- Email: [email protected]
Characterisation of enhanced biological phosphorus removal activated sludges with dissimilar phosphorus removal performances
8)
Pergamon
Wat. Sci. Tech. Vol. 37. No. 4-5. pp. 567-571.1998.
PH: S0273-1223(98)OO161-9
CHARACTERISATION OF ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL ACTIVATED SLUDGES WITH DISSIMILAR PHOSPHORUS REMOVAL PERFORMANCES Philip L. Bond, Jiirg Keller and Linda L. Blackall Advanced Wastewater Management Centre. The University o/Queensland. Brisbane. 4072. Australia
ABSTRACT A sequencing batch reactor (SBR) was operated for enhanced biological phosphorus removal (EBPR) and dramatic differences 10 the P removing capabilities were obtained in different stages of the operation. At one slage extremely poor P removal occurred and it appeared that bacteria inhibiting P removal overwhelmed the rcactor performance. Changes were made to the reactor operation and these led to the development of a sludge with high P removing capability. This laller sludge was analysed by fluorescent in situ hybridisallon (FISH) uSlOg a probe specific for Acinetobacter. Very few cells were detected with this probe indicating that Aci"efobaCleT played an insignificant role in the P removal occurring here. Analysis of the chemical transformations of three sludges supported the biochemical pathways proposed for EBPR and non-EBPR sy.tcms in biological models. A change in operation that led to the improved P removal performance included permilling the pH 10 rise in the anaerobic periods of the SBR cycle. @ 1998 IA WQ. Published by ElseVIer Science Ltd
KEYWORDS
Adnetohacter; activated sludge; bacterial population analysis; EBPR model; phosphorus removal. INTRODUCTION Many investigations have implied that Acinetobacter play an important role in the EBPR process (LOtter. 1985: Wentzel et al.• 1988). Biochemical models have been devised to explain the metabolic features of the enhanced biological phosphorus removal (EBPR) process (Satoh et ai., 1994; Smolders et ai.. 1994). However. details of the metabolic pathways of P removal are not understood. In the initial anaerobic zone it is proposed that bacteria responsible for EBPR accumulate substrate from the .influent wastewater. this is associated with the release of stored phosphate. and it provides the selective advantage for these bacteria in EBPR systems. However. bacteria other than phosphate accumulators may also have mechanisms for anaerobic substrate accumulation and such bacteria have been reported to cause the failure of EBPR (Cech and Hartman, 1993; Satoh etal., 1994).
567
S68
P. L.
BOND~, al.
Subsequently there is interest to reveal details of the bacterial mechanisms pertaining to EBPR and the inhibition of EBPR. In this study bacterial populations with differing P removing capabilities in an EBPR system were investigated for the possible involvement of Acinetobacter in EBPR. The chemical transformations of the different sludges were analysed to assess the validity of EBPR and non-EBPR biochemical models. MATERIALS AND METHODS
Experimental set-up and reactor operation. In this study, two stages of SBR operation (A and B) are described during which the reactor was operated for EBPR. Operational features are given in Table I. The SBR was a 2 I reactor fitted with pH electrodes and a port&ble dissolved oxygen electrode (Bond et al., In preparation ). Table I. Features of the SBRs operated for EBPR. Operation time Medium COD NaCH 3C0 2 .3H20 Peptone P Solids retention time
SBR in stage A 88 days 4OOmg/l 850 mg/l 15 mgll 8d
SBR in stage B 78 days 500mg/l 700mg/l 122 mgll 24 mg/l 6.7 d
Chemical analyses. MLSS, P04 -P, SCOD, acetic acid, total phosphorus, polyhydroxyalkanoate (PHA), and total cellular carbohydrates were determined as described (Bond et al., In preparation). Staining for light microscopy. Staining of sludge for metachromatic granules was carried out using Loeffler methylene blue (Murray et al., 1994) and for lipophilic granules using Sudan black (Jenkins et al., 1984). Cell fixation and fluorescent in situ hybridisation. Immediately after mixed liquor samples were taken from the mid-aerobic stage in the SBR they were fixed according to Wagner et al. (1994). Oligonucleotide probes employed were EUB338, GAM42a, and ACA and these were labelled and used according to previously published methods (Manz et al., 1992; Wagner et aI., 1994). Cells were detected using a Bio-Rad MRC 600 confocal scanning laser microscope mounted on a Zeiss Axioskop fitted with KI1K2 filter sets (Bio-Rad). RESULTS AND DISCUSSION P removal performance during stage A. For the entire operation of the SBR the reactor was operated to achieve EBPR. Throughout stage A the P removal performance was suboptimal. In the term from day 29 to 67 extremely poor P removal occurred (Figure I); all the carbon substrate (acetate) was taken up in the anaerobic periods, but other characteristics of EBPR were not observed. Enhanced phosphorus removal did not occur since the phosphate-P in the influent averaged 15.1 mgtl, while that in the effluent averaged 12.0 mg/l. Very little anaerobic release of phosphate from the sludge occurred in this term and the P content of the sludge was low, at less than 2% of the MLSS (Figure I). On day 61 this was labelled the Q sludge.
