Method and apparatus for measuring use of a substrate in a microbially catalyzed reaction

• US 6,379,914 B1
• Filed: 05/26/1999
• Issued: 04/30/2002
• Est. Priority Date: 11/26/1996
• Status: Expired due to Term

• Current Assignee: Lincoln Ventures Limited
• Original Assignee: Lincoln Ventures Limited
• Inventors: Pasco, Neil
• Primary Examiner(s): Woodward, Michael P.
• Assistant Examiner(s): Moran, Marjorie A
• Application Number:
  US09/308,628
• Time in Patent Office: 1,070 Days
• Field of Search: 435/25, 435/29, 435/287.5, 435/801, 210/606, 210/758, 210/605, 210/750, 210/753, 422/78, 422/79, 422/82.01, 422/82.05, 422/82.02, 71/10
• US Class Current: 435/25
• CPC Class Codes: G01N 33/1806 Biological oxygen demand [BOD] or chemical oxygen demand [COD]
• CPC Class Codes: G01N 33/1866 using microorganisms

Abstract

A sample substrate in a microbially catalysed biochemical reaction is measured by incubating the substrate with an excess of a microorganism and an excess of a co-substrate until the substrate is oxidized to a predetermined end point, and measuring the quantity of reduction of the co-substrate.

Claims

1. A method for measuring the quantity of a sample substrate consumed in a microbially catalysed biochemical reaction which comprises:

conducting said biochemical reaction by incubating said sample substrate in an aqueous medium with an excess of a microorganism and an excess of a mediator which has the ability to reversibly react at an electrode in said biochemical reaction,

continuing said incubation until said sample substrate is oxidised to a predetermined end point,

measuring reduction of said mediator in said incubation, and

correlating said reduction of said mediator to a corresponding oxidation of said sample substrate to thereby measure the quantity of sample substrate consumed in said biochemical reaction.

2. A method as claimed in claim 1 which is used to determine the biochemical oxygen demand (BOD) of said sample substrate.

3. A method as claimed in claim 1 wherein said reduction of said mediator is measured by reoxidising said mediator and measuring a charge required for doing so.

4. A method as claimed in claim 3 wherein said reoxidation of mediator and measurement of charge are carried out in a bulk electrolysis cell.

5. A method as claimed in claim 1 which is used to determine biological toxicity of said sample substrate.

6. A method as claimed in claim 1 wherein said incubation is conducted under anaerobic conditions.

7. A method as claimed in claim 6 wherein said anaerobic conditions are maintained by sparging an incubation container in which said incubation is carried out with oxygen free nitrogen.

8. A method as claimed in claim 1 wherein said predetermined end point is reached when the rate of change in reduced mediator to oxidised substrate ratio is minimal.

9. A method as claimed in claim 1 wherein said sample substrate and said mediator are separated from said microorganism before measuring the reduction of said mediator.

10. A method as claimed in claim 1 wherein said reduction of said mediator is measured by an electrochemical method.

11. A method as claimed in claim 10 wherein the measured charge is converted to a standard BOD measurement.

12. A method as claimed in claim 1 wherein said reduction of said mediator is measured by an optical method.

13. A method as claimed in claim 1 wherein said micro-organism is a member of the group consisting of Escherichia coli, Proteus vulgaris, Torulopsis candida, Bacillus subtilis, Trichosporon cutaneum and Saccharomyces cerevisiae.

14. A method as claimed claim 1 wherein said mediator is a member of the group consisting of benzoquinone, dichlorophenolindophenol, methylene green, methylene blue, phenazine methosulphate, potassium hexacyanoferrate (III), resorufin, thionine, and toluidene-blue-O.

15. A method as claimed in claim 1 wherein said incubation is terminated by filtering said mediator, sample substrate and microorganism through a filter whose pore size is such that the mediator is in the filtrate and the microorganism is in the retentate.