Biological phosphorus removal by pure culture of Lampropedia spp.

~)

Wat. Res. Vol. 31, No. 6, pp. 1317-1324, 1997

Pergamon

PII:S0043-1354(96)00351-X

© 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0043-1354/97 $17.00 + 0.00

BIOLOGICAL PHOSPHORUS REMOVAL BY PURE CULTURE OF L A M P R O P E D I A SPP. L. S T A N T E * , C. M. C E L L A M A R E , F. M A L A S P I N A @, G. B O R T O N E @ a n d A. T I L C H E @ ENEA, Sezione Depurazione e Ciclo dell'Acqua, Via Martiri di Monte Sole, 4 1-40129, Bologna, Italy

(First received March 1996; accepted in revised form October 1996) Abstract--Lampropedia spp. is a Gram-negative, Neisser-positive coccus that was isolated from EBPR (enhanced biological phosphate removal) activated sludge laboratory plants operating on dairy and piggery wa,;tewaters. In aerobic growth tests carried out on sodium acetate, Lampropedia spp. stored PHB up to 12% w/w. Biomass yield was estimated at 0.55gVSS.g -~ HAc and specific growth rate at 0.045 h -~. The experimental maximum acetic acid removal rate was 71.86 mg HAc.g -t VSS.h -~ with a semisaturalion constant of 71.78 mg.l -~. Batch tests were carried out to check whether Lampropedia spp. was capable of enhanced biological phosphorus removal. Under anaerobic conditions, Lampropedia spp. sequestered acetate and stored PHB with an average conversion factor of 0.33 mg PHB.mg -~ HAc. The measured maximum PHB storage capacity was 31% w/w, with a maximum specific PHB accumulation rate of 17mgPHB.g-tVSS.h -L and a specific anaerobic acetate uptake rate of 57 mg HAc.g-t VSS.h -~. The experimental ratio between phosphorus released and acetate taken up was low, on average 0.044mgPO4-P.mg -t HAc, with a specific rate ranging from 1.7 to 3.6mgPO4P.g-~ VSS.h -z at pH 7.5. Despite the low figure, fractionation analyses showed that in anaerobic conditions the released phosphate comes from cell polyphosphate degradation. Therefore, all the results allow us to conclude that Larnpropedia spp. can be classified amongst the phosphorus accumulating bacteria © 1997 Elsevier Science Ltd.

Key words--Lampropedia spp., poly-fl-hydroxybutyrate, enhanced biological phosphorus removal, nutrient removal, polyphosphate accumulating microorganisms

NOMENCLATURE

ADM COD EBPR FID g GC GTA HAc HPIC mod. PHA PHB PHV poly-P rpm

= = = = = = = = = = = = = = = rs = rSa¢ = TSS = VSS = #= gobs =

chemical defined medium chemiczl oxygen demand enhanced biological phosphorus removal flame ionization detector gravity acceleration [L.T -2] gas chromatography green top agar acetic acid high performance ion chromatography modified polyhydroxyalcanoate poly-fl-i~ydroxybutyrate polyhydroxyvalerate polyphosphates revolutions per minute specific substrate removal rate [T ~] specific acetic acid removal rate [T -~] total suspended solids volatile suspended solids specific growth rate [T -~] specific observed growth rate [T -~] INTRODUCTION

In the last few years, m a n y studies o n pure cultures o f activated sludge p o l y p h o s p h a t e accumulating m i c r o o r g a n i s m s (PAO) have been carried out. The c o m m o n characteristics o f this heterogeneous g r o u p o f bacteria are their capability o f synthesizing *Author to whom all correspondence should be addressed.

