NECi Publications
 

Revised and Updated: 02/11/2010                                                               1.888.NITRATE  www.nitrate.com

      The Nitrate Elimination Co., Inc.
    Green Chemistry for Nitrate Detection
                              Biotechnology People Can Use

Campbell, Ellen R. (2009) Poster on nitrate and phosphate test kits for Agriculture, Great Lakes Phosphorus Forum, July 28 – 31, 2009, Windsor, Ontario, Canada.

Campbell, Ellen R. (2009) Speaker, "Green Analytical Chemistry", at the 13th Annual Green Chemistry and Engineering Conference (Am Chemical Society conference), June 23 - 25, 2009 in College Park, Maryland.

Campbell, Ellen R. (2009) Poster and booth on enzyme-based nitrate analysis and test kits, EPA Science Forum Technology Expo, May 19 – 22, 2008, Washington, DC.

Campbell, Ellen R. (2009) Focus group on nitrate test kits for agriculture, run by our marketing partners AuSable LLC (see agnitrogen.com), 15 April 2009, near Lansing, MI.

Campbell, Ellen R. (2009) Poster presentation, PittCon, March 2 – 7, 2008, New Orleans, LA.

Campbell, Ellen R. (2009) Invited speaker on nitrate testing in agriculture, Michigan AgriBusiness Asso Winter Conference, Jan 14 – 16, 2008, Lansing, MI.

Campbell, Ellen R. (2009) Invited speaker on Advances in Nitrate Testing, Michigan AgriBusiness Association Winter Conference, January12 - 14, 2009, Lansing, MI.

Campbell, Ellen R. (2008) Poster and booth on enzyme-based nitrate analysis and test kits, EPA Science Forum Technology Expo, May 19 – 22, 2008, Washington, DC.

Campbell, Ellen R. (2008) Poster presentation, PittCon, March 2 – 7, 2008, New Orleans, LA.

Campbell, Ellen R. (2008) Invited speaker on nitrate testing in agriculture, Michigan AgriBusiness Asso Winter Conference, Jan 14 – 16, 2008, Lansing, MI.

Campbell, Wilbur H., Ellen R. Campbell (2007) Nitrate Analysis Using Different Nitrate Reductase Isozymes. American Laboratory News (In press).

Campbell, Ellen R. (2007) Poster presentation, Agronomy, Crop, & Soils Societies Annual Conference, New Orleans, LA, Nov 4 – 8, 2007.

Campbell, Ellen R. (2007) Invited speaker, NSF Experimental Program to Stimulate Experimental Research (EPSCoR)/USDA Small Business Innovation Research (SBIR) national conference entitled “Enhancing Linkages between Universities and Small Businesses in EPSCoR Jurisdictions,” Portland, Maine on October 15-16, 2007.

Campbell, Ellen R. (2007) Venture Forum, presentation from NIH Commercialization program directed by LARTA, April 30 – May 2, San Francisco, CA.

Campbell, Ellen R. (2007) Poster presentation on enzyme-based nitrate detection for discrete analyzers, Pittsburgh Conference (PittCon), Feb 25-29, 2007, Orlando, FL

2006

Campbell, Wilbur H., P Song, GG Barbier  (2006) Nitrate Reductase for Nitrate Analysis in Water. Environmental Chemistry Letters, 4: 69-73. Download PDF

Campbell, Wilbur H., Ellen R. Campbell, Lynn Egan (2006) Green Chemistry Nitrate Determination: An Alternative Nitrate Analysis Method. American Laboratory, February, 2006.
See: http://www.americanlaboratory.com/

Patton, C. J., et al. (2006) Nitrate Analysis with Nitrate Reductase on the Discrete Analyzer. In preparation
See: http://pubs.acs.org/
 

2005

Barbier, GG & WH Campbell (2005) Viscosity Effects on Eukaryotic Nitrate Reductase Activity. Journal of Biological Chemistry, 280: 26049-54.
 

 Fisher, K, GG Barbier, H-J Hecht, RR Mendel, WH Campbell & G Schwarz (2005) Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site. The Plant Cell, 17: 1167-79.

 

2004

Campbell, WH, T Kinnunen-Skidmore, MJ Brodeur-Campbell & ER Campbell (2004) New and improved nitrate reductase for enzymatic nitrate analysis.  American Laboratory 22(10): 12.  Read this publication
 

Barbier, GG, JC Joshi, ER Campbell & WH Campbell (2004) Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris.  Protein Expression and Purification. 37: 61-71.

