USE OF A UNIQUE BIOBARRIER TO REMEDIATE NITRATE AND PERCHLORATE IN GROUNDWATER

1
USE OF A UNIQUE BIOBARRIER TO REMEDIATE NITRATE
AND PERCHLORATE IN GROUNDWATER

• Presented by Betty A. Strietelmeier
• Los Alamos National laboratory
• To 2001 International Containment and Remediation
  Conference
• Orlando, FL
• June 10-13, 2001
• Co-authors M.L. Espinosa, J.D. Adams, P.A.
  Leonard, and E.M. Hodge

2
Nitrate as a Contaminant

• Nitrate is a highly soluble anion that readily
  transports in groundwater resulting in
  contamination of large subsurface areas
• Many sites are affected by nitrate contamination
• Large number of activities contribute to the
  problem
• Farming, fertilization, animal feedlots, dairy
• Manufacturing of explosives, chemicals
• Nuclear industry, large amounts of nitric acid
  used to dissolve metals and actinides
• Mining industry
• Nitrate health effects can be severe
• Blue baby syndrome from oxygen depletion in
  bloodstream
• Increased rates of gastric cancer in susceptible
  adults

3
Biobarrier Concept

• Cost-effective solution to shallow groundwater
  plumes
• Uses waste material, solves waste disposal
  problem simultaneously
• Simple to use, just place in trench in path of
  plume
• Material is durable, replacement not necessary
  for many years, if at all

4
Biobarrier Concept, contd.

• Can be used with other barrier materials to
  remediate multiple contaminants
• LANL Multi-Barrier system (4 different sections)
• Multi-Barrier system removes colloids, actinides
  and metals, nitrate, perchlorate and other
  biodegradable organic compounds, strontium and
  cesium
• Potential for use with high explosives, petroleum
  hydrocarbons and halogenated organic compounds

5
Biobarrier and Biofilms

• Biobarrier is carbon-based, biofilm forms on
  surface, utilizes carbon for microbial growth,
  destroys nitrate
• Porous material used to prevent plugging of
  biobarrier
• Biofilm growth is not excessive, carbon released
  slowly, provides for growth control
• Development of biofilm takes time, only
  indigenous organisms naturally present are used
• Growth of selected population is based on
  contaminants present, i.e. nitrate enhances
  growth of denitrifiers

6
Nitrate Reduction Enzymes

• Assimilatory nitrate reductases
• Convert nitrate to ammonium compounds
• Provide nitrogen to cell for synthesis of amino
  acids, other amino-based cellular constituents
• Not oxygen-sensitive, present in all microbial
  species
• Dissimilatory nitrate reductases
• Nitrate respiration, acts in place of oxygen as
  electron acceptor in respiration, usually is
  oxygen sensitive
• Second to aerobic respiration in amount of energy
  derived by microbial cell
• Reduction occurs at higher redox potential than
  for redox-active metals and radionuclides

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Dissimilatory Nitrate Reduction

• Two types of DNR, only one is true
  denitrification
• First is carried out by many facultative
  anaerobes
• Nitrate reduced to nitrite and excreted
• Nitrite also can be reduced via hydroxylamine to
  ammonia (nitrate ammonification process)
• Second, true denitrification is carried out by
  denitrifiers
• Nitrate is sequentially reduced to nitrite,
  nitric oxide, nitrous oxide and nitrogen gas
• A reductase enzyme carries out each reduction
  step
• Last three products are gases and can be lost to
  the atmosphere
• Third type recently found in a bacterial species,
  single denitrification enzyme present nitrous
  oxide reductase which converts N2O to N2, does
  not use nitrate or nitrite as substrate

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Denitrification

• (1) (2) (3) (4)
• NO3- ---gt NO2- ---gt NO ---gt N2O ---gt N2
• 5 3 2
  1 0
• (1) Nitrate reductase
• (2) Nitrite reductase
• (3) Nitric oxide reductase
• (4) Nitrous oxide reductase

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Biobarrier Laboratory Study

• Objectives of Study
• Determine effectiveness of carbon-based material
  in supporting growth of a biofilm (i.e. by
  providing carbon nutrient), and in nitrate
  destruction (determine denitrification rates)
• Determine limits for nitrate levels degraded
• Quantify denitrifying microbial populations
• Determine the amounts of nitrite and ammonia
  produced by system with time
• Determine if perchlorate reduction occurs
• Determine the pH in the biobarrier with time

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Biobarrier Laboratory Study, contd.

• Investigated two biobarrier support (nutrient)
  materials
• Pecan shells
• Pecan shells and dog food
• Mortandad Canyon groundwater used in batch
  degradation studies
• Nitrate at natural concentrations (25 mg/L or
  0.5 mM)
• Nitrate spiked up to 600 mg/L (9.7 mM) nitrate

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Biobarrier Laboratory Study, contd.

