University of Helsinki, Department of Applied Chemistry and Microbiology, PO Box 56, FIN-00014 University of Helsinki, Finland1
Helsinki University of Technology, Laboratory of Environmental Engineering, PO Box 6100, FIN-02015 HUT, Finland2
Author for correspondence: Hannes Melasniemi. Tel: +358 9 4513845. Fax: +358 9 4513856. e-mail: hannes.melasniemi{at}hut.fi
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ABSTRACT |
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Keywords: biological-phosphate removal, polyphosphate, activated sludge, yeast spores
Abbreviations: BOD7, seven-day biological oxygen demand; BPR, biological-phosphate removal; COD, chemical oxygen demand; DAPI, 4',6-diamidino-2-phenylindole; PAO, polyphosphate-accumulating organism; polyP, polyphosphate; UCT, University of Cape Town
a Present address: University of Helsinki, Department of Biosciences, Division of General Microbiology, PO Box 56, FIN-00014 University of Helsinki, Finland
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INTRODUCTION |
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Acinetobacter spp. are so far the best known polyP-accumulating bacteria isolated, and for several years Acinetobacter was considered as the principal PAO in BPR. Although polyP accumulation in laboratory-grown Acinetobacter is well established, the significance of this bacterial genus in genuine waste water treating BPR processes has, however, been challenged (Cloete & Steyn, 1988 ; Auling et al., 1991
; Wagner et al., 1994
; Tandoi et al., 1998
). Several other bacteria capable of polyP accumulation have been isolated from BPR sludge (Lötter & Murphy, 1985
; Streichan et al., 1990
; Nakamura et al., 1995
; Stante et al., 1997
). Even though BPR in activated sludge has been known as a phenomenon for 40 years and there have been working full-scale BPR plants for about 20 years, the roles of different bacteria are still obscure (Toerien et al., 1990
; van Loosdrecht et al., 1997
; Mino et al., 1998
), as is the identity of the PAOs involved.
In a previous study (Melasniemi et al., 1998 ) it was shown that acinetobacters did not contain significant amounts of polyP in a UCT (University of Cape Town)-type nutrient-removal activated-sludge process treating municipal waste water. Instead, the stainable polyP in the sludge under process conditions was found almost exclusively in microbial cells corresponding to the descriptions of the grape-like clusters forming PAO so often seen to predominate in real waste water treating BPR processes (Fuhs & Chen, 1975
; Buchan, 1981
, 1983
; Duncan et al., 1988
; Beacham et al., 1990
; Streichan et al., 1990
; Jenkins et al., 1993
). In the present study the organism was assessed in situ in the sludge using differential microscopic methods. Results reported here suggest that the predominant PAO in the process studied was not a bacterium at all.
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METHODS |
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Microscopy and differential stainings.
Fluorescence and phase-contrast microscopy were done with an Olympus AX70 Provis microscope fitted with UV and U-MNG filter blocks and 60x and 100x objectives. Bright-field microscopy was with an Olympus BH-2 microscope fitted with a 100x objective. Slides on Fujichrome 100 were digitized with a Polaroid SprintScan scanner and an Adobe Photoshop 3.0 program was used to handle the images.
PolyP-containing cells were visualized with 4',6-diamidino-2-phenylindole (DAPI, 50 µg ml-1; Allan & Miller, 1980 ) for fluorescence microscopy and with toluidine blue (colour index 52040; Drews, 1983
) for bright-field microscopy. Samples for toluidine blue staining were heat fixed before staining and destained afterwards for 12 min with 1% H2SO4 to reduce staining of the sludge background. Gram staining was done by the rapid method described by Hendrickson & Krenz (1991)
.
Calcofluor white M2R (colour index 40622, Sigma) was used as a stain for ß-1,4-hexapyranose polysaccharides essentially as described by Pringle (1991) . Calcofluor was dissolved by adding 20 µl 0·1 M NaOH to 1 ml of calcofluor suspension (1 mg ml-1) in water, to obtain a solution with a pH of approximately 8. Calcofluor solution (400 µl) was added to sludge sample (200 µl mixed liquor) and the mixture was incubated at room temperature (~22 °C)
30 min. The sludge was centrifuged with a microcentrifuge, washed five times with 1 ml deionized water, resuspended in 200 µl water and inspected by fluorescence microscopy using UV excitation.
Tetramethyl rhodamine-conjugated concanavalin A (Molecular Probes) was used to stain cell-wall mannans essentially as described by Tkacz et al. (1971) . The sludge sample (200 µl mixed liquor) was centrifuged and resuspended in 180 µl 1 M NaCl and 20 µl concanavalin A conjugate stock solution (1 mg ml-1) was added. After 30 min incubation at room temperature the sludge was centrifuged, washed twice with 1 ml 1 M NaCl, resuspended in 200 µl water and inspected by fluorescence microscopy using green excitation.
