Andersen, G.

Borglin, S.E.

Brodie, E.

Chakraborty, R.

Conrad, M.

DeSantis, T.

Faybishenko, B.

Firestone, M.

Hazen, Terry C.

Hubbard, S.

Joyner, D.

Koenigsberg, S.

Long, P.E.

Newcomer, D.R.

Peterson, J.

Tokunaga, T.

Wan, J.

Willett, A.

Williams, K.

UNT Digital Library

Earth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tField Investigations of Lactate-Stimulated Bioreduction of Cr(VI) to Cr(III) at Hanford 100HTerry C. Hazen, B. Faybishenko, E. Brodie, D. Joyner, S. E. Borglin, R. Chakraborty, M. Conrad, T. Tokunaga, J. Wan, S. Hubbard, K. Williams, J. Peterson, M. Firestone, G. Andersen, T. DeSantis, P. E. Long, D. R. Newcomer, A. Willett, and S. KoenigsbergLBNL, UCB, PNNL, RegenesisEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tFe(III) Fe(II)Microbial reductionFe(II) Cr(VI)+ Cr(III) PrecipitationMesoscale Studies on Cr(VI) Bioreduction that led to Field Studies+Jiamin Wan, Tetsu Tokunaga, Mary Firestone and Terry Hazen (NABIR supported 1998-2004)Tokunaga, T. K. J. Wan, M. K. Firestone, T. C. Hazen, K. R. Olson, D. J. Herman, S. R. Sutton, and A. Lanzirotti.  2003.  In-situ reduction of Cr(VI) in heavily contaminated soils through organic carbon amendment. J. Environ. Qual. 32:1641-1649.Tokunaga, T. K., J. Wan, T. C. Hazen, E. Schwartz, M. K. Firestone, S. R. Sutton, M. Newville, K. R. Olson, A. Lanzirotti, and W. Rao.  2003.  Distribution of chromium contamination and microbial activity in soil aggregates.  J. Environ. Qual. 32:541-549.Tokunaga, T. K., J. Wan, M. K. Firestone, T. C. Hazen, E. Schwartz, S. R. Sutton, and M. Newville.  2001.  Chromium diffusion and reduction in soil aggregates.  Environmental Science & Technology 35:3169-3174..00.20.40.60.81fraction of added Cr(VI) unreduced0 100 200 300 400days since OC addition+0 ppm OCk = 7.0 E-8 s-1+800 ppm OCk = 9.6 E-8 s-1+4,000 ppm OCk = 2.0 E-7 s-1+0 ppm OC+800 ppm OC+4,000 ppm OCinitially 1,000 ppm Cr(VI) in pore waters1 0  m m1 0  m m1 0  m m1 ,4 0 01 ,2 0 01 ,0 0 08 0 06 0 04 0 02 0 00+800 ppm OC+80 ppm OC+0 ppm OCnativesoil [Cr]day 31100101 mid-depthoutercentermid-depthoutercentermid-depthoutercenter100101100101+800 ppm OC+80 ppm OC+0 ppm OCdehydrogenase activity, µg/(g soil)  (7 days incubation)Earth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tMultidisciplinary TeamScientific Field LBNL PNNL RegenesisMicrobiology Terry Hazen,Eoin Brodie, Sharon Borglin, Dominique Joyner, Mary FirestoneHydrogeology Boris Faybishenko, Jiamin Wan, Tetsu TokunagaPhilip E. Long, Bruce BjornstadGeophysics Susan Hubbard, Ken Williams, John Peterson,Geochemistry Mark Conrad Tom Resch, Kirk CantrellField and technical supportVictor Gruol, Phil Rizzo Darrell Newcomer Steve Koenigsberg, Anna Willet, Kevin LapusEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tOverall Objective To carry out field investigations to assess the potential for immobilizing Cr(VI) in groundwater using lactate-stimulated bioreduction of Cr(VI) to Cr(III) at the Hanford 100H site, and to determine critical community structure changes and stressors that would enable control and predictions of fundamental biogeochemistry that enables this bioremediation strategy for Cr(VI)Integrated ApproachField MeasurementsHydrogeology Geophysics Geochemistry and Isotopic CompositionLab MeasurementsMicrobiologyEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tThe Cr source is believed to be sodium dichromate (Na2Cr207.2H2O)Hanford 100HCr Concentration Map Lithological ColumnHanford 100H Site CharacterizationHanford 100Dhttp://esd.lbl.gov/ERT/hanford100h/Earth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tLiBr Injection (2/27/2004) LiBr Injection: 15.3 g/liter, 12 liters over 2 hours55 Days No pumping 63 Days Pumping63 Days Pumping + 2nd InjectionLactic Acid MoleculeH+ from waterOH- from waterHRC®(Polylactate Ester)Earth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tLactate-Induced Bioreduction of Cr(IV)Earth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tHRCWater samplersRingold clayInjection of 40 lbs of 13C-labeled HRC Well 699-96-45, August 3, 2004Pumping - 27 daysWell 699-96-44Hanford sandy gravel and gravelly sandPumping Groundwater level Ringold siltInjection at depths of 44 ft to 50 ftField HRC Injection TestEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tPost-HRC Injection Changes in Electrical ConductivityHigh KsatHRC Injection Zone2 Days after HRC injection3 DAYS30 DAYSGroundwater FlowPumpHypothesis: Lactic acidHypothesis: