Grantee Research Project Results

Final Report: Enzymatic Nitrate Elimination Technology for Small Systems

EPA Contract Number: 68D40046
Title: Enzymatic Nitrate Elimination Technology for Small Systems
Investigators: Campbell, Ellen R. , Campbell, Wilbur W.
Small Business: The Nitrate Elimination Company Inc.
EPA Contact:
Phase: I
Project Period: September 1, 1994 through March 1, 1995
Project Amount: $54,735
RFA: Small Business Innovation Research (SBIR) - Phase I (1994) RFA Text |  Recipients Lists
Research Category: SBIR - Water and Wastewater , Watersheds , Small Business Innovation Research (SBIR)

Description:

The purpose of the project was to investigate the potential for practical application of immobilized enzyme technology for removal of nitrate from potable water. The work was based on a paper published in Nature in 1992 (Mellor et al, Nature 355:717-719), of which Dr. WH Campbell, our company president and chief scientist, is a co-author. The technology involves enzyme immobilization and stabilization, as well as development of new methods for driving enzymatic activity in the absence of biological electron donors.

In these enzyme reactors, which we now call EzNET (from Enzymatic Nitrate Elimination Technology), three enzymes work in series to reduce harmful excess nitrate to environmentally benign nitrogen (N₂) gas. The enzymes we used were Nitrate Reductase (NaR, which reduces nitrate to nitrite), Nitrite Reductase (NiR reduces nitrite to nitrous oxide) and Nitrous Oxide Reductase (NoR, reduces nitrous oxide to N₂ gas). We used purified NaR from plants, and semi-purified NiR and NoR from the common soil bacterium Rhodobacter sphaeroides.

The enzymes were immobilized (covalently bound) to an inexpensive, inert support medium. Electron carrier molecules were also immobilized onto the support. The medium was packed between stainless steel electrodes in a small chamber to create the bioreactor. The function of the electrodes is to provide the electrons the enzymes require in order to carry out the chemical reactions of the nitrate reduction steps.

During the project, we built several small scale enzyme bioreactors and evaluated their performance. The prototypes we built and evaluated were designed for use at the home faucet for treatment of drinking and cooking water. The amount of immobilized enzyme material in the reactors was quite small, no more than a few grams (one teaspoon).

We concentrated our efforts on the first enzyme in the system, nitrate reductase (NADH form), for two reasons: 1) it is a fragile enzyme, and we felt it was critical to evaluate its performance in the system to be sure of its potential viability, and 2) since we purify this enzyme we had a dependable source of well-characterized material to work with throughout the project.

Summary/Accomplishments (Outputs/Outcomes):

As demonstrated in the project's final report, our nitrate reductase (NaR) is rugged enough to continue to view this technology as worthwhile. Enzymatic activity lasting more than 30 days under constant flow at ambient temperatures (22 - 30^oC) was achieved. This is a sufficient time frame for practical application at the home faucet. Data on useful lifetime of the NiR and NoR immobilized enzymes was also positive.

The advantage of an enzyme-based system over microorganism-based denitrification is very strong: enzymes are chemical reagents, not living things. They do not need to be fed (no carbon feed is required). There is no biomass waste to dispose of, which is a savings in disposal and maintenance costs. No bacterial metabolites or breakdown products are introduced into the water. When the enzymes are spent, a new immobilized-enzyme module would be installed, similar to maintaining a filtration system. The immobilization media we plan to use is an inert and inexpensive material; spent modules will consist mainly of this material and peptides.

In the past enzymes have been too fragile to be considered for this type of application, but enzyme engineering is now a reality. For example, a mix of thermophilic and halophilic (salt tolerant) nitrate reductases are known and could be mixed in a reactor for a broader range of optimal performance. Or one nitrate reductase isozyme could be modified using molecular biology methods to incorporate these capabilities. NECi is involved in the forefront of this technology; Dr Campbell's publication record in enzyme engineering is strong. NECi hopes to continue making progress in this area under a current Phase II SBIR project, pending with NIH at this time.

Conclusions:

Our main conclusions from the project are that there are two major barriers that need to be addressed to make this technology viable as well as cost-effective. We are working on both of these issues at NECi now:

1. The cost of the enzymes must be reduced dramatically. The enzymes used are not commodity enzymes at this time, and kilograms will be needed in order to build industrial and municipal sized systems. To this end, we are working on expression of nitrate reductase in a yeast expression system, with funding from NIH. Our Phase II is pending at time of writing, following a successful Phase I effort. The other enzymes in the reaction are from a fermentable bacterium, so this barrier should be less of a problem for the nitrite and nitrous oxide reductases.
2. The transfer of electrons to the active sites of the reductase enzymes needs to be improved. We can approach this objective from two angles. First, we are working on nitrate biosensor technology under a new Phase I SBIR from NIH (start date Sept. 99). This project involves investigations into the electrochemistry of nitrate reductase and reductases in general. Second, a variety of electrode materials needs to be tested. New materials, such as pyrolytic graphite, may also help in design of a practical system.

We strongly believe in the potential of this nitrate removal technology. The chemistry of nitrate and NOx compounds is exceedingly complex; potential chemical or thermal methods may produce reaction byproducts of higher toxicity than nitrate, merely complicating an already difficult problem. We can anticipate that the regulatory environment for water and air quality will continue to tighten worldwide as the importance of protecting these resources becomes more manifest. Patience and investment are going to be required, but perhaps it is time to bring the new biotechnologies to bear on the solution of difficult environmental problems, including nitrate remediation. Commercialization Status: Although there is widespread interest in the EzNET system, NECi has not made any serious attempts to commercialize this product. We feel that we need to be in a stronger position, having addressed the technology barriers cited above, before we approach commercialization partners in the water treatment equipment sector.

NECi has been involved in commercialization of its NaR-based nitrate test kits since late 1997. A new Consumer Kit was released in November 1999. These products are part of an effort to raise the awareness of nitrate as an important health and environmental quality issue.

NECi maintains a website that provides information on enzyme-based water testing and treatment, which generates inquiries and comments on a daily basis. Both NECi founders, Ellen R and Wilbur H Campbell, make presentations at technical, business, and academic venues to explain the potential of enzymatic processes for the solution of environmental problems.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Publications Views

Other project views:	All 2 publications	1 publications in selected types	All 1 journal articles

Publications

Type	Citation	Project	Document Sources
Journal Article	Campbell, E.R.(1999) Nitrate removal may call for alternative methods. Water Technology July 99: 64-66.	68D40046 (Final)	not available

Supplemental Keywords:

Biocatalysis, biocatalyst, nutrification, eutrophication, denitrifying bacteria, denitrifiers., RFA, Scientific Discipline, Water, Chemical Engineering, Civil Engineering, Civil/Environmental Engineering, Chemistry, Environmental Chemistry, Engineering, Chemistry, & Physics, Drinking Water, Environmental Engineering, Enzymatic Nitrate Elimination Technology (EzNET), treatment, nitrate contamination, nitrates, exposure, enzymatic nitrate , enzymes, small systems, potable water supplies, exposure and effects, nitrate elimination

SBIR Phase II:

Enzymatic Nitrate Elimination Technology for Small Systems  | 1994 Progress Report  | Final Report