R sludge. Improved P removal was noticed when there was no pH control in the anaerobic period at which
time the pH would rise. The SBR was operated in this mode from day 66 to 117. While an improvement in the P removal was slowly obtained throughout this stage, it was still not optimal compared to the performance of other similar laboratory scale reactors achieving less than I mgtl phosphate-P in the effluent (Appeldoorn et al., 1992; Smolders et al., 1994). The sludge during this phase was called the R sludge.
P removal performance during stage B. Prior to the operation of stage B, the reactor was reseeded with sludge from a full scale EBPR plant. The anaerobic period was slightly shorter than in stage A and peptone was included in the medium (Table I). From day III excellent P removal occurred, effluent phosphate-P was lower than I mg/l and mostly below the detection limit (0.05 mg P/I). From day 121 the average
Enhanced biological phosphorus removal activated sludge
569
anaerobic phosphate-P release had risen to 82.7 mgll and the average P content was 8.8% of the MLSS (Figure I). The sludge generated during stage B was called the P sludge.
Microscopic examination of P sludge. From day 121 to 167 extremely stable and outstanding P removal performance was achieved. Staining of smears of the P sludge mixed liquor from the anaerobic stage indicated that many of the cells contained lipophilic granules. Also cells from the aerobic stage staining for polyphosphate were prolific in the P sludge, and it would be expected that polyphosphate accumulating organisms (PAO) would be abundant in this sludge. As bacteria of the gamma subclass of the proteobacteria, in particular Acinetobacter, are proposed to be important to EBPR, the P sludge bacterial community was investigated using probes specific for these bacteria. In the P sludge, cells detected with the probes GAM42a and ACA made up less than 1% of the cells detected with the general bacterial probe EUB338 (visual estimation). Acinetobacter could not have been significantly involved in the P removal occurring here. This is in agreement with other investigations that have analysed bacterial communities in EBPR sludge using non-culture-dependent techniques (Cloote and Steyn, 1987; Wagner et aI., 1994; Bond et aI., 1995). 120
~ .....E
80
-
60
I I I I I
40
R I
A
.....
100
Q.
ell
I'll
.c
Co
en 0
.c Q.
8
2
I 30
50
90
110
--fI) fI)
...::s .....
c ell u c ell 4 c ..... 80 u
6
!:
20 0
10
90
110
130
150
170
0
~
Q.
0
Day of operation Figure I. Performance of SBR during stage A and stage B of operation. Phosphate-P in the effluent (-0-), phnsphale-P In Ihe end of anaerobic siage mixed liquor (-0-). ll1e P content as a mass percent of the MLSS (-"'-). Labelled arrows indicate when sludges Q. Rand P occurred.
