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Monitoring aerobic sludge digestion by online scanning fluorometry
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Water Research 39 (2005) 1205–1214 www.elsevier.com/locate/watres
Monitoring aerobic sludge digestion by online scanning fluorometry RaviSankar Arunachalam, Hemant K. Shah, Lu-Kwang Ju Department of Chemical Engineering, College of Engineering, The University of Akron, Akron, OH 44325-3906, USA Received 20 September 2002; received in revised form 26 November 2003; accepted 16 November 2004 Available online 23 March 2005
Abstract With sludge samples from two wastewater treatment plants, batch experiments of aerobic sludge digestion were conducted under different dissolved oxygen (DO) and solids concentrations. A fluorometer capable of online excitation and emission scanning was used to monitor the digestion process. Three major fluorescence peaks were observed. The peak at excitation/emission maxima of 290/350 nm was attributed to the fluorescence of proteinaceous materials in the sludge, with tryptophan residues being the primary contributor. The sources for the other two peaks (at 370/430 nm and 430/510 nm) remain unknown. The well-known biological fluorescence from reduced nicotinamide adenine dinucleotides (NADH and NADPH), at excitation/emission maxima of 340/460 nm, was found very weak in the aerobic digestion systems studied. It was buried under the broad peak at 370/430 nm and was detectable only in the early stage of the experiment that had the highest solids loading (at 4.8%) and was operated under low DO (0.2–1.0 mg/ L) conditions. On the other hand, the profile of the protein fluorescence (PF) correlated well with that of the volatile solids (VS) reduction in all the experiments. A semi-empirical exponential decay function was developed, which described well the profiles of both normalized VS and normalized PF. The feasibility of following the real-time performance of aerobic sludge digestion by monitoring PF was clearly demonstrated. r 2005 Elsevier Ltd. All rights reserved. Keywords: Sludge digestion; Fluorescence; Proteins; Tryptophan; NAD(P)H; Dissolved oxygen
1. Introduction Energy consumption for aeration of the aerobic sludge digesters represents a major running cost to the plant. To minimize the aeration cost, it is desirable to monitor the real-time progress of the digestion process so that the supply and demand of aeration can be better matched, which depending on the process designs, may be achieved by adjusting the aeration rate, optimizing the cyclic timing between oxic and anoxic operations Corresponding author. Tel.: +1 330 972 7252; fax: +1 330 972 5856. E-mail address: [email protected] (L.-K. Ju).
(Al-Ghusain and Hao, 1995; Daigger and Bailey, 2000; Hao et al., 1991), and/or shortening the treatment period according to the indicated reach of the desired extent of digestion. The real-time process information also offers peace-of-mind to the plant operators and managers during normal operation and the warning for remedial actions if process upsets should occur. Previous researchers have used parameters such as dissolved oxygen (DO), oxidation-reduction potential (ORP), and pH to monitor the process of sludge digestion (AlGhusain and Hao, 1995). However, the measuring devices for these parameters require rather frequent calibration because they are based on electrochemical reactions that inevitably change the properties of the
0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2004.11.028
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electrolytes and/or electrode(s) employed. They are therefore not ideal for long-term, continuous process monitoring. In addition, these parameters describe the conditions of the water phase, instead of the sludge itself. Because the organisms in the sludge are the real ‘‘workers’’ performing the digestion and the state of the sludge is the true property of concern in the digestion process, these water-phase parameters are not definitely indicative of the process performance, especially under often fluctuating process conditions. Recognizing the above limitations, we explored in this work the possibility of monitoring sludge digestion processes using the culture fluorescence technology that employs much more stable optical sensors and detects the fluorescence directly associated with the sludge. Compared to the electrodes for measuring DO, ORP and pH, optical fluorometers offer superior long-term stability and eliminate the need for frequent recalibration and maintenance, thus being more suitable for the continuous operation in wastewater treatment plants (Ju et al., 1995). The technology is based on the following principle (Sharma and Schulman, 1999): When a molecule absorbs light (at the excitation wavelengths), it must emit a quantity of energy equivalent to that absorbed if it is to return to its stable ground state. The energy can be released in forms of light, heat, resonance radiation, Rayleigh scattering, and Raman scattering. When light is emitted (at the emission wavelengths), the phenomenon is referred to as luminescence, which includes both fluorescence and phosphorescence. Molecules that fluoresce are called fluorophores. Many fluorophores, both intracellular and extracellular, are present in biological systems (Marose et al., 1998; Udenfriend, 1962). The concentration variations of some of these fluorophores are closely related to the biological activities and, therefore, can be used as indicators of important process parameters such as biomass concentration and metabolic state (e.g. Li et al., 1991; Marose et al., 1998; Trivedi and Ju, 1994; Zabriskie, 1979). On-line monitoring of these fluorescence changes may lead to better understanding of the complex processes of sludge digestion and development of valuable process control and optimization strategies. In an earlier study of aerobic digestion of a mixed primary and secondary sludge, we showed that the fluorescence at excitation and emission (ex/em) maxima of 340/440 nm exhibited a characteristic profile that correlated well with the progress of the digestion (Li and Ju, 1999). The above fluorescence occurred at ex/em maxima near those from the reduced nicotinamide adenine dinucleotides, NADH and NADPH, hereafter referred to as NAD(P)H. NAD(P)H, with ex/em maxima at 340/ 460 nm, are among the best-studied biological fluorophores (e.g. Ju and Trivedi, 1992; Ju et al., 1995; Kwong and Rao, 1994; Siano and Mutharasan, 1990). They,
and their non-fluorescent oxidized counterparts NADðPÞþ ; are present in all living organisms as the coenzymes for many metabolic reactions (Madigan et al., 1997). The levels of NAD(P)H, and their fluorescence intensity, in a biological system depend on the live cell concentration, the energy inventory within each cell, and the metabolic activity. Proteins are another important source of culture fluorescence (Li et al., 1991; Marose et al., 1998; Udenfriend, 1962). While NAD(P)H fluorescence is observed only in live organisms with active metabolism, the proteins fluoresce regardless of their being associated with live or dead organisms. Accordingly, the profile of protein fluorescence in a digestion process would directly reflect the change of the sludge’s proteinaceous content. Because the proteinaceous materials are destroyed along the volatile solids (VS) reduction, it is feasible to follow the progress of VS reduction by monitoring the profile of protein fluorescence in the digestion process. In this study, a multi-channel online fluorescence sensor (BioViews from Biobalance Technology, Inc., Denmark) was used to monitor the batch experiments of aerobic sludge digestion. The fluorometer was equipped with 16 excitation channels (270–530 nm) and 16 emission channels (290–570 nm), stepped at 20-nm intervals. The wide sensing ranges cover the fluorescence from the majority of important fluorophores known to be present in biological systems, including those from NAD(P)H and proteins (Marose et al., 1998; Udenfriend, 1962). The digestion experiments were made under different DO and solids concentrations with the sludge samples from two different plants: Akron, OH and Los Lunas, NM. The results confirmed the applicability of the on-line fluorescence technology in different process conditions.
