Enhanced biological phosphorus removal (EBPR) activated sludge has been applied for decades to economically reduce wastewater effluent phosphorus (P) levels. Using molecular techniques, as yet uncultured organisms related to “Candidatus
Accumulibacter phosphatis” (hereafter referred to as Accumulibacter) are frequently identified as the major polyphosphate accumulating organisms (PAOs) in lab-scale EBPR systems fed with acetate or propionate (Hesselmann et al., 1999
; Crocetti et al., 2000
) and some full-scale EBPR facilities (Zilles et al., 2002
; Kong et al., 2004
; He et al., 2008
). A rich diversity of Accumulibacter can be organized into two main Types (I and II), using the polyphosphate kinase gene (ppk1
) as a genetic marker. Each Type is comprised of several distinct clades (He et al., 2007
; Peterson et al., 2008
). Lab-scale acetate-fed reactors studied to date have exclusively contained clades IA, IIA, and IID (Peterson et al., 2008
; Wilmes et al., 2008
). Accumulibacter has been reported to be able to use nitrate as an electron acceptor to achieve anoxic P-uptake, based on work conducted on activated sludge communities (Dabert et al., 2001
; Zeng et al., 2003
; Kong et al., 2004
). However, a metagenomic analysis of clade IIA-enriched EBPR sludges indicated a lack of any clearly identifiable respiratory nitrate reductase homolog in Accumulibacter (Garcia Martin et al., 2006
). Recently, Carvalho and colleagues (2007)
reported varying nitrate reduction capabilities in two lab-scale reactors fed with acetate and propionate, respectively, and containing two morphologically distinct Accumulibacter populations. Further work determined that Accumulibacter populations established in a lab-scale bioreactor under nitrite reducing conditions could not reduce nitrate (Guisasola et al., 2009
). The differences in nitrate reduction activities suggested finer phenotypic and ecological differences among members of the Accumulibacter lineage. Therefore, we hypothesized that clade IA can use nitrate as electron acceptor for anoxic P-uptake, while clade IIA cannot.
Two lab-scale acetate-fed EPBR sequencing batch reactors (R1 and R2), with good and stable P removal performance were operated in parallel under essentially identical conditions. R1 was inoculated from a local EBPR wastewater treatment plant, and R2 was inoculated from R1. Detailed operational conditions were described elsewhere (He et al., 2006
). Briefly, the influent contained (mg/L total feed): CH3
O (425); casamino acids (31), yeast extract (8.6), NaH2
O (62.3) and other mineral salts to achieve the chemical oxygen demand (COD) to P ratio of 14. The pH was controlled at 7.0–7.5 and allyl-thiourea was added to the reactors at 4 mg/L to inhibit nitrification (Hooper and Terry, 1973
). As a result, both reactors had not been exposed to nitrate prior to this study.
The relative abundance of Accumulibacter was estimated by 16S rRNA-targeted fluorescent in situ hybridization (FISH) with the PAOMIX probes (PAO 462, PAO 651 and PAO 846) (Crocetti et al., 2000
) and counterstaining total cells with 1 μg/ml of 4′6-diamidino-2-phenylindole (DAPI). The slides were viewed and a total of 10 images per sample were captured using a Zeiss Axioplan 2 epifluorescent microscope (Carl Zeiss, Thornwood, N.Y.) equipped with an Olympus DP70 color camera (Center Valley, PA). The images were analyzed using DP Controller v2.2.1 (Olympus, Center Valley, PA). To quantify the % Accumulibacter of total DAPI-stained cells, the images for each sample were manually counted. Both reactors were enriched (>50% of total DAPI-stained cells) in Accumulibacter. Originally, Accumulibacter clades were defined based on the ppk
genes (He et al., 2007
). Later, the 16S rRNA sequence of clade IIA was identified from its finished genome, which also contains the clade IIA ppk
(JGI img/m: http://img.jgi.doe.gov/cgi-bin/m/main.cgi
). Therefore, in addition, two probes (Acc-I-444 and Acc-II-444) () were designed to differentiate between these two clades in our reactors. They target the same site in the 16S rRNA, and are different from each other by two adjacent nucleotides. Based on an in silico prediction of formamide denaturation profiles according to Yilmaz and Noguera (2007)
, the melting formamide concentrations are approximately 35% and 42% for Acc-I-444 and Acc-II-444, respectively. Accordingly, experiments were performed with 35% formamide, to maximize specificity while gaining sufficient signal intensity from hybridized cells. When applied independently, a non-labeled competitor probe targeting the other Type was added to eliminate cross hybridization. shows the FISH images hybridized by the two probes and the PAOMIX on the same sample.
