Title: USE OF A UNIQUE BIOBARRIER TO REMEDIATE NITRATE AND PERCHLORATE IN GROUNDWATER
1USE 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
2Nitrate 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
3Biobarrier 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
4Biobarrier 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
5Biobarrier 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
6Nitrate 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
7Dissimilatory 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
8Denitrification
- (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
9Biobarrier 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
10Biobarrier 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
11Biobarrier 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
12Biobarrier 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
13Analytical Methods
- Nitrate, nitrite, ammonia and perchlorate - Ion
Chromatography (IC) - Microbial cell counts - Most Probable Number
(MPN) technique with denitrifying medium - pH - hydrogen ion electrode
14Results of Study 9.7 mM (600 ppm) Nitrate with
Pecan shells
15Results of Study, 9.7 mM (600 ppm) Nitrate with
Pecan shells and Dog Food
16Nitrate Degradation Rates, Sterile Control with
Pecan Shells, Background to 600 ppm Nitrate
17Nitrate Degradation, Non-Sterile Test with Pecan
Shells, Background to 600 ppm Nitrate
18Nitrate Degradation, Sterile Control with Pecan
Shells and Dog Food, Background to 600 ppm Nitrate
19Nitrate Degradation, Non-Sterile Test with Pecan
Shells and Dog Food, Background to 600 ppm Nitrate
20Accumulation 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
21Accumulation 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
22Perchlorate Biodegradation
23Microbial 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
24pH 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
25Conclusions
- 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
26Future 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