• Experimental set-up, 3 controls, 1 test
• 1A/B - Water and support material are sterile
• 2A/B - Support material sterile, water
  non-sterile
• 3A/B - Water sterile, support material
  non-sterile
• 4A/B - Both water and support material
  non-sterile
• 5A/B - Sample of water used in experiment
• Nitrate Concentration Range
• Background (30 ppm) to 600 ppm nitrate

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Biobarrier Laboratory Study, contd.

• Studies conducted in batch mode
• Ratio of 10 ml water to 1 g solid
• Ratio of 1 g pecan shells to 0.1 g dog food
• Two sizes of containers, 20 ml or 100 ml
• Incubation at room temperature for up to 21 days
• Sampled periodically and analyzed for nitrate,
  nitrite, ammonia, perchlorate and pH

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Analytical Methods

• Nitrate, nitrite, ammonia and perchlorate - Ion
  Chromatography (IC)
• Microbial cell counts - Most Probable Number
  (MPN) technique with denitrifying medium
• pH - hydrogen ion electrode

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Results of Study 9.7 mM (600 ppm) Nitrate with
Pecan shells
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Results of Study, 9.7 mM (600 ppm) Nitrate with
Pecan shells and Dog Food
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Nitrate Degradation Rates, Sterile Control with
Pecan Shells, Background to 600 ppm Nitrate
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Nitrate Degradation, Non-Sterile Test with Pecan
Shells, Background to 600 ppm Nitrate
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Nitrate Degradation, Sterile Control with Pecan
Shells and Dog Food, Background to 600 ppm Nitrate
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Nitrate Degradation, Non-Sterile Test with Pecan
Shells and Dog Food, Background to 600 ppm Nitrate
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Accumulation of Nitrate Degradation Products

• Pecan shell biobarrier system
• Pecan Shell 9.7 mM nitrate in MCO-5 Water
• Day Nitrate (mM) Nitrite (mM) Ammonia (µM)
• 1 9.20 0 11
• 2 9.10 0 11
• 7 4.10 1.3 100
• 14 3.00 0.7 133
• 21 1.50 1.3 106

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Accumulation of Nitrate Degradation Products

• Pecan shell/dog food biobarrier system
• Pecan Shell and Dog Food 9.7 mM nitrate in
  MCO-5 Water
• Day Nitrate (mM) Nitrite (mM) Ammonia (µM)
• 1 6.22 2.3 156
• 2 2.58 4.1 217
• 7 0.01 0.9 778
• 14 0.02 1.5 794
• 21 0.02 1.9 806

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Perchlorate Biodegradation
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Microbial Quantitation (MPNs)

• Denitrifying Most Probable Number (MPN) cell
  counts (cells/mL) in MCO-5 water unamended with
  nitrate (i.e. 30 ppm)
• Sample Identification Day 1 Day 7
  Day 14 Day 21
• 1A-Sterile Control 9.3E04 9.3E04 2.4E05
  1.1E08
• 1B-Sterile Control 9.0E03 0.0E00
  0.0E00 4.6E07
• 2A-PS Sterile, H2O not 1.1E04 gt1.1E07
  gt1.1E07 gt1.1E08
• 2B-PS Sterile, H2O not 3.4E04 gt1.1E07
  gt1.1E07 gt1.1E08
• 3A-H2O Sterile, PS not gt1.1E06 gt1.1E07
  gt1.1E07 gt1.1E08
• 3B-H2O Sterile, PS not gt1.1E06 gt1.1E07
  gt1.1E07 gt1.1E08
• 4A-PS H2O Unsterile gt1.1E06 gt1.1E07
  gt1.1E07 gt1.1E08
• 4B-PS H2O Unsterile gt1.1E06 gt1.1E07
  gt1.1E07 gt1.1E08

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pH Measurements

• The pH of the Sterile Control (1A/B) and the
  Unsterile Cultures (4A/B), pecan shells and pecan
  shell/dog food systems in MCO-5 water, 9.7 mM
  nitrate (600 ppm)
• Pecan Shells Pecan Shell Dog Food
• Day pH 1A/B pH 4A/B pH 1A/B pH 4A/B
• 1 5.7 7.3 5.7 nd
• 2 5.4 7.4 5.6 6.1
• 7 7.3 8.0 5.5 5.8
• 14 7.4 8.4 5.5 5.3
• 21 7.2 8.2 7.1 5.6
• nd not determined
• not determined

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Conclusions

• Either biobarrier material will effectively
  degrade nitrate up to 9.7 mM (600 mg/L) in under
  two weeks
• Addition of dog food enhances the rate of nitrate
  destruction, but increases production of ammonia
• Healthy microbial population is present by day 7
  (gt108 cells/ml)
• Perchlorate is reduced in biobarrier, but more
  investigation of interferences is needed

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Future Work

• Investigate more fully the effectiveness of
  biobarrier in reduction of perchlorate
• Determine perchlorate concentration range over
  which biobarrier is effective
• Investigate effectiveness of biobarrier in
  destruction of other biodegradable organics such
  as petroleum hydrocarbons, high explosives,
  chlorinated hydrocarbons and PAHs
• Investigate microbial populations and community
  structure using molecular tools