Determining Microbiological Content In Fouling Deposits

By Todd Stimpson Guardian-IPCO, Inc. Birmingham, AL (205) 991-5316

     The open-recirculating system is an ideal environment for growth for a variety of
microorganisms including bacteria, algae, fungi, etc. These microorganisms are
responsible for the formation of slime, corrosion of metal, and degradation of cellulose.
Analyzing fouling deposits reveals a wealth of information in determining the problems
that the system is experiencing. By spectrophotometric means, the biological content of
a fouling deposit can be estimated. This is essential in determining the extent of the
organic content in a deposit that is a result of microbiological fouling. The method
proposed is the Coomassie Protein Assay. This is a quantitative method of determining
total protein in a sample. The relationship between total protein and determining
the40"microbiological content is mathematical. Algae and bacteria contain 12-15
percent total protein. (1) Thus by knowing the total protein of a given foulingdeposit,
the microbiological content can be determined.

     The materials needed to perform this assay are as follows: a spectrophotometer
capable of measuring absorbance at 595 nm; reaction tubes or 16 X 100 mm test tubes;
and pipettes capable of accurately dispensing 0.1 ml, 1.0 ml, and 5.0 ml. The protein
assay reagent is available commercially or can be prepared in a lab. Sensitivity,
interfering substances, and the degree of difficulty in performing the assay should be
considered when choosing a protein assay reagent.

     The Coomassie Protein Assay reagent is based on the Bradford Method which
exercises an absorbance shift from 465 to 595 nm. This occurs when Coomassie Blue
G-250 Protein Reagent binds to protein in an acidic solution resulting in the intensity of
the color change from reddish-brown to brilliant blue. Thus the intensity of the color
developed is proportional to and becomes a measure of the amount of protein that is
present. However, the color esponse is non-linear over a wide range of protein
concentrations. Therefore, it is required that a standard curve be performed. Standard
curves should always be prepared as part of a colorimetric assay.

     "Molecular associations or dissociations of the color compound mayaffect its

abilityto absorb light. Hence, it is necessary to determine that the amount of light
absorbed by a compound is indeed directly proportional to its concentration before
using absorbance as a measure of concentration." (2) A brief description of the
procedure is in order. Standards are performed from known concentrations. A known
quantity of the fouling deposit is digested with reagents compatible to the Protein Assay
Reagent. An aliquot of the supernatent liquid is diluted in a reaction tube. The
Coomassie Protein Assay Reagent is added. The standard protein and unknown protein
absorbances are read versus deionized water at 595 nm.

     Determining the microbiological content is performed in a series of mathematical

steps. The following information needs to be obtained from the assay: The weight of the
sample in grams, the total protein expressed in milligrams and the aliquot expressed in
milliliters. To determine thepercent protein of the sample, use the following formula:

          Total Protein (mg) x (100)                                 Sample (g) x (Aliquot/ 10) x
(1000)
     Knowing that algae and bacteria contain 12-15 percent total protein, the
microbiological content can be determined. For instance, if a sample was found to
contain 1.5 percent protein, it is estimated that microbiological content is 10 percent.

     It has been shown that the Coomassie Protein Assay is a beneficial method for
determining total protein concentrations in fouling deposits. It is a highly reproducible
and sensitive Protein Assay. Thus, protein analysis, because it is indicative of the
biological fouling content, is a vital tool in measuring the success of the biocide
program