2. Materials and methods 2.1. Sludge samples Four batch experiments were carried out in this study. The first run was made with the fresh sludge sample from the nearby Akron wastewater treatment plant. The sludge samples used in the other three runs were from a plant in Los Lunas, NM. The three batches of samples were shipped overnight in an iced container and studied immediately upon receiving to minimize the change of sludge properties during the shipment. With an average flow of 70–80 million gallons per day (MGD), i.e., ð2:6 3:0Þ 105 m3 =d; the Akron plant uses aeration ditches for secondary biological treatment ðHRT6:5 hÞ; to remove ammonium and organic substances. The primary influent had the following average properties: BOD5 (biological oxygen demand)—100 mg/
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L, TSS (total suspended solids)—160 mg/L, TKN (total Kjeldahl nitrogen)—20 mg/L, and NHþ 4 —N—12 mg/L. The influent to secondary treatment had an average BOD5 of approximately 70 mg/L and TSS of approximately 55 mg/L (N information unavailable). The solids concentration of the Akron plant’s secondary sludge was typically around 2% (w/v). The much smaller Los Lunas plant has a design capacity of 0.7 MGD (i.e., 2650 m3 =d) with a peak flow capacity of 2.1 MGD (i.e., 7950 m3 =d). The plant has no primary clarifiers and employs an activated sludge process with an extended-aeration configuration ðHRT20 hÞ: The typical influent values for the plant were as follows: BOD5 —270 mg/L, TSS—220 mg/L, TKN—33 mg/L, and NHþ 4 –N—37 mg/L. (The seemingly erroneous larger TKN than NHþ 4 –N concentrations were caused by measurements made on samples from different periods.) The waste sludge was thickened to about 5% in a gravity belt thickener. The processes and operating conditions of the two plants were very different. The thickened Los Lunas sludge was further mixed with the plant’s fresh sludge (details given later) to give different solids concentrations and sludge properties. These arrangements were made so that the applicability of the on-line fluorescence technology to different sludge could be evaluated.
2.2. Digestion experiments All experiments were carried out at room temperature. The Akron sludge used in the first run had an initial total solids (TS) concentration of 1.6%. The DO was maintained at 3–4 mg/L in this run, for fully aerobic operation. The pH was controlled at 6.9–7.2 by automatic addition of 0:5 N Na2 CO3 : The second run was made under the same conditions but with the Los Lunas sludge having initial TS of 2.3%. Runs 3 and 4 were carried out at low DO, in the range of 0.2–1 mg/L. The initial TS were 4.8% in Run 3 and 2.8% in Run 4. No pH control was necessary in these low DO runs, as the pH never went below 6.5. As described earlier, the sludge at the Los Lunas plant was typically thickened to about 5% (w/v) before digestion. While the thickened sludge was used in Run 3, the sludge samples used in Runs 2 and 4 were prepared by mixing the thickened sludge with the fresh sludge from the secondary clarifier. The digestion was carried out in a 2-L plastic container. The fluorometer, DO and pH probes, as well as ports for aeration and sampling were introduced through the container lid. Continuous aeration was provided by bubbling humidified air through a diffuser placed at the bottom of the reactor. Agitation was provided by magnetic stir bar for the first two experiments. The stirring speed was adjusted daily to maintain DO in the designed range. For the last two low
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DO experiments, the aeration rate was decreased significantly. To compensate for the less mixing provided by the reduced aeration, a marine impeller driven by a variable-speed motor was used for agitation. The Los Lunas sludge tended to foam, especially during the early stage of the digestion. The foams were broken periodically and the trapped flocs returned to the sludge. The water loss by evaporation was compensated almost daily by addition of tap water. 2.3. Analyses Samples were taken daily for various analyses: 10 mL for TS and VS, 10 mL for NHþ 4 2N; NOX 2N (i.e., combined NO3 –N and NO2 –N) and NO 2 –N, and 10 mL for fecal coliform density. To make sure that the samples were representative, the sludge was stirred thoroughly (but briefly) before sampling. The analyses were performed according to the standard methods (APHA, 1995): Procedures 2540B and 2540E for TS and VS, respectively; Procedures 4500-NH3 F and 4500 þ NO 3 for NH4 –N and NOX –N, using an ammoniaselective electrode; Procedure 4500B for NO 2 –N; Procedure 9221B for fecal coliform density, by the multiple-tube fermentation technique; and Procedure 2710B for oxygen uptake rate (OUR), using a DO probe. The specific OUR (SOUR) was then calculated by dividing OUR by TS. The OUR was not measured in Run 3 where the very high solids loading (4.8% TS) made it difficult to raise the DO to high enough levels for adequate measurement by the dynamic method. The sludge supernatant required for some of the above analyses were collected after centrifuging the sludge sample at 14; 000g for 10 min.
3. Results and discussion 3.1. Fluorescence spectra The typical fluorescence spectra obtained in the digestion experiments, for one scanning cycle, are shown in Fig. 1. Three major peaks always appeared around the ex/em maxima of 290/350 nm, 370/430 nm, and 430/ 510 nm. The peak at 290/350 nm has the same maxima as the aromatic amino acid tryptophan, which has much higher absorption and fluorescence efficiencies than the other two common aromatic amino acids (tyrosine and phenylalanine) (Udenfriend, 1962). This peak is therefore attributed to the fluorescence from the proteinaceous materials in the sludge. The responsible fluorophores for the other two peaks are, however, unknown. Further, the fluorescence from NAD(P)H (ex/ em maxima: 340/460 nm) appeared to be too weak and was buried under the broad peak at 370/430 nm. Some
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more discussion about the NAD(P)H fluorescence is given later.
