Low Acetate Concentrations Favor Polyphosphate-Accumulating

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Low Acetate Concentrations Favor Polyphosphate-Accumulating Organisms over Glycogen-Accumulating Organisms in Enhanced Biological Phosphorus Removal from Wastewater Yunjie Tu and Andrew J. Schuler* Department of Civil Engineering, The University of New Mexico, Albuquerque, New Mexico 87131, United States S Supporting Information *

ABSTRACT: Glycogen-accumulating organisms (GAOs) are thought to compete with polyphosphate-accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) wastewater treatment systems. A laboratory sequencing batch reactor (SBR) was operated for one year to test the hypothesis that PAOs have a competitive advantage at low acetate concentrations, with a focus on low pH conditions previously shown to favor GAOs. PAOs dominated the system under conventional SBR operation with rapid acetate addition (producing high in-reactor concentrations) and pH values of 7.4−8.4. GAOs dominated when the pH was decreased (6.4−7.0). Decreasing the acetate addition rate led to very low reactor acetate concentrations, and PAOs recovered, supporting the study hypothesis. When the acetate feed rate was increased, EBPR failed again. Dominant PAOs and GAOs were Candidatus Accumulibacter phosphatis and Defluviicoccus Cluster 2, respectively, according to fluorescent in situ hybridization and 454 pyrosequencing. Surprisingly, GAOs were not the immediate causes of PAO failures, based on functional and population measurements. Pyrosequencing results suggested Dechloromonas and Tetrasphaera spp. may have also been PAOs, and additional potential GAOs were also identified. Full-scale systems typically have lower in-reactor acetate concentrations than laboratory SBRs, and so, previous laboratory studies may have overestimated the practical importance of GAOs as causes of EBPR failure.

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INTRODUCTION Enhanced biological phosphorus removal (EBPR), a modification of the activated sludge process, enriches for polyphosphate-accumulating organisms (PAOs) by cycling them through anaerobic and aerobic environments, thereby producing low effluent phosphorus (P) concentrations.1 In the anaerobic phase, PAOs take up volatile fatty acids (primarily acetate) and store them as polyhydroxyalkanoates (primarily poly-β-hydroxybutyrate (PHB)), with polyphosphate hydrolysis as a source of ATP and consequent release of orthophosphate from the cell. Glycogen is degraded to provide a source of reducing equivalents during the anaerobic phase. In the following aerobic phase, stored PHB is metabolized for growth and energy, and polyphosphate is replenished.2 Glycogen-accumulating organisms (GAOs) are thought to be a cause of EBPR failure because, under some conditions, they can out-compete PAOs for acetate uptake in the anaerobic phase of EBPR.3−7 GAOs utilize a similar metabolism to PAOs, but they do not accumulate polyphosphate.8,9 Conditions thought to favor GAOs include low pH values (less than approximately 7.25), warmer temperatures (greater than approximately 25 °C), P limitation, glucose addition, and longer solids residence times (SRTs).4,7,10−21 The role of GAOs in full-scale systems is unclear: although they are thought to be detrimental to EBPR, they are commonly noted in fullscale systems without EBPR failure.22−25 It has been suggested © 2013 American Chemical Society

that their presence may actually indicate well-functioning EBPR, since low effluent P concentrations indicates P limitation of PAO activity, allowing GAOs to grow on acetate present in excess of PAO requirements.26 This study tested the hypothesis that in-reactor (as opposed to influent) acetate concentration affects PAO/GAO competition, with PAOs having a competitive advantage over GAOs at lower concentrations. This hypothesis was based in part on previous work in which reactor measurements indicated that PAOs produce sufficient ATP during the EBPR anaerobic phase for use in active transport of acetate into the cell, while GAOs had little or no ATP available for acetate transport.27 This could enable PAOs to better scavenge low acetate concentrations than GAOs, which may rely on passive transport and/or have less energy for transport. Others came to a similar conclusion based on testing of laboratory systems with chemical inhibitors.28 The effects of in-reactor acetate concentrations on PAO/ GAO competition have not been studied previously, although reactor type and configuration, in addition to influent concentrations, can greatly affect these concentrations. Most Received: Revised: Accepted: Published: 3816

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Table 1. Reactor Operating and Performance Characteristicsa stage 1: high acetate rate, high pH 2: high acetate rate, low pH 3: low acetate rate, low pH 4: high acetate rate, low pH a

acetate feed rate and duration/pH

day range (duration)

20 mL/min for 10 min/7.4−8.4

0−161 (161)

Pns/TSS (mg/mg)

Pns/VSS (mg/mg)

anaerobic P release/acetate uptake (mol/C-mol)

