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aerobic sequencing batch reactors
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WaL Sci. Tech. Vol. 3'. No. I. pp. "3-160. 1997. Copyright Ci 1996 IAWQ. Published by Elsevier Science Ltd Printed In Great Britain. All rights reserved. 0273-1223197 $17'00 +0'00
Pergamon
PH: S0273-1223(96)OO891-8
THE EFFECT OF VOLATILE FATTY ACIDS ON ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL AND POPULATION STRUCTURE IN ANAEROBIC/AEROBIC SEQUENCING BATCH REACTORS Andrew Amis Randal1*, Larry D. Benefield**, William E. Hill***, Jean-Paul Nicol**, Gerald K. Boman** and Shuh-Ren Jingt * Department o/Civil and Environmental Engineering, P.O. Box 162450, University o/Central Florida. Orlando, FL 32816-2450. USA .. do Col/ege 0/ Engineering, 108 Ramsey Hall. Aubum University, Aubum, AL 36849, USA *** Depanment o/Chemistry, 179 Chemistry Building, Auburn University, Auburn, AL 36849, USA t Chia-Nan Jr. College 0/ Pharmacy, Department 0/ Envr. Engr., Tainan, Taiwan, Republic of China
ABSTRACf Three anaerobic/aerobic sequencing balch reaclors (SBRs) were operaled for S 1/2 years. Volatile fatty acids (VFAs) in influent wastewater for two of the SBRs (the Glucosel and 2 SBRs) resulted in optimization of enhanced biological phosphorus removal (EBPR), and a bacterial population capable of increasing phosphorus (P) removals in response to increased VFA or P concentration. Another SBR not receiving VFAs (the Starch SBR) showed marginal EBPR and was incapable of either response. All three anaerobic/aerobic sequencing batch reactors (SBRs) showed bounded oscillations in P removal that did not correspond to any operational or environmental change. The oscillations were probably associated with interspecies population dynamics intensified due to the periodic. unsteady-state. nature of the SBR process. The glucose SBRs also showed an additional type of variability associated with EBPR. probably from competition between poly-P and "G" bacteria for readily available substrate (Le. glucose. VFAs) during anaerobiosis. The predominant bacterial isolates from the glucose SBRs were Pseudomonas and Bacillus while Auomonas was isolated most frequently from the Starch SBR. The relatively slow growth rate of Pseudomonas may have contributed to the high variability of EBPR observed. Fractal analysis indicated overall variability may have been chaotic. but was inconclusive. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd
KEYWORDS
Chaotic behavior; enhanced biological phosphorus removal (EBPR); fractal analysis; poly-P bacteria; polyphosphate (PPn); population dynamics; Pseudomonas; sequencing batch reactor (SBR). 1S3
A. A. RANDALL et al.
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INTRODUCTION A Brief E3plaoatjon of the EBPR Phenomenon Enhanced biological phosphorus removal (EBPR), or luxury uptake, is phosphorus uptake by bacteria that exceeds the 2.3% phosphorus by weight typical of conventional wastewater treatment system biomass. The EBPR phenomenon occurs when anaerobic/aerobic sequencing of an activated sludge system (continuous or batch) is used. In addition fermentation products must be present during the anaerobic phase. The role of fermentation products, in particular the short carbon-chain carboxylic acids (ie. volatile fatty acids or VFAs), in EBPR is well known and has been investigated by a number of researchers (Abu-ghararah and Randall, 1991; Arunetal., 1989; Comeau etal., 1987a; Jones etal., 1987; Randall etal., 1994; Randalletal., 1995). The two basic models for EBPR were presented by Comeau et al. (1987b), and Arun et al. (1989). The models are almost identical. It is thought that polyphosphate (PPn) supplies energy for the anaerobic uptake of fermentation products (e.g. VFAs) during anaerobiosis. The fermentation products are then stored as polyhydroxyalkanoates (PHA), of which polyhydroxybutyrate (PHB) is the best known member. During subsequent aerobiosis the PHAs are metabolized, and the energy derived from them is stored as polyphosphate, which accounts for the luxury uptake observed. Reducing power must be supplied to synthesize PHA from fermentation products, however, and it is here where the models differ. In the model presented by Comeau et al. (1987) the reducing power is supplied by the anaerobic operation of the TCA cycle. In the model presented by Arun et al. (1989), reducing power is supplied by the utilization of glycogen.
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EBPR and Population Structure The importance of the bacterial population with respect to EBPR has been noted in a variety of papers. Kavanaugh (1991) found that bacteria belonging to AeromonasIVibrio, coliforms, Pseudomonas, and Acinetobacter were present in a continuous flow EBPR system which was also operated to achieve biological nitrogen removal. Acinetobacter only accounted for 5% of the population, with the other three groups being the predominant bacteria. Aeromonas sp. (hydrophila), Pseudomonas sp., and Acinetobacter sp. were all shown to accumulate phosphorus far in excess of conventional accumulation levels (2.3% P) using VFAs as substrate, and all formed polyphosphate (PPn) granules. Pseudomonas sp. were capable of storing 10 to 30% by weight of polyphosphate. She did not analyze gram positive bacteria such as Bacillus, but noted that they had been isolated in EBPR systems before, and could be important to the phenomena. Okada et al. (1992) used quinone profiles as well as phosphorus uptake data obtained during short term disturbance test of SBRs to show that the slow recovery of EBPR following short term disturbances was because of species succession of bacteria, and not due to metabolic slow downs. Acinetobacter and Pseudomonas were the predominant species and had the highest phosphorus removal activities in the system, so apparently they were responsible for EBPR. Both had a slow specific growth rate which resulted in slow recovery from disturbances which eroded their predominance. Coliforms were also present in significant numbers but showed low phosphorus removal activities. Hiraishi et al. (1989) used quinone profiles also, and found that the introduction of anaerobic/aerobic sequencing alone had little effect on the bacterial community structure, which suggests that influent characteristics might be more important in selecting poly-P bacteria. Cech and Hartman (1990) and Cech et al. (1993) observed that there were two competing populations in EBPR systems. One group, organisms which accumulated PPn (ie. poly-P bacteria), were responsible for EBPR. They competed for anaerobic substrate with so-called "G" bacteria, which were predominant when glucose was a significant portion of the influent substrate. The "G" bacteria sequestered anaerobic substrate without the use or subsequent production of PPn'
