Biotechnol. Prog. 1995, 1 1, 545-551
Monitoring of the Biological Nutrient Removal Process by an On-Line NAD(P)HFluorometer Lu-Kwang Ju* Department of Chemical Engineering, The University of Akron, Akron, Ohio 44325
Xin Yang and Jaw F. Lee BioChem Technology, Inc., 100 Ross Road, King of Prussia, Pennsylvania 19406
William B. Armiger Biocontrol Systems Corp., 21 Woodstream Drive, Wayne, Pennsylvania 19087
An on-line NAD(P)H fluorometer has been used to investigate the fluorescence profile of a simulated anaerobidanoxidoxic biological nutrient removal (BNR) process and its dependency o n operating parameters, such as the concentrations of organic substrate (acetate) and activated sludge. As expected from the different rates of NAD(P)H oxidation a t different stages, the order of fluorescence levels was found t o be anaerobic > anoxic > oxic. The effects of the acetate and sludge concentrations were observed most clearly at the anoxic stage: a n increase in either concentration led to faster recovery of fluorescence from the drop caused by Nos- addition. A higher acetate concentration also resulted in a more significant, continuous rise in fluorescence, corresponding well with the enhanced Pod3- release, a t the anaerobic stage and a slower initial fluorescence decrease at the oxic stage, indicating the dependency of the decrease on substrate availability. The ratio of the two fluorescence drops caused by Nos- addition and aeration, Al/Az, obtained with the plant’s sludge was found to 0.1, which is similar to those of pure nitrate-respiring cultures (e.g., be 0.7 Pseudomonas aeruginosa and Escherichia coli). The dominant majority of nonnitrifiers in the sludge therefore are capable of nitrate respiration, and the nitrifiers either contribute little to the overall fluorescence or perform nitrate respiration as the rest of the sludge population.
Introduction Clean water is a priority of our industrialized society. There is an ever increasing need to process and purify water from industrial operations and municipal sources prior to discharge into natural water systems. Biological wastewater treatment plants (WWTPs) have been utilized to address this problem. In these plants, microorganisms are the true bioreactors treating the water. Consequently, plant management and process performance can benefit much from information on the activity of the microorganisms. All living cells contain NAD(P) coenzymes, i.e., nicotinamide adenine dinucleotide (phosphate), which serve as the major intermediate electron carriers in cellular metabolism. The cyclic nature of NAD(P) for heterotrophs is summarized in Figure 1. Accompanying substrate catabolism, the oxidized forms of coenzymes, NAD(PI+,are reduced to NAD(P)H. NADPH primarily serves as the reducing power in biosynthetic reactions (anabolism). NADH, on the other hand, is oxidized back to NAD+ through the following reactions: In aerobic respiration (reaction 3 in Figure 11, NADH is directly involved in oxidative phosphorylation for ATP generation. Under anoxic conditions, certain microbial species can use oxidants such as nitratehitrite (also, Fe3+, Cod CO&, S042-, So, and some organic compounds) as the terminal electron acceptors. The NAD(P) cycle for nitrate/ nitrite respiration thus is very similar to that for aerobic
* Author to whom all correspondence
should be addressed.