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conducted under different operating conditions. No consistent correlation with the change of other measured parameters can be identified. More studies are needed to understand their origins and, perhaps, their relations with the digestion process. The profiles of the measured process parameters were mostly expected. Rapid pathogen destruction was observed in the experiments. Active nitrification occurred in the high DO digestion experiments (Figs. 2 and 3), resulting in negligible ammonium concentration and increasing NO x concentration. On the other hand, the digestion at low DO involved slower nitrification and simultaneous denitrification, which in one of the low DO experiments (Fig. 5) caused a distinct three-phase phenomenon in the ammonium and NO x profiles: the ammonium concentration increased during the first 50 h, followed by a sharp decline to negligible levels; the NO x concentration remained very low during 0–50 h, increased to (and fluctuated around) 18 mg/L NO x –N during 50–170 h, and de-
creased steadily after 170 h. The phenomena can be explained as follows: During the first 50 h, the higher respiration demand kept the DO very low in the digester, about 0.1–0.2 mg/ L. At such low DO, nitrification was very slow and simultaneous denitrification took place, resulting in negligible NO x concentrations. Meanwhile, the rate of ammonium generation from the rapid initial VS digestion exceeded the rate of nitrification, causing the observed increase of ammonium concentration during this period. After 50 h, DO rose gradually and fluctuated around 0.5–1.0 mg/L (data not shown). The increased DO led to faster nitrification and slower denitrification, which in turn brought about the subsequent drop of ammonium concentration and increase of NO x concentration. After about 170 h, the VS digestion slowed down significantly. The decrease in the accompanying ammonium release would lower the NO x generation by nitrification, causing the eventual slow decline of the NO x concentration.
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3.3. Correlation between protein fluorescence and VS profiles As mentioned above, the observed profiles suggested the possibility of developing mathematical correlations between VS and PF as well as between SOUR and PF. The correlation between VS and PF is fundamentally plausible because both relate directly to the proteinaceous contents of the sludge. The SOUR, on the other hand, reflects only the respiring population. Furthermore, the correlation between SOUR and PF may be very plant-specific because SOUR depends on the population composition of the sludge and any plant conditions (e.g. temperature and pH) that affect the cellular metabolism. Consequently, only the development of quantitative correlation between VS and PF is pursued in this work. VS degradation is normally described by a first-order kinetics (Krishnamoorthy and Loehr, 1989; Metcalf and Eddy, 1991), i.e., dðVSb Þ ¼ kd ðVSb Þ, dt
where VSb is the biodigestible VS (¼ VS VSf ; VSf being the hypothetically ‘‘inert,’’ ‘‘non-biodigestible’’ portion of VS) and kd is the decay constant ðh1 Þ: On integration, the above equation gives an exponential decay function of VS, VS VSf ¼ ekd t VSi VSf
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or VS ¼ VSf þ ðVSi VSf Þekd t , where VSi is the initial VS and ðVS VSf Þ=ðVSi VSf Þ represents the dimensionless, normalized VS. Because PF followed similar time profiles as VS, the same kinetics may be assumed analogously for PF, i.e., Normalized PF ¼
PF PFf ¼ ekd;PF t PFi PFf
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If the same decay constant applies to both profiles (i.e., kd ¼ kd;PF ), an identical equation describes the profiles of both normalized VS and normalized PF, and the two normalized properties correspond to each
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other directly, i.e., VS VSf PF PFf ¼ . VSi VSf PFi PFf
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This is shown to be practically valid for the experimental results obtained in this study, as described in the following: The VS data from each experiment were first regressed with Eq. (1) to obtain the best-fit parameters: kd ; VSi and ðVSf =VSi Þ: Similar regression was then performed with the PF data, according to Eq. (2), but with the kd value fixed as that obtained from the VS regression. The best-fit PFi and ðPFf =PFi ) were obtained accordingly. All the best-fit parameters obtained are summarized in Table 1. The Akron sludge had a relatively high nonbiodigestible VS fraction (Arunachalam et al., 2004). The values of ðVSf =VSi Þ for the Los Lunas sludge were similar in the two low DO experiments (Runs 3 and 4), while that in the high-DO Run 2 was lower. Besides the DO effect, the more complete digestion in Run 2 might have benefited from the use of a mixture of thickened and fresh sludge with a lower initial solids concentration
(Arunachalam et al., 2004). Although larger, the corresponding residual PF fractions, PFf =PFi ; followed the same trend as ðVSf =VSi Þ; with the largest value found with the Akron sludge (Run 1). The only exception was with Run 3, which had a relatively high ðVSf =VSi Þ but the lowest ðPFf =PFi Þ: The discrepancy likely reflected the highly nonlinear behavior of the optical signals, e.g. the fluorescence detected in Run 3 came from a much smaller ‘‘sensed’’ volume because of the limited light penetration in the sludge of a significantly higher solids concentration. Use of the normalized PF would reduce the nonlinear effects to certain extent. With these best-fit parameters, the raw data of VS and PF were converted to normalized VS and normalized PF, and subsequently plotted against time. The resultant profiles are shown in Fig. 6. It is clear that the simple, semi-empirical approach is adequate in correlating VS reduction with the PF profile, at least as a reasonable estimation. This approach may be applied in the plants for process monitoring and/or controlling purposes, although some adjustment may be needed to account for
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Table 1 The best-fit parameters for the exponential decay function used to correlate both protein fluorescence and VS, and the times for reaching Class B sludge requirements in the four experimental runs
Best-fit parameters kd ðh1 Þ VSi ðg=LÞ VSf =VSi PFi PFf =PFi
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0:012 0:001 11:35 0:26 0:47 0:02 1773 7 0:617 0:002
0:008 0:001 17:90 0:57 0:22 0:06 2013 6 0:449 0:003
0:004 0:001 34:69 0:79 0:36 0:05 1334 2 0:349 0:002
0:006 0:001 22:44 0:73 0:33 0:04 1883 4 0:509 0:003
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the different process kinetics between the batch operation and the plant’s design, e.g. continuous-stir tank operation. For example, the sludge can be sampled and analyzed for determining the VS decay constant ðkd Þ in
the plant conditions. PFi can be monitored in the waste sludge fed to the digester. Another PF value can be obtained in the digester, with known treatment time ðtÞ: Plugging in PFi ; PF; kd and t; Eq. (2) (or the equivalent
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As the NAD(P)H fluorescence appeared to be buried under the broad peak of 370/430 nm in this study, we performed the above test of switch between aerobic and anoxic/anaerobic conditions periodically in all the experiments. The characteristic response of NAD(P)H fluorescence was clearly observed (Fig. 7) only in the early stage of Run 3, with high TS and low DO. The NAD(P)H fluorescence was therefore very weak in the aerobic sludge digestion systems studied. The observation can be explained as follows: In aerobic digestion systems, the heterotrophic organisms were starved for the organic materials that drive their energy-producing catabolism. Consequently, the generation of NAD(P)H by heterotrophic catabolism is expected to be minimal. Nitrifiers might be more active and survived longer than heterotrophs in the digestion systems (Hao et al., 1991; Li and Ju, 2002). However, their autochemotrophic nature of energy generation (Prosser, 1986) does not favor for maintaining a high intracellular NAD(P)H concentration, and the NAD(P)H fluorescence in nitrifying cultures has been shown to be undetectably low (Ju et al., 1995; Ju and Nallagatla, 2003). The NAD(P)H fluorescence was only detectable in the early stage of Run 3 presumably because a sizable portion of the initial heterotrophic population introduced with the high solids loading was still viable during the early stage, and the low DO condition slowed the NAD(P)H oxidation.