0.15 ± 0.01

0.20 ± 0.02

0.68 ± 0.05

20 mL/min for 10 min/6.4−7.0

162−206 (44)

0.035 ± 0.003

0.036 ± 0.004

0.041 ± 0.015

1.7 mL/min for 120 min/6.4−7.0

207−325 (118)

0.16 ± 0.01

0.28 ± 0.02

0.55 ± 0.04

20 mL/min for 10 min/6.4−7.0

326−367 (41)

0.024 ± 0.001

0.025 ± 0.001

0.019 ± 0.005

Average values after reaching approximate steady states for each stage of operation are shown.

mg/L). Acetate feed was added separately to allow variation of the acetate feed rate. The reactors were deoxygenated by bubbling with N2 gas for 15 min, simultaneous with nutrient feed addition, and then the acetate feed was added. The pH was continuously controlled (Chemcadet pH meter/controller, Cole−Parmer Instrument Company, Vernon Hills, IL) using 0.1 M HCl and 0.4 M Na2CO3 solutions in the ranges described below. The temperature was controlled at 22 ± 1 °C in a water bath. The reactor was operated in four stages, with pH and rates of acetate addition as experimental variables (Table 1). Stage 1 began on day 0 when the reactor was seeded with SWRF activated sludge. Stage 1 conditions included rapid acetate feed addition at the beginning of the anaerobic phase (over 10 min) and relatively high pH conditions (7.4−8.4), similar to those used in many previous laboratory-scale studies of PAOs.4,11,18,20,29−33 During stage 2, rapid acetate addition was maintained, but the pH was decreased to the range 6.4−7.0, with the objective of inducing EBPR failure as in previous studies.4,11,14,16,19,34 During stage 3, the pH was unchanged, but the rate of acetate feed was decreased (acetate addition occurred over 120 min rather than 10 min), to decrease acetate concentrations in the reactor while maintaining the same mass added per cycle. In stage 4, the reactor conditions were returned to those of stage 2 to determine whether any observed changes in going from stage 2 to 3 were reversible. Chemical Analyses. TSS and volatile suspended solids (VSS) were determined by Standard Methods 2540B and 2540E, respectively,35 on end-aerobic phase samples. Soluble P was measured on GF/C-filtered samples by Standard Method 4500-P C.35 Total P was determined by the persulfate digestion method (Standard Method 4500-P B.535). Pns was calculated as the difference between total and soluble P on samples taken at the end of the aerobic phase. Acetate was analyzed on GF/Cfiltered, phosphoric acid acidified samples by flame ionization detector gas chromatography, using a J&W Scientific DB-FFAP 0.53 mm capillary column, 2 μL injection volumes, and a detector temperature of 250 °C. The oven temperature ramped from 90 to 150 °C at 40 °C/min and was held at 150 °C for 8 min. Filtered samples were analyzed immediately or stored at 4 °C prior to acidification and analysis. Bacteria Characterization. PAOs were identified by fluorescence in situ hybridization (FISH) microscopy using probes PAO462, PAO641, and PAO846 to target Accumulibacter spp. as described by others.36 GAOs were identified using the probes GAOQ98937 and GB_G238 to target Competibacter spp.: DF1MIX (TFO_DF218 plus TFO_DF618) for the cluster 1 Defluviicoccus spp.39 and DF2MIX (DF988, DF1020 plus helper probes H966 and H1038) for GAO cluster 2 Defluviicoccus spp.40 All microscopy

previous PAO/GAO studies have been performed using laboratory-scale sequencing batch reactors (SBRs), typically with cyclic phases of feeding (with rapid addition of acetate and nutrients), anaerobic conditions, aeration, settling, and removal of settled effluent. The batch nature of these systems, with rapid addition of feed, leads to high acetate concentrations at the beginning of the anaerobic phase. While full-scale EBPR systems sometimes utilize SBRs, they more typically include completely mixed flow reactors (CMFRs) in series, in which influent is continuously fed and immediately diluted, leading to lower acetate concentrations than in a SBR. The primary objective of this research was to determine the effect of in-reactor acetate concentrations on PAO/GAO competition, with a focus on lower pH values previously shown to favor GAOs. The approach was to operate a laboratory-scale EBPR system, to cause PAO failure by decreasing the pH, and to reduce in-reactor acetate concentration (but not the mass of acetate added per cycle) by slowing the acetate addition rate, with monitoring of system performance and microbial populations.

