Influence of dissolved oxygen and oxidation-reduction potential on phosphate release and uptake by activated sludge from sewage plants with enhanced biological phosphorus removal

War. Res.Vol.27,No. 3,pp. 349-354,1993 Printed in Great Britain. All rights reserved

0043-1354/93$6.00+0.00 Copyright © 1993 Pergamon Press Ltd

INFLUENCE OF DISSOLVED OXYGEN AND OXIDATION-REDUCTION POTENTIAL ON PHOSPHATE RELEASE AND UPTAKE BY ACTIVATED SLUDGE FROM SEWAGE PLANTS WITH ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL GEORG SCHON*, SUSANNEGEYWrrZ and FRANK MERTENS Institut f'tir Biologie II, Mikrobiologie, Sch/inzlestr. 1, D-7800 Freiburg, Germany

(First received April 1992; accepted in revised form September 1992) Al~a'act--In wastewater treatment plants with enhanced biological phosphorus removal, the rate of aerobic phosphate uptake by activated sludge depends on previous phosphate release by polyphosphateaccumulating bacteria under anaerobic conditions. However, there has been disagreement as to whether the PO 4 release is caused either directly by low concentrations of dissolved oxygen (DO) or by the lowered redox potential (ORP) at zero DO. The results from experiments in a laboratory-scale treatment plant with activated sludge from different full-scale plants indicated a direct DO dependence of phosphate uptake and release. In contrast, the ORP modified the rate of phosphate release only under certain conditions. Phosphate release began at values between 0.1 and 0.5 mg O2/1, depending on sludge conditions. At limiting DO concentrations, nitrate reduced the phosphate release in sludge from lowly loaded plants (with nitrification and denitrification zones) but not in sludge from highly loaded plants (without nitrification). In a pure culture of a polyphosphate-accumulating Acinetobacter strain (Br-2) phosphate release started only in completely anaerobic conditions. In sludge from sewage plants and in a pure culture of Acinetobacter, enhanced phosphate uptake began as soon as O 2 was available for respiration.

Key words--activated sludge, biological phosphorus removal, phosphate uptake, phosphate release, DO influence, nitrate effect, Acinetobacter

INTRODUCTION

investigation we therefore tested to what extent the content of dissolved oxygen and the o x i d a t i o n In sewage plantswith enhanced biologicalphosphorus reduction potential influence phosphate release in removal, the activated sludge must pass alternately activated sludge of different composition (with and through anaerobic and aerobic stages. In nitrifying without nitrate), as well as in pure cultures of an plants, an additionalanoxic stage for nitrateremoval Acinetobacter strain. Some of the results have already is also necessary (Comcau et al., 1987). During the been presented at the conference of the G e r m a n anaerobic phase, the phosphate that had previously Society of General and Applied Microbiology in been taken up aerobicallyby the biomass and stored Freiburg, 1991 (Summary, Mertens et al., 1991).

as polyphosphatc is partiallyreleased.The higher the anaerobic release,the more effectivethe subsequent aerobic phosphorus removal (Barnard, 1976; Nicolls and Osborn, 1979). As the triggering factor for the phosphate release, Randall et al. (1970) proposed a deficiencyin dissolved oxygen. In other studies,however, a correlation was found between phosphate release and low oxidation-reduction potential(ORP) (Shapiro et aL, 1967; Koch and Oldham, 1985). Earlier investigations had shown that nitrate can influencephosphate releasein activated sludge differently. This depends on the composition of the bacterialflora,which in turn can change with alterations in the processes used for phosphorus removal, the sewage composition and other environmental conditions (Sch6n and Streichan, 1989). In the present *Author to whom all correspondence should be addressed.

MATERIALS AND METHODS Activated sludge Sludges were obtained from different full-scale municipal sewage treatment plants in Germany which operate with anaerobic-aerobic or anaerobic-anoxic-aerobic activated sludge processes for enhanced biological phosphorus elimination. The plants in Berlin-Ruhleben and Hildesheim had a low BOD sludge loading (lower than 0.15kg BODs/kg SS/d) and therefore exhibit nitrification. Most of this nitrate produced is eliminated by an internal recycling of the mixed liquor from the aeration tank to an anoxic zone. The plants in Berlin-Marienfelde, Braunschweig and Wyk have a high BOD sludge loading (approx. 0.6 kg BODs/kg SS/d) and no nitrification or only in a secondary aeration tank.

Acinetobacter The polyphosphate-accumulating Acinetobacter strain Br-2 was isolated from the activated sludge of the plant in Braunschweig (Streichan et al., 1990).

349

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GEORGSCH6N et al.