respiration, with NO,- replacing the role of oxygen (reaction 2 in Figure 1). In anaerobic fermentation, no externally supplied electron acceptor is required. The regeneration of NAD(P)+from NAD(P)H is coupled with the reduction of an organic compound that is formed during catabolism. For example, the anaerobic uptake of acetate in a biological nutrient removal (BNR) process may involve the reduction of NAD+ to NADH with the conversion of glycogen to acetyl-coA via the EMP (Embden-Meyerhof-Parnas) pathway and the oxidation of NADH with the synthesis of poly(B-hydroxybutyrate) (PHB) from acetyl-coA (reaction 1 in Figure 1)(Arun et al., 1988). In any case, the concentration ratio of NAD(P)H to NAD(P)+ is determined by the balance between the rates of reduction and oxidation, thus reflecting the metabolic activity of the organisms. NAD(P)H fluoresces at 460 nm when irradiated with 340-nm light, while NAD(P)+ does not (Chance and Baltscheffsky, 1956). Monitoring of the fluorescence of intracellular NAD(P)H therefore is an effective way to obtain information on biological activity. The fluorescence technique has been applied t o many microbial fermentations and animal cell cultures to generate information on cellular metabolism and to provide better process control in large-scale industrial operations (e.g., Armiger et al., 1986;Groom et al., 1988;J u and Trivedi, 1992;Li and Humphrey, 1989;MacMichael et al., 1987; Rao and Mutharasan, 1988;Trivedi and Ju, 1994). It can be especially suitable for BNR processes, where cell growth is normally regulated to minimize sludge generation and the water is treated in a series of bioreactors
8756-7938/95/3011-0545$09.00/00 1995 American Chemical Society and American Institute of Chemical Engineers
Biofechnol. Prog., 1995, Vol. 11, No. 5
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Metabolic Intermediates
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under different redox (Le., anaerobic, anoxic, and oxic) conditions. The mixed effects on NAD(P)H fluorescence due to changes in both cell concentration and metabolism, which are often observed (and are sometimes troublesome for clear signal interpretation) in other biological processes, do not exist in BNR processes. Furthermore, plant operation can be monitored effectively on the basis of marked differences in fluorescence levels anticipated in the bioreactors at different redox conditions: higher in anaerobic zones and lower in anoxic/oxic ( N O )zones because of the much weaker oxidizing power of the organic compounds used for NAD(P)H oxidation in fermentation than those of nitrate and oxygen in anaerobic and aerobic respiration. In this work, an on-line NAD(P)H fluorometer (the BioGuide system, BioChem Technology, Inc., King of Prussia, PA) was used to study the biological activity of a BNR process simulated with a bench-scale bioreactor. The bioreactor was operated under a sequential batch mode to simulate the N O process with denitrification (Air Products, Allentown, PA), employed by an 8.5 MGD (million gallons per day) wastewater treatment plant (Oaks, PA). In the plant, the wastewater flows through the anaerobic, anoxic, and oxic zones in series (Figure 2). The activated sludge collected from the final clarifiers is returned to the first stage of the anaerobic zone and
mixed with the primary effluent (PE), which is the influent wastewater after pretreatment and sedimentation to remove noncolloidal solid particles. An internal loop recycles a portion of the mixed liquor from the final stage of the oxic zone to the first stage of the anoxic zone. This study aims to establish the general NAD(P)H fluorescence profile in the BNR process and to correlate the profile with the process information offered by offline analyses and wastewater microbiology. Together, they provide a background for interpretation and analysis of the on-line signals generated by the fluorometers implanted in the Oaks plant.
Materials and Methods The sequential batch experiments were performed in a 1-L double-side-arm Celstir bioreactor (Wheaton Scientific, Millville,NJ). Water was circulated from a water bath through the glass jacket of the bioreactor for temperature control. The fluorometer accessed the wastewater through an optical well mounted on the reactor wall. The bioreactor was equipped with a pH electrode (Model A320, Ingold Electrodes, Inc., Wilmington, MA), a galvanic dissolved-oxygen electrode (Model A316, Associated Bioengineering and Consultant, Inc., Lehigh Valley, PA), a redox combination electrode (Markson Science, Inc., Phoenix, AZ),and an RTD thermometer.