Antibiotic susceptibility.
The minimal Acinetobacter enrichment medium described by Towner (1992) was modified by replacing the original mineral base with the trace element solution of Kotai (1971
; 1 ml l-1) and raising the pH of the medium to 7·0. The medium was used with addition of ampicillin (1 g l-1) or cycloheximide (50 mg l-1), or without antibiotics. The medium (5 ml) was inoculated with sludge (0·5 ml mixed liquor) and the cultures were incubated at 28 °C shaken at 150 r.p.m. Changes in the sludge were monitored microscopically over several days.
Cell-wall digestion.
Sludge (1 ml mixed liquor) was stained for polyP with DAPI, washed with Tris/HCl buffer (25 mM, pH 7·0) and resuspended in 1 ml of the same buffer. Lysozyme from egg white (Sigma) or lysing enzymes from Trichoderma harzianum (Sigma) were added to 0·5 ml aliquots (final concentrations 26 and 16 mg ml-1, respectively) and the mixtures were incubated at room temperature for 6 and 2 d, respectively. Changes in the sludge were monitored microscopically.
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RESULTS |
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Morphological transformation
When fresh sludge was transferred to liquid acetate minimal medium containing ampicillin, the original PAO cells tightly packed in the grape-like clusters were converted to considerably bigger cells containing polyP (Fig. 3). The transformation started unobtrusively and asynchronously at various points on the edges of the clusters, but after a few days the clusters of the principal PAO had been replaced by clusters of the still bigger cells. Other parts of the sludge seemed to remain fairly unchanged and to retain the original flock structure. The length of the cells produced by this morphological transformation was approximately 5 µm or more, but less than 10 µm, and the appearance of the cells was that of vegetative yeast cells (Fig. 3
). If ampicillin was replaced by cycloheximide, sludge flock structure disintegrated, the principal PAO clusters vanished, and there was an abundant growth of free bacteria, many containing polyP (Fig. 4
). Finally, if both antibiotics were omitted, no striking changes were observed. Initial principal PAO cell clusters, although showing some swelling and staining less intensely for polyP, could still be found even after incubation for a month (not shown).
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DISCUSSION |
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Established PAO characteristics match with fungal spores
The PAOs mediating BPR have been conventionally thought of as polyP-accumulating bacteria. There is, however, no a priori reason why BPR had to rely (solely) on bacteria. The ability to accumulate polyP intracellularly is common among different types of micro-organisms (Kulaev, 1979 ). Bacteria certainly are numerically predominant in activated sludge, but higher organisms, algae, fungi, protozoa and invertebrates are also known to occur in the sludge.
Cells of the principal PAO were the largest cells abounding in the sludge. Yeast cells, even in spore form, are larger than bacterial cells, and numerous yeast species live in aquatic environments (Hagler & Ahearn, 1987 ). Although typical yeast spores are bigger than the principal PAO cells, there are examples of fungal spores in the size range 12 µm (Kreger-van Rij & Veenhuis, 1974
; Asano et al., 1999
). The cells of the principal PAO occurred in clusters as fungal spores often do, simply because of their mode of formation. In addition, cell-to-cell adhesion and flocculation are usual among yeasts (Calleja, 1987
). The oval-to-coccoid form of the PAO cells is typical of fungal spores, as is also their tendency towards swelling.
The first organism shown to contain polyP was the bakers yeast Saccharomyces cerevisiae (Liebermann, 1888 ). PolyP has since been found in several yeast species (Kulaev & Vagabov, 1983
). As accumulation of polyP and spore production serve the same goal, survival of the organism, it is not surprising that especially high polyP content in fungal cells has been found in the spores (Bajaj et al., 1954
). Vegetative growth and full-scale metabolism cease temporarily in sporulation and the fungus goes over to a stationary state with reduced metabolism. Reduced metabolism of the clustered PAO was reported by Beacham et al. (1990)
and reasons why polyP accumulation in BPR sludge, especially in a highly clustered PAO, can be expected to take place under stationary growth conditions were discussed by Melasniemi et al. (1998)
.
Cell surface with yeast attributes
The cell envelope of the principal PAO was not of the character typical of bacteria. The cell wall of the principal PAO stained anomalously in Gram staining and it was not affected by the enzyme lysozyme, which degrades the peptidoglycan of most bacteria. Weak staining by the predominantly chitin-binding fluorescent dye calcofluor (Pringle, 1991 ) suggested the presence of chitin in the principal PAO. Chitin is a common constituent of fungal cell walls, being absent in bacteria. In the cell wall of S. cerevisiae spores the deacetylated analogue of chitin, chitosan, is shielded from efficient staining (Briza et al., 1988
), whereas in asexual spores of Mucor rouxii the relative amount of chitin is low, only one-quarter of that in the vegetative cells (Bartnicki-Garcia, 1968
). The presence of chitin-like ß-1,4-hexapyranose polysaccharide in the cell wall of the principal PAO was corroborated by the fact that the PAO cells were converted to spheroplasts by lysing enzymes, containing chitinase, cellulase and protease. This enzyme cocktail is commonly used for the digestion of yeast cell walls. The cell surface of the principal PAO also bound concanavalin A, a protein specifically binding to branched
-mannans and
-glucans (Tkacz et al., 1971
). Consequently, the principal PAO should have on its surface either or both of these polysaccharides, characteristically found on the surface of yeasts.