equation for the plant’s process design) can be solved for PFf : With known PFi and PFf ; Eq. (3) can then be used to estimate/follow the extent of VS reduction achieved at real time using the online protein fluorescence signal PF. The correlation can be updated with process parameters measured regularly. Changes of the PF intensity or profile over time indicate variations in the process performance. With time the PF profile corresponding to normal plant operation, including the seasonal and diurnal fluctuations, can be established. Deviations from the normal profile may then be used to alert the plant personnel for remedial actions if necessary.
3.4. NAD(P)H fluorescence NAD(P)H exist in all living organisms. Therefore, their fluorescence at 340/460 nm should be part of the fluorescence spectra obtained in the experiments. One easy way to detect the NAD(P)H fluorescence is by observing how the fluorescence intensity responds to the change between aerobic and anoxic/anaerobic conditions. This is because the concentrations of intracellular NAD(P)H depend on the dynamic balance between their generation by catabolism and their consumption by respiration and anabolism (Armiger et al., 1986; Ju and Trivedi, 1998; Madigan et al., 1997). Under aerobic conditions, the NADH generated by catabolism is rapidly oxidized by aerobic respiration, leaving a low level of NAD(P)H. Under anoxic/anaerobic conditions, the NADH oxidation is much slower, through either anaerobic respiration (such as denitrification) or some fermentative pathways, depending on the metabolism of the organisms. The NAD(P)H fluorescence of a heterotrophic culture thus typically exhibits an immediate step drop responding to the switch from anaerobic/anoxic to aerobic conditions, and a fast increase if switched from aerobic to anoxic/anaerobic conditions (Armiger et al., 1986; Ju and Trivedi, 1992; Ju et al., 1995).
4. Conclusions The online fluorometer revealed three major fluorescence peaks in the aerobic sludge digestion systems studied. The peak at 290/350 nm was attributed to the fluorescence of proteinaceous materials in the sludge. The sources for the other two peaks (at 370/430 nm and 430/510 nm) remain unknown. The protein fluorescence 1450
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Fig. 7. Responses of fluorescence at excitation/emission wavelengths of 350/450 nm to switches between aerobic and anoxic/anaerobic conditions, observed in the early stage of Run 3. The characteristics step responses of NAD(P)H fluorescence did not occur as clearly when the switches were made in the later stage of Run 3 or in any other runs.
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(PF) profile was found to correlate well with the profile of VS reduction in all four experiments. Using a simple semi-empirical approach, the same exponential decay function can be used to describe the profiles of both normalized PF and normalized VS. The feasibility of following the real-time performance of aerobic sludge digestion by monitoring culture fluorescence was clearly demonstrated. On the other hand, the NAD(P)H fluorescence was found very weak in the digestion systems studied. It was buried under the broad peak at 370/430 nm. Even with the switch between aerobic and anoxic/anaerobic conditions, the characteristic step response of NAD(P)H fluorescence could be detected only in the early stage of Run 3, which had the highest solids loading and was operated under low DO conditions. The applicability of NAD(P)H fluorescence in aerobic sludge digestion processes was probably more limited than that in the biological wastewater treatment processes.
Acknowledgements The work was supported by Enviroquip, Inc. (Austin, TX). The online fluorometer was loaned, without charge, from BioBalance Technology, Inc. (Denmark) during the study period. The authors were especially grateful to Mr. Erik Skibsted (BioBalance) for the technical assistance in fluorometer set-up as well as acquisition and interpretation of the fluorescence signals.
References Al-Ghusain, I., Hao, O.J., 1995. Use of pH as control parameter for aerobic/anoxic sludge digestion. J. Environ. Eng. ASCE 121 (3), 225–235. APHA, 1995. Standard Methods for the Examination of Water and Wastewater, Nineteenth ed. American Public Health Association, Washington, DC. Armiger, W.B., Forro, J.F., Montalvo, L.M., Lee, J.F., 1986. The interpretation of on-line process measurements of intracellular NADH in fermentation processes. Chem. Eng. Commun. 45, 197–206. Arunachalam, R., Shah, H.K., Ju, L.-K., 2004. Aerobic sludge digestion under low dissolved oxygen concentrations. Water Environ. Res. 76, 453–462. Daigger, G.T., Bailey, E., 2000. Improving aerobic digestion by prethickening, staged operation, and aerobic-anoxic operation: four full-scale demonstrations. Water Environ. Res. 72, 260–270.
