The Nitrate Elimination Co., Inc.

Transcription

1 The Nitrate Elimination Co., Inc. NECi Lake Linden, Michigan NECi: Clean Water. Fertile Soil. Serious Science.

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3 NECi is an environmental biotechnology company We are dedicated to the application of biotechnology to the solution of environmental problems. NECi s niche: Reagent Grade Enzymes for Green Analytical Chemistry.

4 What s an Enzyme? How does it work?

5 How Nitrate Reductase Works Nitrate reductase (NaR) catalyzes the reduction of nitrate to nitrite. The B vitamin NADH is cofactor. The resulting nitrite reacts with the Griess color reagents to produce a highly colored product. Absorbance measured at 540nm. NaR and NADH replace the toxic cadmium or zinc reagents used in conventional nitrate analysis methods. *Minimal change is required from standard methods

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7 Analytical Advantages of Enzymes Selectivity Find target in complex mixtures Sensitivity Low detection limits in complex mixtures Specificity False negatives and false positives are rare Safety For shipping, storage, handling, and disposal Enzyme-based analytical chemistry is accurate, reliable, and environmentally benign.

8 Enzymes are great analytical tools Active site only binds nitrate High specificity, low sensitivity, rare interferences in complex matrices Nitrate reductase naturally occurring enzyme in plants Non-toxic for lab personnel + no hazardous shipping or waste fees Nitrate reductase simply replaces cadmium Griess reaction follows nitrate reduction = minimal training to switch Recombinant enzyme design & production Stringent quality control, specified traits, batch consistency

9 Recombinant Enzymes are Better Increased production capacity Guaranteed lot-to-lot reproducibility Reasonable production costs Engineered for improved stability Allows storage at controlled RT Stable during analytical runs Shipping at ambient

10 NECi Production of Nitrate Reductase by Fermentation

11 Method Validation Data Interagency round-robin studies showing accuracy + reliability of nitrate reductase method for nitrate-n analysis

12 Sample Types in EPA and ASTM Validations Sample Type Filtered Acid Denver area treatment plant Influent wastewater Yes Yes Denver area treatment plant Wastewater effluent #1 Yes Yes Denver area treatment plant Wastewater effluent #2 Yes Yes Michigan paper mill waste stream effluent Yes Yes Denver area metal finisher waste stream effluent Yes Yes Denver area Commercial laundry waste effluent Yes Yes Environmental Resources Associates #507 Hardness WasteWatR reference material Yes Yes Michigan Confined Animal Feeding Operation Yes Yes (CAFO) effluent from tiled field Low-nutrient seawater (collected offshore Hawaii) Yes No ERA # 608 Reference Standard Yes Yes USGS PE N-116 (low nutrient-fortified river water) Yes No USGS PE N-115 (high nutrient-fortified river water) Yes No Tap water at each lab Tap water plus added Chlorine

13 Table 4: Enzymatic Reduction Efficiency Summary Acceptance Standard is 90% or greater reduction efficiency. 2nd Source = Nitrate Standard in mg N/L Nitrite Standard = mg N/L Lab 1 2nd Source Nitrite Std Lab 2 2nd Source Nitrite Std Lab 3 2nd Source Nitrite Std Lab 4 2nd Source Nitrite Std Lab 5 2nd Source Nitrite Std Lab 6 2nd Source Nitrite Std Lab 7 2nd Source Nitrite Std Lab 8 2nd Source Nitrite Std Lab 9* 2nd Source [550nm] Nitrite Std Lab 10 2nd Source Nitrite Std A-540 nm mg N/L Reduction Efficiency 102.1% 101.3% 107.2% 116.6% 94.9% 103.8% 103.7% 102.3% 98.6% 105.0%

14 Table 5 from EPA Validation Report: Initial Performance and Recovery (IPR) Summary Lab # Analyses Mean Recovery (%) RSD (%) Minimum Recovery (%) Max Recovery (%)

15 Table 7: Method Detection Limit (MDL) Summary Abbreviations: DA = Discrete Analyzer; NA = Not Analyzed. *One replicate discarded due to issue with blank Lab Method MDL Replicates Spike Spike/MDL mg N/L mg N/L Ratio 1 DA DA (7)* DA DA DA DA DA DA NA 9 DA DA

16 Nitrate + Nitrite by DA: Enzyme vs Cadmium Sample NaR CdR RPD mg N/L mg N/L % WW-1* WW WW WW-4* WW WW WW-7* WW SW-1* SR SRM SRM

17 Nitrate + Nitrite Concentration in mg-n/l (NaR) Line of equal correlation Nitrate + Nitrite Concentration in mg-n/l (CdR)

18 NECi HANDHELD PHOTOMETER Dual Wavelength for reliable nitrate and phosphate data anywhere Data is stored and tracked on Android devices for export and further analysis User-friendly, non-toxic test kits with improved mobile app interface Applications for alcohol & glycerol in biodiesel, fuel, and beverages in progress

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20 WHICH ONE WILL YIELD BETTER DATA?

21 TEST KITS for On-Site or Plant Spot Checks Fast Easy Versatile Accurate Economical Safe Results in 20 minutes Prepackaged, premeasured, pre-calculated Nitrate (and phosphate) in many sample types Strong correlation to laboratory data ~ $5 per sample (varies with test kit size) Non-toxic reagents for handling & disposal

22 VARIOUS SAMPLE TYPES Well + Ag Water Standard Range Levels Environmental Water Low Range Levels Soil Plant Petioles Green or Dry Animal Forage

23 21 st CENTURY TEST KITS Legacy Test Kits Add Reagents to 5-10 ml Sample Cadmium or Zinc Powder Round Glass Vials 2+ Points of Manual Data Entry/Transfer Acidification or Complex Preparation NECi Photometer + Test Kits 50 µl Sample into Buffer Freeze-dried Enzyme Flat-Sided Cuvettes Automated Data Input, Storage, Transfer Water Extraction Sample Preparation Manual Sample Detail Entry Sample Collection Time + GPS Stamp

24 NSF FUNDED OPEN-SOURCE PHOTOMETER Collaborative Effort with Michigan Technological University Open Source 3-D Printed Case Design by Dr. Joshua Pierce s Graduate Team SBIR/STTR program award #IIP $65 in materials to build your own PLOS One publication summer 2015

25 COMMERCIAL HANDHELD PHOTOMETER Dual Wavelength for reliable nitrate and phosphate data anywhere Data is stored and tracked on Android devices for export and further analysis User-friendly, non-toxic test kits with improved mobile app interface Applications for alcohol & glycerol in biodiesel, fuel, and beverages in progress

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27 NECi Superior Enzymes Thanks the Following for Their Work and Support: Small Business Innovation Research programs of The US Dept of Agriculture (nitrate and phosphate reagents and test kits) The National Science Foundation (photometer) Michigan s ETF program (travel to conferences) Validation partners at ASTM International, US EPA, & US Geological Survey The people who produced this work: Dr JM Pearce and students at MI Technological University Dr CJ Patton, USGS in Denver NECi Technicians Find more information at