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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. 319
Object Description
Purdue Identification Number | ETRIWC199435 |
Title | Advanced bioassay for nitrifier identification |
Author |
Saraswat, Nirmala Alleman, James E. |
Date of Original | 1994 |
Conference Title | Proceedings of the 49th Industrial Waste Conference |
Conference Front Matter (copy and paste) | http://e-archives.lib.purdue.edu/u?/engext,44602 |
Extent of Original | p. 319-326 |
Collection Title | Engineering Technical Reports Collection, Purdue University |
Repository | Purdue University Libraries |
Rights Statement | Digital object copyright Purdue University. All rights reserved. |
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Color Depth | 8 bit |
Description
Title | page 319 |
Collection Title | Engineering Technical Reports Collection, Purdue University |
Repository | Purdue University Libraries |
Rights Statement | Digital copyright Purdue University. All rights reserved. |
Language | eng |
Type (DCMI) | text |
Format | JP2 |
Capture Device | Fujitsu fi-5650C |
Capture Details | ScandAll 21 |
Transcript | 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. 319 |
Resolution | 300 ppi |
Color Depth | 8 bit |
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