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MATERIALS AND METHODS Reactor Operation. A SBR with a 2 L working volume was operated with an 8 h cycle composed of the following sequential phases: anaerobic, 158 min; aerobic, 247 min; settle, 45 min; draw (remove settled effluent), feed, and N2(g) strip, 30 min. The hydraulic residence time was 16 h (1/2 of the reactor volume was removed and replaced with feed each cycle). The mean cell residence time of 7 days was maintained by daily manual wasting, including solids lost in effluent. The SBRs were seeded with mixed liquor taken from the Southside Water Reclamation Facility (SWRF; Albuquerque, NM), a nitrification/denitrification activated sludge plant (modified Ludzack−Ettinger configuration) that also performs EBPR, as indicated by a large PAO population (shown by Neisser staining), and an elevated activated sludge nonsoluble phosphorus (Pns)/total suspended solids (TSS) value of 0.043 mg/mg. Synthetic feed was added each cycle as separate concentrated acetate (200 mL) and nutrient (200 mL) feed solutions, with 600 mL of deionized water. The acetate feed contained (mg/L of total feed, including deionized water) CH3COONa·3H2O (425) and casamino acids (30), giving an acetate concentration of 200 mg/L. Nutrient feed contained (mg/L total feed) NaH 2 PO 4 ·2H 2 O (142), KCl (117), NH 4 Cl (199), MgCl2·6H2O (219), MgSO4·7H2O (14.4), CaCl2 (45.9), yeast extract (8.3), H3BO3 (0.061), ZnSO4·7H2O (0.305), KI (0.015), CuSO4·5H2O (0.061), Co(NO3)2·6H2O (0.075), Na2MoO4·2H2O (0.031), MnSO4·H2O (0.342), FeSO4·7H2O (0.304), and a nitrification inhibitor (N-allylthiourea, 98%, 4 3817

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was performed using an epifluorescent Olympus BX51 microscope. Pyrosequencing of DNA from activated sludge samples was performed by Research and Testing Laboratories (Lubbock, TX). Samples were washed in phosphate buffer solution and shipped frozen. Genomic DNA was extracted using a QIAamp stool DNA mini kit (Qiagen, Valencia, CA), quantified using a Nanodrop spectrophotometer (Nyxor Biotech, Paris, France), and the quality was confirmed using a Bio-Rad Experion system (Bio-Rad, Hercules, CA). They were diluted to 100 ng/μL, and 1 μL of each was used for 50 μL polymerase chain reaction (PCR) reactions. Bacterial tag-encoded FLX amplicon pyrosequencing was performed as described previously,41 with small modifications to utilize the Titanium sequencing platform (Roche Applied Science, Indianapolis, IN). A single 35 cycle PCR step with Qiagen HotStar master mix and addition of 0.5 U of HotStar HiFidelity polymerase were used in each reaction (Qiagen, Valencia, CA). The primers were 28f (5′-GAG TTT GAT CNT GGC TCA G-3′) and 519r (5′-GTN TTA CNG CGG CKG CTG-3′) (Escherichia coli 16S gene numbering), extending across V1 and into the V3 ribosomal regions. Pyrosequence reads were analyzed using AmpliconNoise 1.2542 to remove low quality sequences and denoise the sequencing reads, which included sequences less than 200 bp in length, with an average quality score of less than 25, containing ambiguous characters, and/or without the correct primer sequence. A workflow script in QIIME 1.543 was used to pick operational taxonomic units (OTUs) at the 97% sequence identity level. Representative sequences from each OTU were identified in Qiime using the Ribosomal Database Project44 classification method, with assignment of taxonomic identities using the Greengenes 16s rRNA gene database.45 Some sequences were manually cross-checked using the National Center for Biotechnological Information nr collection of databases with the Basic Local Alignment Search Tool for nucleotides, including some sequences that were not identified at the genus level by QIIME, and also Accumulibacter OTUs, which were not in the Greengenes database.

Figure 1. Typical acetate and phosphorus profiles in anaerobic and aerobic phases with (A) rapid acetate addition in stage I (day 58), when acetate was added continuously over the first 10 min of the anaerobic phase, and (B) slow acetate addition in stage 3 (day 310), when acetate feed was added continuously over the first 120 min of the anaerobic phase.

