USE OF A UNIQUE BIOBARRIER TO REMEDIATE USE OF A UNIQUE BIOBARRIER TO REMEDIATE NITRATE AND PERCHLORATE IN GROUNDWATER NITRATE AND PERCHLORATE IN GROUNDWATER Presented by Betty A. Strietelmeier Presented by Betty A. Strietelmeier Los Alamos National laboratory Los Alamos National laboratory To 2001 International Containment and To 2001 International Containment and Remediation Conference Remediation Conference Orlando, FL Orlando, FL June 10-13, 2001 June 10-13, 2001 Co-authors: M.L. Espinosa, J.D. Adams, Co-authors: M.L. Espinosa, J.D. Adams, P.A. Leonard, and E.M. Hodge P.A. Leonard, and E.M. Hodge
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USE OF A UNIQUE BIOBARRIER TO REMEDIATE NITRATE AND PERCHLORATE IN GROUNDWATER Presented by Betty A. Strietelmeier Los Alamos National laboratory To 2001.
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USE OF A UNIQUE BIOBARRIER TO REMEDIATE USE OF A UNIQUE BIOBARRIER TO REMEDIATE NITRATE AND PERCHLORATE IN GROUNDWATERNITRATE AND PERCHLORATE IN GROUNDWATER
Presented by Betty A. StrietelmeierPresented by Betty A. Strietelmeier
Los Alamos National laboratoryLos Alamos National laboratory
To 2001 International Containment and Remediation To 2001 International Containment and Remediation ConferenceConference
Orlando, FLOrlando, FL
June 10-13, 2001June 10-13, 2001
Co-authors: M.L. Espinosa, J.D. Adams, P.A. Leonard, and Co-authors: M.L. Espinosa, J.D. Adams, P.A. Leonard, and E.M. Hodge E.M. Hodge
Nitrate as a ContaminantNitrate as a Contaminant
Nitrate is a highly soluble anion that readily transports in Nitrate is a highly soluble anion that readily transports in groundwater resulting in contamination of large subsurface areasgroundwater resulting in contamination of large subsurface areas
Many sites are affected by nitrate contaminationMany sites are affected by nitrate contamination Large number of activities contribute to the problem:Large number of activities contribute to the problem:
– Manufacturing of explosives, chemicalsManufacturing of explosives, chemicals
– Nuclear industry, large amounts of nitric acid used to dissolve Nuclear industry, large amounts of nitric acid used to dissolve metals and actinidesmetals and actinides
– Mining industryMining industry Nitrate health effects can be severeNitrate health effects can be severe
– ““Blue baby syndrome” from oxygen depletion in bloodstreamBlue baby syndrome” from oxygen depletion in bloodstream
– Increased rates of gastric cancer in susceptible adultsIncreased rates of gastric cancer in susceptible adults
Can be used with other barrier materials to Can be used with other barrier materials to remediate multiple contaminantsremediate multiple contaminants– LANL Multi-Barrier system (4 different LANL Multi-Barrier system (4 different
sections)sections)– Multi-Barrier system removes colloids, Multi-Barrier system removes colloids,
actinides and metals, nitrate, perchlorate and actinides and metals, nitrate, perchlorate and other biodegradable organic compounds, other biodegradable organic compounds, strontium and cesiumstrontium and cesium
– Potential for use with high explosives, Potential for use with high explosives, petroleum hydrocarbons and halogenated petroleum hydrocarbons and halogenated organic compoundsorganic compounds
Biobarrier and BiofilmsBiobarrier and Biofilms
Biobarrier is carbon-based, biofilm forms on surface, Biobarrier is carbon-based, biofilm forms on surface, utilizes carbon for microbial growth, destroys nitrateutilizes carbon for microbial growth, destroys nitrate
Porous material used to prevent plugging of biobarrierPorous material used to prevent plugging of biobarrier Biofilm growth is not excessive, carbon released Biofilm growth is not excessive, carbon released
slowly, provides for growth controlslowly, provides for growth control Development of biofilm takes time, only indigenous Development of biofilm takes time, only indigenous
organisms naturally present are usedorganisms naturally present are used Growth of selected population is based on Growth of selected population is based on
contaminants present, i.e. nitrate enhances growth of contaminants present, i.e. nitrate enhances growth of denitrifiersdenitrifiers
Assimilatory nitrate reductasesAssimilatory nitrate reductases– Convert nitrate to ammonium compoundsConvert nitrate to ammonium compounds
– Provide nitrogen to cell for synthesis of amino acids, Provide nitrogen to cell for synthesis of amino acids, other amino-based cellular constituentsother amino-based cellular constituents
– Not oxygen-sensitive, present in all microbial speciesNot oxygen-sensitive, present in all microbial species
Dissimilatory nitrate reductasesDissimilatory nitrate reductases– Nitrate respiration, acts in place of oxygen as electron Nitrate respiration, acts in place of oxygen as electron
