The overall objective of this paper is to carry out field investigations to assess the potential for immobilizing and detoxifying chromium-contaminated groundwater using lactate-stimulated bioreduction of Cr(VI) to Cr(III) at the Hanford 100H site

Borglin, S.

Brodie, E.

Faybishenko, B.

Firestone, M.

Hazen, T. C.

Hubbard, S.

Joyner, D.

Koenigsberg, S.

Long, P. E.

Newcomer, D.

Peterson, J.

Resch, C.T.

Tokunaga, T.

Wan, J.

Willet, A. C. T. Resch, and D. Newcomer (Pacifific Northwest National Lab), S. Koenigsberg and A. Willet (Regenesis, Ltd.) Field Investigations of Lactate-Stimulated Bioreduction of Cr(VI) at Hanford 100H

Williams, K.

UNT Digital Library

T. C. Hazen, B. Faybishenko, D. Joyner, S. Borglin, E. Brodie, S.Hubbard, K. Williams, J. Peterson, J. Wan, T. Tokunaga, M.Firestone (LBNL, University of California-Berkeley), P. E. Long,C. T. Resch, and D. Newcomer (Pacific Northwest National Lab),S. Koenigsberg and A. Willet (Regenesis, Ltd.)Field Investigations of Lactate-StimulatedBioreduction of Cr(VI) at Hanford 100HOverall ObjectiveTo carry out field investigations to assessthe potential for immobilizing anddetoxifying chromium-contaminatedgroundwater using lactate-stimulatedbioreduction of Cr(VI) to Cr(III) at theHanford 100H site 	       		   4/26/05   ©T. C. Hazen #3Center forEnvironmentalBiotechnologyTimeDistance from SourceDominant Terminal Electron Accepting Process+100-10Electron AcceptorspEAerobicRespirationO2OrganicsO2SO4SulfateReductionSO4H2SMethanogenesisCO2CH4H2DenitrificationNO3NO3Iron (III)ReductionFe (III)Fe (II)Chemical SpeciesEquivalentsCritical BiogeochemistryPCE/TCEMn (IV)Cr (VI)U (VI)The Cr source is believed to besodium dichromate (Na2Cr207.2H2O)Hanford 100HCr Concentration MapLithological ColumnHanford 100H Site CharacterizationHanford 100DOn-Site Data CollectionFlow cell with Br sensorFlow cell with a multiparameter probe(pH, DO, conductivity, temperature)Sampling portKs=3.7-7.4 m/dayEffective porosity = 0.2-0.26Br-Injection and Pumping TestsWater travel time between the injection and pumping wells vs. the pumping ratey  = 2.25x-1051015200 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Pumping rate (gpm)Water travel time (days)PumpingstartedTracerinjectionFor a distance of 4 m from the pumping well,q=0.35 gpm, water travel time is 2.75 days012345678-200 -150 -100 -50 0 50 100 150 200 250Elapsed Time Since Tracer Injected (hr)Br Concentration (ppm)Br Ion-Selective Electrode MeasurementsFlow-Through CellFlow-Through CellLactic Acid MoleculeH+ from waterOH- from waterHRC®(Polylactate Ester)4/26/05   ©T. C. Hazen #8Center forEnvironmentalBiotechnology Lactate-Induced Bioreduction of Cr(IV)HRCWater samplersRingold clayInjection of 40 lbs of 13C-labeled HRC           Well 699-96-45, August 3, 2004 Pumping - 27 days     Well 699-96-44Hanford sandy graveland gravelly sandPumping Groundwater level Ringold siltInjection at depths of 44 ft to 50 ft Field HRC Injection TestPost-HRC Injection Changes in Electrical ConductivityHigh KsatHRC Injection Zone2 Days afterHRC injection3 DAYS30 DAYSGroundwater FlowPumpHypothesis: Lactic acidHypothesis: Reaction halodue to formation ofprecipitatesLower Ksat42’45’42’45’42’45’Results of HRC BiostimulationRedox dropped from 240 to -130 mVDO dropped from 9 mg/l (~100%)to 0.35 mg/l (4.5%)D. vulgaris (directfluorescent antibody)Acetate