Biotechnology Research Centre, La Trobe University, Bendigo, 3552 Victoria, Australia1
Murray-Darling Freshwater Research Centre and CRC for Freshwater Ecology, Albury, 2640 NSW, Australia2
Author for correspondence: Robert J. Seviour. Tel: +61 35 444 7459. Fax: +61 35 444 7476. e-mail: r.seviour{at}latrobe.edu.au
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ABSTRACT |
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Keywords: enhanced biological phosphorus removal (EBPR), G-bacteria, fluorescence in situ hybridization/microautoradiography (FISH/MAR), Rhodocyclus
Abbreviations: EBPR, enhanced biological phosphorus removal; FISH, fluorescence in situ hybridization; GAB, glycogen-accumulating bacteria; MAR, microautoradiography; PAB, phosphorus-accumulating bacteria; P/C, phosphorus/carbon; PHA, poly ß-hydroxyalkanoate; PHB, poly ß-hydroxybutyrate; SBR, sequencing batch reactor
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INTRODUCTION |
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As well as the PAB, other physiological groups of bacteria can dominate aerobicanaerobic activated sludge systems and these include the so-called G-bacteria, a term used here to describe a morphotype of cocci usually arranged in tetrads and/or clusters. These G-bacteria are now known to be phylogenetically diverse (Seviour et al., 2000 ). Cocci fitting this morphotype description and seen in large numbers in some aerobicanaerobic activated sludge systems have been referred to as glycogen-accumulating bacteria (GAB) (Mino et al., 1998
), even though their abilities to accumulate glycogen have not been determined. Heterotrophic lactic acid bacteria have also been detected as dominating populations in SBR systems fed a mixture of glucose and acetate (Kong et al., 2001
). Understanding the possible relationships between the PAB and G-bacteria has attracted interest, since G-bacteria have been considered responsible, in some studies, for the failure of EBPR systems by out-competing the PAB for substrates in the anaerobic phase of these aerobicanaerobic systems (Cech & Hartman, 1993
). However, which of the different G-bacteria, if any, might be responsible for EBPR failure is not known (Seviour et al., 2000
). The original G-bacteria candidate of Cech & Hartman (1993)
, Amaricoccus kaplicensis (Maszenan et al., 1997
), is absent from aerobicanaerobic SBRs fed a mixture of glucose and acetate and showing no EBPR (Kong et al., 2001
); pure cultures of this organism also fail to demonstrate any ability for anaerobic glucose and acetate assimilation (Falvo et al., 2001
; Y. H. Kong & R. J. Seviour, unpublished data).
Liu et al. (1997) proposed that the GAB and PAB can coexist in some aerobicanaerobic SBR systems. Their relative dominance was determined by the phosphorus/carbon (P/C) ratios in the influent feed. Higher P/C ratios favoured the PAB, whereas lower ones favoured the GAB. These bacteria were not identified, although cells resembling G-bacteria dominated these communities upon microscopic examination. No molecular methods were used in the study of Liu et al. (1997)
to analyse the composition of the two different microbial communities, hence the identities of the PAB and GAB were not resolved. Furthermore, data linking the chemical changes occurring in the reactors fed different influents to specific populations in the biomass were not obtained. In this study, fluorescence in situ hybridization (FISH)/microautoradiography (MAR) was used to elucidate the relationships between the structure and function of bacterial communities within aerobicanaerobic SBR systems (Gray & Head, 2001
), to resolve the composition of the PAB and G-bacterial populations and to understand their roles in anaerobic substrate assimilation and storage-polymer production.
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METHODS |
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Anaerobic batch trials.
These were performed, as described previously (Kong et al., 2001 ), to measure the capacity of the biomass taken from the SBR run with different feed P/C ratios for anaerobic glucose assimilation. Samples were taken for analyses and handled and stored in the same way as those used for samples from the SBR cycles as described previously (Kong et al., 2001
).
Chemical analyses of samples.
All of the methods used for the chemical analyses of samples taken from the SBR and anaerobic batch trials, including those for poly ß-hydroxyalkanoate (PHA) and glycogen, were as described previously (Kong et al., 2001 ).
FISH of biomass samples.
The 16S-rRNA-targeted oligonucleotide probe sequences used for FISH in this study are listed in Table 1. The conditions of stringency and permeabilization procedures, if required, were those described originally for each probe. Biomass samples for FISH were fixed in paraformaldehyde (Amann, 1995
) or in absolute ethanol (Roller et al., 1994
) for Gram-negative and Gram-positive bacteria, respectively. All FISH procedures were performed according to Amann (1995)
.
