35    ADVANCED BIOASSAY FOR NITRIFIER IDENTIFICATION
Nirmala Saras wat, Doctoral Candidate
James E. Alleman, Professor
School of Civil Engineering
Purdue University
West Lafayette, Indiana 47907
BACKGROUND
Nitrification represents an important metabolic reaction within many industrial wastewater treatment systems. In turn, the presence and performance of the involved nitrifying bacteria warrants
considerable operator attention in terms of process monitoring. Obtaining accurate measurements
with these unique bacteria, however, has proven to be an exceedingly difficult task. First, a typical
mixed culture contains very few of these cells, with nitrifying populations generally amounting to only
a few percent of the total viable cell mass. Second, nitrifiers reproduce very slowly, thereby complicating attempts at direct cell counting.
In short, the available analytical procedures for measuring nitrifying microorganisms are considerably more qualitative than quantitative. This paper, therefore, explores a new approach to enumerating
nitrifiers based on enzyme-labeled immunoassay technology which should offer substantial improvements in terms of overall utility.
The three basic analytical options for enumerating nitrifying bacteria presently in widespread use
are as follows:
1. Selective plating procedures,
2. Activity-based measurement of substrate loss and/or product formation, and
3. Selective antibody bioassay procedures.
As with most bacteria, the 'selective plating' method historically represents the traditional approach
to counting nitrifiers. However, as documented by Mateluvich, et. al.1 this strategy is subject to
several complications which seriously degrade its reliability. Most importantly, the test requires considerably long incubation periods, ranging from several weeks to months. In fact, there does not seem
to be any set time requirement for incubation, with various researchers holding their Nitrosomonas
cultures for up to eight weeks, and Nitrobacter cultures for even longer incubation periods. Rather
obviously, these sorts of lag times for analytical data are unacceptable in terms of providing useful
process control information.
Srinath, et. al.2 pioneered the use of an 'activity-based' procedure for evaluating nitrifier densities
within mixed cultures. In this case, the goal is to measure either substrate (i.e., ammonia) removal or
product (i.e., nitrate) formation as direct indicators of nitrifier activity. One of the clear advantages to
this method is speed, in that a rapid assessment of nitrification rates can be derived within a few
hours. However, at the same time, it must be recognized that this method is based on an indirect assay,
as opposed to specific cell enumeration. In turn, this sort of 'activity-based' measurement may be
subject to error caused by sub-par metabolic activity associated with such factors as environmental
stress, etc.
The third analytical alternative uses specially prepared antibodies developed for their ability to
selectively bind to individual nitrifiers. Schmidt and Bankole3 originally demonstrated this approach
over three decades ago using a fluorescent antibody (FA) procedure to identify a particular nitrite-
oxidizer, Nitrobacter, with a fluorescein-bound antibody.
Belser and Schmidt4 followed with the first use of this so-called 'FA' method for detecting
ammonia-oxidizers. After testing several different genera of nitrifiers, these researchers concluded
that their bacteria had low cross-reactivities. By 1980, Ward and Perry5 had used the same procedure
to detect a marine ammonia-oxidizer, Nitrosococcus oceanus. Their bioassay reportedly yielded quantitative results comparable to hemacytometer and acridine orange counts, with several additional
49th Purdue Industrial Waste Conference Proceedings, 1994 Lewis Publishers, Chelsea, Michigan 48118. Printed in
U.S.A.
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