increase0204060801001201408/2 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)Sulfur reductionNitrate decreaseBiogeochemical Evidence of Microbial Metabolism in Groundwater01020304050608/2 8/12 8/22 9/1 9/11 9/21Phosphate (ppm)Phosphate increaseIsotopic Composition of NitrateIndicates DenitrificationAfter Kendall and McDonnell, 1998Nitrate       NitriteBiogeochemical Evidence of Microbial Metabolism in GroundwaterIncrease in Ferrous Concentrationafter the HRC InjectionHRC injectionPumping stopped1.E-031.E-021.E-011.E+001.E+011.E+028/2 8/9 8/16 8/23 8/30 9/6 9/13 9/20 9/27 10/4 10/11Soluble Iron Fe(II) (ppm)Injection wellDowngradient Monitoring  wellUpgradient Monitoring  well i j ti Pumping stoppedBackground Fe(II)Fe(III) Fe(II)Microbial reductionFe(II)      Cr(VI)+ Cr(III) PrecipitationGeochemical Evidence of Microbial Metabolism in Groundwater+Acridine orange direct counts:Injection well - 45Sampling dateAug  Sep  Oct  Nov  Dec  Jan  btil ll l1e+41e+51e+61e+71e+845.0 ft "yellow" 49.5 ft "black" 52.5 ft "white" 56.5 ft "blue" Acridine orange direct counts:Monitoring well - 44Sampling dateJul  Aug  Sep  Oct  Nov  Dec  Jan  Feb  btil ll l1e+41e+51e+61e+71e+8pump 45.0 ft "yellow" 49.5 ft "black" 52.5 ft "white" 56.5 ft "blue" PLFAbiomassestimatesPLFA biomass estimates, extraction well 441.00E+051.01E+072.01E+073.01E+074.01E+075.01E+076.01E+078/1/04 8/21/04 9/10/04 9/30/04 10/20/04 11/9/04 11/29/04 12/19/04datecells/ml sampleyellowblackwhitebluepumpPLFA biomass estimates, injection well 451.00E+052.01E+074.01E+076.01E+078.01E+071.00E+081.20E+081.40E+088/1/04 8/21/04 9/10/04 9/30/04 10/20/04 11/9/04 11/29/04 12/19/04cells/mlyellowblackwhitebluePLFA:  Community structure44, yellow, community structure0.000.100.200.300.400.500.600.700.800.901.009 11 13 15 17 20 23 27 43time (days)mole fraction lipid20:020:4w 5,8,11,14c18:018:1w 5c18:1w 7c18:1w 9c18:2w 9,12t18:2w 9,12c18:3w 6,9,1218:3w 9,12'1518:1iso17:017:1w 6c17:1w 8c17:0 anteiso17:0 isoa17:1w 9ci17:1w 9c15:0 ISO 3OH16:016:115:0 iso 2 OH16:1w 5c16:1w 7c16:1w 9c16:0 iso14:0 3OH16:1 iso H16:1 w 7c alcohol14:0 2OH15:015:0 anteisoPLFA:  Selected Biomarkerswell 44, yellow depth0.000.200.400.600.801.009 11 13 15 17 20 23 27 43dayFraction PseudomonasDesulfovibrio sp.Geobacterwell 44, black depth0.000.200.400.600.801.009 13 15 17 20 23 27 43dayFraction PseudomonasDesulfovibrio sp.Geobacterwell 44, white depth0.000.200.400.600.801.009 13 15 17 20 23 27 43dayFraction PseudomonasDesulfovibrio sp.Geobacterwell 44, blue depth0.000.200.400.600.801.009 13 15 17 20 23 43dayFraction PseudomonasDesulfovibrio sp.Geobacter4/26/05   ©T. C. Hazen #20Center forEnvironmentalBiotechnology DOE 16s rDNA microarray• Rapidly detect the compositionand diversity of microbes in anenvironmental sample• Massive parallelism - 550,000probes in a 1.28 cm2 array•  all 9,900 species in 16S rDNAdatabase•  Single nucleotide mismatchresolutionMATCHMISMATCHcctagcatgCattctgcatacctagcatgGattctgcataDays since HRC injection0 11 17 27050010001500200025003000 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 later16S Microarray is Quantitative:when comparing same OTU over time/treatmentnormalized intensity~107 16S gene copies~1011 16S gene copies16S rDNA GeneChip Archaea Only Crenarchaeotes (non-thermophilic) detected Dominated by one type – no cultured relative. Bacteria Initial enrichment of denitrifiers