Effect of volatile fally acids on enhanced biological phosphorus removal
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MATERIALS AND METHODS Three identical sequencing batch reactors (SBRs) were operated and two of these were monitored constantly over a 5 1/2 year period during which an anaerobic/aerobic sequence was used in order to obtain enhanced biological phosphorus removal (EBPR). The systems received a synthetic wastewater consisting of nutrient broth, yeast, and inorganic salts to provide nitrogen, phosphorus (P), and micronutrients. The treatment cycle for all 3 SBRs consisted of a 2 hour anaerobic phase, followed by a 4 hour aerobic phase, and a 2 hour period for settling, draining of supernatant, and a brief idle period. This yielded an 8 hour treatment cycle, with 3 cycles per day. The only significant difference among the three SBRs was that 2 systems received glucose as an additional influent carbon source (the Glucose 1 and Glucose2 SBRs) while 1 system received an equal amount of starch rather than glucose (the Starch SBR). The synthetic wastewater without the supplemental carbon source will be referred to as the "base influent" in this paper. Average mean cell residence times (MCRTs) were approximately 6 days for all 3 SBRs. Batch tests began at the end of an anaerobic/aerobic cycle by dividing the mixed liquor of an SBR into six small reactors. Base influent was added and then each reactor spiked with the desired concentration of the substrate to be studied. The normal anaerobic/aerobic cycle was then duplicated and the final phosphorus concentration obtained for each reactor. Chemical P removal was eliminated as a significant mechanism of P removal from pH data and batch experiment results. Bacteria from the SBRs were cultured on agar containing the C.-C s carboxylic acids and autoclaved base influent and mixed liquor. The most numerous isolates (Le. colonies) were identified by testing their ability to oxidize a variety of substrates in a 96 well microplate. Additional tests such as oxidase and catalase production. or motility and morphology, were used when necessary. RESULTS AND DISCUSSION During the first 3 1/2 years (day 0 to day 1314) of observations the influent total phosphorus (TPinf) to the SBRs was 10 mgll. During the last 2 years of the study (day 1314 to day 2050) TPinf was increased to 20 mgll. The change in TPinf was the only operational or environmental change to take place during the 5 1/2 year period. There was one brief period where the Glucose2 SBR was operated with a 3 hourl3 hour anaerobic/aerobic cycle, but this resulted in very poor phosphorus removals. Contrast of SBR PQPulations: PhQsphQrus RemQvals and ResPQnse to Influent TP Throughout the study it was observed that the glucose SBRs typically had superior P removal compared to the Starch SBR (Table I). Table 1. Average phosphorus removal and reliability for three SBRs (influent of 10 and 20 mglL total phosphorus) Influent Phos. (mg/l)
System Starch Glucose1 Glucose2
10 10 10
Starch Glucose1 Glucose2
20 20 20
• •• •••
------Phosphorus Removal In MG/L-----90\·· Average 50\· 5.9 6.5
4.4
5.3
11.3
10.6
•••
5.9 7.2
2.8 2.8
4.8
2.8
10.5 10.6
7.4
6.0
50\ of all observations were greater than the value shown 90\ of all observations were greater than the value shown There were insufficient observations to obtain meaningful comparisons for the Glucose2 SBR at 10 mg/l
A. A. RANDALL ~t al.
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The superior P removals observed in the glucose SBRs were shown to be the result of VFAs in the influent (Randall et al., 1994; Randall et al., 1995). The VFAs resulted from prefermentation of glucose at ambient temperature (23 degrees C) in the feed jars prior to entering the SBRs. Starch was not observed to preferment in this way. Since feed was made once a day, all the SBRs received feed aged a minimum of 0 hours, 8 hours, and 16 hours at the start of the first, second, and third daily treatment cycles. At 8 hours the influent glucose was typically 50% fermented, largely to organic acids (e.g. VFAs). At 16 hours the fermentation was almost complete. Thus the glucose SBR routinely received significant concentrations of VFAs throughout the 5 112 year period, while the starch SBR did not. This had important implications with respect to the average P removal observed, the variability of the P removal, the P removal capacity of the bacterial populations when influent TP was increased, and the bacterial population structures observed. A simple statistical analysis of P removal data from the entire study is shown in Table I. The most important observation was the dramatic increase in P removal in the glucose SBRs when TPinf doubled, while the starch system remained unaffected. The P removal in the glucosel SBR was always superior to that of the starch SBR, but not reliably so at IO mg/l TPinf (compare the 50% and 90% columns in the table). However, once the TPinf was changed, the glucose system removals increased dramatically, even at the 90% level, while starch system removals remained almost the same. The location and molecular weight of PPn was compared within the biomass cells using nuclear magnetic resonance during the period of the study in which TP inf equalled 10 mg/l. A significant difference between the G1ucosel and Starch SBR biomasses was seen (Hill et al., 1989; ling et al., 1992). Direct measurements of MLVSS phosphorus, and P mass balances, during the period when TPinf equalled 20 mg/l, showed that the glucose SBR biomasses clearly had EBPR with average P contents of 6.2%, while Starch SBR P content was only marginally within the EBPR range at 3.7%. Contrast of SBR Populations: Response to YEAs Batch tests using different ratios of glucose and acetate in the feed were conducted for all three SBRs (Figure I). The glucose SBR biomasses showed a linear improvement in P removals of roughly 6 mg/l as the acetate/glucose ratio changed from 0.2 to 2.6 (mg/mg at a constant combined concentration of 113 mgll). The starch SBR biomass showed relatively little change in removal over the same range.
! 2O,I.::;;;;::;LUCOS:;;;;;;;:;;;E;":;;S:;;;BR;;O::;Gl;;'::;UCOS;;:;;;;E2;;::S;'BR;=.=;ST;;AR;;:;CH;:;'SBR;;:;:L, ~
-IACETATE+GlUCOSE HEW CONS 'ANT
~ 10
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---8--~--------
AT 113 MGII.
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0
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+---=:::u--"rl-------+---I -I-_~--~-~--~-~_--I
o
0.5
,
1.5
2
ACETATEIGLUCOSE RATIO
2.5
3
Figure I. Variations in SBR P removals as a functioll of acetate/glucose ratio.