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Air supply and sampling lines were introduced through the reactor cap. In addition to fluorescence, the data of pH, DO (dissolved oxygen), redox potential, and temperature were recorded by computer data acquisition and analysis software (BioGuide Software, BioChem Technology). To closely simulate plant operation, the experimental conditions, such as the rate and mechanism of agitation and aeration, the temperature, etc., were similar to those in the Oaks plant, and the experiments were conducted by allowing the culture to go through sequential periods of anaerobic, anoxic, and oxic stages. The lengths of these periods, typically 1,0.5, and 3 h, respectively, also corresponded to the hydraulic residence time of the anaerobic, anoxic, and oxic zones in the plant. A typical run is described as follows. Fresh samples of PE and the returned activated sludge (RAS) from the final clarifiers were collected from the plant. A measured amount of PE was transferred to the bioreactor. Organic substrates, phosphate, and ammonium were sometimes added to investigate the effects of their concentrations. The RAS/PE ratios used in most runs corresponded to the mixed-liquor suspended solid (MLSS) levels used in the plant. Some were varied to study the effects of sludge concentration (or F/M ratio). The actual MLSS in the bioreactor was determined by the standard dry weight measurement. Without aeration, the mixed liquor reached the anaerobic state rapidly due to microbial consumption of the dissolved oxygen in PE. At the scheduled end of the anaerobic stage, a known amount of NaN03 solution was added to the bioreactor to simulate the anoxic stage. In the plant, NO3- was introduced to the anoxic zone by the mixed liquor recycled from the final stage of the oxic zone. The high NO3- content in the recycled mixed liquor resulted from nitrification, i.e., sequential aerobic conversion of N H 3 to NOy and then to NO3- by microorganisms like Nitrosomonas and Nitrobacter, respectively, in the oxic zone. Following the anoxic stage, air was sparged into the bioreactor to create oxic conditions. Suitable air flow rates were employed to raise the DO level of the mixed liquor to about 20-40% air saturation. Samples were taken periodically along the experiment and centrifuged immediately to remove the microorganisms. A portion of the supernatant was used for analyses of NO3- and N&+ concentrations. The rest was frozen and analyzed later, within 12 h, for its Pod3- concentration and organic matter content in terms of chemical oxygen demand (COD). The ammonium concentrations were measured using an ammonia-selective electrode (Clesceri et al., 1989). The nitrate concentrations were determined by the titanous chloride reduction method (Clesceri et al., 1989). Results given by the method were actually the combined concentrations of N03--N (nitrate nitrogen) and N02--N (nitrite nitrogen). The latter, however, had been found to be negligible in the systems studied. CODs and phosphate contents of the samples were analyzed with the closed reflux colorimetric method and the ascorbic acid method, respectively, as described in the standard methods (Clesceri et al., 1989).
Results and Discussion Although many (>lo) runs have been made in this study, the detailed data of only two of them are shown in Figures 3 and 4, respectively, for ease of presentation. The description and discussion given in the following sections are generally consistent with the observations in other runs. The results shown in Figure 3 correspond to a run of 25%RAS (MLSS = 2.2 g/L)in raw, unsupplemented PE, thus representing the typical F/M value employed in the Oaks plant. Those in Figure 4 are,
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however, from a run of 25% RAS (MLSS = 2.6 g/L) in PE added with 200 ppm sodium acetate and 5 ppm N&+-N (ammonium nitrogen). Fluorescence Profile of the AM0 Process. The fluorescence profile shown in Figure 3 is first described here, together with the off-line analysis results of COD, Pod3--P (phosphate phosphorus), N03--N, and NH3-N,
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Anaerobic Stage. The background fluorescence from PE was first measured to be about 128 NFU (normalized fluorescence units). Immediately after the addition of RAS into the mildly agitated reactor, the fluorescence jumped to 143.5 NFU, mainly due to the introduction of NAD(P)H present in the added microorganisms. In the absence of aeration, DO and redox potential decreased drastically. The fluorescence remained at that level for a very short period of time before making the second leap to about 146 NFU. The fluorescence then increased at much slower rates to a maximum value, Le., 148.5 NFU, and stayed at that value until the end of the anaerobic stage. Phosphate release and nutrient uptake (indicated by the decrease in COD) were found to occur simultaneously in the anaerobic stage (Figure 3b). The Nos- concentration was also reduced to below the detectable level, ca. 0.3 ppm (Figure 3c). It should be noted that the intermediate fluorescence level of 143.5 NFU was the same as that observed in the later anoxic state (Figure 3a), and the second leap occurred when the NO3- originally present in PE was depleted (Figure 312). The second leap in fluorescence thus can be attributed to the rapid change in the cells' NAD(P)H levels, corresponding to the shift from the anoxic to anaerobic states. The high DO readings, ca. 5-40% saturation observed in the intermediate anoxic stage indicated the much slower response of the DO electrode than the fluorometer. Anoxic Stage. The anoxic stage was then initiated by the addition of NaNO3 solution to make 2.5 ppm NOS-N, which was the average value found in the first anoxic stage of the Oaks plant. Different groups of bacteria exploit nitrate in different ways (Cole, 1988):
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to establish the general correlation between the fluorescence profile and metabolic activity. Comparison with the profile in Figure 4 will be made later to emphasize the effects of substrate concentration.