Probes designed to distinguish Accumulibacter clade IA from IIA
Figure 1 FISH images of sludge from reactor enriched in Accumulibacter clade IIA (R2). Images in panels (A) and (B) were collected in the same field, and panels (C) and (D) were collected in the same field. Cells in panels (B) and (D) were hybridized with PAOMIX (more ...)
To determine the relative abundance of Accumulibacter clades IA and IIA accurately, quantitative real-time PCR (qPCR) with Accumulibacter clade-specific primers targeting ppk1
was performed as described (He et al., 2007
). The distribution patterns of Accumulibacter in the two reactors were correlated (correlation coefficient = 0.75, p<0.001) from 37 paired data points collected from R1 and R2 during over two years of operation before October 2007, despite seemingly random shifts in the Accumulibacter community composition. However, for about 3 months after December, 2007 R1 was dominated by clade IA, and R2 was dominated by clade IIA, although they achieved equally good P removal performance (data not shown). The relative abundances of clades IA and IIA measured by ppk1
-qPCR were qualitatively verified by FISH with probes Acc-I-444 and Acc-II-444. No explanation as to why the two reactors exhibited very different Accumulibacter composition has been determined, nor do we have the ability to intentionally change the operational conditions to enrich for either clade. However, the contrasting clade distribution in the two reactors gave the opportunity to evaluate the ability of each clade to denitrify, and therefore test a previously published hypothesis that the two clades are physiologically different (He et al., 2007
During the period with differential enrichment, batch tests were conducted to investigate whether the two sludges could couple nitrate reduction with P-uptake (). Briefly, 500 mL of sludge was collected from each bioreactor at the end of the anaerobic phase when soluble P was at its maximum, to establish four batch reactors, each consisting of 250 mL of sludge. Nitrogen gas was sparged into all four batch reactors to maintain an oxygen-free environment. Sodium nitrate was added to one batch reactor derived from R1 (BR1-N) and one batch reactor derived from R2 (BR2-N) to reach an initial nitrate concentration of 25 mg/L as nitrogen (NO3
-N). The remaining two batch reactors (BR1-C and BR2-C) were maintained under anaerobic conditions as controls. During the 24-h incubation, pH was maintained at 7.3±0.1 and dissolved oxygen (DO) was measured at intervals to assure that DO was below detection in all batch experiments. Soluble inorganic phosphate (Pi
) (by modified ascorbic acid method 4500-P E; (APHA et al., 1995
)), acetate, NO3
-N, and NO2
-N [by a Shimadzu high performance liquid chromatography (Kyoto, Japan) equipped with an Alltech Previal Organic Acid Column (Deerfield, Illinois) and a photodiode array detector set at 210 and 214nm] were monitored at regular intervals. Total suspended solids (TSS) and volatile suspended solids (VSS) were measured to estimate biomass concentration (APHA et al., 1995
) at the end of the batch tests.
Summary of Accumulibacter distribution and batch test results
In the controls we observed secondary P-release instead of P-uptake, and the release was not coupled to acetate uptake since acetate was below detection at the start of the batch test. This phenomenon was documented previously (Stephens and Stensel, 1998
). In the nitrate-amended batch reactors (BR1-N and BR2-N), both sludges consumed nitrate (), but a much higher effective nitrate consumption rate was observed in BR1-N across the entire experiment (Samples BNA in ). No nitrite accumulation was observed during the batch tests, unlike as previously observed by Carvalho and colleagues (2007)
. Net P-uptake only occurred in BR1-N (), with both initial nitrate consumption and P-uptake rates comparable to those obtained by Carvalho and colleagues (2007)
. BR2-N did not exhibit a net P-uptake ( and ). However, the differences between BR2-N and BR2-C suggested that P-uptake and secondary P-release occurred simultaneously, probably mediated by the two respective populations of Accumulibacter. The lack of net P-uptake in BR2-N was not likely due to the limitation of carbon storage, since P-uptake occurred rapidly when oxygen was introduced after the 24-hour incubation (data not shown). Nearly identical results were obtained during another set of experiments conducted under similarly enriched conditions (no statistically significant difference in rates) (data not shown).