Batch tests were conducted to quantify the effects of VFAs and other substrates on P removal and are discussed in detail in prior publications (Randall et al., 1994; Randall et al., 1995). While the starch SBR did not respond to any substrate added, the glucose SBR P removals were improved significantly by most of the VFAs, but were adversely affected by glucose. VFAs clearly were important in selecting and inducing EBPR in the SBR bacterial populations.
Effect of volatile fany acids on enhanced biological phosphorus removal
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SBR Phosphorus Removal Yariabi1ity The three anaerobic/aerobic sequencing batch reactors (SBRs) studied showed bounded oscillations in phosphorus (P) removal that did not correspond to any operational or environmental change (Figures 2a, b, and e).
-I rEJ l20
.."'"'- TPiof
II. - -
1000
1500
OAY~R
200lI
1-.r - - - - - - -
2500
Figure 2lH:. Longlerm P removal dala - slarch receiving SBR(a) and glucose receiving SBRs I(b) and 2(c).
From day 0 to day 1314 both SBRs showed bounded oscillations in P removal (Figure 2a and 2b). Most StarCh SBR P removals oscillated between 4 to 8 mgll. However, within these boundaries removals were highly unpredictable, even during a short period of time. The Glucosel SBR also showed bounded variabi1ity mostly between 6 to 10 mgll, plus an additional type of variability characterized by longterm shifts or trends to new removal levels. This can be seen in Figure 2b during days 800 to 1000, and days 1150 to 1250 (Figure 2b) when EBPR was "lost" and removals ranged from 2 to 6 mgll. Both types of variabi1ity were also manifest when influent TP went to 20 mgll. Both glucose systems experienced significant increases in removal following the change in TP inf. Then, in the glucose 1 SBR there was a long downward trend from days 1600 to 2050 in which P removal dropped from roughly 18 to 7 mgll. The glucose2 SBR experienced a downward trend from 18 mgll to 8 mgll from day 1550 to day 1700, followed by an upward trend in P removal from 8 to 12 mgll during days 1700 to 2050 (Figure 2c). Additional analysis of the glucose system batch tests over time showed longterm shifts or trends in the response of the population to a given substrate (Figure 3). This change in batch experiment P removals was analogous to the changes observed in the glucose system P removals (Figures 2a and b), but did not correspond precisely with the trends occurring in the system. Fractal Analysis for Chaotic Behavior In SBR Phosphorus Remoyal Data Rescaled range analysis is a vehicle to determine if there is a fractal nature to a data set. This method of analysis was first applied by Hurst (1951) to a time series of streamflow data. The most recent interest in rescaled range analysis has been in the geophysical arena, where the independent variable has been a spatial
A. A. RANDALL et al.
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coordinate rather than time. RlS is the rescaled range divided by the standard deviation of a data set. R is found, for a given lag or offset, by plotting the cumulative or graded values of the data and adding the maximum positive and negative deviations from the data trend. By performing the analysis for a series of lags, a set of RlS values is obtained. A more detailed discussion is given by Molz and Boman (1993). If a plot of In(RlS) vs In (lag) is linear, then there is long term correlation within the data; this is called Hurst's phenomenon. The slope of the line is Hurst's coefficient (H) and falls between 0.5 and 1.0 for positively correlated natural processes. The presence of Hurst's phenomenon in a data set indicates that the data set has a fractal nature. A time series with a fractal nature is symptomatic of chaos in the underlying dynamic system. It should be noted that this type of analysis is designed to be performed upon evenly spaced data.
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c
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0
II!
•
•
~
-2
.•
-4+--~--_--~-~-__I
lMO
1800
1_ 2000 DAY NUMBER
zoeo
2100
Figure 3. P removal data from gIucose2 SBR batch experiments: base influent + 48 mgll glucose.
Rescaled range analysis was applied to the time dependant phosphorus removal data. Although this analysis method assumes a uniform measurement interval, it was applied to these admittedly non-uniformly spaced data in hopes of getting at least an indication of the fractal nature of the data. The data set corresponding to the glucose I reactor with an TPinf of 20 mg/l gave the best results (Figure 4). The data appear to be linear, with a slope (H) of 0.95. This is, at least, a hint that chaotic dynamics may be a factor in this reactor. The other data sets analyzed gave results that were largely linear, but with more significant deviations from linearity.
Set 111 offort.12 slope = .95, 1 set with 87 data
4,-----,-......--r--.--..,.-----, 3
~2
.El
°
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......-...l2'---......3----~4---~
In(1ag) Figure 4. Fractal analysis of glucose I SBR days 1320 to 20S0; TPinF 20 mgll.