In assimilative nitrate reduction, nitrate is reduced to ammonia for use as a nitrogen source for growth; in dissimilative nitrate reduction, nitrate is used as an alternative electron acceptor for anaerobic respiration. In the latter case, NOS- effectively oxidizes NADH to NAD+. As shown in Figure 3, the fluorescence dropped instantaneously upon NaN03 addition, indicating the presence of nitrate-respiring bacteria in the plant's activated sludge. It then remained constant at that value until the added nitrate was completely removed (Figure 3c). The fluorescence signal subsequently tended to rise back to the previous anaerobic level. The concentrations of influent organic materials and RAS employed in this run were slightly lower than the normal plant values. The fluorescence did not recover fully within the scheduled 30-min anoxic operation. However, in other similar runs of 25% RAS in PE, the fluorescence would generally reach the previous anaerobic values at the end of the anoxic stage. Dissimilative nitrate reduction is coupled with bacterial catabolism; nitrate is reduced by accepting the electrons originally donated by the oxidized substrates. The observed removal of carbonaceous materials during anoxic treatment thus was anticipated (Figure 3b). The slight increase in NH3-N concentration (Figure 3c) indicated the presence of the bacteria performing dissimilative nitrate ammonification, although the majority of the added NOs- was reduced through denitrification. The gaseous products of denitrification, i.e., nitric oxide (NO), nitrous oxide (NzO)and nitrogen (Nz),would escape into
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the atmosphere. The anoxic treatment thus led to reduction of the total nitrogen content in wastewater. Oxic Stage. The oxic condition was created by sparging air into the mixed liquor. The fluorescence was found to drop sharply, followed by a continued slower decrease. The uptake of phosphate and the remaining carbonaceous substrates in the aerobic process are clearly shown in Figure 3b. The phosphate uptake observed in the later stage, after the extracellular substrates had been exhausted, was driven by the energy produced by the aerobic digestion of the stored intracellular carbohydrates, such as PHB and glycogen. ATP was formed first in the process, and then a portion of ATP was converted to polyphosphate by the enzyme polyphosphate kinase, which regulated the ATP/ADP ratio in the cells (Tracy and Flammino, 1987). As shown in Figure 3b, a net reduction in the phosphorus content in wastewater was achieved as a result of cell growth. Capable of accumulatingintracellular polyphosphates, the microbial population developed in the A/A/Oprocess can remove more phosphate than a conventional aerobic activated sludge at the same sludge generation rate. The ammonium concentration also decreased in the oxic stage (Figure 312). Part of the ammonium was assimilated into growing cells. However, considering the regulated biomass generation in wastewater treatment, the ammonium was primarily removed by the two-step process of nitrification: NH,
+ 20, + NADH - NO,- + 2H,O + NAD' NO,- + '/,O, - NO,-
This is confirmed by the observed symmetric profiles of ammonium reduction and nitrate formation (Figure 3c). Effects of Substrate Concentration. The basic features of the fluorescence profile in Figure 4, where PE has been supplemented with 200 ppm sodium acetate and 5 ppm NH4+-N,remain the same as those in Figure 3. The most significant difference occurred at the anoxic stage. While in Figure 3 the fluorescence just began to climb up from the anoxic level at the end of a 30-min anoxic operation, in Figure 4 the fluorescence reached back to the previous anaerobic level in about 13min. The nitrate removal rate was shown to be strongly dependent on the concentration of organic substrate, which serves as the electron donor to drive the dissimilative NO,reduction. At the anaerobic stage, the high substrate concentration caused a slow continuous increase in fluorescence in Figure 4, as opposed to the leveling off in Figure 3. This corresponded well with the different extents of phosphate release observed in these cases (Figures 3b and 4b) and may be tentatively explained as follows. The AIM0 process selects for certain aerobic microorganisms that are able to rapidly take up and store organic substrates under anaerobic conditions, utilizing the energy provided by the breakdown of intracellular polyphosphates. The energy generation by polyphosphate hydrolysis is much faster and more efficient than that by substrate-level phosphorylation in normal fermentative metabolism, such as the EMP pathway (Tracy and Flammino, 19871, thus denying the access of other competing organisms to the limited substrates available. In oxic and, possibly, anoxic zones, the consumed polyphosphate pool is replenished to conserve part of the energy generated from oxidative phosphorylation. It is generally believed that, under anaerobic conditions, the organic materials, typically acetate, are taken up by these microorganisms and converted to acetyl-coA, with the energy for this conversion coming from the