Figure 2 Batch test results using sludge from R1 and R2 one day before (BNA) and one day after nitrate acclimation (ANA), showing P profiles in the nitrate-amended (■) and control (□) batches, and NO3-N profiles in the nitrate-amended batch (▲). (more ...)
Previous studies suggested that nitrate reduction rates were higher following acclimatization with nitrate (Zeng et al., 2003
; Carvalho et al., 2007
). To evaluate this possibility, batch tests were also conducted on sludges that had been exposed to nitrate by addition of 4 mg/L NO3
-N into both reactors at the beginning of each anaerobic phase for four consecutive cycles (24 hours) prior to the batch tests. This concentration was chosen because we had previously determined that 4 mg/L NO3
-N did not affect reactor P removal performance (unpublished data). To minimize the effect of potential microbial community composition change, we compared the batch tests one day before (Samples BNA) and one day after the nitrate acclimatization (Samples ANA). Initial P-uptake rates in both BR1-N and BR2-N batch tests were found to increase significantly relative to the BNA experiments, after the acclimatization (p < 0.01 for both BR1-N and BR2-N, respectively) ( and ). Indeed, BR2-N exhibited a small net P-uptake by the end of the 24 hour testing period. Interestingly, the initial P-uptake rates for BR1-N and BR2-N after acclimation were not statistically different from one another. However, the uptake profile for BR2-N appeared to be strongly biphasic with a significantly shallower slope after 6 hours ().
The assumption that clade IIA is not able to reduce nitrate is further supported by the fact that nitrate consumption continued throughout the entire 24 hour cycle period for clade IA-dominant sludge () while nitrate uptake rate decreased during phase 2 in clade IIA-dominant sludge () indicating a limiting nitrate reduction capacity. We hypothesize that the period of more rapid P-uptake and nitrate reduction during phase 1 was controlled by the clade IA population that quickly depleted its PHA reserves, upon which P-uptake and nitrate reduction slowed substantially (phase 2).
Taking the genomic and experimental evidence together, we conclude that members of clade IA, but not clade IIA, can use nitrate as an electron acceptor for P-uptake. It is possible that clade IIA can use nitrite generated by clade IA as a denitrification intermediate for P-uptake, because genes enabling nitrite reduction to N2
were found in the genome of clade IIA (Garcia Martin et al., 2006
), but how significant this contributed to the overall P-uptake cannot be clearly assessed by this study.
As noted in , roughly 20–30% of the microbial community in our bioreactors was not Accumulibacter. While it is possible that some of this flanking community can reduce nitrate, the operational conditions in the bioreactor likely excluded a significant contribution of the flanking community to nitrate reduction since all of the readily available carbon (acetate) was sequestered during the previous anaerobic phase. This ability to sequester carbon anaerobically provides Accumulibacter a competitive advantage activated sludge systems (Oehmen et al., 2007
). We cannot rule out the presence of glycogen accumulating non-polyphosphate organisms capable of sequestering acetate as PHA and using it to fuel denitrification, but high calculated ratios of anaerobic P-release to acetate-uptake in our systems (0.61 ± 0.02 mmole-P/mmole-C) provide strong evidence that Accumulibacter sequestered nearly all of the acetate (Schuler and Jenkins, 2003
The observed difference in anoxic P uptake supports the previously proposed hypothesis that different Accumulibacter clades have different metabolic capabilities (Carvalho et al., 2007
; He et al., 2007
). Therefore, we emphasize the importance of studying both genetic and physiological differences among different Accumulibacter populations. It also confirms the lack of nitrate reduction capability of clade IIA, which was inferred from the metagenomic analysis (Garcia Martin et al., 2006
). The hypothesis that some Accumulibacter clades can denitrify, while others cannot, resolves conflicting observations about Accumulibacter’s denitrification capabilities previously reported in the literature (Kong et al., 2004
; Freitas et al., 2005
; Carvalho et al., 2007
). A better understanding of Accumulibacter ecophysiology will help to better explain and predict the clade distribution in EBPR systems, and provides important information about whether or not simultaneous P and total nitrogen removal can be achieved under certain Accumulibacter community compositions.