ABSTRACT:

NAD(P)H:nitrate reductase (NaR, EC 1.7.1.1-3) is a useful enzyme in biotechnological applications, but it is very complex in structure and contains three cofactors-flavin adenine dinucleotide, heme-Fe, and molybdenum-molybdopterin (Mo-MPT). A simplified nitrate reductase (S-NaR1) consisting of Mo-MPT-binding site and nitrate-reducing active site was engineered from yeast Pichia angusta NaR cDNA (YNaR1).      S-NaR1 was cytosolically expressed in high-density fermenter culture of methylotrophic yeast Pichia pastoris. Total amount of S-NaR1 protein produced was approximately 0.5 g per 10 L fermenter run, and methanol phase productivity was 5 microg protein/g wet cell weight/h. Gene copy number in genomic DNA of different clones showed direct correlation with the expression level. S-NaR1 was purified to homogeneity in one step by immobilized metal affinity chromatography (IMAC) and total amount of purified protein per run of fermentation was approximately 180 mg. Polypeptide size was approximately 55 kDa from electrophoretic analysis, and S-NaR1 was mainly homo-tetrameric in its active form, as shown by gel filtration.      S-NaR1 accepted electrons efficiently from reduced bromphenol blue (kcat = 2081 s(-1)) and less so from reduced methyl viologen (kcat = 159 s(-1)). The nitrate Km for S-NaR1 was 30 +/- 3 microM, which is very similar to YNaR1. S-NaR1 is capable of specific nitrate reduction, and direct electric current, as shown by catalytic nitrate reduction using protein film cyclic voltammetry, can drive this reaction. Thus, S-NaR1 is an ideal form of this enzyme for commercial applications, such as an enzymatic nitrate biosensor formulated with S-NaR1 interfaced to an electrode system.

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2002 and earlier

ABSTRACT:

Development, characterization, and operational details of an enzymatic, air-segmented continuous-flow analysis method for colorimetric determination of nitrate + nitrite in natural water is described.  This method is similar to U.S. Environmental Protection Agency method 353.2 and U.S. Geological Survey method I-2545-90 except that nitrate is reduced to nitrite by soluble, NADH: nitrate reductase (NaR, EC 1.6.6.1) rather than a packed-bed cadmium reactor (CdR).  A 3-channel, air-segmented, continuous-flow analyzer—configured for simultaneous determination of nitrite (0.020-1.000 mg-N/L) and nitrate + nitrite (0.05-5.00 mg-N/L) by the NaR-and CdR-reduction methods—was used to characterize analytical performance of the NaR-reduction method.  At an analysis rate of 90 hr^-1, sample interaction was less than one percent for all three methods.  Method detection levels were 0.001 mg NO₂^--N/L for nitrite, 0.003 mg NO₃^-+ NO₂^--N/L for nitrate + nitrite by the CdR-reduction method, and 0.006 mg NO₃^-+ NO₂^--N/L for nitrate + nitrite by the NaR-reduction method.  Reduction of nitrate to nitrite by the CdR and NaR methods were both greater than 95 percent over the entire calibration range.  Nitrate + nitrite concentrations determined by the NaR-reduction method in 124 natural water samples were statistically equivalent (p_0.001) to those determined simultaneously by the CdR-reduction method.

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Campbell, E. R., T. Kinnunen-Skidmore, L. A. Winowiecki, and W. H. Campbell (2001) A New Trend in Nitrate Analysis: An Enzyme-based Field Test for Nitrate. American Laboratory (News Edition) 33 (4): 90-92.

see http://www.americanlaboratory.com/

Campbell, E.R. (2000) Nitrate and Health. Focus 10,000, Minnesota's Lakeside Mag.. Fall 2000: 8-9.

ABSTRACT: see article

ABSTRACT: The enzyme nitrate reductase is used to catalyze the reduction of nitrate to nitrite in a protocol requiring microliter volumes of field samples.   Nitrate reductase is commercially available in a stabilized form.  One advantage of the enzyme based nitrate assay is that the reduction of nitrate is easily driven to completion, resulting in a more reliable nitrate determination.

ABSTRACT: Nitrate is routinely measured in a variety of substrates - water, tissues, soils, and foods - both in the field and in laboratory settings. The most commonly used nitrate test methods involve the reduction of nitrate to nitrite via a copper-cadmium reagent, followed by reaction of the nitrite with the Griess dye reagents. The resulting color is translated into a nitrate concentration by comparison with a calibrated color chart or comparator, or by reading the absorbance in a spectrophotometer. This basic method is reliable and sufficiently sensitive for many applications. However, the cadmium reagent is quite toxic. The trend today is for continued increase in concern for worker health and safety; in addition, there are increasing costs and logistical problems associated with regulatory constraints on transport and disposal of hazardous materials. Some suppliers have substituted a zinc-based reagent powder for the cadmium in an effort to reduce toxicity. We describe here an enzyme-based nitrate detection method as an improvement on the basic Griess method that demonstrates equal or superior sensitivity, superior selectivity, and is more environmentally benign. Comparisons between the enzyme-based method and some standard field test kits being used today are made.

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