polyphosphates (poly-P) a n d polyhydroxyalkanoates as internal storage c o m p o u n d s ; p o l y p h o s p h a t e s are formed during the aerobic phase, while P H A s are stored during the anaerobic phase ( C o m e a u et al., 1986; Wentzel et al., 1986; M i n o et al., 1987, 1994). M a n y different kinds o f poly-P accumulating bacteria have been isolated from activated sludge. Brodisch a n d Joyner (1983) f o u n d t h a t in several E B P R plants p h o s p h o r u s removal was mainly carried out by bacteria belonging to the genera Aeromonas a n d Pseudomonas, representing up to 50% of the microbial population. The presence of those two genera has been confirmed in other studies by Florentz a n d H a r t e m a n n (1984) a n d L6tter a n d M u r p h y (1985). Activated sludge bacteria, belonging to the genus Acinetobacter, were isolated for the first time by Fuhs a n d C h e n (1975), who d e m o n s t r a t e d in b a t c h tests t h a t Acinetobacter was able to take up p h o s p h a t e a n d to degrade poly-fl-hydroxybutyrate (PHB) u n d e r aerobic conditions, releasing soluble o r t h o p h o s p h a t e in anaerobic conditions. On the other h a n d , they did n o t give any figure o n acetate u p t a k e in anaerobic conditions. O t h e r tests have been carried out by D i e n e m a et al. (1980, 1985); in their experience, Acinetobacter cultures released soluble o r t h o p h o s p h a t e without synthesizing P H B u n d e r anaerobic conditions, using

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L. Stante et al.

acetate, lactate and ethanol as the c a r b o n source. T a n d o i et al. (1987) revealed incomplete C O D uptake a n d p h o s p h o r u s release with pure cultures of Acinetobacter lwoffi, m a i n t a i n e d in anaerobic/aerobic alternate conditions. H e y m a n n et al. (1989) emphasized the distinction between aerobic or microaerophilic growth. In the latter, Acinetobacter lwoffi was rich in either P H B or poly-P, while the aerobic cultures had a lower poly-P content. Bayly et al. (1990) observed p h o s p h o r u s release without acetate uptake in a n Acinetobacter culture that, in spite of this behaviour, showed enhanced poly-P storage capacity. Some tests with Acinetobacter calcoaceticus failed. A significant a n a e r o b i c acetate uptake was observed without any correlation to p h o s p h a t e release (Ohtake et al., 1985). However, it is n o w clear t h a t in full-scale E B P R plants, m a n y different bacterial groups are responsible for e n h a n c e d biological p h o s p h a t e removal (Wagner et al., 1994; Bond et al., 1995). N a k a m u r a et al. (1991) worked in microaerophilic conditions with pure cultures o f a bacteria t h o u g h t to belong to the genus Microcuccus a n d then assigned to a new genus and species Microlunatus phosphorovorus ( N a k a m u r a et al., 1995). These bacteria were capable of p h o s p h a t e uptake in oxidative conditions without organic substrate in the medium, and released p h o s p h a t e when fed with glucose in anaerobic conditions. M o r e recently, U b u k a t a a n d Takii (1994) hypothesised that the enzymatic system for the take up of p h o s p h a t e could be induced by at least two anaerobic/aerobic cycles. In t h a t particular case, p h o s p h o r u s release has been observed with a c o n c o m i t a n t uptake of casamino acid after the induction of the cell culture. The present study regards a Gram-negative coccus, Neisser-positive in aerobic conditions a n d Neissernegative in anaerobic conditions, t h a t was observed from E B P R activated sludge laboratory plants operating on dairy a n d piggery wastewaters; it was supposed to be capable o f ~nhanced biological p h o s p h a t e removal. The microorganism was morphologically identified as belonging to the genus Lampropedia. The striking quality of these bacteria is u n d o u b t e d l y the large cell sheets they build b o t h in solid a n d liquid medium, a property which gives to L a m p r o p e d i a the rare prerogative of being immediately recognized u n d e r the microscope (Murray, 1984). In this paper, isolation procedures, growth culture conditions a n d alternate anaerobic/oxic b a t c h tests for evaluating the e n h a n c e d biological phosp h o r u s removal capacity of L a m p r o p e d i a spp. are described. MATERIALS AND METHODS