Morphological transformation to yeast-like cells
Short-chain fatty acids are thought to play a central role in BPR (van Loosdrecht et al., 1997 ; Mino et al., 1998
). The PAOs should thus be able to take up acetate and use it as a substrate for growth. In accordance with this, the principal PAO cells present in untreated sludge were transformed to considerably bigger cells with a different appearance, i.e. yeast spores germinated to produce vegetative yeast cells, when sludge was transferred to acetate minimal medium containing ampicillin. The size of the transformed PAO cells was clearly bigger than the size of any of the bacteria seen in the same medium containing cycloheximide, or any of the bacteria isolated from sludge of the same process in a previous study (Melasniemi et al., 1998
). Further evidence of the non-bacterial nature of the PAO is given by the fact that the transformation took place in the presence of a bacterial antibiotic (ampicillin) and did not take place in the presence of a yeast antibiotic (cycloheximide). In the presence of cycloheximide, yeast spores lost their polyP, died and disappeared, whilst at the same time diverse bacteria started to grow and synthesize polyP de novo. In a rich medium favouring bacterial growth (e.g. nutrient broth), sludge disintegrated and bacteria took over the culture even in the absence of cycloheximide (data not shown). The difficulty of perceiving the simultaneous degradation and synthesis of polyP in sludge grown further in the laboratory, together with the fact that Acinetobacter is favoured by acetate and forms polyP in culture, has probably been a major factor contributing to the birth of the Acinetobacter hypothesis of BPR.
Bacterial PAOs in BPR processes
Considering the undisputed fact that many bacteria are capable of polyP accumulation, the idea of the involvement of several bacterial PAO groups in BPR (Mino et al., 1998 ) might well be true. This opinion is based on the results of culture-independent studies (e.g. Wagner et al., 1994
; Bond et al., 1995
, 1999
; Kämpfer et al., 1996
; Kawaharasaki et al., 1999
), which have suggested ß-proteobacteria, Gram-positive bacteria with a high G+C content in their DNA and
-proteobacteria as PAOs. However, the salient role of any bacterial group as PAOs in BPR taking place at actual waste-water treatment plants has as yet not been clearly proven. Although one may perhaps justifiably expect to find PAOs in higher numbers in BPR than in non-BPR sludge, mere enrichment of a specific bacterial group under BPR conditions does not imply that these bacteria were PAOs. Methods based on nucleotide sequences only can only give information on the phylogenetic structures of the communities studied. PolyP accumulation must be shown by other means.
We are aware of only four studies where PAOs have been assigned to a specific bacterial group by combining phylogenetic in situ data with microscopic observation of polyP. Two of the studies (Kawaharasaki et al., 1999 ; Bond et al., 1999
) were based, however, on sequencing batch reactors fed in the laboratory with pure bacteriological culture media (synthetic waste waters), which, although dilute, were nonetheless totally soluble and of fixed formula. Only two of the studies (Wagner et al., 1994
; Kämpfer et al., 1996
) relate to processes (Phoredox and A/O, respectively) treating genuine waste water with an ever-fluctuating composition. In these studies metachromatic granules were found in cell morphotypes that bound the probe for high G+C bacteria. With the exception of the Microthrix parvicella morphotype, the high G+C morphotypes were, however, not described. In both cases polyP granules were especially evident in M. parvicella, a well-known filamentous polyP bacterium often associated with sludge bulking (Jenkins et al., 1993
) and often encountered in BPR processes.
Neither Wagner et al. (1994) nor Kämpfer et al. (1996)
reported polyP in the distinct grape-like clusters forming PAO that have so often been found at other BPR waste-water treatment plants to contain massive amounts of polyP. Thus, it seems that this PAO (or group of PAOs) has so far not been associated with any other bacterial group except (incorrectly) with Acinetobacter. The Acinetobacter hypotheses of BPR of the past two decades has become ever less attractive in recent years. However, the even more widely held presumption of the ubiquitously predominant role of prokaryotes, i.e. polyP bacteria, in BPR has not been challenged until now.
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ACKNOWLEDGEMENTS |
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Received 8 September 1999;
revised 22 November 1999;
accepted 9 December 1999.
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