acceptor in respiration, usually is oxygen sensitiveacceptor in respiration, usually is oxygen sensitive
– Second to aerobic respiration in amount of energy Second to aerobic respiration in amount of energy derived by microbial cellderived by microbial cell
– Reduction occurs at higher redox potential than for Reduction occurs at higher redox potential than for redox-active metals and radionuclidesredox-active metals and radionuclides
Dissimilatory Nitrate ReductionDissimilatory Nitrate Reduction Two types of DNR, only one is true “denitrification”Two types of DNR, only one is true “denitrification” First is carried out by many facultative anaerobesFirst is carried out by many facultative anaerobes
– Nitrate reduced to nitrite and excretedNitrate reduced to nitrite and excreted
– Nitrite also can be reduced via hydroxylamine to ammonia Nitrite also can be reduced via hydroxylamine to ammonia (nitrate ammonification process)(nitrate ammonification process)
Second, true “denitrification” is carried out by denitrifiersSecond, true “denitrification” is carried out by denitrifiers
– Nitrate is sequentially reduced to nitrite, nitric oxide, nitrous Nitrate is sequentially reduced to nitrite, nitric oxide, nitrous oxide and nitrogen gasoxide and nitrogen gas
– A reductase enzyme carries out each reduction stepA reductase enzyme carries out each reduction step
– Last three products are gases and can be lost to the atmosphereLast three products are gases and can be lost to the atmosphere Third type recently found in a bacterial species, single Third type recently found in a bacterial species, single
denitrification enzyme present: nitrous oxide reductase which denitrification enzyme present: nitrous oxide reductase which
converts Nconverts N22O to NO to N22, , does not use nitrate or nitrite as substratedoes not use nitrate or nitrite as substrate
DenitrificationDenitrification
(1) (2) (3) (4)
NO3- ---> NO2
- ---> [NO] ---> N2O ---> N2
+5 +3 +2 +1 0
(1) Nitrate reductase
(2) Nitrite reductase
(3) Nitric oxide reductase
(4) Nitrous oxide reductase
Biobarrier Laboratory StudyBiobarrier Laboratory Study Objectives of StudyObjectives of Study
– Determine effectiveness of carbon-based Determine effectiveness of carbon-based material in supporting growth of a biofilm (i.e. material in supporting growth of a biofilm (i.e. by providing carbon nutrient), and in nitrate by providing carbon nutrient), and in nitrate destruction (determine denitrification rates)destruction (determine denitrification rates)
– Determine limits for nitrate levels degradedDetermine limits for nitrate levels degraded– Quantify denitrifying microbial populationsQuantify denitrifying microbial populations– Determine the amounts of nitrite and ammonia Determine the amounts of nitrite and ammonia
produced by system with timeproduced by system with time– Determine if perchlorate reduction occursDetermine if perchlorate reduction occurs– Determine the pH in the biobarrier with timeDetermine the pH in the biobarrier with time
Investigated two biobarrier support Investigated two biobarrier support (nutrient) materials(nutrient) materials– Pecan shellsPecan shells– Pecan shells and dog foodPecan shells and dog food
Mortandad Canyon groundwater used in Mortandad Canyon groundwater used in batch degradation studiesbatch degradation studies– Nitrate at natural concentrations (~25 mg/L or Nitrate at natural concentrations (~25 mg/L or
0.5 mM)0.5 mM)– Nitrate spiked up to 600 mg/L (9.7 mM) nitrateNitrate spiked up to 600 mg/L (9.7 mM) nitrate
Experimental set-up, 3 controls, 1 testExperimental set-up, 3 controls, 1 test– 1A/B - Water and support material are sterile1A/B - Water and support material are sterile– 2A/B - Support material sterile, water non-sterile2A/B - Support material sterile, water non-sterile– 3A/B - Water sterile, support material non-sterile3A/B - Water sterile, support material non-sterile– 4A/B - Both water and support material non-sterile4A/B - Both water and support material non-sterile– 5A/B - Sample of water used in experiment5A/B - Sample of water used in experiment
Nitrate Concentration RangeNitrate Concentration Range– Background (~30 ppm) to 600 ppm nitrateBackground (~30 ppm) to 600 ppm nitrate
Studies conducted in batch modeStudies conducted in batch mode– Ratio of 10 ml water to 1 g solidRatio of 10 ml water to 1 g solid– Ratio of 1 g pecan shells to 0.1 g dog foodRatio of 1 g pecan shells to 0.1 g dog food
Two sizes of containers, 20 ml or 100 mlTwo sizes of containers, 20 ml or 100 ml Incubation at room temperature for up to 21 Incubation at room temperature for up to 21
daysdays Sampled periodically and analyzed for Sampled periodically and analyzed for