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Identification of bacterial populations accumulating PHA.
The methods used to identify cells in biomass samples taken from the end of the SBR anaerobic cycle that were capable of accumulating poly ß-hydroxybutyrate (PHB) were those described by Liu et al. (2001) .
Staining methods and microscopy.
The Gram and methylene blue staining protocols of Lindrea et al. (1999) were used in this study. Stained samples were examined under a Nikon microscope (Eclipse E800). All specimens for FISH were mounted in Vectashield (Vector Laboratories) and viewed under a Nikon epifluorescence microscope with the appropriate filter blocks for the fluorochromes used. Colour photographs and Nomarski photographs were taken automatically with a Nikon V-TP micrography module.
Enumeration of bacteria after FISH probing.
Digital images for quantifying the bacteria that responded positively to probes were captured with a CompuScope CCD 1600 12HS digital camera installed on a Nikon microscope, which was used with an oil immersion objective lens. The digital camera was controlled by ProControl software (High Performance 32 bit Scientific Image Processing), and the images obtained were prepared in Adobe Photoshop before being analysed with NIH image (Kong et al., 2001 ). Cells fluorescing with a given FISH probe were expressed as a percentage of the total area of bacteria giving a signal with the EUB 338 probe of Amann et al. (1995)
or the EUB mix probes of Daims et al. (1999)
in the same field. The percentage of the total number of cells detected with 4',6-diamidino-2-phenyindole (DAPI) that fluoresced with the EUB 338 and the EUB mix probes was very similar in these biomass samples, being 96·1% (±5%) and 96·6% (±9·8%), respectively (n=25). At least eight randomly selected fields containing an estimated more than two thousand cells in total were examined for each sample and probe. The numbers obtained were averaged and expressed as a percentage of the total DAPI-stained bacteria in the microbial community. An allowance was made for the larger
- and
-proteobacterial G-bacteria in these determinations, as detailed by Kong et al. (2001)
.
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RESULTS |
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DISCUSSION |
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The microbial community composition in the SBRs operated with acetate as sole carbon source differed markedly from those seen in SBRs fed a mixture of glucose and acetate and showing no EBPR (Fig. 2), which were described by Kong et al. (2001)
. In the SBRs of Kong and colleagues, the tetrad-forming cocci or G-bacteria dominated, whereas the ß-Proteobacteria, which were dominant in the three EBPR biomasses described in this study, were numerically insignificant (Kong et al., 2001
). Furthermore, two of the major populations of bacteria detected by Kong et al. (2001)
in the SBRs showing no EBPR i.e. the unidentified lactic-acid-producing low-G+C Gram-positive bacteria and the high-G+C Gram-positive bacteria, many of which responded to the probe for M. glycogenica, a known glycogen-accumulating bacterium (Shintani et al., 2000
) either were not seen at all in the EBPR communities examined in this study or were only detected in small numbers.
The FISH/MAR data presented here strongly support the view that the Rhodocyclus-related bacteria are the major contributors to the EBPR recorded in SBRs and that they behave in accordance with current biochemical models. Their abundance at each feed P/C ratio correlated well with both biomass phosphorus content and anaerobic phosphorus release (Fig. 4). These results, together with a failure to detect any Acinetobacter cells using FISH, add to the increasing body of data (Bond et al., 1999
) that suggest the major PAB in EBPR activated sludge systems, including full-scale plants (Crocetti et al., 2000
; Y. H. Kong & R. J. Seviour, unpublished data), regardless of their geographical location or operational configuration, belong to the Rhodocyclus group. The FISH data also demonstrate that the Rhodocyclus-related PAB are pleiomorphic members of phylogenetically closely related populations, since all cells, regardless of their size, fluoresced with a single probe designed for this group (Crocetti et al., 2000
).
FISH/MAR results suggested that the Rhodocyclus-related bacteria were not the only PAB in our EBPR communities, since some of the high-G+C Gram-positive bacteria could also accumulate phosphorus aerobically. However, numerically they contributed less to these microbial communities than in other (Bond et al., 1995 , 1999
; Kämpfer et al., 1996
; Kawaharasaki et al., 1999
; Liu et al., 2001
; Wagner et al., 1994
), if not all other, similar EBPR communities (e.g. Hesselmann et al., 1999
). None of the Gram-positive PAB detected in this study appeared to be Tetrasphaera spp., which have been considered as potential PAB (Maszenan et al., 2000a
) and were detected by FISH in an SBR EBPR community (Liu et al., 2001
). Hence, the identity of the high-G+C Gram-positive PAB present in our system remains unknown.