Effect of volatile fatty acids on enhanced biological phosphorus removal
IS9
Thus fractal analysis (Molz and Boman. 1993) of the glucose I and starch P removal data indicated that the data sets were not inconsistent with chaotic behavior. The glucose I system. at an influent TP of 20 mgll. conformed the best with chaotic behavior. and it is interesting 10 note this was when EBPR was strongest and both types of variability discussed previously were operative. If confIrmed by subsequent research. truly chaotic behavior could have important implications for our understanding of SBR performance and the limitations of predicting that performance (chaotic dynamics are deterministic. and therefore predictable over short time frames. and should not be confused with random fluctuations or "noise"; they are also very sensitive to initial conditions). At this point the data merely suggest an interesting path of inquiry. Contrast Of SBR Populations; StructurelPredomjnant Bacterial Isolates The predominant bacterial isolates were identified and compared for the starch and glucose I SBRs during operations at 20 mgll influent TP. SignifIcant differences were observed. In the glucose I SBR. Pseudomonas was the most frequent genus isolated. Bacillus was also frequently seen. In the starch system Pseudomonas was never isolated. This is not surprising since Pseudomonas does not have the ability to hydrolyze starch (Brock. 1994; Holt et ai.• 1984). and also does not have the ability to ferment substrates. Thus the prefermentation of glucose in the glucose system influents created conditions where Pseudomonas could proliferate. In the starch system influent. unhydrolyzed starch was inaccessible to Pseudomonas. The genus most frequently isolated in the starch SBR system. Aeromonas. is capable of starch hydrolysis. Acinetobacter. the organism traditionally credited with EBPR capabilities. was only isolated and identified once, and that was in the starch system. Bacteria resembling "0" bacteria were identified in all three systems by Neisser stain and morphological characteristics and it is likely they were always present, although they could not be quantified. CONCLUSIONS The presence of VFAs was critical in the selection of a bacterial population with a high capacity for EBPR, and for maintenance of the EBPR phenomena in the glucose SBRs. Identical anaerobic/aerobic sequencing without VFAs. in contrast. selected a population with very marginal EBPR capabilities in the Starch SBR. While proper anaerobic/aerobic sequencing was necessary for EBPR. the presence of VFAs was just as critical. Phosphorus removal data, and batch experiments with VFAs. indicated that the glucose and Starch populations were metabolically different with respect to P removal. This was either because the Starch population was not acclimated to VFAs. or the populations were structurally different. The contrast in the SBR responses to changes in TPinf• as well as differences in P removal variability and the predominant bacterial isolates. strengthened the hypothesis that there was a different population structure. Increased TPinf may have given a selective advantage to poly-P bacteria over their competitors (e.g. "0" bacteria). explaining the observed glucose SBR responses. Variability in the glucose SBRs P removal data was consistent with two distinct bacterial populations competing for anaerobic substrate. a dynamic that was not observed in the Starch SBR data. Probably. during periods when EBPR was "lost" in the Olucosel SBR. "0" bacteria were predominant. and during periods of high removal poly-P bacteria were predominant. This tenuous predominance of the poly-P bacteria may have been the result of the relatively slow growth rate of Pseudomonas, if they were in fact an active genus in glucose system EBPR. The type of variability that might result from such shifts in predominance was also observed as longterm trends in batch experiment results. A mechanistic model contrasting the metabolism of poly-P bacteria and their competitors has been proposed by Matsuo et al. (1992) and Satoh et al. (1992). In it the organisms competing for fermentation products with the poly-P bacteria use glycogen rather than polyphosphate as an energy storage product and thus do not participate in luxury uptake of phosphorus. Longterm P removal data showed bounded oscillations in removal which implied that population dynamics had a signifIcant effect on P removal in all three SBRs. and fractal analysis indicated that this variability was not inconsistent with chaotic behavior. The underlying variability common to all three SBRs was apparently a characteristic of the mixed culture, periodic. and non-steady state. SBR system. This type of competition .lI/ST
l~t ...
A. A. RANDAll et al.
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(and the resulting variability) is probably present in any mixed culture system (e.g. continuous-flow activated sludge systems), but may be intensified in SBR systems due to the constant changes in environment with time. ACKNOWLEDGEMENTS This research was partially funded by the Auburn University Water Resources Research Institute.
REFERENCES Abu-ghararah, Z.H., and Randall. C.W. (1991) The Effect Of Organic Compounds On Biological Phosphorus Removal. Wat. Sci. Tech. n(4-6). 585. Arun, V.• Mino, T., Matsuo, T. (1989) Metabolism of Carboxylic Acids Located in and Around the Glycolytic Pathway and the TCA Cycle in the Biological Phosphorus Removal Process. Water Sci. Tech. 21(4/5),363. Brock, T.D. (1994) Biology ofMicroorganismJ, 7th Ed. Prentice-Hall, Inc., Englewood Cliffs, New Jersey, USA. Cech, J.S., and Hartman, P. (1990) Glucose Induced Break Down Of Enhanced Biological Phosphate Removal. Environmental Technology, 11, 651. Cech, J.S., Hartman, P.. Wanner. J. (1993) Competition between polyp and non-polyp bacteria in an enhanced phosphate removal system. Water Environment Research, ~, 690. Comeau, Y., Oldham, W.K., Hall, KJ. (l987a) Dynamics of Carbon Reserves in Biological Dephosphatation of Wastewater. In Biological Phosphate Removal From Wastewaters. (Adv. Wat. Pollut. Control. no. 4) R. Ramadori (Ed.). Pergamon Press. New York, New York, USA, 39. Comeau. Y., Rabinowitz, B., Hall, KJ.• Oldham. W.K. (1987b) Phosphate Release and Uptake in Enhanced Biological Phosphorus Removal from Wastewater. Journal WPCF. ~, 707. Hill, W.E.• Benefield. L.D., Jing, S.R. (1989) 3Ip_NMR Spectroscopy Characterization of Polyphosphates in Activated Sludge Exhibiting Enhanced Phosphorus Removal. Water Res. 1177. Hiraishi, A., Masamune, K., Kitamura, H. (1989) Characterization of the Bacterial Population Structure in an Anaerobic-Aerobic Activated Sludge System on the Basis of Respiratory Quinone Profiles. Applied and Environmental Microbiology, 897. Holt, J.G., et al. (Eds.) (1984) Bergey's Manual of Systematic Bacteriology, Volumes I and 2. Williams & Wilkins, Baltimore, Maryland. Hurst, RE. (1951) Long-term storage capacity ofreservoirs. Trans. Am. Soc. Civ. Eng., ill, 770. Jing, S.R., Benefield, L.D., Hill, W.E. (1992) Observations Relating to Enhanced Phosphorus Removal in Biological Systems. Water Res. 22. 213. Jones, P.H., Tadwalker, A.D., and Hsu, C.L. (1987) Enhanced Uptake of Phosphorus by Activated Sludge - Effect of Substrate Addition. Water Res. 21, 301. Kavanaugh. R.G. (1991) Investigation of bacterial populations in a biological nutrient removal system. Ph.D. dissertation, Dept. Civ. Eng.. Virginia Tech. Blacksburg. Virginia, USA. Matsuo, T., Mino, T.. Sato, H. (1992) Metabolism Of Organic Substances In Anaerobic Phase Of Biological Phosphate Uptake Process. Wat. Sci. Tech., ~(6). 83. Molz, Fred J., Boman. G.K. (1993) A fractal-based stochastic interpolation scheme in subsurface hydrology. Water Resources Research, Z2U.ll, 3769. Okada, M., Lin, C.K.. Katayama. Y., Murakami. A. (1992) Stability of phosphorus removal and population of bio-p-bacteria under shan term disturbances in sequencing batch reactor activated sludge process. Wat. Sci. Tech., 22(3-4),483. Randall, A.A., Benefield. L.D., Hill, W.E. (1994) The effect of fermentation products on enhanced biological phosphorus removal, polyphosphate storage. and microbial population dynamics. Wat. Sci. Tech., :ll!(6), 213. Randall. A., Benefield, L.D., Hill. W.E. (1995) The Effect of Fermentation Products On Enhanced Biological Phosphorus Removal Capacity, Polyphosphate Storage, Bacterial Population Structure, and the Long Term Performance Characteristics Of Anaerobic/Aerobic Sequencing Batch Reactors. Water Environment Federation (formerly WPCF) 68th Annual Conference, Washington. D.C., I. Satoh. H.• Mino, T., Matsuo. T. (1992) Uptake Of Organic Substrates And Accumulation Of Polyhydroxyalkanoates Linked With Glycolysis Of Intracellular Carbohydrates Under Anaerobic Conditions In The Biological Excess Phosphate Removal Processes. Wat. Sci. Tech.• ~5-6), 933.
n.