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hydrolysis of polyphosphate (Arun et al., 1988; Cameau et al., 1986; Matsuo, 1985). The more significant phosphate release observed in Figure 4, where a higher acetate concentration was available, therefore is understandable. Acetyl-coA is further converted to PHB for storage. The NADH required (as the reducing power) for this conversion is obtained from the consumption of intracellular carbohydrates, such as glycogen, via the EMP pathway (Arun et al., 1988). The carbohydrate consumption was found to be greater than the theoretical requirement for PHB synthesis, thus producing excess NADH in the process. This may account for the slow fluorescence increase observed in the study after the anaerobic conditions had been reached. As the need for PHB storage would increase with increasing acetate concentration, the more significant fluorescence increase in Figure 4 than in Figure 3 is expected, if glycogen is present in sufficient quantities. The substrate addition also led to a different fluorescence profile at the oxic stage. In addition to the initial supplement, 100 ppm sodium acetate was added right before aeration in this run,as indicated by a COD peak in Figure 4b. Following the sharp drop due to aeration, the fluorescence initially decreased more slowly (Figure 4). The phenomenon was confirmed by several other runs involving the addition of different concentrations of acetate right before aeration (data not shown). With higher acetate concentrations, the fluorescence could even rise slightly before the trend of slow decrease sets in. The exact mechanism responsible is uncertain. Although a brief period of cell growth is possible, it could not be confirmed by the insensitive dry weight measurement. Nevertheless, the dependency of the fluorescence decrease in the oxic stage on the substrate (extracellular and intracellular) concentration is clearly indicated. Further study is needed to examine the possibility of online assessment of substrate removal by the BNR process, based on the profile of fluorescence decrease in the oxic stage. Effects of Sludge Concentration. The fluorescence profiles of three runs made with different sludge concentrations, i.e., 15%, 25%, and 40% RAS, are shown in Figure 5. The runs were conducted on different days with fresh, unsupplemented PE samples taken from the plant, thus involving the daily variations of the influent wastewater and the activated sludge of the plant. The variations are clearly indicated with the background fluorescence of PE before RAS addition. Consequently, the overall fluorescence levels shown in Figure 5 are not in the order corresponding to the sludge concentrations: the 15% RAS run had higher fluorescence than the 25% RAS run. The change in fluorescence caused by different
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redox conditions and microbial metabsolism were found to ride on the changes in background fluorescence, which may or may not be significant depending on its sources. For the Oaks plant, the background fluorescence shows clear diurnal and weekly cycles, but much less change along the treatment process, except the mixing/dispersion effects. In Figure 5, the effects of sludge concentration are shown most clearly in the anoxic stage: while the fluorescence of the 40% RAS run returned to the anaerobic level in 15 min, that of the 15% RAS run never recovered. This is plausible because the nitrate removal rate would be proportional to the concentration of nitraterespiring microorganisms, if the specific rate of dissimilative nitrate reduction of these microbes remained relatively constant. The capability of the fluorescence technique to monitor the nitratehitrite removal process is clearly demonstrated. The effects of sludge concentration on the more subtle changes in fluorescence within the same operation stage, such as the slow rise in the anaerobic stage and the decrease in the oxic stage discussed in previous sections, cannot be confidently concluded. The rather fast decline in fluorescenceobserved in the oxic stage in the 15%RAS