Medium and growth conditions In phosphate release experiments mixed liquor or pure culture samples (400ml in a 0.5 or 1.01. fermentor) were kept aerobically for 3 h (to accumulate polyphosphate) in Tris-buffered mineral medium without ammonium to prevent nitrate synthesisby nitrification. Medium composition has been described previously (Streichan et al., 1990). To induce phosphate release, substrate (Na-acetate, 0.5 g/l) was added and the DO reduced stepwise. Completely anaerobic conditions were achieved with N2 gas. In phosphate uptake experiments the samples were aerated stepwise.

Phosphate analysis For determination of phosphate, samples of sludge or bacterial cultures were harvested by low-speed centrifugation. The o-phosphate concentration in the supernatant was measured as molybdenum-vanadate phosphoric acid at 405urn (Merck, Spectroquant, 14 842, Darmstadt, Germany).

released phosphate when the DO content was reduced to 0.5 mg O2/1. At the onset the oxidationreduction potential (ORP) was positive, but the rate of phosphate release remained about the same after the ORP fell to negative values ( - 1 5 0 mV). In another experiment in which the ORP was markedly lower at the start of phosphate release (about zero), there was similarly no significant difference to detect at the beginning of phosphate release by the biomass [Fig. I(B)]. In activated sludge from another lowly loaded sewage plant (Hildesheim) phosphate was released at approx. 0.3 rng O2/1 (data not shown). In activated sludge from a highly loaded sewage plant (Wyk, without nitrification in the aeration basin) phosphate release began only at a DO value of 0.1 rag O2/1 (Fig. 2). Moreover, the maximum release rate was not reached until there was no more measurable DO. The course of phosphate release was similar in other plants with high sludge loading, although the decline in the ORP varied (Berlin-Marienfelde: Fig. 3; Braunschweig, data not shown).

RESULTS

Influence o f DO on phosphate release in the presence o f nitrate

DO and ORP determination The concentration of dissolvedoxygen was measured with a DO electrode (CLARKE-type) and the ORP with an Ag/AgCl-redoxelectrode. A measured DO concentration of 0.0mg O2/1 during very low aeration means that 02 was taken up faster by microorganisms for respiration than by the electrode.

Influence o f DO on phosphate release in activated sludge without nitrate

In activated sludge from sewage plants with and without nitrification, nitrate has a different influence on phosphate release under oxygen-free conditions (Sch6n and Streichan, 1989).

Activated sludge from sewage plants with nitrification and denitrification (Berlin-Ruhleben, Fig. 1)

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Fig. 1(A) and (B). Influenceof the dissolvedoxygen(DO) concentration on phosphate releaseand oxidationreduction potential (ORP) in two different activated sludge samples from the lowly loaded plant (BerlinRuhleben). (A) Automatic control of DO and (B) manual DO control. At the start of the experiments the batch was saturated with oxygen; when DO was lowered to 0.5 mg O2/1 substrate was again added in excess (0.5-1.0 g/1 Na acetate); 0.0 mg O,/1 indicates that, in spite of aeration, no oxygen was detectable; complete anaerobiosis occurred only after N2 gasing. - - , DO; - - - , ORP; , dissolved phosphate.

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Fig. 2. Influence of the dissolved oxygen (DO) concentration on phosphate release and oxidationreduction potential (ORP) in activated sludge from the highly loaded plant (Wyk) (for further details see Fig. 1). - - . , DO; - - - , OR_P; , dissolved phosphate. even somewhat higher (at 0.0 mg 02/0, although the oxidation-reduction potential in the medium with nitrate was 80-100mV less negative than without nitrate. After the nitrate was exhausted, the ORP values reached similar levels.

The influence of nitrate on the composition of the sludge bacteria can also be observed for phosphate release under limiting DO content. Activated sludge from a lowly loaded plant (Berlin-Ruhleben) with nitrification and denitrification released less phosphate in the presence of nitrate (with substrate) than without nitrate [Fig. 4(A)]. Release began at a DO concentration, on average, of approx. 0.1 mg O5/1. Within the floes, however, a deficiency of DO will already have set in, since a significant nitrate reduction could also be measured. After nitrate was exhausted by anoxic respiration, the rate of phosphate release increased markedly. Activated sludge from a highly loaded plant without nitrification (Berlin-Marienfelde) showed three times as high a rate of aerobic phosphate uptake as lowly loaded sludge [Fig. 4(B)]. On the other hand, in the presence of nitrate, phosphate release by activated sludge was not reduced at limiting DO, although a nitrate reduction was also observed. The rate of phosphate release was approximately the same or

Dependence of phosphate uptake on DO After a complete anaerobic phosphate release in the sludge from Berlin-Marienfelde, the oxygen content was increased stepwise (Fig. 5). Phosphate uptake already started with maximum rate at a measured DO content of 0.1 mg O5/1 (OR]) approx. - 2 0 0 mV). Similar results were obtained with activated sludge from other plants (Hildesheim and Wyk). The maximum phosphate uptake began between 0.1 and 0.2mg O5/1.