.u,
WaL Sci. Tech. Vol. 3'. No. I. pp. "3-160. 1997. Copyright Ci 1996 IAWQ. Published by Elsevier Science Ltd Printed In Great Britain. All rights reserved. 0273-1223197 $17'00 +0'00
Pergamon
PH: S0273-1223(96)OO891-8
THE EFFECT OF VOLATILE FATTY ACIDS ON ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL AND POPULATION STRUCTURE IN ANAEROBIC/AEROBIC SEQUENCING BATCH REACTORS Andrew Amis Randal1*, Larry D. Benefield**, William E. Hill***, Jean-Paul Nicol**, Gerald K. Boman** and Shuh-Ren Jingt * Department o/Civil and Environmental Engineering, P.O. Box 162450, University o/Central Florida. Orlando, FL 32816-2450. USA .. do Col/ege 0/ Engineering, 108 Ramsey Hall. Aubum University, Aubum, AL 36849, USA *** Depanment o/Chemistry, 179 Chemistry Building, Auburn University, Auburn, AL 36849, USA t Chia-Nan Jr. College 0/ Pharmacy, Department 0/ Envr. Engr., Tainan, Taiwan, Republic of China
ABSTRACf Three anaerobic/aerobic sequencing balch reaclors (SBRs) were operaled for S 1/2 years. Volatile fatty acids (VFAs) in influent wastewater for two of the SBRs (the Glucosel and 2 SBRs) resulted in optimization of enhanced biological phosphorus removal (EBPR), and a bacterial population capable of increasing phosphorus (P) removals in response to increased VFA or P concentration. Another SBR not receiving VFAs (the Starch SBR) showed marginal EBPR and was incapable of either response. All three anaerobic/aerobic sequencing batch reactors (SBRs) showed bounded oscillations in P removal that did not correspond to any operational or environmental change. The oscillations were probably associated with interspecies population dynamics intensified due to the periodic. unsteady-state. nature of the SBR process. The glucose SBRs also showed an additional type of variability associated with EBPR. probably from competition between poly-P and "G" bacteria for readily available substrate (Le. glucose. VFAs) during anaerobiosis. The predominant bacterial isolates from the glucose SBRs were Pseudomonas and Bacillus while Auomonas was isolated most frequently from the Starch SBR. The relatively slow growth rate of Pseudomonas may have contributed to the high variability of EBPR observed. Fractal analysis indicated overall variability may have been chaotic. but was inconclusive. Copyright © 1996 IAWQ. Published by Elsevier Science Ltd
KEYWORDS
Chaotic behavior; enhanced biological phosphorus removal (EBPR); fractal analysis; poly-P bacteria; polyphosphate (PPn); population dynamics; Pseudomonas; sequencing batch reactor (SBR). 1S3
A. A. RANDALL et al.
154
INTRODUCTION A Brief E3plaoatjon of the EBPR Phenomenon Enhanced biological phosphorus removal (EBPR), or luxury uptake, is phosphorus uptake by bacteria that exceeds the 2.3% phosphorus by weight typical of conventional wastewater treatment system biomass. The EBPR phenomenon occurs when anaerobic/aerobic sequencing of an activated sludge system (continuous or batch) is used. In addition fermentation products must be present during the anaerobic phase. The role of fermentation products, in particular the short carbon-chain carboxylic acids (ie. volatile fatty acids or VFAs), in EBPR is well known and has been investigated by a number of researchers (Abu-ghararah and Randall, 1991; Arunetal., 1989; Comeau etal., 1987a; Jones etal., 1987; Randall etal., 1994; Randalletal., 1995). The two basic models for EBPR were presented by Comeau et al. (1987b), and Arun et al. (1989). The models are almost identical. It is thought that polyphosphate (PPn) supplies energy for the anaerobic uptake of fermentation products (e.g. VFAs) during anaerobiosis. The fermentation products are then stored as polyhydroxyalkanoates (PHA), of which polyhydroxybutyrate (PHB) is the best known member. During subsequent aerobiosis the PHAs are metabolized, and the energy derived from them is stored as polyphosphate, which accounts for the luxury uptake observed. Reducing power must be supplied to synthesize PHA from fermentation products, however, and it is here where the models differ. In the model presented by Comeau et al. (1987) the reducing power is supplied by the anaerobic operation of the TCA cycle. In the model presented by Arun et al. (1989), reducing power is supplied by the utilization of glycogen.
