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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 International Containment and Remediation Conference Orlando, FL June 10-13, 2001
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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 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
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
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
Biobarrier Concept, cont’d. • 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
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
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
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
Denitrification (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 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
Biobarrier Laboratory Study, cont’d. • 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
Biobarrier Laboratory Study, cont’d. • 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
Biobarrier Laboratory Study, cont’d. • 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
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
Results of Study, 9.7 mM (600 ppm) Nitrate with Pecan shells and Dog Food
Nitrate Degradation Rates, Sterile Control with Pecan Shells, Background to 600 ppm Nitrate
Nitrate Degradation, Non-Sterile Test with Pecan Shells, Background to 600 ppm Nitrate
Nitrate Degradation, Sterile Control with Pecan Shells and Dog Food, Background to 600 ppm Nitrate
Nitrate Degradation, Non-Sterile Test with Pecan Shells and Dog Food, Background to 600 ppm Nitrate
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
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
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 9.3E+04 9.3E+04 2.4E+05 1.1E+08 1B-Sterile Control 9.0E+03 0.0E+00 0.0E+00 4.6E+07 2A-PS Sterile, H2O not 1.1E+04 >1.1E+07 >1.1E+07 >1.1E+08 2B-PS Sterile, H2O not 3.4E+04 >1.1E+07 >1.1E+07 >1.1E+08 3A-H2O Sterile, PS not >1.1E+06 >1.1E+07 >1.1E+07 >1.1E+08 3B-H2O Sterile, PS not >1.1E+06 >1.1E+07 >1.1E+07 >1.1E+08 4A-PS + H2O Unsterile >1.1E+06 >1.1E+07 >1.1E+07 >1.1E+08 4B-PS + H2O Unsterile >1.1E+06 >1.1E+07 >1.1E+07 >1.1E+08
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 8.27.1 5.6 nd = not determined not determined
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 (>108 cells/ml) • Perchlorate is reduced in biobarrier, but more investigation of interferences is needed
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