nitrate, nitrite, ammonia, perchlorate and nitrate, nitrite, ammonia, perchlorate and pHpH
Analytical MethodsAnalytical Methods
Nitrate, nitrite, ammonia and perchlorate - Nitrate, nitrite, ammonia and perchlorate - Ion Chromatography (IC)Ion Chromatography (IC)
Microbial cell counts - Most Probable Microbial cell counts - Most Probable Number (MPN) technique with denitrifying Number (MPN) technique with denitrifying mediummedium
pH - hydrogen ion electrodepH - hydrogen ion electrode
Results of Study 9.7 mM (600 ppm) Results of Study 9.7 mM (600 ppm) Nitrate with Pecan shellsNitrate with Pecan shells
0
2
4
6
8
10
12
1 6 11 16 21
Incubation Period, Days
Nit
rate
Co
ncen
trati
on
, m
M
Sterile Control Sterile PS, Unsterile H2O Unsterile PS, Sterile H2OUnsterile Control Filtered H2O
Results of Study, 9.7 mM (600 ppm) Nitrate Results of Study, 9.7 mM (600 ppm) Nitrate with Pecan shells and Dog Foodwith Pecan shells and Dog Food
Nitrate Degradation Rates, Sterile Control with Nitrate Degradation Rates, Sterile Control with Pecan Shells, Background to 600 ppm NitratePecan Shells, Background to 600 ppm Nitrate
Nitrate Degradation, Non-Sterile Test with Nitrate Degradation, Non-Sterile Test with Pecan Shells, Background to 600 ppm NitratePecan Shells, Background to 600 ppm Nitrate
0
100
200
300
400
500
600
1 2 7 14 21
Incubation Period, Days
30 ppm
200 ppm
400 ppm
600 ppm
Nitrate Degradation, Sterile Control with Pecan Shells Nitrate Degradation, Sterile Control with Pecan Shells and Dog Food, Background to 600 ppm Nitrateand Dog Food, Background to 600 ppm Nitrate
0
100
200
300
400
500
600
700
1 2 7 14 21
Incubation Period, Days
30 ppm
400 ppm
600 ppm
Nitrate Degradation, Non-Sterile Test with Pecan Nitrate Degradation, Non-Sterile Test with Pecan Shells and Dog Food, Background to 600 ppm NitrateShells and Dog Food, Background to 600 ppm Nitrate
0
50
100
150
200
250
300
350
400
450
1 2 7 14 21
Incubation Period, Days
30 ppm
400 ppm
600 ppm
Accumulation of Nitrate Accumulation of Nitrate Degradation ProductsDegradation Products
Pecan shell biobarrier systemPecan shell biobarrier system
Pecan Shell – 9.7 mM nitrate in MCO-5 WaterDay Nitrate (mM) Nitrite (mM)
Denitrifying Most Probable Number (MPN) cell counts (cells/mL) in Denitrifying Most Probable Number (MPN) cell counts (cells/mL) in MCO-5 water unamended with nitrate (i.e. ~30 ppm)MCO-5 water unamended with nitrate (i.e. ~30 ppm)
Sample Identification Day 1 Day 7 Day 14 Day 211A-Sterile Control 9.3E+04 9.3E+04 2.4E+05 1.1E+08
1B-Sterile Control1B-Sterile Control 9.0E+03 0.0E+00 0.0E+00 4.6E+072A-PS Sterile, H2O not 1.1E+04 >1.1E+07 >1.1E+07 >1.1E+082B-PS Sterile, H2O not 3.4E+04 >1.1E+07 >1.1E+07 >1.1E+083A-H2O Sterile, PS not >1.1E+06 >1.1E+07 >1.1E+07 >1.1E+083B-H2O Sterile, PS not >1.1E+06 >1.1E+07 >1.1E+07 >1.1E+084A-PS + H2O Unsterile >1.1E+06 >1.1E+07 >1.1E+07 >1.1E+084B-PS + H2O Unsterile >1.1E+06 >1.1E+07 >1.1E+07 >1.1E+08
pH MeasurementspH Measurements
The pH of the Sterile Control (1A/B) and the Unsterile The pH of the Sterile Control (1A/B) and the Unsterile Cultures (4A/B), pecan shells and pecan shell/dog food Cultures (4A/B), pecan shells and pecan shell/dog food systems in MCO-5 water, 9.7 mM nitrate (600 ppm)systems in MCO-5 water, 9.7 mM nitrate (600 ppm)
Either biobarrier material will effectively Either biobarrier material will effectively degrade nitrate up to 9.7 mM (600 mg/L) in degrade nitrate up to 9.7 mM (600 mg/L) in under two weeksunder two weeks
Addition of dog food enhances the rate of Addition of dog food enhances the rate of nitrate destruction, but increases production nitrate destruction, but increases production of ammoniaof ammonia
Healthy microbial population is present by Healthy microbial population is present by day 7 (>10day 7 (>1088 cells/ml) cells/ml)
Perchlorate is reduced in biobarrier, but more Perchlorate is reduced in biobarrier, but more investigation of interferences is neededinvestigation of interferences is needed
Future WorkFuture Work Investigate more fully the effectiveness of Investigate more fully the effectiveness of
biobarrier in reduction of perchloratebiobarrier in reduction of perchlorate Determine perchlorate concentration range over Determine perchlorate concentration range over
which biobarrier is effectivewhich biobarrier is effective Investigate effectiveness of biobarrier in Investigate effectiveness of biobarrier in
destruction of other biodegradable organics such destruction of other biodegradable organics such as petroleum hydrocarbons, high explosives, as petroleum hydrocarbons, high explosives, chlorinated hydrocarbons and PAHschlorinated hydrocarbons and PAHs
Investigate microbial populations and Investigate microbial populations and community structure using molecular toolscommunity structure using molecular tools