Farrell, J.B., Bhide, V., Smith Jr., J.E., 1996. Development of EPAs new methods to quantify vector attraction of wastewater sludges. Water Environ. Res. 68, 286–294. Hao, O.J., Kim, M.H., Al-Ghusain, I., 1991. Alternating aerobic and anoxic digestion of waste activated sludge. J. Chem. Tech. Biotechnol. 52, 457–472. Ju, L.-K., Nallagatla, P.B., 2003. Monitoring and kinetic study of nitrification using dissolved oxygen electrode and NAD(P)H fluorometer. Water Res. 37, 453–462. Ju, L.-K., Trivedi, H.K., 1992. Monitoring of denitrification by Pseudomonas aeruginosa using on-line fluorescence technique. Biotechnol. Tech. 6, 549–554. Ju, L.-K., Trivedi, H.K., 1998. Oscillation of NAD(P)H fluorescence in Escherichia coli cultures performing dissimilative nitrate/nitrite reduction. World J. Microbiol. Biotechnol. 14, 263–269. Ju, L.-K., Yang, X., Lee, J.F., Armiger, W.B., 1995. Monitoring of the biological nutrient removal process by an on-line NAD(P)H fluorometer. Biotechnol. Prog. 11, 545–551. Krishnamoorthy, R., Loehr, R.C., 1989. Aerobic sludge stabilization—Factors affecting kinetics. J. Environ. Eng. 115, 283–301. Kwong, S.C.W., Rao, G., 1994. Metabolic monitoring by using the rate of change of NAD(P)H fluorescence. Biotechnol. Bioeng. 44, 453–459. Li, X., Ju, L.-K., 1999. On-line fluorescence profile of aerobic sludge digestion. Biotechnol. Prog. 15, 1125–1132. Li, X., Ju, L.-K., 2002. Two-phase decay of aerobic sludge shown by online fluorescence and modeled with interaction of heterotrophs and nitrifiers. Environ. Eng. Sci. 19, 79–87. Li, J.-K., Asali, E.C., Humphrey, A.E., 1991. Monitoring cell concentration and activity by multiple excitation fluorometry. Biotechnol. Prog. 7, 21–27. Madigan, M.T., Martinko, J.M., Parker, J., 1997. Brock Biology of Microorganisms. Prentice-Hall, Upper Saddle River, NJ. Marose, S., Lindemann, C., Scheper, T., 1998. Two-dimensional fluorescence spectroscopy: a new tool for on-line bioprocess monitoring. Biotechnol. Prog. 14, 63–74. Metcalf & Eddy, Inc. (revised by Tchobanoglous, G. and Burton, F.L.), 1991. Wastewater Engineering: Treatment, Disposal, and Reuse, third ed. McGraw Hill, New York. Prosser, J.I. (Ed.), 1986. Nitrification. IRL Press, Oxford, UK. Sharma, A., Schulman, S.G., 1999. Introduction to Fluorescence Spectroscopy. Wiley, New York. Siano, S.A., Mutharasan, R., 1990. NADH fluorescence and oxygen-uptake responses of hybridoma cultures to substrate pulse and step changes. Biotechnol. Lett. 37, 141–159. Trivedi, H.K., Ju, L.-K., 1994. Study of nitrate metabolism of Escherichia coli using fluorescence. Biotechnol. Prog. 10, 421–427. Udenfriend, S., 1962. Fluorescence Assay in Biology and Medicine. Academic Press, New York. Zabriskie, D.W., 1979. Use of culture fluorescence for monitoring of fermentation systems. Biotechnol. Bioeng. Symp. 9, 117–123.
Water Research 39 (2005) 1205–1214 www.elsevier.com/locate/watres
Monitoring aerobic sludge digestion by online scanning fluorometry RaviSankar Arunachalam, Hemant K. Shah, Lu-Kwang Ju Department of Chemical Engineering, College of Engineering, The University of Akron, Akron, OH 44325-3906, USA Received 20 September 2002; received in revised form 26 November 2003; accepted 16 November 2004 Available online 23 March 2005
Abstract With sludge samples from two wastewater treatment plants, batch experiments of aerobic sludge digestion were conducted under different dissolved oxygen (DO) and solids concentrations. A fluorometer capable of online excitation and emission scanning was used to monitor the digestion process. Three major fluorescence peaks were observed. The peak at excitation/emission maxima of 290/350 nm was attributed to the fluorescence of proteinaceous materials in the sludge, with tryptophan residues being the primary contributor. The sources for the other two peaks (at 370/430 nm and 430/510 nm) remain unknown. The well-known biological fluorescence from reduced nicotinamide adenine dinucleotides (NADH and NADPH), at excitation/emission maxima of 340/460 nm, was found very weak in the aerobic digestion systems studied. It was buried under the broad peak at 370/430 nm and was detectable only in the early stage of the experiment that had the highest solids loading (at 4.8%) and was operated under low DO (0.2–1.0 mg/ L) conditions. On the other hand, the profile of the protein fluorescence (PF) correlated well with that of the volatile solids (VS) reduction in all the experiments. A semi-empirical exponential decay function was developed, which described well the profiles of both normalized VS and normalized PF. The feasibility of following the real-time performance of aerobic sludge digestion by monitoring PF was clearly demonstrated. r 2005 Elsevier Ltd. All rights reserved. Keywords: Sludge digestion; Fluorescence; Proteins; Tryptophan; NAD(P)H; Dissolved oxygen
1. Introduction Energy consumption for aeration of the aerobic sludge digesters represents a major running cost to the plant. To minimize the aeration cost, it is desirable to monitor the real-time progress of the digestion process so that the supply and demand of aeration can be better matched, which depending on the process designs, may be achieved by adjusting the aeration rate, optimizing the cyclic timing between oxic and anoxic operations Corresponding author. Tel.: +1 330 972 7252; fax: +1 330 972 5856. E-mail address: [email protected] (L.-K. Ju).