DO influence on phosphate release and uptake by Acinetobacter In a pure culture of Acinetobacter strain Br-2, isolated from activated sludge, phosphate uptake also

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Fig. 3. Influence of the dissolved oxygen (DO) concentration on phosphate release and oxidationreduction potential (ORP) in activated sludge from the highly loaded plant (Bcrlin-Marienfelde) (for further details see Fig. 1). , DO; - - - , ORP; , dissolved phosphate.

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Fig. 4(A) and (B). Rate of phosphate uptake and release in the presence of nitrate. (A) Lowly loaded plant (Berfin-Ruhleben) and (B) highly loaded plant (Berlin-Marienfelde). Columns indicate the rate (per hour) of change in the concentration of the dissolved phosphate during the various phases of aeration, positive values show release, negative values show uptake of phosphate (m, with nitrate; ~ , control without nitrate); lines show the corresponding average ORP [VI. I"1, with nitrate (l-q* at this point nitrate was completely exhausted); ~ O, control without nitrate]. occurred at such a low aeration that DO was no longer measurable with an oxygen electrode (Fig. 6). At this time, the ORP was below - 150 mV. The rate of phosphate uptake, however, was reduced with decreasing DO content. A slight but significant phosphate release began only at completely anaerobic conditions (N 2 gas phase). The phosphate release rate was low and could not be increased further by reducing the ORP with Nadithionite. When the culture was incubated anaerobically (16 h) and the DO content subsequently increased stepwise, phosphate uptake (6.0rag P/1.h) and polyphosphate-accumulation already began at very low DO (below 0.1 mg O2/1). The full rate of

phosphate uptake (8.7 mg P/I. h), however, was only reached at approx. 0.5 mg O2/1 (Table 1). DISCUSSION

Activated sludge from sewage plants with enhanced biological phosphorus removal, which has accumulated polyphosphate aerobically, releases phosphate as soon as oxygen deficiency sets in. At a limiting content of dissolved oxygen, phosphate uptake would probably stilloccur on the surface of the sludge floe, while in the interior,phosphate is released.Consistent with thisassumption, in the case of large,compact sludge floes(e.g.Berlin-Ruhleben), phosphate release by the biomass began earlierthan

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Fig. 5. Influence of the dissolved oxygen (DO) concentration on phosphate release and oxidationreduction potential (ORP) without addition of substrate in activated sludge from the highly loaded plant (Berlin-Marienfelde) after previous anaerobic phosphate release; at the start of the experiment aeration without detectable DO (O.Omg 02/I). - - , DO; - - - , ORP; , dissolved phosphate.

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Fig. 6. Influence of the dissolved oxygen (DO) concentration on phosphate uptake, phosphate release and oxidation-reduction potential (ORP) in a pure culture of Acinetobacter, strain Br-2; after 1 h of complete anaerobiosis ORP was lowered further by addition of 0.6 mM sodium dithionite , DO; - - - , ORP; , dissolved phosphate. with smaller, uncompact sludge flocs (e.g. Wyk). In activated sludge, to which no substrate was added after shifting to aerobic conditions, there was only a low oxygen consumption of oxygen, and thus no anaerobic zones were present in the flocs. Hence, phosphate uptake already began at minimum oxygen concentrations (between 0.0 and 0.1 mg O2/1). These results also indicate that the measured oxygen concentrations at phosphate uptake or release depend on a gradient of oxygen within the sludge flocs. Investigations of pure cultures of Acinetobacter also show that as long as there is a functional respiratory metabolism, the cells will not release any phosphate into the medium. The ORP had no direct influence on inducing phosphate release. The variable effects of nitrate on phosphate release in activated sludge from sewage plants with and without nitrification and denitrification also support the proposal that, under anoxic conditions, it is probably not the nitrate-induced increase in the ORP that has an inhibitory effect on phosphate release. Together with earlier investigations (Sch6n and Streichan, 1989) these experiments show, however, that nitrate can influence phosphate metabolism directly: if nitrate-respiring polyphosphate-accumulating bacteria are present in the sludge, phosphate uptake is due to these strains. The observed low anoxic (net) phosphate uptake is consequently the difference between the phosphate release of the polyphosphate-accumulating bacteria incapable of nitrate respiration and the phosphate uptake by strains which can use nitrate as Table 1. Phosphateuptakeand oxidation-reductionpotential(ORP) in a purecultureof Acinetobacterstrain Br-2;anaerobicprecuhure 3 h Dissolved oxygen (mg/l) 0.0 0.1 0.5 6.0 Phosphate uptake -0.95 6.0+ !.5 9.2± 1.2 8.0±0 (ms Pfl) ORP (mV) -160 -140 -70 -10