Bacteria Bacteria were isolated from the activated sludge of a sequencing batch reactor (SBR) treating dairy and piggery wastewater as described in the isolation procedure. The single cells have a rounded, almost cubical shape, and they

are arranged in square tablets of 16-64 or more individual cells. They divide synchronously in a sheet and alternately in two planes. Staining and microscopic observation Gram and Sudan Black B staining were carried out as reported by Jenkins et al. (1986); metachromatic granules were stained both with the Pergola method (Pasquinelli, 1981), Albert method (Tandoi et al., 1990) and Neisser method (Jenkins et al., 1986); Blue Nile A staining was done as reported by Ostle and Hot (1982). Light microscopic observations were carried out using a Jenalumar A/D contrast light microscope (1000 × magnification) in bright field, phase contrast, Nomarski interferential contrast and bright field coupled to fluorescence epimicroscopy; scanning electron microscope (SEM) observations were carried out using a Philips XL 20. Media Two kinds of media were used for the tests: a modified green top agar (mod.GTA) and a chemical defined medium (ADM). All media were sterilized by autoclaving at 121°C for 15 min. Modified green top agar (based on green top agar by Pringsheim (1980) was made with: 2 g of yeast extract, 1 g of peptone, 1 g of sodium acetate, 100 ml of filtered and sterilized effluent from dairy wastewater treatment plant, 15 g of agar (only 2 g for semisolid medium), distilled water to 1 I volume and pH adjusted to 7.2. This medium was used as solid substrate during isolation and maintenance on Petri plates (100 mm) and tubes (16 x 200mm). The modified green top agar without agar was used for growth, PHB storage and phosphorus uptake tests. ADM was composed of 160mg of NH4CI, 64mg of KH2PO4, 2 ml of a microelement stock solution (Puttlitz and Seeley, 1968), and distilled water to 11 volume. After sterilization, 1 mg of biotine, 1/~g of tiamine and a variable amount of sodium acetate (from 500 to 10000mg, depending on test conditions) were added after filtration on 0.22/~m sterile filters. This method was also used for growth tests. Sodium acetate in data tests was expressed as acetic acid. The ADM medium without KHzPO4 was used in phosphorus releasing tests. Instruments and analytical methods Analyses of TSS and VSS were performed on the residue retained by GF filter following Standard Methods (APHA, 1989). The different forms of phosphorus were measured according to the same manual. Phosphorus fractionation was carried out as reported by De Haas (1989). Nitrate, orthophosphate and ammonia were determined using an HPIC (Dionex 4000i). Acetic acid was determined gas-chromatographically, using a DANI 8510 GC equipped with a 25 m, 0.53 mm, 1.2 pm capillary wide bore column (Alltech SO FA bound FSOT) and an FID detector; hydrogen was used as carrier gas and 2,2-dimethylbutyric acid as internal standard. GC working conditions were as follows: oven temperature from 107 to 140°C with an increasing rate of 6°C.min-~, injector temperature of 220°C and FID port temperature of 230°C. PHB was determined by GC with a modified method as proposed by Braunegg et al. (1978) and Gerhardt et al. (1994). A 10 ml sample of culture media was centrifuged at 4000 g x 15 min at 4°C. After centrifugation, the supernatant was discharged and replaced with an equal volume of a solution of sodium hypochlorite; after incubation at 37°C for 1 h, the sample was centrifuged at 9000 g x 15 min at 4°C. The solid fraction was washed with water and centrifuged at 9000 g × 15 min at 4°C. The solid fraction was then mixed with 2 ml of acidified methanol (3 % HzSO4) and 1 ml of chloroform and then heated for 3.5 h at 100°C. After the digestion/methylation, 1 ml of water and 25/tl of internal standard (4000 mg.l -~ 2,2-dimethylbutyric acid

Biological P removal by Lampropedia spp. solution) were added to the sample; after 10 min of vigorous shaking, phase separation follows. A 0.1 #1 sample of the chloroform fraction is injected into the GC, equipped with the same column and detector described above, and hydrogen was used as carrier gas. GC working conditions w e r e : oven temperature from 60 to 120°C with an increasing rate of 4.7°C. rain- t injector temperature of 220°C, and FID port temperature of 230°C. Pure PHB (Sigma) was used for GC calibration.