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EBPR and Population Structure The importance of the bacterial population with respect to EBPR has been noted in a variety of papers. Kavanaugh (1991) found that bacteria belonging to AeromonasIVibrio, coliforms, Pseudomonas, and Acinetobacter were present in a continuous flow EBPR system which was also operated to achieve biological nitrogen removal. Acinetobacter only accounted for 5% of the population, with the other three groups being the predominant bacteria. Aeromonas sp. (hydrophila), Pseudomonas sp., and Acinetobacter sp. were all shown to accumulate phosphorus far in excess of conventional accumulation levels (2.3% P) using VFAs as substrate, and all formed polyphosphate (PPn) granules. Pseudomonas sp. were capable of storing 10 to 30% by weight of polyphosphate. She did not analyze gram positive bacteria such as Bacillus, but noted that they had been isolated in EBPR systems before, and could be important to the phenomena. Okada et al. (1992) used quinone profiles as well as phosphorus uptake data obtained during short term disturbance test of SBRs to show that the slow recovery of EBPR following short term disturbances was because of species succession of bacteria, and not due to metabolic slow downs. Acinetobacter and Pseudomonas were the predominant species and had the highest phosphorus removal activities in the system, so apparently they were responsible for EBPR. Both had a slow specific growth rate which resulted in slow recovery from disturbances which eroded their predominance. Coliforms were also present in significant numbers but showed low phosphorus removal activities. Hiraishi et al. (1989) used quinone profiles also, and found that the introduction of anaerobic/aerobic sequencing alone had little effect on the bacterial community structure, which suggests that influent characteristics might be more important in selecting poly-P bacteria. Cech and Hartman (1990) and Cech et al. (1993) observed that there were two competing populations in EBPR systems. One group, organisms which accumulated PPn (ie. poly-P bacteria), were responsible for EBPR. They competed for anaerobic substrate with so-called "G" bacteria, which were predominant when glucose was a significant portion of the influent substrate. The "G" bacteria sequestered anaerobic substrate without the use or subsequent production of PPn'
Effect of volatile fally acids on enhanced biological phosphorus removal
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MATERIALS AND METHODS Three identical sequencing batch reactors (SBRs) were operated and two of these were monitored constantly over a 5 1/2 year period during which an anaerobic/aerobic sequence was used in order to obtain enhanced biological phosphorus removal (EBPR). The systems received a synthetic wastewater consisting of nutrient broth, yeast, and inorganic salts to provide nitrogen, phosphorus (P), and micronutrients. The treatment cycle for all 3 SBRs consisted of a 2 hour anaerobic phase, followed by a 4 hour aerobic phase, and a 2 hour period for settling, draining of supernatant, and a brief idle period. This yielded an 8 hour treatment cycle, with 3 cycles per day. The only significant difference among the three SBRs was that 2 systems received glucose as an additional influent carbon source (the Glucose 1 and Glucose2 SBRs) while 1 system received an equal amount of starch rather than glucose (the Starch SBR). The synthetic wastewater without the supplemental carbon source will be referred to as the "base influent" in this paper. Average mean cell residence times (MCRTs) were approximately 6 days for all 3 SBRs. Batch tests began at the end of an anaerobic/aerobic cycle by dividing the mixed liquor of an SBR into six small reactors. Base influent was added and then each reactor spiked with the desired concentration of the substrate to be studied. The normal anaerobic/aerobic cycle was then duplicated and the final phosphorus concentration obtained for each reactor. Chemical P removal was eliminated as a significant mechanism of P removal from pH data and batch experiment results. Bacteria from the SBRs were cultured on agar containing the C.-C s carboxylic acids and autoclaved base influent and mixed liquor. The most numerous isolates (Le. colonies) were identified by testing their ability to oxidize a variety of substrates in a 96 well microplate. Additional tests such as oxidase and catalase production. or motility and morphology, were used when necessary. RESULTS AND DISCUSSION During the first 3 1/2 years (day 0 to day 1314) of observations the influent total phosphorus (TPinf) to the SBRs was 10 mgll. During the last 2 years of the study (day 1314 to day 2050) TPinf was increased to 20 mgll. The change in TPinf was the only operational or environmental change to take place during the 5 1/2 year period. There was one brief period where the Glucose2 SBR was operated with a 3 hourl3 hour anaerobic/aerobic cycle, but this resulted in very poor phosphorus removals. Contrast of SBR PQPulations: PhQsphQrus RemQvals and ResPQnse to Influent TP Throughout the study it was observed that the glucose SBRs typically had superior P removal compared to the Starch SBR (Table I). Table 1. Average phosphorus removal and reliability for three SBRs (influent of 10 and 20 mglL total phosphorus) Influent Phos. (mg/l)
System Starch Glucose1 Glucose2
10 10 10
Starch Glucose1 Glucose2
20 20 20
• •• •••
------Phosphorus Removal In MG/L-----90\·· Average 50\· 5.9 6.5
4.4
5.3
11.3
10.6
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5.9 7.2
2.8 2.8
4.8
2.8
10.5 10.6
7.4
6.0
50\ of all observations were greater than the value shown 90\ of all observations were greater than the value shown There were insufficient observations to obtain meaningful comparisons for the Glucose2 SBR at 10 mg/l
A. A. RANDALL ~t al.
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The superior P removals observed in the glucose SBRs were shown to be the result of VFAs in the influent (Randall et al., 1994; Randall et al., 1995). The VFAs resulted from prefermentation of glucose at ambient temperature (23 degrees C) in the feed jars prior to entering the SBRs. Starch was not observed to preferment in this way. Since feed was made once a day, all the SBRs received feed aged a minimum of 0 hours, 8 hours, and 16 hours at the start of the first, second, and third daily treatment cycles. At 8 hours the influent glucose was typically 50% fermented, largely to organic acids (e.g. VFAs). At 16 hours the fermentation was almost complete. Thus the glucose SBR routinely received significant concentrations of VFAs throughout the 5 112 year period, while the starch SBR did not. This had important implications with respect to the average P removal observed, the variability of the P removal, the P removal capacity of the bacterial populations when influent TP was increased, and the bacterial population structures observed. A simple statistical analysis of P removal data from the entire study is shown in Table I. The most important observation was the dramatic increase in P removal in the glucose SBRs when TPinf doubled, while the starch system remained unaffected. The P removal in the glucosel SBR was always superior to that of the starch SBR, but not reliably so at IO mg/l TPinf (compare the 50% and 90% columns in the table). However, once the TPinf was changed, the glucose system removals increased dramatically, even at the 90% level, while starch system removals remained almost the same. The location and molecular weight of PPn was compared within the biomass cells using nuclear magnetic resonance during the period of the study in which TP inf equalled 10 mg/l. A significant difference between the G1ucosel and Starch SBR biomasses was seen (Hill et al., 1989; ling et al., 1992). Direct measurements of MLVSS phosphorus, and P mass balances, during the period when TPinf equalled 20 mg/l, showed that the glucose SBR biomasses clearly had EBPR with average P contents of 6.2%, while Starch SBR P content was only marginally within the EBPR range at 3.7%. Contrast of SBR Populations: Response to YEAs Batch tests using different ratios of glucose and acetate in the feed were conducted for all three SBRs (Figure I). The glucose SBR biomasses showed a linear improvement in P removals of roughly 6 mg/l as the acetate/glucose ratio changed from 0.2 to 2.6 (mg/mg at a constant combined concentration of 113 mgll). The starch SBR biomass showed relatively little change in removal over the same range.
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Figure I. Variations in SBR P removals as a functioll of acetate/glucose ratio.