run possibly was linked to the removal of some background fluorophores originally present in PE. The separation of contributions to fluorescence from NAD(P)H and background fluorophores remains a significant challenge. Implications on Sludge Populations. While both nitrate addition and aeration caused a rapid decrease in fluorescence, the drop effected by nitrate addition was smaller than that by aeration. The presence of bubbles in the aerated water was not responsible because nitrogen sparging had been found to have no effect on the fluorescence a t the anaerobic stage. Instead, the difference can be accounted for by the following two factors. First, the activated sludge consists of many different species of organisms. Some (e.g., most nitrifiers) are incapable of nitrate respiration and, therefore, do not respond to nitrate addition. The observed ratio of the two fluorescence drops (Al/AZ), one corresponding to the shift from the anaerobic t o anoxic states and the other from the anaerobic to oxic states,
- _ - NFUanaerobic
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may contain information about the population fraction of nitrate-respiring bacteria in the sludge. While a ratio of 0, i.e., no response to NO3- addition, indicates the absence of a NOs--respiring population, a large ratio corresponds to a large population fraction. Secondly, the reduction potentials for denitrification (i.e., NO3-/Nz: +0.74 V) and dissimilative ammonification (i.e., N03-/NH3: +0.70 V) are lower than that for respiration (i.e., 1/z02/H~O:+0.82 V) (Brock and Madigan, 1988). The smaller fluorescence drop following nitrate addition therefore can be partly attributed to the slower NADH oxidation rate associated with nitrate reduction than that with aerobic respiration. This has been confirmed in the recent studies with pure cultures of Pseudomonas aeruginosa (a denitrifier) and Escherichia coli (a nitrate-respiring bacterium via ammonification) (Ju and Trivedi, 1992; Trivedi and Ju, 1994). The ratios of fluorescence drops (A1/A2) for P. aeruginosa (0.65-0.85) and E . coli (0.70 f 0.14) were both found to be lower than 1. It should be noted that the values of AJAz observed in this study (0.7 f 0.1) with the activated sludge from a BNR plant are about the same as those found with pure
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nitrate-respiring cultures, i.e., P. aeruginosa and E. coli. As the presence of nitrifiers is clearly indicated by the effective conversion of NH3 to NO3- (Figures 3c and 4c), the preceding finding implies the following: (1) The dominant majority of the non-nitrifying population in the activated sludge established in the BNR process are capable of nitrate respiration. (2) The nitrifiers either contribute insignificantly to the overall fluorescence or perform NAD(P)H-dependent nitrate reduction as the rest population. Most nitrifiers are obligate lithotrophs that use ammonia and nitrite, respectively, as the sole energy source (Bock et al., 1986). As the redox potentials for NOy/ NH4+(+0.34V) and N03-/N02-(+0.43 V) are much more positive than that for NAD+/NADH(-0.32 V) (Brock and Madigan, 1988; Wood, 19861, the lithotrophic nitrifiers cannot use NADH as the intermediate electron carrier. Instead, the electrons released from ammonia and nitrite enter the respiratory chain at the level of cytochrome al (Bock et al., 1986). Part of the ATP (or proton motive force) thus generated is then consumed to reduce NAD(PI+to NAD(P)H in the process of reversed electron flow (Wood, 1986). Because of the less important roles of NAD(P),these lithotrophic nitrifiers could have a smaller pool size of the coenzymes than heterotrophic microorganisms. The NAD(P)H fraction in nitrifiers could also be smaller because the reduction of NAD(P)+ to NAD(P)H consumes the limited energy derived from nitrification. Together, they could support the possibility of a very minor contribution from nitrifiers to the overall fluorescence. Nitrobacter represents the only known exception to the obligate lithotrophy of nitrifiers. It is able to grow both lithotrophically and heterotrophically (Bock et al., 1986). Anaerobic nitrate respiration of Nitrobacter in media containing simple organic materials, such as pyruvate, acetate, and glycerol, has also been demonstrated (Bock et al., 1986). As Nitrobacter is the dominant nitrite oxidizer in WWTPs (Belser, 1979), the NAD(P)H fluorescence from a significant fraction of the nitrifiers may respond to nitrate addition and aeration similar to the denitrifylng non-nitrifiers. Nevertheless, the possibilities to assess the fraction of denitrifiers in the non-nitrifying population and to monitor the long-term stability of the sludge in wastewater treatment plants based on the aforementioned ratio of fluorescence drops (A1IAz) certainly warrant further study. Comparison with Plant Results. Figure 6 shows the typical fluorescence profiles observed in different zones of the Oaks plant (Armiger et al., 1990). The locations of the fluorometric detectors are given in Figure 2. The results obtained in this simulated study using a sequential batch reactor compare reasonably well with the plant observations, considering that the plant results