Test methods The growth tests were carried out under aerobic conditions in a 2 1 well mixed glass bottle (magnetic stirrer at 60 rpm) with 0.5 1 of media (modified GTA or ADM) covered with a cotton lid. The inoculum was taken from a stock plate colony. 7temperature was kept at 25°C and the initial pH was 7.5. Cells for phosphorus release tests were cultured batchwise under aerobic condition in modified GTA medium. Phosphorus release and PHB storage tests were carried out on centrifuged cells (4000g x 15rain) washed with phosphorus-free media. The washed cells were resuspended in 0.3~).5 1of ADM without phosphate in a 2 1 glass bottle. Anaerobic conditions were obtained by bubbling nitrogen gas sterilized by filtration on a 0.22 #m sterile filter. Temperature was kept at 20°C and, during the tests, pH reached 8.3-8.5.

RESULTS AND DISCUSSION

Isolation and identification After Neisser staining, these bacteria showed the presence of metachromatic granules; in addition, they were Sudan Black positive (Murray, 1963) and the isolate was also positive to Albert staining (Tandoi et al., 1990) which, together with Neisser staining, is recommended for ,evidencing the presence of volutine granules (Jenkins et al., 1986). On the basis of these characteristics, it was supposed that Lampropedia might belong to the functional group of poly-P accumulating microorganisms. Activated sludge was sampled from two SBR reactors operating on dairy and piggery wastewater and observed by a phase-contrast light microscope. Some particular ,;haped bacteria belonging to the genus Lampropedia were observed. Other authors before (Standridge, 1981) have identified Lampropedia spp. in an activated sludge trickling filter plant treating cheese factory wastes. Lampropedia hyalina was isolated for the first time by Schroeter (1886) and studied in more detail by Pringsheim (1955).

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Lampropedia cells are enclosed in a Gram-negative type of cell wall; no flagella occur, they are strictly aerobic, chemo-organotrophic and usually include PHB granules, as described in Bergey's manual. Lampropedia was first observed in muddy water and probably its name originated because they appear like tablets "glistening". They have been isolated in an organic-matter-rich environment, although a definite ecological niche is not clearly definable. Growth occurs as a thin, hydrophobic and extended cell pellicle spreading on a solid surface or liquid media (Murray, 1963). Each cell is usually linked to the near cells by a remarkable proteic surface structure or S layer (Murray, 1963; Austin and Murray, 1990). Murray (1984) and K h u n and Starr (1965) reported the presence of refractile PHB inclusions when cells grow in the presence of sodium acetate. Several isolation techniques have been tested; among them, the most reliable was the direct spreading of sludge samples on to agar plates. Sequential steps on to further agar plates allowed culture isolation. The solid medium was modified G T A , kept at 25°C in aerobic conditions. Once Lampropedia colonies were isolated from the rest of the sludge population, several cultural enrichments on a liquid medium containing 2 g.1 ~ agar were tested. In this liquid medium, Lampropedia cells were mainly growing on the liquid surface. The pure cultures of Lampropedia were renewed almost daily in new agar plates or in test tubes; the latter were more stable than plates, since this kind of environment is slightly damp, as Lampropedia requires (Murray, 1963). The properties of Lampropedia hyalina as originally described by Puttlitz and Seeley (1968) correspond to the used strain. Characterization tests, a group of those enumerated in Bergey's manual (Table 1), were carried out in order to confirm the visual identification of Lampropedia. According to test results, the used strain corresponds to Lampropedia. The species' attributes however, are not clearly defined. To check the presence of PHB granules in the cells (Kuhn and Starr, 1965), Sudan Black B and the more specific Blue Nile A stains were used for microscopic observations. PHB was also measured by G C analysis.

Table 1. Identification tests for Lampropediaspp. Characteristic Gram staining Neisser slaining Growth under anaerobic conditions Intracellular PHB formed C a t a l a s e test Nitrate

reduction

Motility S h a p e characteristic

Cells arranged in square tablets of 16-64 cells Final pH of culture media "

Reaction or result obtained + +

Reaction or result for Lampropediaspp. (Bergey's) Not reported +

+

+

-

-

-

-

Cocci in sheet +

Cocci in sheet +

8.3-8.5

8.4-8.6

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L. Stante et al.

Fig. 1. Lampropedia spp. in pure culture (Sudan Black stain; white bar is 10/~m).