Fulvimonas, Pseudomonas, Hyphomicrobium,Acidovorax, Aquaspirillum,Thauera, Azoarcus,Comamonas, Dechloromonas, Clostridium. Followed by enrichment of sulfate reducer(s)13C of Dissolved Inorganic Carbon is Byproductof HRC MetabolismPossible CO2 production Biogeochemical Evidence of Microbial Metabolism in Groundwater1.E-031.E-021.E-011.E+008/2 8/9 8/16 8/23 8/30 9/6 9/13 9/20 9/27 10/4 10/11Cr(VI) ppmInjection wellDowngradient Monitoring  wellUpgradient Monitoring  wellHRC injection Pumping stoppedChanges in Cr(VI) Concentrations after the HRC InjectionConclusions1.  Bacteria in Hanford groundwater is low but includes Arthrobacter, Oxalobacter, Sporomusa andPseudomonas species.  Under background conditions, the total microbial population is <105 cells g-1.2.  Lactate increases biomass to >108 cells g-1, generates highly reducing conditions, and enhancesCr(VI) removal from the pore solution.3.  Bacteria densities and diversity continued to increase for the first 6 weeks, followed by a decrease inthe microbial diversity and density after 3 months.4.  DO dropped from 8.2 to 0.35 mg/l, redox potential from 240 to -130 mV, and pH from 8.9 to 6.5,followed by reduction of nitrate to non detect, and finally sulfate reduction. DO and nitrate began toreturn to background concentrations two months after HRC injection, despite ground water bacterialdensities remaining high (>107 cells/ml).5.  Geophysical investigations show that HRC products (such as lactic acids) injected into groundwatercan be detected using radar and seismic survey, and that even small variations in hydrogeologicalheterogeneity may influence the distribution of the amendment and its products.6. 13C ratios in dissolved inorganic carbon confirmed microbial metabolism of HRC. 13C ratios remainabove background values after 6 months. Increases in carbon isotope ratios of DIC in Well 44 arecoincident with increases in bromide, chloride and acetate and decreases in nitrate.  The source ofchloride is likely from the HRC.7.  Hydrogen sulfide production was first observed after about 20 days post-injection, which correspondswith the enrichment of a Desulfovibrio species (sulfate reducer) identified using 16S rDNAmicroarray and monitored by direct fluorescent antibodies.8.  Cr(VI) concentrations in the monitoring and pumping wells decreased significantly and remainedbelow up-gradient concentrations even after 6 months, when redox conditions and microbial densitieshad returned to background levels.Future ResearchWell 699-96-45 Well 699-96-44• Mass transfer between highand low permeability zones• Changes in hydraulicproperties of sediments afterHRC injection• Evaluation of the potential forCr(III) reoxidation• Development of a numericalcode TOUGH Bio-React• Monitoring and new field testsCoring4/26/05   ©T. C. Hazen #28Center forEnvironmentalBiotechnology ContactsDr. Terry C. Hazen          tchazen@lbl.govHanford 100H Chromium Projecthttp://www-esd.lbl.gov/ERT/hanford100h/Center for Environmental Biotechnologyhttp://www-esd.lbl.gov/CEBEnvironmental Remediation Technology Programhttp://www-esd.lbl.gov/ERTEcology Departmenthttp://www-esd.lbl.gov/ECODOE Natural and Accelerated Bioremediation Research Programhttp://www.lbl.gov/NABIR

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

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

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