(Al-Ghusain and Hao, 1995; Daigger and Bailey, 2000; Hao et al., 1991), and/or shortening the treatment period according to the indicated reach of the desired extent of digestion. The real-time process information also offers peace-of-mind to the plant operators and managers during normal operation and the warning for remedial actions if process upsets should occur. Previous researchers have used parameters such as dissolved oxygen (DO), oxidation-reduction potential (ORP), and pH to monitor the process of sludge digestion (AlGhusain and Hao, 1995). However, the measuring devices for these parameters require rather frequent calibration because they are based on electrochemical reactions that inevitably change the properties of the
0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2004.11.028
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electrolytes and/or electrode(s) employed. They are therefore not ideal for long-term, continuous process monitoring. In addition, these parameters describe the conditions of the water phase, instead of the sludge itself. Because the organisms in the sludge are the real ‘‘workers’’ performing the digestion and the state of the sludge is the true property of concern in the digestion process, these water-phase parameters are not definitely indicative of the process performance, especially under often fluctuating process conditions. Recognizing the above limitations, we explored in this work the possibility of monitoring sludge digestion processes using the culture fluorescence technology that employs much more stable optical sensors and detects the fluorescence directly associated with the sludge. Compared to the electrodes for measuring DO, ORP and pH, optical fluorometers offer superior long-term stability and eliminate the need for frequent recalibration and maintenance, thus being more suitable for the continuous operation in wastewater treatment plants (Ju et al., 1995). The technology is based on the following principle (Sharma and Schulman, 1999): When a molecule absorbs light (at the excitation wavelengths), it must emit a quantity of energy equivalent to that absorbed if it is to return to its stable ground state. The energy can be released in forms of light, heat, resonance radiation, Rayleigh scattering, and Raman scattering. When light is emitted (at the emission wavelengths), the phenomenon is referred to as luminescence, which includes both fluorescence and phosphorescence. Molecules that fluoresce are called fluorophores. Many fluorophores, both intracellular and extracellular, are present in biological systems (Marose et al., 1998; Udenfriend, 1962). The concentration variations of some of these fluorophores are closely related to the biological activities and, therefore, can be used as indicators of important process parameters such as biomass concentration and metabolic state (e.g. Li et al., 1991; Marose et al., 1998; Trivedi and Ju, 1994; Zabriskie, 1979). On-line monitoring of these fluorescence changes may lead to better understanding of the complex processes of sludge digestion and development of valuable process control and optimization strategies. In an earlier study of aerobic digestion of a mixed primary and secondary sludge, we showed that the fluorescence at excitation and emission (ex/em) maxima of 340/440 nm exhibited a characteristic profile that correlated well with the progress of the digestion (Li and Ju, 1999). The above fluorescence occurred at ex/em maxima near those from the reduced nicotinamide adenine dinucleotides, NADH and NADPH, hereafter referred to as NAD(P)H. NAD(P)H, with ex/em maxima at 340/ 460 nm, are among the best-studied biological fluorophores (e.g. Ju and Trivedi, 1992; Ju et al., 1995; Kwong and Rao, 1994; Siano and Mutharasan, 1990). They,
and their non-fluorescent oxidized counterparts NADðPÞþ ; are present in all living organisms as the coenzymes for many metabolic reactions (Madigan et al., 1997). The levels of NAD(P)H, and their fluorescence intensity, in a biological system depend on the live cell concentration, the energy inventory within each cell, and the metabolic activity. Proteins are another important source of culture fluorescence (Li et al., 1991; Marose et al., 1998; Udenfriend, 1962). While NAD(P)H fluorescence is observed only in live organisms with active metabolism, the proteins fluoresce regardless of their being associated with live or dead organisms. Accordingly, the profile of protein fluorescence in a digestion process would directly reflect the change of the sludge’s proteinaceous content. Because the proteinaceous materials are destroyed along the volatile solids (VS) reduction, it is feasible to follow the progress of VS reduction by monitoring the profile of protein fluorescence in the digestion process. In this study, a multi-channel online fluorescence sensor (BioViews from Biobalance Technology, Inc., Denmark) was used to monitor the batch experiments of aerobic sludge digestion. The fluorometer was equipped with 16 excitation channels (270–530 nm) and 16 emission channels (290–570 nm), stepped at 20-nm intervals. The wide sensing ranges cover the fluorescence from the majority of important fluorophores known to be present in biological systems, including those from NAD(P)H and proteins (Marose et al., 1998; Udenfriend, 1962). The digestion experiments were made under different DO and solids concentrations with the sludge samples from two different plants: Akron, OH and Los Lunas, NM. The results confirmed the applicability of the on-line fluorescence technology in different process conditions.
2. Materials and methods 2.1. Sludge samples Four batch experiments were carried out in this study. The first run was made with the fresh sludge sample from the nearby Akron wastewater treatment plant. The sludge samples used in the other three runs were from a plant in Los Lunas, NM. The three batches of samples were shipped overnight in an iced container and studied immediately upon receiving to minimize the change of sludge properties during the shipment. With an average flow of 70–80 million gallons per day (MGD), i.e., ð2:6 3:0Þ 105 m3 =d; the Akron plant uses aeration ditches for secondary biological treatment ðHRT6:5 hÞ; to remove ammonium and organic substances. The primary influent had the following average properties: BOD5 (biological oxygen demand)—100 mg/
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L, TSS (total suspended solids)—160 mg/L, TKN (total Kjeldahl nitrogen)—20 mg/L, and NHþ 4 —N—12 mg/L. The influent to secondary treatment had an average BOD5 of approximately 70 mg/L and TSS of approximately 55 mg/L (N information unavailable). The solids concentration of the Akron plant’s secondary sludge was typically around 2% (w/v). The much smaller Los Lunas plant has a design capacity of 0.7 MGD (i.e., 2650 m3 =d) with a peak flow capacity of 2.1 MGD (i.e., 7950 m3 =d). The plant has no primary clarifiers and employs an activated sludge process with an extended-aeration configuration ðHRT20 hÞ: The typical influent values for the plant were as follows: BOD5 —270 mg/L, TSS—220 mg/L, TKN—33 mg/L, and NHþ 4 –N—37 mg/L. (The seemingly erroneous larger TKN than NHþ 4 –N concentrations were caused by measurements made on samples from different periods.) The waste sludge was thickened to about 5% in a gravity belt thickener. The processes and operating conditions of the two plants were very different. The thickened Los Lunas sludge was further mixed with the plant’s fresh sludge (details given later) to give different solids concentrations and sludge properties. These arrangements were made so that the applicability of the on-line fluorescence technology to different sludge could be evaluated.
2.2. Digestion experiments All experiments were carried out at room temperature. The Akron sludge used in the first run had an initial total solids (TS) concentration of 1.6%. The DO was maintained at 3–4 mg/L in this run, for fully aerobic operation. The pH was controlled at 6.9–7.2 by automatic addition of 0:5 N Na2 CO3 : The second run was made under the same conditions but with the Los Lunas sludge having initial TS of 2.3%. Runs 3 and 4 were carried out at low DO, in the range of 0.2–1 mg/L. The initial TS were 4.8% in Run 3 and 2.8% in Run 4. No pH control was necessary in these low DO runs, as the pH never went below 6.5. As described earlier, the sludge at the Los Lunas plant was typically thickened to about 5% (w/v) before digestion. While the thickened sludge was used in Run 3, the sludge samples used in Runs 2 and 4 were prepared by mixing the thickened sludge with the fresh sludge from the secondary clarifier. The digestion was carried out in a 2-L plastic container. The fluorometer, DO and pH probes, as well as ports for aeration and sampling were introduced through the container lid. Continuous aeration was provided by bubbling humidified air through a diffuser placed at the bottom of the reactor. Agitation was provided by magnetic stir bar for the first two experiments. The stirring speed was adjusted daily to maintain DO in the designed range. For the last two low
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DO experiments, the aeration rate was decreased significantly. To compensate for the less mixing provided by the reduced aeration, a marine impeller driven by a variable-speed motor was used for agitation. The Los Lunas sludge tended to foam, especially during the early stage of the digestion. The foams were broken periodically and the trapped flocs returned to the sludge. The water loss by evaporation was compensated almost daily by addition of tap water. 2.3. Analyses Samples were taken daily for various analyses: 10 mL for TS and VS, 10 mL for NHþ 4 2N; NOX 2N (i.e., combined NO3 –N and NO2 –N) and NO 2 –N, and 10 mL for fecal coliform density. To make sure that the samples were representative, the sludge was stirred thoroughly (but briefly) before sampling. The analyses were performed according to the standard methods (APHA, 1995): Procedures 2540B and 2540E for TS and VS, respectively; Procedures 4500-NH3 F and 4500 þ NO 3 for NH4 –N and NOX –N, using an ammoniaselective electrode; Procedure 4500B for NO 2 –N; Procedure 9221B for fecal coliform density, by the multiple-tube fermentation technique; and Procedure 2710B for oxygen uptake rate (OUR), using a DO probe. The specific OUR (SOUR) was then calculated by dividing OUR by TS. The OUR was not measured in Run 3 where the very high solids loading (4.8% TS) made it difficult to raise the DO to high enough levels for adequate measurement by the dynamic method. The sludge supernatant required for some of the above analyses were collected after centrifuging the sludge sample at 14; 000g for 10 min.