Growth

Growth tests have been carried out in aerobic conditions on enriched (mod. GTA) and minimal media (ADM), to calculate stoichiometric and kinetic parameters. Trends of substrate (sodium acetate) and biomass (calculated from VSS, not including the weight of PHB) during nine growth tests showed a decrease of substrate concentration with corresponding biomass growth. In some tests, a slight biomass growth after substrate consumption, probably due to degradation of stored PHB, was observed. The same trend was observed with different initial substrate concentrations; however growth rates varied depending on substrate/biomass ratios. PHB was also stored during aerobic cell growth, particularly with high substrate/biomass ratios (Fig. 3). This phenomenon has been already described for other microorganisms in unbalanced growth conditions (Dawes and Senior, 1973). PHB ranged from 3 to 12% w/w, with initial acetic acid concentrations ranging from 450 to 3500 mg.1 ' and initial acetic acid/VSS ratios of 3.5 and of 8.8 g HAc.g -~ VSS, respectively. PHB/acetate conversion factors were 6.5 and 16% at the acetic acid

concentrations reported above. Figure 4 shows that VSS increase as a function of the removed substrate. The curve slope represents the average biomass yield (0.55 g-~ VSS.g-' HAc). The observed COD:nitrogen ratio consumed for growth was on average 100:2. On enriched medium, the specific growth rate (/~ob~) was quite stable in the substrate concentration range from 100 to 550 mg.l ' of acetic acid, with an average value of 0.045 h ~. Similar results were obtained on minimal medium. The specific substrate removal rate (rsa~) followed a Michaelis-Menten curve, as reported in Fig. 5. From data interpolation, the estimated maximum rate was 71.9 mg HAc.g -~ VSS.h -~ and the semisaturation constant 71.8 mg HAc.I '. Tests were carried out to evaluate the capability of Lampropedia spp. to grow with nitrate as the final electron acceptor instead of oxygen: no growth was detected. Substrate uptake and PHB storage under anaerobic conditions

To evaluate stoichiometric and kinetic parameters in anaerobic conditions, tests without any inorganic electron acceptor and with sodium acetate as the sole substrate were carried out. From Fig. 6 it can be seen that acetate uptake is strictly related to PHB storage. The average PHB yield, calculated as the slope of the curve in Fig. 7, was about 0.33 mg P H B . m g - ' HAc; this value is lower than the one reported by Comeau et al. (1986) and by Smolders et al. (1994) for mixed cultures, but it is quite similar to those reported by Fukase et al. (1982) and Arun et al. (1988) in pure culture. This discrepancy could partially depend on incomplete extraction of PHB and on the fact that the analytical method allowed only the estimation of PHB and not of other PHAs. The chromatograms showed qualitatively the presence of other methylvolatile fatty acids. PHB was stored in the cells in considerable amounts, about 31% of VSS dry weight. The specific PHB accumulation rate was strongly dependent on initial acetic acid concentration, with higher values obtained at higher substrate concentration. The maximum rate obtained was 1 7 m g P H B . g VSS.h-'. In accordance to this, specific acetic acid uptake rate (31-57 mg HAc.g -~ VSS.h -~) increased as a function of initial acetic acid concentration. Phosphorus release and uptake

Fig. 2. Lampropedia spp. (scanning electron microscope; bar corresponds to 2/~m).

Tests on phosphorus release under anaerobic conditions were carried out using acetic acid as substrate. The rate of phosphorus release and acetic acid uptake were always higher at the beginning of the test (Fig. 8). Figure 9 shows the relationship between released orthophosphate and acetic acid taken up in one representative test; the slope represents the average stoichiometric ratio obtained. The ratios between released orthophosphate and acetic acid removed in the various tests ranged from

Lampropediaspp.

Biological P removal by

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Fig, 3. Trend of PHB/VSS and acetic acid during a growth test in aerobic conditions.

0.5.

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I I I ! slope = 0.55 mgVSS/mgNAc

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Fig. 5. Acetic acid specific removal rate during growth as a function of acetic acid concentration (averaged values of three parallel tests).

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Fig. 6. Trends of acetic acid and PHB during a PHB storage test.