an electron acceptor in substrate oxidation under O2-free conditions. Nevertheless, in the activated sludge of full-scale sewage treatment plants with enhanced biological phosphorus removal, low ORP has a certain relevance for phosphate release. Particularly in lowly loaded plants, if no more nitrate respiration occurs, short-chain organic acids and alcohols are released during the fermentative metabolism of facultatively anaerobic bacteria. Phosphate-accumulating bacteria can subsequently take up and store these fermentation end-products. The required metabolic energy is provided by cellular polyphosphate and thus phosphate is released into the medium. In sewage treatment plants with nitrification, denitrification and enhanced biological phosphorus removal, the oxidation-reduction potential (ORP) can be used to assay the various states of the sludge (aerobic, anoxic, anaerobic) and for optimizing the aeration rate (Koch and Oldham, 1985; Charpentier et al., 1989; de la M~nardi~re et al., 1991). In highly loaded plants (without nitrification) an ORP measurement is not as suitable for the regulation of phosphate release or uptake, because without denitrification there is no distinct step in ORP, and also, under these operating conditions, the content of dissolved oxygen is primarily responsible for phosphate removal. CONCLUSIONS The results show that phosphate release in activated sludge first sets in at a relatively low content of dissolved oxygen. Nonetheless, in many sewage plants a phosphate release--at least in local zones of the aerated basins---can occur, since the DO, as a rule, is kept low for economic reasons and usually measured only at the end of the aerated basin where the easily degradable organic pollutants have already been oxidized.

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GF.ORG SCH6~ et al.

During secondary clarification in plants without nitrification (and thus without nitrate inhibition) a relatively high phosphate release must be expected, even when all the more easily degradable substrates from the inflow have been oxidized. Therefore, in plants with enhanced biological phosphorus removal an adequate aeration in all parts of the activated sludge basin must be guaranteed, and the residence time in the secondary clarifier must be kept as short as possible. Acknowledgement--This investigation was supported by

a grant from the Bundesministcrium ffir Forschung und Technologie in Germany (No. 02 WA 8807).

REFERENCES

Barnard J. L. (1976) A review of biological phosphorus removal in the activated sludge process. War. Sth Aft. 2, 136-144. Charpentier J., Godart H., Martin G. and Mogno Y. (1989) Oxidation-reduction potential (ORP) regulation as a way to optimize aeration and C, N and P removal: Experimental basis and various full-scale examples. War. Sci. Technol. 21, 1209-1223. Comeau Y., Rabinowitz B., Hall K. J. and Oldham W. K. (1987) Phosphate release and uptake in enhanced biologi-

cal phosphorus removal from wastewater. J. War. Pollut. Control Fed. 59, 707-715. Koch F. A. and Oldham W. K. (1985) Oxidation-reduction potential--a tool for monitoring, control and optimization for biological nutrient removal systems. Bat. Sci. Technol. 17, 259-281. de la M6nardi6re M., Charpentier J., Vachon A. and Martin G. (1991) ORP as a control parameter in a single sludge biological nitrogen and phosphorus removal activated sludge system. Wat. Sth Afr. 17, 123-132. Mertens F., Geywitz S. and Sch6n G. (1991) Influence of O2-concentration and oxidation-reduction potential on phosphate uptake and release by activated sludge from sewage plants. Bioforum 14, 94. Nicholls H. A. and Osborn D. W. (1979) Bacterial stress, a prerequisite for biological removal of phosphorus. J. War. Pollut. Control Fed. 51, 557-569. Randall C. W., Marshall D. W. and King P. H. (1970) Phosphate release in activated sludge process. J. sanit. Engng Die., Am. Soc. cir. Engrs 96 (SA2) 395-408. Sch6n (3. and Streichan M. (1989) Anoxische Phosphataufnahme und Phosphatabgabe dutch belebten Schlamm aus Kl.~ranlagen mit biologischer Phosphorentfernung. GWF-Waas./Abwaas. 130, 67-73. Shapiro J., Levin (3. V. and Zea G. (1967) Anoxically induced release of phosphate in wastewater treatment. J. War. Pollut. Control Fed. 39, 1810-1818. Streichan M., Golecki J. R. and Sch6n G. (1990) Polyphosphate-accumulating bacteria from sewage plants with different processes for biological phosphorus removal. F E M S Microbiol. Ecol. 73, 113-124.