Correlation (~r sludge performance with biochemical models. The anaerobic transformations of compounds that are reported to be important to EBPR were determined for the Q, Rand P sludges during cycles of the SBR operation (Figure I and Table 2). For all three sludges rapid uptake of acetate occurred along with accumulation of cellular PHA and degradation of carbohydrate occurred in the anaerobic stage of the SBR. These events are characteristic of EBPR sludge, however very little anaerobic phosphate release was detected for the Q sludge (Table 2). For the P sludge there was considerable anaerobic phosphate release. The transformations that occurred in the P sludge correlated remarkably well with transformations suggested by an EBPR model (Table 2). The R sludge, which had medial P removing performance, carried out transformations that were intermediate those of the Q and P sludges (Table 2). The transformations observed here support the suggestion that certain bacteria could compete with PAO in EBPR activated sludge systems. However, cells matching the description of the G-bacteria, organisms reported to inhibit EBPR (Cech and Hartman, 1993), were not observed in this Q sludge.
Op('fational clul/Iges leading to the improved EBPR performance. Although it is possible that there was a lack of bactcrial diversity in the sludge during stage A, and left reseeding prior to stage B introduced bactcria important to EBPR, one operational change that seemed to have an immediate effect during stage A was when the pH control was turned off in the anaerobic periods.
S70
P. L. BOND et al.
Table 2. Anaerobic transfonnations detected in the SBR, compared with theoretical values as molar ratios. Sludge type b
Non-EBPR
Q R P
Acetate uptake 6
6 6 6
consumed-
PHA units produced
2.6
4.4
Cell carbohydrate
2.5
2.1
1.23 1
5.5
3.6 3.9
Phosphate released
o
0.27 2.7
5.6
4 6 EBPRb 6 • Molar units are calculated as glycogen units b Theoretical ratios for non-EBPR (Satoh et al., 1994) and EBPR models (pH 7.0) (Smolders et aL, 1994) CONCLUSIONS
The perfonnance of an SBR activated sludge system operating for EBPR was evaluated by chemical and microscopic examination during stages of poor through to good phosphate removal. The transfonnations observed in the SBR cycles matched well with the theoretical values proposed by the models explaining EBPR and non-EBPR behaviour in these systems (Satoh et al., 1994; Smolders et al., 1994), and the results supported the biochemical pathways proposed in biological models. However, details and confinnation of the biochemical pathways are still required for progress in the understanding of the EBPR process, particularly for the development of conditions that favour PAO over non-P removing organisms. For example, if the pH has some influence on the anaerobic phosphate release and subsequent selection for PAO, as suggested here, this occurrence is not explained by the current EBPR biological models. The low numbers of Acinetobacter cells detected in the EBPR sludge suggest that bacteria other than Acinetobacter species were responsible for the EBPR that occurred here. Bacteria other than Acinetobacter and gamma proteobacteria should be investigated for polyphosphate accumulation and a possible role in EBPR. ACKNOWLEDGMENTS This work was funded by the CRC for Waste Management and Pollution Control Ltd., a centre established and supported under the Australian Government's Cooperative Research Centres Program. REFERENCES Appeldoom. K. J.• Kortstee. G. J. J. and Zehnder. A. J. B. (1992). Biological phosphate removal by activated sludge under defined conditions. Water Res. 26, 453·460. Bond. P. L.• Erhart. R.• Keller. J. and Blackall. L. L. (In preparation). Phosphorus removing capabilities of defined bacterial commUnities in activated sludge. Bond. P. L.• Hugenholtz. P.• Keller. J. and Blackall. L. L. (1995). Bacterial community structures of phosphate-removing and non· phosphate·removing activated sludges from sequencing batch reactors. Appl. Environ. Microbiol. 61. 1910-1916. Cech. 1. S. and Hartman. P. (1993). Competition between polyphosphate and polysaccharide accumulating bacteria in enhanced hiological phosphate removal system. Water Res. 27. 1219·1225. Cloete. T. E. and Steyn. P. L. (1987). A combined fluorescent antibody-membrane tilter technique for enumerating Acinetobacter in activated sludge. In Biological Phosphate Removal From Wastewater.., pp. 335·338. Edited by R. Ramadori. Oxford. Pergamon Press. Jenkins, 0 .. Richard. M. G. and Diagger. G. T. (1984). Manual on the Causes and Control of Activated Sludge Bulking and Foaming. Water Research Commission. Pretoria. Ulner, L. H. (1985). The role of bacterial phosphate metabolism in enhanced phosphorus removal from the activated sludge process. War. Sci. Tech. 17. 127·138. Manz. W.• Amann, R.. LudWig. W.• Wagner. M. and Schleifer, K. H. (1992). Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst. AppL Microbiol. 15. S93-600.