3. Results and discussion 3.1. Fluorescence spectra The typical fluorescence spectra obtained in the digestion experiments, for one scanning cycle, are shown in Fig. 1. Three major peaks always appeared around the ex/em maxima of 290/350 nm, 370/430 nm, and 430/ 510 nm. The peak at 290/350 nm has the same maxima as the aromatic amino acid tryptophan, which has much higher absorption and fluorescence efficiencies than the other two common aromatic amino acids (tyrosine and phenylalanine) (Udenfriend, 1962). This peak is therefore attributed to the fluorescence from the proteinaceous materials in the sludge. The responsible fluorophores for the other two peaks are, however, unknown. Further, the fluorescence from NAD(P)H (ex/ em maxima: 340/460 nm) appeared to be too weak and was buried under the broad peak at 370/430 nm. Some
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conducted under different operating conditions. No consistent correlation with the change of other measured parameters can be identified. More studies are needed to understand their origins and, perhaps, their relations with the digestion process. The profiles of the measured process parameters were mostly expected. Rapid pathogen destruction was observed in the experiments. Active nitrification occurred in the high DO digestion experiments (Figs. 2 and 3), resulting in negligible ammonium concentration and increasing NO x concentration. On the other hand, the digestion at low DO involved slower nitrification and simultaneous denitrification, which in one of the low DO experiments (Fig. 5) caused a distinct three-phase phenomenon in the ammonium and NO x profiles: the ammonium concentration increased during the first 50 h, followed by a sharp decline to negligible levels; the NO x concentration remained very low during 0–50 h, increased to (and fluctuated around) 18 mg/L NO x –N during 50–170 h, and de-
creased steadily after 170 h. The phenomena can be explained as follows: During the first 50 h, the higher respiration demand kept the DO very low in the digester, about 0.1–0.2 mg/ L. At such low DO, nitrification was very slow and simultaneous denitrification took place, resulting in negligible NO x concentrations. Meanwhile, the rate of ammonium generation from the rapid initial VS digestion exceeded the rate of nitrification, causing the observed increase of ammonium concentration during this period. After 50 h, DO rose gradually and fluctuated around 0.5–1.0 mg/L (data not shown). The increased DO led to faster nitrification and slower denitrification, which in turn brought about the subsequent drop of ammonium concentration and increase of NO x concentration. After about 170 h, the VS digestion slowed down significantly. The decrease in the accompanying ammonium release would lower the NO x generation by nitrification, causing the eventual slow decline of the NO x concentration.
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3.3. Correlation between protein fluorescence and VS profiles As mentioned above, the observed profiles suggested the possibility of developing mathematical correlations between VS and PF as well as between SOUR and PF. The correlation between VS and PF is fundamentally plausible because both relate directly to the proteinaceous contents of the sludge. The SOUR, on the other hand, reflects only the respiring population. Furthermore, the correlation between SOUR and PF may be very plant-specific because SOUR depends on the population composition of the sludge and any plant conditions (e.g. temperature and pH) that affect the cellular metabolism. Consequently, only the development of quantitative correlation between VS and PF is pursued in this work. VS degradation is normally described by a first-order kinetics (Krishnamoorthy and Loehr, 1989; Metcalf and Eddy, 1991), i.e., dðVSb Þ ¼ kd ðVSb Þ, dt
where VSb is the biodigestible VS (¼ VS VSf ; VSf being the hypothetically ‘‘inert,’’ ‘‘non-biodigestible’’ portion of VS) and kd is the decay constant ðh1 Þ: On integration, the above equation gives an exponential decay function of VS, VS VSf ¼ ekd t VSi VSf
(1)
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If the same decay constant applies to both profiles (i.e., kd ¼ kd;PF ), an identical equation describes the profiles of both normalized VS and normalized PF, and the two normalized properties correspond to each
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other directly, i.e., VS VSf PF PFf ¼ . VSi VSf PFi PFf
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This is shown to be practically valid for the experimental results obtained in this study, as described in the following: The VS data from each experiment were first regressed with Eq. (1) to obtain the best-fit parameters: kd ; VSi and ðVSf =VSi Þ: Similar regression was then performed with the PF data, according to Eq. (2), but with the kd value fixed as that obtained from the VS regression. The best-fit PFi and ðPFf =PFi ) were obtained accordingly. All the best-fit parameters obtained are summarized in Table 1. The Akron sludge had a relatively high nonbiodigestible VS fraction (Arunachalam et al., 2004). The values of ðVSf =VSi Þ for the Los Lunas sludge were similar in the two low DO experiments (Runs 3 and 4), while that in the high-DO Run 2 was lower. Besides the DO effect, the more complete digestion in Run 2 might have benefited from the use of a mixture of thickened and fresh sludge with a lower initial solids concentration
(Arunachalam et al., 2004). Although larger, the corresponding residual PF fractions, PFf =PFi ; followed the same trend as ðVSf =VSi Þ; with the largest value found with the Akron sludge (Run 1). The only exception was with Run 3, which had a relatively high ðVSf =VSi Þ but the lowest ðPFf =PFi Þ: The discrepancy likely reflected the highly nonlinear behavior of the optical signals, e.g. the fluorescence detected in Run 3 came from a much smaller ‘‘sensed’’ volume because of the limited light penetration in the sludge of a significantly higher solids concentration. Use of the normalized PF would reduce the nonlinear effects to certain extent. With these best-fit parameters, the raw data of VS and PF were converted to normalized VS and normalized PF, and subsequently plotted against time. The resultant profiles are shown in Fig. 6. It is clear that the simple, semi-empirical approach is adequate in correlating VS reduction with the PF profile, at least as a reasonable estimation. This approach may be applied in the plants for process monitoring and/or controlling purposes, although some adjustment may be needed to account for
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Table 1 The best-fit parameters for the exponential decay function used to correlate both protein fluorescence and VS, and the times for reaching Class B sludge requirements in the four experimental runs
Best-fit parameters kd ðh1 Þ VSi ðg=LÞ VSf =VSi PFi PFf =PFi
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0:012 0:001 11:35 0:26 0:47 0:02 1773 7 0:617 0:002
0:008 0:001 17:90 0:57 0:22 0:06 2013 6 0:449 0:003
0:004 0:001 34:69 0:79 0:36 0:05 1334 2 0:349 0:002
0:006 0:001 22:44 0:73 0:33 0:04 1883 4 0:509 0:003
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Time for reaching Class B requirements (h) 96 VSa SOURb 110 Note: (Farrell et al., 1996). a VS reduction 438%: b SOUR o1:5 mg O2 =g TS-h.