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were under the influence of local mixing within each reactor (Figure 2) and were obtained with separately calibrated fluorometers. The mixing effect is shown clearly in Figure 6, with the much reduced diurnal variations in fluorescence in the anoxic and, especially, oxic zones. Although more quantitative and precise interpretation of the plant results may require complicated modeling to incorporate the variations in hydraulic flow rate and background fluorescence of the influent water, as well as the mixing effects of the plant’s operation, these factors are expected and confirmed to have cyclic regularities for each plant. The fluorescence profiles corresponding to normal, acceptable operating conditions for the specific plant can still be established, Deviations from the normal profiles indicate the need for careful examination of plant operation for abrupt or gradual process upsets or changes. With greater understanding of the relationship between the fluorescence profiles and the plant operating conditions, possible causes of the deviations can be identified. The potential of the on-line fluorescence technique in facilitating process monitoring and plant management of BNR wastewater treatment is clearly indicated.
Conclusions The fluorescence profiles of a simulated anaerobic/ anoxidoxic BNR process were obtained using an on-line NAD(P)H fluorometer. As expected from the faster NAD(P)H oxidation by NO3- and 0 2 , lower fluorescence levels were observed in the anoxic and oxic stages. The profiles of the runs conducted with F/M values similar to those used in the Oaks plant compare well with the profiles obtained in the plant, considering that the plant results were subject to the variations in the influent water and RAS as well as the mixing effects within a series of reactors in the treatment process. An increase in the acetate concentration of the water resulted in a very different profile at the anoxic stage: the fluorescence recovered much faster from the anoxic level following the addition of the same amount of Nos-, reflecting more active nitrate reduction. A continuous rise in fluorescence at the anaerobic stage, as opposed to the gradual leveling off observed in unsupplemented runs, and a slower initial fluorescence decrease at the oxic stage were also effected by the acetate addition. While the latter indicates the dependency of the fluorescence decrease at the oxic stage on substrate availability, the former is attributed to NADH formation by glycogen degradation for converting acetate to PHB as food storage. The effects of different sludge concentrations were found to be most evident at the anoxic stage: the higher the sludge concentration, the faster the nitrate reduction and, consequently, the fluorescence recovery from the drop caused by NO3- addition. Together with the observations in the study of substrate effects, this clearly established the capability of the fluorescence technique in monitoring the N03-/N02- removal process. The ratio of the two fluorescence drops caused by NO3addition a n d aeration, Al/Az, contains information about the population fraction of the nitrate-reducing organisms (mainly denitrifiers) in the sludge. The value of AJAz for the sludge (0.7 f 0.1) was found to be similar to those of Pseudomonas aeruginosa (a pure denitrifier, 0.650.85) and Escherichia coli (a nitrate-respiring bacterium via ammonification, 0.70 f 0.14). As the presence of nitrifiers in the sludge is clearly shown with the conversion of NH3 to NO3- at the oxic stage, it implies that (1) the dominant majority of non-nitrifiers in the sludge are capable of nitrate respiration and (2) the nitrifiers either
contribute insignificantly to the overall fluorescence or perform nitrate respiration as the rest of the sludge population. The potential of the on-line fluorescence technique in facilitating process monitoring and plant management of BNR wastewater treatment clearly is indicated.
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