Enhanced biological phosphorus removal activated sludge
571
Murray. R. G. E.• Doetsch, R. N. and Robinow, C. F. (1994). Determinative and cytological microscopy. In Methods for General and Molecular Bacteriology, pp. 21-36. Edited by P. Gerhardt. R. G. E. Murray. W. A. Wood and N. R. Krieg. Washington, D.C.: American Society for Microbiology. Satoh, H., Mino, T. and Matsuo, T. (1994). Deterioration of enhanced biological phosphorus removal by the domination of microorganisms without polyphosphate accumulation. Wat. Sci. Tech. 30, 203-211. Smolders. G. 1. F.• van der Meij, J.• van Loosdrecht, M. C. M. and Heijnen, J. J. (1994). Model of the anaerobic metabolism of the biological phosphorus removal process: stoichiometry and pH influence. Biotechnol. Bioengng. 43. 461-470. Wagner. M.• Erhan. R., Manz. W., Amann. R.. Lemmer. H., Wedi, D. and Schleifer. K. H. (1994). Development of an rRNA• targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Appl. Environ. Microbiol. 60, 792-800. Wentzel. M. C., Loewenthal. R. E., Ekama, G. A. and Marais, G. R. (1988). Enhanced polyphosphate organism cultures in activated sludge systems - Part I: enhanced culture development. Water SA 14, 81-92.
Pergamon
Wat. Sci. Tech. Vol. 37. No. 4-5. pp. 567-571.1998.
PH: S0273-1223(98)OO161-9
CHARACTERISATION OF ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL ACTIVATED SLUDGES WITH DISSIMILAR PHOSPHORUS REMOVAL PERFORMANCES Philip L. Bond, Jiirg Keller and Linda L. Blackall Advanced Wastewater Management Centre. The University o/Queensland. Brisbane. 4072. Australia
ABSTRACT A sequencing batch reactor (SBR) was operated for enhanced biological phosphorus removal (EBPR) and dramatic differences 10 the P removing capabilities were obtained in different stages of the operation. At one slage extremely poor P removal occurred and it appeared that bacteria inhibiting P removal overwhelmed the rcactor performance. Changes were made to the reactor operation and these led to the development of a sludge with high P removing capability. This laller sludge was analysed by fluorescent in situ hybridisallon (FISH) uSlOg a probe specific for Acinetobacter. Very few cells were detected with this probe indicating that Aci"efobaCleT played an insignificant role in the P removal occurring here. Analysis of the chemical transformations of three sludges supported the biochemical pathways proposed for EBPR and non-EBPR sy.tcms in biological models. A change in operation that led to the improved P removal performance included permilling the pH 10 rise in the anaerobic periods of the SBR cycle. @ 1998 IA WQ. Published by ElseVIer Science Ltd
KEYWORDS
Adnetohacter; activated sludge; bacterial population analysis; EBPR model; phosphorus removal. INTRODUCTION Many investigations have implied that Acinetobacter play an important role in the EBPR process (LOtter. 1985: Wentzel et al.• 1988). Biochemical models have been devised to explain the metabolic features of the enhanced biological phosphorus removal (EBPR) process (Satoh et ai., 1994; Smolders et ai.. 1994). However. details of the metabolic pathways of P removal are not understood. In the initial anaerobic zone it is proposed that bacteria responsible for EBPR accumulate substrate from the .influent wastewater. this is associated with the release of stored phosphate. and it provides the selective advantage for these bacteria in EBPR systems. However. bacteria other than phosphate accumulators may also have mechanisms for anaerobic substrate accumulation and such bacteria have been reported to cause the failure of EBPR (Cech and Hartman, 1993; Satoh etal., 1994).