Batch tests were conducted to quantify the effects of VFAs and other substrates on P removal and are discussed in detail in prior publications (Randall et al., 1994; Randall et al., 1995). While the starch SBR did not respond to any substrate added, the glucose SBR P removals were improved significantly by most of the VFAs, but were adversely affected by glucose. VFAs clearly were important in selecting and inducing EBPR in the SBR bacterial populations.
Effect of volatile fany acids on enhanced biological phosphorus removal
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SBR Phosphorus Removal Yariabi1ity The three anaerobic/aerobic sequencing batch reactors (SBRs) studied showed bounded oscillations in phosphorus (P) removal that did not correspond to any operational or environmental change (Figures 2a, b, and e).
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1000
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Figure 2lH:. Longlerm P removal dala - slarch receiving SBR(a) and glucose receiving SBRs I(b) and 2(c).
From day 0 to day 1314 both SBRs showed bounded oscillations in P removal (Figure 2a and 2b). Most StarCh SBR P removals oscillated between 4 to 8 mgll. However, within these boundaries removals were highly unpredictable, even during a short period of time. The Glucosel SBR also showed bounded variabi1ity mostly between 6 to 10 mgll, plus an additional type of variability characterized by longterm shifts or trends to new removal levels. This can be seen in Figure 2b during days 800 to 1000, and days 1150 to 1250 (Figure 2b) when EBPR was "lost" and removals ranged from 2 to 6 mgll. Both types of variabi1ity were also manifest when influent TP went to 20 mgll. Both glucose systems experienced significant increases in removal following the change in TP inf. Then, in the glucose 1 SBR there was a long downward trend from days 1600 to 2050 in which P removal dropped from roughly 18 to 7 mgll. The glucose2 SBR experienced a downward trend from 18 mgll to 8 mgll from day 1550 to day 1700, followed by an upward trend in P removal from 8 to 12 mgll during days 1700 to 2050 (Figure 2c). Additional analysis of the glucose system batch tests over time showed longterm shifts or trends in the response of the population to a given substrate (Figure 3). This change in batch experiment P removals was analogous to the changes observed in the glucose system P removals (Figures 2a and b), but did not correspond precisely with the trends occurring in the system. Fractal Analysis for Chaotic Behavior In SBR Phosphorus Remoyal Data Rescaled range analysis is a vehicle to determine if there is a fractal nature to a data set. This method of analysis was first applied by Hurst (1951) to a time series of streamflow data. The most recent interest in rescaled range analysis has been in the geophysical arena, where the independent variable has been a spatial
A. A. RANDALL et al.
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coordinate rather than time. RlS is the rescaled range divided by the standard deviation of a data set. R is found, for a given lag or offset, by plotting the cumulative or graded values of the data and adding the maximum positive and negative deviations from the data trend. By performing the analysis for a series of lags, a set of RlS values is obtained. A more detailed discussion is given by Molz and Boman (1993). If a plot of In(RlS) vs In (lag) is linear, then there is long term correlation within the data; this is called Hurst's phenomenon. The slope of the line is Hurst's coefficient (H) and falls between 0.5 and 1.0 for positively correlated natural processes. The presence of Hurst's phenomenon in a data set indicates that the data set has a fractal nature. A time series with a fractal nature is symptomatic of chaos in the underlying dynamic system. It should be noted that this type of analysis is designed to be performed upon evenly spaced data.
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Figure 3. P removal data from gIucose2 SBR batch experiments: base influent + 48 mgll glucose.
Rescaled range analysis was applied to the time dependant phosphorus removal data. Although this analysis method assumes a uniform measurement interval, it was applied to these admittedly non-uniformly spaced data in hopes of getting at least an indication of the fractal nature of the data. The data set corresponding to the glucose I reactor with an TPinf of 20 mg/l gave the best results (Figure 4). The data appear to be linear, with a slope (H) of 0.95. This is, at least, a hint that chaotic dynamics may be a factor in this reactor. The other data sets analyzed gave results that were largely linear, but with more significant deviations from linearity.
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Effect of volatile fatty acids on enhanced biological phosphorus removal
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Thus fractal analysis (Molz and Boman. 1993) of the glucose I and starch P removal data indicated that the data sets were not inconsistent with chaotic behavior. The glucose I system. at an influent TP of 20 mgll. conformed the best with chaotic behavior. and it is interesting 10 note this was when EBPR was strongest and both types of variability discussed previously were operative. If confIrmed by subsequent research. truly chaotic behavior could have important implications for our understanding of SBR performance and the limitations of predicting that performance (chaotic dynamics are deterministic. and therefore predictable over short time frames. and should not be confused with random fluctuations or "noise"; they are also very sensitive to initial conditions). At this point the data merely suggest an interesting path of inquiry. Contrast Of SBR Populations; StructurelPredomjnant Bacterial Isolates The predominant bacterial isolates were identified and compared for the starch and glucose I SBRs during operations at 20 mgll influent TP. SignifIcant differences were observed. In the glucose I SBR. Pseudomonas was the most frequent genus isolated. Bacillus was also frequently seen. In the starch system Pseudomonas was never isolated. This is not surprising since Pseudomonas does not have the ability to hydrolyze starch (Brock. 1994; Holt et ai.• 1984). and also does not have the ability to ferment substrates. Thus the prefermentation of glucose in the glucose system influents created conditions where Pseudomonas could proliferate. In the starch system influent. unhydrolyzed starch was inaccessible to Pseudomonas. The genus most frequently isolated in the starch SBR system. Aeromonas. is capable of starch hydrolysis. Acinetobacter. the organism traditionally credited with EBPR capabilities. was only isolated and identified once, and that was in the starch system. Bacteria resembling "0" bacteria were identified in all three systems by Neisser stain and morphological characteristics and it is likely they were always present, although they could not be quantified. CONCLUSIONS The presence of VFAs was critical in the selection of a bacterial population with a high capacity for EBPR, and for maintenance of the EBPR phenomena in the glucose SBRs. Identical anaerobic/aerobic sequencing without VFAs. in contrast. selected a population with very marginal EBPR capabilities in the Starch SBR. While proper anaerobic/aerobic sequencing was necessary for EBPR. the presence of VFAs was just as critical. Phosphorus removal data, and batch experiments with VFAs. indicated that the glucose and Starch populations were metabolically different with respect to P removal. This was either because the Starch population was not acclimated to VFAs. or the populations were structurally different. The contrast in the SBR responses to changes in TPinf• as well as differences in P removal variability and the predominant bacterial isolates. strengthened the hypothesis that there was a different population structure. Increased TPinf may have given a selective advantage to poly-P bacteria over their competitors (e.g. "0" bacteria). explaining the observed glucose SBR responses. Variability in the glucose SBRs P removal data was consistent with two distinct bacterial populations competing for anaerobic substrate. a dynamic that was not observed in the Starch SBR data. Probably. during periods when EBPR was "lost" in the Olucosel SBR. "0" bacteria were predominant. and during periods of high removal poly-P bacteria were predominant. This tenuous predominance of the poly-P bacteria may have been the result of the relatively slow growth rate of Pseudomonas, if they were in fact an active genus in glucose system EBPR. The type of variability that might result from such shifts in predominance was also observed as longterm trends in batch experiment results. A mechanistic model contrasting the metabolism of poly-P bacteria and their competitors has been proposed by Matsuo et al. (1992) and Satoh et al. (1992). In it the organisms competing for fermentation products with the poly-P bacteria use glycogen rather than polyphosphate as an energy storage product and thus do not participate in luxury uptake of phosphorus. Longterm P removal data showed bounded oscillations in removal which implied that population dynamics had a signifIcant effect on P removal in all three SBRs. and fractal analysis indicated that this variability was not inconsistent with chaotic behavior. The underlying variability common to all three SBRs was apparently a characteristic of the mixed culture, periodic. and non-steady state. SBR system. This type of competition .lI/ST
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A. A. RANDAll et al.
