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. StrietelmeierLos Alamos National laboratoryLos Alamos National laboratory

To 2001 International Containment and Remediation To 2001 International Containment and Remediation ConferenceConference

Orlando, FLOrlando, FLJune 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:

– Farming, fertilization, animal feedlots, dairyFarming, fertilization, animal feedlots, dairy– 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

Biobarrier ConceptBiobarrier Concept

Cost-effective solution to shallow groundwater Cost-effective solution to shallow groundwater plumesplumes– Uses waste material, solves waste disposal Uses waste material, solves waste disposal

problem simultaneouslyproblem simultaneously– Simple to use, just place in trench in path of Simple to use, just place in trench in path of

plumeplume– Material is durable, replacement not necessary Material is durable, replacement not necessary

for many years, if at allfor many years, if at all

Biobarrier Concept, cont’d.Biobarrier Concept, cont’d.

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

Nitrate Reduction EnzymesNitrate Reduction Enzymes

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

Biobarrier Laboratory Study, cont’d.Biobarrier Laboratory Study, cont’d. 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

Biobarrier Laboratory Study, cont’d.Biobarrier Laboratory Study, cont’d.

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

Biobarrier Laboratory Study, cont’d.Biobarrier Laboratory Study, cont’d.

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 pHnitrate, nitrite, ammonia, perchlorate and pH

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 21Incubation Period, Days

Nit

rate

Con

cent

rati

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

0

2

4

6

8

10

12

1 6 11 16 21Incubation Period, Days

Nit

rate

Con

cent

rati

on, m

M

Sterile Control Sterile PS + DF, Unsterile H2OUnsterile PS + DF, Sterile H2O Unsterile ControlFiltered H2O

0

100

200

300

400

500

600

700

1 2 7 14 21Incubation Period, Days

30 ppm200 ppm400 ppm600 ppm

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 21Incubation Period, Days

30 ppm200 ppm400 ppm600 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 21Incubation 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 21Incubation Period, Days

30 ppm400 ppm600 ppm

Accumulation of Nitrate Accumulation of Nitrate Degradation ProductsDegradation Products

Pecan shell biobarrier systemPecan shell biobarrier systemPecan Shell – 9.7 mM nitrate in MCO-5 WaterDay Nitrate (mM) Nitrite (mM)

Ammonia (µM)1 9.20 0 112 9.10 0 117 4.10 1.3 10014 3.00 0.7 13321 1.50 1.3 106

Accumulation of Nitrate Accumulation of Nitrate Degradation ProductsDegradation Products

Pecan shell/dog food biobarrier Pecan shell/dog food biobarrier systemsystem

Pecan Shell and Dog Food – 9.7 mM nitrate in MCO-5 Water

Day Nitrate (mM) Nitrite (mM) Ammonia (µM)

1 6.22 2.3 1562 2.58 4.1 2177 0.01 0.9 778140.02 1.5 794210.02 1.9 806

Perchlorate BiodegradationPerchlorate Biodegradation

0

20

40

60

80

100

120

140

1 6 11 16 21

Incubation Period, Days .

Perc

hlor

ate

Conc

entr

atio

n, µ

M

Sterile Control Sterile PS + DF, Unsterile H2OUnsterile PS + DF, Sterile H2O Unsterile PS + DF and H2OFiltered H2O

Microbial Quantitation (MPNs)Microbial Quantitation (MPNs)

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+081B-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 MeasurementsThe 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)

Pecan Shells Pecan Shell + Dog FoodDay pH – 1A/BpH – 1A/B pH – 4A/B pH –1A/B pH –

4A/B1 5.7 7.3 5.7 nd2 5.4 7.4 5.6 6.17 7.3 8.0 5.5 5.814 7.4 8.4 5.5 5.321 7.2 8.2 7.1 5.6nd = not determinednot determined

ConclusionsConclusions 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 of nitrate destruction, but increases production of ammoniaammonia

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