1322

L. Stante et al. 250

! I slope = 0.333 mgPH8 /

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Fig. 8. Trend of acetic acid and phosphorus during one of the P release tests. 0.02 to 0.11mgPro,.mg t HAcrem, with biomass concentration ranging from 0.2 to 0.64 g - ' VSS.1 t. The average value was 0.044mgPre,.mg ~HAc .... These low values are comparable to those reported for Acinetobacter calcoaceticus by Ohtake et al. (1985) and for an isolated Gram-positive coccus by Ubukata and Takii (1994), but lower than data obtained from poly-P activated sludge mixed cultures. On average, the amount of P-PO4 released per gram of biomass during the anaerobic phase was around 4 mg, in accordance with values of 2.5 ~ , mg reported for Acinetobacter calcoaceticus by Ohtake et al. (1985). Biomass phosphorus fractionation demonstrated the presence of significant amounts of polyphosphates. The cell culture showed a relevant difference in polyphosphate content (5 mg P.g-~ VSS) between

the end of the anaerobic phase and the end of the aerobic one (Table 2). It is possible to observe that poly-P content drastically increases during the anaerobic/aerobic tests. These figures confirm the previous values on phosphorus release reported above and are also similar to the values reported by Ohtake et al. (1985). In anaerobic conditions, the specific PO4-P release rate ranged from 1.7 to 3.6 mgP-PO4.g -t VSS.h -~, with a pH near neutrality (pH = 7.5). After about 5 h, with only slight differences among different tests, the rate slows down to values below 0.1 mg PO4P.g-] VSS.h-L During all tests, the specific rate of PO4-P release did not show statistically significant correlation with acetic acid and phosphorus concentrations in the media, even if the highest values were obtained with the highest substrate concentration and

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185

190

195

200

215

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Fig. 9. Phosphorus release vs acetic acid uptake during one of the P release tests.

Biological P removal by Lampropedia spp. Table 2. Phosphorus fractionation in Lampropedia cells exposed to alternate aerobic/anaerobic conditions, compared to cells before alternate exposure Polyphosphate Other cellular phosphorus phosphorus Phase (mgP.g -I VSS) (mgP.g n VSS) End of anaerobic 2.42 I 1.42 End of aerobic 7.42 22.56 Difference 5 11.14 Not exposed cells 1.6 11.4

the lowest PO4-P c o n c e n t r a t i o n at the beginning of each test. O n the contrary, the specific acetic acid removal rate showed a significant correlation to substrate concentration. The highest value obtained was 70 m g H A c . g -~ V S S . h -~. P h o s p h o r u s uptake u n d e r aerobic conditions showed a specific rate from 0.25 to 0.54 m g PO4-P.g -1 V S S . h -1. This leads to a n a c c u m u l a t i o n o f p h o s p h o r u s in the biomass up to 3.5% o f VSS.

CONCLUSION E n h a n c e d biological p h o s p h a t e removal is extensively reported in literature o n mixed culture, but very few works h~Lve been successfully performed in pure culture, a n d most o f them do not report complete data o n the whole uptake/release mechanism o f acetate a n d p h o s p h o r u s . In the present work, a bacterium belonging to the genus Lamproped,~a isolated from E B P R activated sludge has been sl:udied for its capacity to perform e n h a n c e d biological p h o s p h a t e removal. Acetate uptake, P H B f o r m a t i o n a n d p h o s p h a t e release in anaerobic conditions as well as p h o s p h a t e u p t a k e a n d P H B c o n s u m p t i o n in aerobic conditions have been recorded. Stoichiometric a n d kinetic values were usually lower t h a n those o b t a i n a b l e in mixed culture, but cell p h o s p h o r u s fractionation analyses d e m o n strated a cyclic increase a n d decrease of the poly-P fraction from aerobic to anaerobic conditions. All this evidence allo~'s us to conclude that Lampropedia spp. can be classified as a poly-P accumulating microorganism. The easiness of its recognition a n d its isolation in pure culture m a k e Lampropedia spp. a candidate bacterium for future pure culture experiments. Its study m i g h t contribute to a better c o m p r e h e n s i o n of the biochemical mechanism involved in e n h a n c e d biological p h o s p h a t e removal processes. Acknowledgements--The authors wish to thank B. Biavati for his useful suggestions, and A. Tarozzi and S. Gemelli of the Montecatini Environment Research Center for their collaboration in this research.

REFERENCES

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