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(and the resulting variability) is probably present in any mixed culture system (e.g. continuous-flow activated sludge systems), but may be intensified in SBR systems due to the constant changes in environment with time. ACKNOWLEDGEMENTS This research was partially funded by the Auburn University Water Resources Research Institute.
REFERENCES Abu-ghararah, Z.H., and Randall. C.W. (1991) The Effect Of Organic Compounds On Biological Phosphorus Removal. Wat. Sci. Tech. n(4-6). 585. Arun, V.• Mino, T., Matsuo, T. (1989) Metabolism of Carboxylic Acids Located in and Around the Glycolytic Pathway and the TCA Cycle in the Biological Phosphorus Removal Process. Water Sci. Tech. 21(4/5),363. Brock, T.D. (1994) Biology ofMicroorganismJ, 7th Ed. Prentice-Hall, Inc., Englewood Cliffs, New Jersey, USA. Cech, J.S., and Hartman, P. (1990) Glucose Induced Break Down Of Enhanced Biological Phosphate Removal. Environmental Technology, 11, 651. Cech, J.S., Hartman, P.. Wanner. J. (1993) Competition between polyp and non-polyp bacteria in an enhanced phosphate removal system. Water Environment Research, ~, 690. Comeau, Y., Oldham, W.K., Hall, KJ. (l987a) Dynamics of Carbon Reserves in Biological Dephosphatation of Wastewater. In Biological Phosphate Removal From Wastewaters. (Adv. Wat. Pollut. Control. no. 4) R. Ramadori (Ed.). Pergamon Press. New York, New York, USA, 39. Comeau. Y., Rabinowitz, B., Hall, KJ.• Oldham. W.K. (1987b) Phosphate Release and Uptake in Enhanced Biological Phosphorus Removal from Wastewater. Journal WPCF. ~, 707. Hill, W.E.• Benefield. L.D., Jing, S.R. (1989) 3Ip_NMR Spectroscopy Characterization of Polyphosphates in Activated Sludge Exhibiting Enhanced Phosphorus Removal. Water Res. 1177. Hiraishi, A., Masamune, K., Kitamura, H. (1989) Characterization of the Bacterial Population Structure in an Anaerobic-Aerobic Activated Sludge System on the Basis of Respiratory Quinone Profiles. Applied and Environmental Microbiology, 897. Holt, J.G., et al. (Eds.) (1984) Bergey's Manual of Systematic Bacteriology, Volumes I and 2. Williams & Wilkins, Baltimore, Maryland. Hurst, RE. (1951) Long-term storage capacity ofreservoirs. Trans. Am. Soc. Civ. Eng., ill, 770. Jing, S.R., Benefield, L.D., Hill, W.E. (1992) Observations Relating to Enhanced Phosphorus Removal in Biological Systems. Water Res. 22. 213. Jones, P.H., Tadwalker, A.D., and Hsu, C.L. (1987) Enhanced Uptake of Phosphorus by Activated Sludge - Effect of Substrate Addition. Water Res. 21, 301. Kavanaugh. R.G. (1991) Investigation of bacterial populations in a biological nutrient removal system. Ph.D. dissertation, Dept. Civ. Eng.. Virginia Tech. Blacksburg. Virginia, USA. Matsuo, T., Mino, T.. Sato, H. (1992) Metabolism Of Organic Substances In Anaerobic Phase Of Biological Phosphate Uptake Process. Wat. Sci. Tech., ~(6). 83. Molz, Fred J., Boman. G.K. (1993) A fractal-based stochastic interpolation scheme in subsurface hydrology. Water Resources Research, Z2U.ll, 3769. Okada, M., Lin, C.K.. Katayama. Y., Murakami. A. (1992) Stability of phosphorus removal and population of bio-p-bacteria under shan term disturbances in sequencing batch reactor activated sludge process. Wat. Sci. Tech., 22(3-4),483. Randall, A.A., Benefield. L.D., Hill, W.E. (1994) The effect of fermentation products on enhanced biological phosphorus removal, polyphosphate storage. and microbial population dynamics. Wat. Sci. Tech., :ll!(6), 213. Randall. A., Benefield, L.D., Hill. W.E. (1995) The Effect of Fermentation Products On Enhanced Biological Phosphorus Removal Capacity, Polyphosphate Storage, Bacterial Population Structure, and the Long Term Performance Characteristics Of Anaerobic/Aerobic Sequencing Batch Reactors. Water Environment Federation (formerly WPCF) 68th Annual Conference, Washington. D.C., I. Satoh. H.• Mino, T., Matsuo. T. (1992) Uptake Of Organic Substrates And Accumulation Of Polyhydroxyalkanoates Linked With Glycolysis Of Intracellular Carbohydrates Under Anaerobic Conditions In The Biological Excess Phosphate Removal Processes. Wat. Sci. Tech.• ~5-6), 933.
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