Whereas the numbers of Rhodocyclus-related PAB were lower when the SBR was operated at the low P/C feed ratio, the numbers of -Proteobacteria and to a lesser extent
-Proteobacteria, most of which possessed the G-bacteria morphotype, increased (Fig. 2
). The highest P/C ratio at which EBPR still occurred here was 1:10; above this ratio EBPR failed (Y. H. Kong, unpublished data). Thus, high P removal was achieved even when ca 15% of the total bacterial cells in the community were the
-proteobacterial G-bacteria, which suggests that PAB and G-bacteria can co-exist in EBPR systems. This agrees with observations made by Liu et al. (1996
, 1997
). It also questions whether these G-bacteria necessarily indicate failing EBPR, although this question must remain unanswered until the identities of the G-bacteria in EBPR systems are satisfactorily resolved. It is not clear from our data whether the
-proteobacterial G-bacteria are a single phylogenetically homogeneous population or if they comprise a range of related but distinct bacteria. If the latter is true, then the changes seen in their numbers with different P/C ratios may reflect a shift in the different populations within this group.
Equally importantly, as the numbers of -proteobacterial G-bacteria increased with a lowered P/C ratio feed, the biomass glycogen levels also increased (Table 2
). For example, in Runs P1, P2 and P3 the percentages of
-Proteobacteria present (calculated from FISH analysis) were ca 15, 23 and 48%, respectively, with biomass glycogen levels (expressed as a percentage of the total biomass content) of ca 8, 12 and 17% (w/w), respectively. The
-Proteobacteria were at the limit of detection in the biomasses from Runs P1 and P2, where glycogen levels were also high, so these are unlikely to be the main bacteria responsible for biomass glycogen storage. The same argument would also apply to the high-G+C Gram-positive bacteria, including the known glycogen-accumulating bacterium M. glycogenica (Shintani et al., 2000
) and the cocci of Crocetti et al. (2001)
neither of which were detected here by FISH. Furthermore, although the biochemical models suggest a role for glycogen synthesis in the PAB (Mino et al., 1998
), the Rhodocyclus-related PAB were considered unlikely to be responsible for the increase in the glycogen content of the biomass, which corresponded to a drop in their numbers and an overall decrease in EBPR (Fig. 4
). Thus, the evidence strongly suggests that it is the
-proteobacterial G-bacteria that are the major GAB responsible for the measured increases in biomass glycogen levels. However, from the FISH data neither Amaricoccus spp. nor Defluvicoccus vanus, previously isolated and characterized
-proteobacterial G-bacteria (Maszenan et al., 1997
; A. M. Maszenan, unpublished data), were numerically important. It is possible that some of the
-proteobacterial G-bacteria seen in such large numbers in these communities are members of the genus Amaricoccus that do not respond to the FISH probes used here for members of this genus (Maszenan et al., 2000b
), but instead represent undescribed bacteria (M. Beer, unpublished data). As well as being phylogenetically diverse, the
-proteobacterial G-bacteria seen in these aerobicanaerobic SBR systems also appear to be physiologically different. None assimilated glucose anaerobically, yet most of those seen in the SBR communities with no EBPR could assimilate both glucose and acetate (Kong et al., 2001
).
The decrease in Rhodocyclus-related PAB and the corresponding increase in the - and
-Proteobacteria at the low P/C feed ratio was not accompanied by any discernible change in the acetate uptake rates of these communities (Table 2
). These results contrast with those of Liu et al. (1996)
, who showed a much lower acetate uptake rate in a G-bacteria-dominated biomass than in one where the PAB dominated. They suggested that acetate uptake was an important determinant as to which of these populations ultimately dominated. This result might suggest that their G-bacteria are different to those seen in this study. However, if the acetate uptake rate is not a key selective factor in determining the relative abundances of the PAB and G-bacterial populations, as seems to be the case from these data, then the question as to what is remains unanswered.
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ACKNOWLEDGEMENTS |
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Received 21 December 2001;
revised 27 March 2002;
accepted 4 April 2002.