Reaction halo due to formation of precipitatesLower Ksat42’45’42’45’42’45’Earth Sciences Division, Ecology Departmentrt  i  i i i , l  rt t0.111010010007/5 7/12 7/19 7/26 8/2 8/9 8/16 8/23 8/30 9/6RedoxDO1.E+051.E+061.E+071.E+087/5 7/12 7/19 7/26 8/2 8/9 8/16 8/23 8/30 9/6Biomass Pumping waterWell 44 - pumpingWell 45 - injection wellHRC injection and pumping beganpump shut offMaximum biomassResults of HRC Biostimulation66.577.588.599.5107/5 7/12 7/19 7/26 8/2 8/9 8/16 8/23 8/30 9/6pHRedox dropped from 240 to -130 mVDO dropped from 9 mg/l (~100%) to 0.35 mg/l (4.5%)D. vulgaris (direct fluorescent antibody)Earth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tDissolved Oxygen (mg/l)0123456789108/1/04 10/31/04 1/30/05 5/1/05 7/31/05 10/30/05HRC injectionPumpingInjection wellDowngradient monitoring wellUpgradient monitoring wellRedox Potential (mV-200-10001002003004008/1/04 10/31/04 1/30/05 5/1/05 7/31/05 10/30/05HRC injectionPumpingpH56789108/1/04 10/31/04 1/30/05 5/1/05 7/31/05 10/30/05HRC injectionPumpingElectrical Conductivity (μS/cm)2003004005006007008009008/1/04 10/31/04 1/30/05 5/1/05 7/31/05 10/30/05HRC injectionPumpingEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt t-20-1001020304050608/1/04 10/1/04 12/1/04 1/31/05 4/2/05 6/2/05 8/2/05Sample Dated13C DIC45 ft49.5 ft52.5 ft56.5 ftd13C of HRCd13C of Backgroundd13C of Dissolved Inorganic Carbon is Byproduct of HRC MetabolismBiogeochemical Evidence of Microbial Metabolism in GroundwaterEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt t0204060801001201408/2 8/12 8/22 9/1 9/11 9/21Acetate (ppm)45 ft49.5 ft52.5 ft56.5 ft0204060801008/2 8/12 8/22 9/1 9/11 9/21Nitrate (ppm)0204060801001201401601802008/2 8/12 8/22 9/1 9/11 9/21Sulfate (ppm)Sulfate reductionNitrate decrease Acetate IncreaseBiogeochemical Evidence of Microbial Metabolism in Groundwater1.E-041.E-031.E-021.E-011.E+001.E+011.E+028/2/04 9/1/04 10/2/04 11/1/04 12/2/04 1/1/05 2/1/05 3/3/05 4/3/05 5/3/05 6/3/05 7/3/05 8/3/05 9/2/05Fe(II) Concentration, mg/L44 pump4549.552.556.5Well 43Fe(II) IncreaseEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tChanges of Cr(VI) Concentration in Groundwater after HRC InjectionAverage Soluble Cr(VI) Concentration1.E-031.E-021.E-011.E+008/1/04 10/1/04 12/1/04 1/31/05 4/2/05 6/2/05 8/2/05CrVI concentration (ppm)Injection wellDowngradient monitoring  wellUpgradient monitoring  wellAverage Total Chromium Concentration1.E-031.E-021.E-011.E+008/1/04 10/1/04 12/1/04 1/31/05 4/2/05 6/2/05 8/2/05Total chromium (ppm)Injection wellDowngradient monitoring  wellUpgradient monitoring  wellEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tApproach• Statistical analysis & data reduction used to mine vast quantities of data• Organisms assigned to groups based on covariance (similar response to treatment)• Combined with geochemical data and PLFA  this yields insight into functional role/nicheof specific organismsCombined High Density Microarray Analysis and 13C Phospholipid AnalysisDays since HRC injection0 11 17 27Corrected hybridization intensity050010001500200025003000 Desulfovibrio halophilus Geobacter metallireducens Dechloromonas agitatus Pseudomonas putida Microarray analysis of bacterial community changesduring Cr(VI) remediation at Hanford 100H site:Dynamics of some significant organisms.Injection Day30 days laterEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tLow HighHierarchical clustering and heatmap plot of 16S GeneChip analysis of microbial community sub-families detected during chromate bioremediation. PCA groups are indicated by brackets.1234Bacteria and Archaea DetectedGrouped according to response to HRC during chromate remediationGroup1 organisms declinePseudomonas, Burkholderia (Denitrifiers) Acidithiobacillus, Thiothrix (Sulfur oxidizers)Leptothrix (Iron oxidizer)Group2 organisms increase then declineAcidovorax, Thauera (denitrifiers)Flavobacteria (aerobes, use glycerol)Group3 organisms decline then returnMainly oligotrophic bacteriaGroup 4 organisms increase in late stagesLegionella, Chlamydophila, Flectobacillus.High Density Microarray AnalysisEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tEuryarchaeota(Methanogens)Intensity (a.u.)010002000300045ft 49.5ft 52.5ft 56.5ft 0100020003000Injection well9         17        216       312Monitoring well9         17        216       312Days post HRC Injection Days post HRC InjectionComment – methanogenic conditions 312 days after single HRC injectionEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tIntensity (a.u.)010002000300045ft 49.5ft 52.5ft 56.5ft Intensity (a.u.)010002000300045ft 49.5ft 52.5ft 56.5ft Bacteroidetes(Flavobacteriaceae)Injection well9         17        216       312Monitoring well9         17        216       312Days post HRC Injection Days post HRC InjectionComment - Initial enrichment of Flavobacteria but declining over timeDo not use lactate – but use glycerol – hence no 13C detected in their PLFAsEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tIntensity (a.u.)010002000300045ft 49.5ft 52.5ft 56.5ft Intensity (a.u.)010002000300045ft 49.5ft 52.5ft 56.5ft Deltaproteobacteria(Desulfovibrionaceae)Injection well Monitoring well9         17        216       312 9         17        216       312Days post HRC Injection Days post HRC InjectionComment – continuous presence of Desulfovibrio –may help maintain chromium reducing conditions by producing H2S - observedEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tIntensity (a.u.)010002000300045ft 49.5ft 52.5ft 56.5ft Intensity (a.u.)010002000300045ft 49.5ft 52.5ft 56.5ft Deltaproteobacteria(Geobacteraceae)Injection well Monitoring well9         17        216       312 9         17        216       312Days post HRC Injection Days post HRC InjectionComment – continuous presence of Geobacter –may help maintain chromium reducing conditions by producing Fe(II) – observed alsoEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt t13C Phospholipid Analysis• General bacterial biomarkers indicate rapid enrichment in 13C• 13C ratio is greater than expected (overall spiked HRC ratio was 15 per mil)• 13C polylactate used as spike it is not esterified to glycerol backbone• it is released and consumed more rapidly• Biomarkers for Flavobacteriaceae increased following injection but showed minimal enrichment with 13C.• Flavobacteria do NOT typically utilize lactate, but may use glycerol (backbone, unlabeled)Earth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tMajor Findings to Date Despite low initial microbial densities (<105 cells g-1), HRC injection in the groundwater stimulated increase in the biomass up to 107 - 108 cells ml-1 Highly reducing conditions were achieved quickly with hierarchical depletion of electron acceptors O, NO3, and Fe (III) (SO4 was reduced but never depleted except transiently months later), sulfate reduction has been sustained to for the last 20 months SIP analysis confirmed microbial metabolism of HRC and PLFA indicated which group of organisms was utilizing the electron donor Geophysical measurements were capable of characterizing hydrogeological conditions and monitoring the HRC distribution in groundwater Biostimulation has not yet had an effect on subsurface flow Cr(VI) was reduced to drinking water standards after increases in Fe(II), and has remained low for the last 20 months. Microbial community structure changes indicate dominance by sulfate reducers and iron reducers that are apparently maintaining Fe(II) and Cr(VI) reductionEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tFuture ResearchWell 699-96-45Well 699-96-44• Metagenome Sequence by JGI• Metagenome (large Insert and small insert clone libraries using MDA) by Diversa• Isolation and sequencing of Desulfovibrio strains by JGI in the Lab Sequencing Program• Mass transfer between high and low permeability zones• Changes in hydraulic properties of sediments after HRC injection• Evaluation of the potential for Cr(III) reoxidation• Development of a numerical code TOUGH Bio-React• Monitoring and new field tests (2 new wells in May).CoringEarth Sciences Division, Ecology Departmentrt  i  i i i , l  rt tContacts and PublicationsDr. Terry C. Hazen   tchazen@lbl.govHanford Project  http://esd.lbl.gov/ERT/hanford100h/ERSP  http://www.lbl.gov/ERSPHazen Lab  http://www-esd.lbl.gov/ECO/Hazenlab/index.htmEcology Department  http://www-esd.lbl.gov/ECOCenter for Environmental Biotechnology  http://www-esd.lbl.gov/CEBVirtual Institute for Microbial Stress and Survival  http://vimss.lbl.gov.  .    l l. j   // .l l. / / /  // .l l. /   // .l l. / / l /i .l    // .l l. /  i l i l   // .l l. /i l I i   i i l   i l  // i .l l.

Field Investigations of Lactate-Stimulated Bioreduction of Cr(VI) to Cr(III) at Hanford 100H

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Field Investigations of Lactate-Stimulated Bioreduction of Cr(VI) to Cr(III) at Hanford 100H

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