567
S68
P. L.
BOND~, al.
Subsequently there is interest to reveal details of the bacterial mechanisms pertaining to EBPR and the inhibition of EBPR. In this study bacterial populations with differing P removing capabilities in an EBPR system were investigated for the possible involvement of Acinetobacter in EBPR. The chemical transformations of the different sludges were analysed to assess the validity of EBPR and non-EBPR biochemical models. MATERIALS AND METHODS
Experimental set-up and reactor operation. In this study, two stages of SBR operation (A and B) are described during which the reactor was operated for EBPR. Operational features are given in Table I. The SBR was a 2 I reactor fitted with pH electrodes and a port&ble dissolved oxygen electrode (Bond et al., In preparation ). Table I. Features of the SBRs operated for EBPR. Operation time Medium COD NaCH 3C0 2 .3H20 Peptone P Solids retention time
SBR in stage A 88 days 4OOmg/l 850 mg/l 15 mgll 8d
SBR in stage B 78 days 500mg/l 700mg/l 122 mgll 24 mg/l 6.7 d
Chemical analyses. MLSS, P04 -P, SCOD, acetic acid, total phosphorus, polyhydroxyalkanoate (PHA), and total cellular carbohydrates were determined as described (Bond et al., In preparation). Staining for light microscopy. Staining of sludge for metachromatic granules was carried out using Loeffler methylene blue (Murray et al., 1994) and for lipophilic granules using Sudan black (Jenkins et al., 1984). Cell fixation and fluorescent in situ hybridisation. Immediately after mixed liquor samples were taken from the mid-aerobic stage in the SBR they were fixed according to Wagner et al. (1994). Oligonucleotide probes employed were EUB338, GAM42a, and ACA and these were labelled and used according to previously published methods (Manz et al., 1992; Wagner et aI., 1994). Cells were detected using a Bio-Rad MRC 600 confocal scanning laser microscope mounted on a Zeiss Axioskop fitted with KI1K2 filter sets (Bio-Rad). RESULTS AND DISCUSSION P removal performance during stage A. For the entire operation of the SBR the reactor was operated to achieve EBPR. Throughout stage A the P removal performance was suboptimal. In the term from day 29 to 67 extremely poor P removal occurred (Figure I); all the carbon substrate (acetate) was taken up in the anaerobic periods, but other characteristics of EBPR were not observed. Enhanced phosphorus removal did not occur since the phosphate-P in the influent averaged 15.1 mgtl, while that in the effluent averaged 12.0 mg/l. Very little anaerobic release of phosphate from the sludge occurred in this term and the P content of the sludge was low, at less than 2% of the MLSS (Figure I). On day 61 this was labelled the Q sludge.
R sludge. Improved P removal was noticed when there was no pH control in the anaerobic period at which
time the pH would rise. The SBR was operated in this mode from day 66 to 117. While an improvement in the P removal was slowly obtained throughout this stage, it was still not optimal compared to the performance of other similar laboratory scale reactors achieving less than I mgtl phosphate-P in the effluent (Appeldoorn et al., 1992; Smolders et al., 1994). The sludge during this phase was called the R sludge.
P removal performance during stage B. Prior to the operation of stage B, the reactor was reseeded with sludge from a full scale EBPR plant. The anaerobic period was slightly shorter than in stage A and peptone was included in the medium (Table I). From day III excellent P removal occurred, effluent phosphate-P was lower than I mg/l and mostly below the detection limit (0.05 mg P/I). From day 121 the average
Enhanced biological phosphorus removal activated sludge
569
anaerobic phosphate-P release had risen to 82.7 mgll and the average P content was 8.8% of the MLSS (Figure I). The sludge generated during stage B was called the P sludge.