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Fig. 6. Best-fit correlation of both normalized VS ð&Þ and normalized PF ð Þ with the same exponential decay function ð2Þ: The bestfit parameters are summarized in Table 1.
the different process kinetics between the batch operation and the plant’s design, e.g. continuous-stir tank operation. For example, the sludge can be sampled and analyzed for determining the VS decay constant ðkd Þ in
the plant conditions. PFi can be monitored in the waste sludge fed to the digester. Another PF value can be obtained in the digester, with known treatment time ðtÞ: Plugging in PFi ; PF; kd and t; Eq. (2) (or the equivalent
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As the NAD(P)H fluorescence appeared to be buried under the broad peak of 370/430 nm in this study, we performed the above test of switch between aerobic and anoxic/anaerobic conditions periodically in all the experiments. The characteristic response of NAD(P)H fluorescence was clearly observed (Fig. 7) only in the early stage of Run 3, with high TS and low DO. The NAD(P)H fluorescence was therefore very weak in the aerobic sludge digestion systems studied. The observation can be explained as follows: In aerobic digestion systems, the heterotrophic organisms were starved for the organic materials that drive their energy-producing catabolism. Consequently, the generation of NAD(P)H by heterotrophic catabolism is expected to be minimal. Nitrifiers might be more active and survived longer than heterotrophs in the digestion systems (Hao et al., 1991; Li and Ju, 2002). However, their autochemotrophic nature of energy generation (Prosser, 1986) does not favor for maintaining a high intracellular NAD(P)H concentration, and the NAD(P)H fluorescence in nitrifying cultures has been shown to be undetectably low (Ju et al., 1995; Ju and Nallagatla, 2003). The NAD(P)H fluorescence was only detectable in the early stage of Run 3 presumably because a sizable portion of the initial heterotrophic population introduced with the high solids loading was still viable during the early stage, and the low DO condition slowed the NAD(P)H oxidation.
equation for the plant’s process design) can be solved for PFf : With known PFi and PFf ; Eq. (3) can then be used to estimate/follow the extent of VS reduction achieved at real time using the online protein fluorescence signal PF. The correlation can be updated with process parameters measured regularly. Changes of the PF intensity or profile over time indicate variations in the process performance. With time the PF profile corresponding to normal plant operation, including the seasonal and diurnal fluctuations, can be established. Deviations from the normal profile may then be used to alert the plant personnel for remedial actions if necessary.
3.4. NAD(P)H fluorescence NAD(P)H exist in all living organisms. Therefore, their fluorescence at 340/460 nm should be part of the fluorescence spectra obtained in the experiments. One easy way to detect the NAD(P)H fluorescence is by observing how the fluorescence intensity responds to the change between aerobic and anoxic/anaerobic conditions. This is because the concentrations of intracellular NAD(P)H depend on the dynamic balance between their generation by catabolism and their consumption by respiration and anabolism (Armiger et al., 1986; Ju and Trivedi, 1998; Madigan et al., 1997). Under aerobic conditions, the NADH generated by catabolism is rapidly oxidized by aerobic respiration, leaving a low level of NAD(P)H. Under anoxic/anaerobic conditions, the NADH oxidation is much slower, through either anaerobic respiration (such as denitrification) or some fermentative pathways, depending on the metabolism of the organisms. The NAD(P)H fluorescence of a heterotrophic culture thus typically exhibits an immediate step drop responding to the switch from anaerobic/anoxic to aerobic conditions, and a fast increase if switched from aerobic to anoxic/anaerobic conditions (Armiger et al., 1986; Ju and Trivedi, 1992; Ju et al., 1995).
4. Conclusions The online fluorometer revealed three major fluorescence peaks in the aerobic sludge digestion systems studied. The peak at 290/350 nm was attributed to the fluorescence of proteinaceous materials in the sludge. The sources for the other two peaks (at 370/430 nm and 430/510 nm) remain unknown. The protein fluorescence 1450
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(PF) profile was found to correlate well with the profile of VS reduction in all four experiments. Using a simple semi-empirical approach, the same exponential decay function can be used to describe the profiles of both normalized PF and normalized VS. The feasibility of following the real-time performance of aerobic sludge digestion by monitoring culture fluorescence was clearly demonstrated. On the other hand, the NAD(P)H fluorescence was found very weak in the digestion systems studied. It was buried under the broad peak at 370/430 nm. Even with the switch between aerobic and anoxic/anaerobic conditions, the characteristic step response of NAD(P)H fluorescence could be detected only in the early stage of Run 3, which had the highest solids loading and was operated under low DO conditions. The applicability of NAD(P)H fluorescence in aerobic sludge digestion processes was probably more limited than that in the biological wastewater treatment processes.
Acknowledgements The work was supported by Enviroquip, Inc. (Austin, TX). The online fluorometer was loaned, without charge, from BioBalance Technology, Inc. (Denmark) during the study period. The authors were especially grateful to Mr. Erik Skibsted (BioBalance) for the technical assistance in fluorometer set-up as well as acquisition and interpretation of the fluorescence signals.
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