial
biofilms. Proceedings, 10th international Symposium on Bioluminescence and Chemiluminescence. 1998

Burlage, R. Gene expression from  bacteria under biofilm conditions. DOE NABIR workshop. Reston, VA. January, 1999

Burlage, R.S.. Visualizing gene expression in situ. SPIE Photonics East, Boston, MA. October, 1998

Burlage,  R., R. Palmer,  and D. White.  Bioreporter bacteria in biofilm  communities.  DOE  Bioremediation  Workshop.
Washington, DC. September, 1998
                                               Page 54 of 54