Microscopic examination of P sludge. From day 121 to 167 extremely stable and outstanding P removal performance was achieved. Staining of smears of the P sludge mixed liquor from the anaerobic stage indicated that many of the cells contained lipophilic granules. Also cells from the aerobic stage staining for polyphosphate were prolific in the P sludge, and it would be expected that polyphosphate accumulating organisms (PAO) would be abundant in this sludge. As bacteria of the gamma subclass of the proteobacteria, in particular Acinetobacter, are proposed to be important to EBPR, the P sludge bacterial community was investigated using probes specific for these bacteria. In the P sludge, cells detected with the probes GAM42a and ACA made up less than 1% of the cells detected with the general bacterial probe EUB338 (visual estimation). Acinetobacter could not have been significantly involved in the P removal occurring here. This is in agreement with other investigations that have analysed bacterial communities in EBPR sludge using non-culture-dependent techniques (Cloote and Steyn, 1987; Wagner et aI., 1994; Bond et aI., 1995). 120
~ .....E
80
-
60
I I I I I
40
R I
A
.....
100
Q.
ell
I'll
.c
Co
en 0
.c Q.
8
2
I 30
50
90
110
--fI) fI)
...::s .....
c ell u c ell 4 c ..... 80 u
6
!:
20 0
10
90
110
130
150
170
0
~
Q.
0
Day of operation Figure I. Performance of SBR during stage A and stage B of operation. Phosphate-P in the effluent (-0-), phnsphale-P In Ihe end of anaerobic siage mixed liquor (-0-). ll1e P content as a mass percent of the MLSS (-"'-). Labelled arrows indicate when sludges Q. Rand P occurred.
Correlation (~r sludge performance with biochemical models. The anaerobic transformations of compounds that are reported to be important to EBPR were determined for the Q, Rand P sludges during cycles of the SBR operation (Figure I and Table 2). For all three sludges rapid uptake of acetate occurred along with accumulation of cellular PHA and degradation of carbohydrate occurred in the anaerobic stage of the SBR. These events are characteristic of EBPR sludge, however very little anaerobic phosphate release was detected for the Q sludge (Table 2). For the P sludge there was considerable anaerobic phosphate release. The transformations that occurred in the P sludge correlated remarkably well with transformations suggested by an EBPR model (Table 2). The R sludge, which had medial P removing performance, carried out transformations that were intermediate those of the Q and P sludges (Table 2). The transformations observed here support the suggestion that certain bacteria could compete with PAO in EBPR activated sludge systems. However, cells matching the description of the G-bacteria, organisms reported to inhibit EBPR (Cech and Hartman, 1993), were not observed in this Q sludge.
Op('fational clul/Iges leading to the improved EBPR performance. Although it is possible that there was a lack of bactcrial diversity in the sludge during stage A, and left reseeding prior to stage B introduced bactcria important to EBPR, one operational change that seemed to have an immediate effect during stage A was when the pH control was turned off in the anaerobic periods.
S70
P. L. BOND et al.
Table 2. Anaerobic transfonnations detected in the SBR, compared with theoretical values as molar ratios. Sludge type b
Non-EBPR
Q R P
Acetate uptake 6
6 6 6
consumed-
PHA units produced
2.6
4.4
Cell carbohydrate
2.5
2.1
1.23 1
5.5
3.6 3.9
Phosphate released
o
0.27 2.7
5.6
4 6 EBPRb 6 • Molar units are calculated as glycogen units b Theoretical ratios for non-EBPR (Satoh et al., 1994) and EBPR models (pH 7.0) (Smolders et aL, 1994) CONCLUSIONS
The perfonnance of an SBR activated sludge system operating for EBPR was evaluated by chemical and microscopic examination during stages of poor through to good phosphate removal. The transfonnations observed in the SBR cycles matched well with the theoretical values proposed by the models explaining EBPR and non-EBPR behaviour in these systems (Satoh et al., 1994; Smolders et al., 1994), and the results supported the biochemical pathways proposed in biological models. However, details and confinnation of the biochemical pathways are still required for progress in the understanding of the EBPR process, particularly for the development of conditions that favour PAO over non-P removing organisms. For example, if the pH has some influence on the anaerobic phosphate release and subsequent selection for PAO, as suggested here, this occurrence is not explained by the current EBPR biological models. The low numbers of Acinetobacter cells detected in the EBPR sludge suggest that bacteria other than Acinetobacter species were responsible for the EBPR that occurred here. Bacteria other than Acinetobacter and gamma proteobacteria should be investigated for polyphosphate accumulation and a possible role in EBPR. ACKNOWLEDGMENTS This work was funded by the CRC for Waste Management and Pollution Control Ltd., a centre established and supported under the Australian Government's Cooperative Research Centres Program. REFERENCES Appeldoom. K. J.• Kortstee. G. J. J. and Zehnder. A. J. B. (1992). Biological phosphate removal by activated sludge under defined conditions. Water Res. 26, 453·460. Bond. P. L.• Erhart. R.• Keller. J. and Blackall. L. L. (In preparation). Phosphorus removing capabilities of defined bacterial commUnities in activated sludge. Bond. P. L.• Hugenholtz. P.• Keller. J. and Blackall. L. L. (1995). Bacterial community structures of phosphate-removing and non· phosphate·removing activated sludges from sequencing batch reactors. Appl. Environ. Microbiol. 61. 1910-1916. Cech. 1. S. and Hartman. P. (1993). Competition between polyphosphate and polysaccharide accumulating bacteria in enhanced hiological phosphate removal system. Water Res. 27. 1219·1225. Cloete. T. E. and Steyn. P. L. (1987). A combined fluorescent antibody-membrane tilter technique for enumerating Acinetobacter in activated sludge. In Biological Phosphate Removal From Wastewater.., pp. 335·338. Edited by R. Ramadori. Oxford. Pergamon Press. Jenkins, 0 .. Richard. M. G. and Diagger. G. T. (1984). Manual on the Causes and Control of Activated Sludge Bulking and Foaming. Water Research Commission. Pretoria. Ulner, L. H. (1985). The role of bacterial phosphate metabolism in enhanced phosphorus removal from the activated sludge process. War. Sci. Tech. 17. 127·138. Manz. W.• Amann, R.. LudWig. W.• Wagner. M. and Schleifer, K. H. (1992). Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst. AppL Microbiol. 15. S93-600.
Enhanced biological phosphorus removal activated sludge
571
Murray. R. G. E.• Doetsch, R. N. and Robinow, C. F. (1994). Determinative and cytological microscopy. In Methods for General and Molecular Bacteriology, pp. 21-36. Edited by P. Gerhardt. R. G. E. Murray. W. A. Wood and N. R. Krieg. Washington, D.C.: American Society for Microbiology. Satoh, H., Mino, T. and Matsuo, T. (1994). Deterioration of enhanced biological phosphorus removal by the domination of microorganisms without polyphosphate accumulation. Wat. Sci. Tech. 30, 203-211. Smolders. G. 1. F.• van der Meij, J.• van Loosdrecht, M. C. M. and Heijnen, J. J. (1994). Model of the anaerobic metabolism of the biological phosphorus removal process: stoichiometry and pH influence. Biotechnol. Bioengng. 43. 461-470. Wagner. M.• Erhan. R., Manz. W., Amann. R.. Lemmer. H., Wedi, D. and Schleifer. K. H. (1994). Development of an rRNA• targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Appl. Environ. Microbiol. 60, 792-800. Wentzel. M. C., Loewenthal. R. E., Ekama, G. A. and Marais, G. R. (1988). Enhanced polyphosphate organism cultures in activated sludge systems - Part I: enhanced culture development. Water SA 14, 81-92.