of the bacteria detected by the EUB probe were positive for the PAOmix probe targeting Accumulibacter spp. (Figure 3B, Table 2). The GAOs Competibacter spp. (probes GAOQ989 and GBG2) and cluster 1 Defluviicoccus spp. (probe DF1MIX) were absent, while cluster 2 Defluviicoccus spp. (probe DF2MIX) was present at 4.0 ± 2.0% of the EUB-positive bacteria. Stage 2: Low pH, Rapid Acetate Addition. Stage 2 was initiated by decreasing the reactor pH to the range 6.4−7.0, while maintaining the same high rate of acetate feed addition (Table 1). This led to a decline in PAO activity, as indicated by a drop in the Pns/TSS, Pns/VSS, and Prel/Ac values (Figure 2 and Table 1). Acetate immediately began to “leak” into the aerobic phase after the pH was decreased (Figure 2B), also indicating decreased PAO activity. End anaerobic phase acetate concentrations increased from zero to a maximum of 69 mg/L 7 days after the pH change, indicating that only 31 of the total 100 mg/L acetate added was taken up prior to the aerobic phase. Acetate uptake recovered by day 198 but without increased P removal, implying GAO activity. From day 198 through the end of stage 2, the average Pns/TSS and Prel/Ac uptake values remained low (Table 1), also suggesting that GAOs dominated the culture after day 198. Consistent with these results, bacteria with the tetrad morphology characteristic of many GAOs began to dominate the reactor after approximately day 198 (Figure 4A), but in earlier samples, there were few to no tetrads (image not shown). Similarly, quantitative FISH indicated that cluster 2 Defluviicoccus spp. represented 33 ± 7% of the EUB-positive bacteria on day 205 (Table 2, Figure 4B), while cluster 1 Defluviicoccus spp. comprised 2.1 ± 0.7% of the EUB-positive

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RESULTS Stage 1: High pH, Rapid Acetate Addition. Typical stage 1 anaerobic and aerobic phase acetate and P concentrations during a single cycle are shown in Figure 1. Anaerobic acetate uptake and P release and aerobic P uptake were consistent with the PAO phenotype and EBPR. Consistent with typical SBR behavior, acetate concentrations were initially high in the anaerobic phase; at the end of the 10 min acetate addition period, they were approximately 79 mg/L. Reactor performance is shown in Figure 2 and summarized in Table 1. Several measurements indicated that the stage 1 culture was highly enriched with PAOs. First, the biomass P content (Pns/TSS) value of 0.15 ± 0.01 mg/mg was much greater than the typical biomass value of approximately 0.02 mg/mg,26 indicating high polyphosphate storage. Second, the average anaerobic P release/acetate uptake (Prel/Ac) ratio of 0.68 ± 0.05 mol/C-mol was greater than the 0.5 mol/C-mol value suggested as a benchmark for PAO dominance over GAOs.26 This coupled with the complete acetate uptake during the anaerobic phase throughout stage 1 (Figure 2B) suggested that PAOs were responsible for most or all acetate consumption. Third, Neisser staining indicated a large quantity of polyphosphate-containing cocci (Figure 3A), consistent with the PAO phenotype. Finally, FISH analyses indicated 82 ± 11% 3818

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Figure 2. Reactor performance with respect to (A) phosphorus content of the biomass (Pns/TSS) and (B) anaerobic P release/acetate uptake and end anaerobic phase acetate concentrations during four stages of operation with variable pH and rates of acetate addition.

Table 2. FISH Data with Comparison to Pyrosequencing Dataa Accumulibacter stage

FISH

pyroseq

stage 1 (day 128)

82 ± 11%

NA

stage 2-early (day 190)

0%

NA

stage 2-late (day 205)

0%

NA

stage 3-late (day 283, FISH; day 286, pyroseq)

77 ± 11%

80%

stage 4-early (day 334)

0%

0%

stage 4-late (day 356)

0%

0.04%

Defluviicoccus FISH

pyroseq

0%b 4.0 ± 2.0%c 0%b 0%c 2.1 ± 0.7%b 33 ± 7%c 0%b 5.0 ± 1.1%c 0%b 0%c 3.3 ± 0.4%b 31 ± 7%c

NA NA NA 1.7% 0.5% 33%

a

FISH counts are reported as percent of EUB-positive cells, and pyrosequencing is reported as percent of total OTUs. Pyrosequencing was not performed during stages 1 and 2 (“NA”), and results in stages 3 and 4 are shown only for samples coinciding with FISH analyses. The GAO Competibacter was also evaluated but was not detected by FISH or pyrosequencing. bDefluviicoccus cluster 1 probe. cDefluviicoccus cluster 2 probe. Figure 3. Typical stage 1 microscopy images (day 128): (A) Neisser stain, dark cells indicate polyphosphate storage (PAOs); (B) FISH with probes EUBMIX (FAM) targeting most bacteria60 and PAOMIX (Cy3) targeting Accumulibacter spp. (cells positive for both probes appear orange). FISH images throughout this paper were collected at two different filter sets corresponding to the fluorescent labels, artificially colored, and overlaid.

bacteria. Competibacter spp. and Accumulibacter spp. were absent. Stage 3: Low pH, Slow Acetate Addition. In stage 3, the acetate feed flow rate was decreased so that the same volume was added over the course of 112 min, rather than over 10 min as in stages 1 and 2, with the pH unchanged (6.4−7.0). The decreased rate of acetate feed addition accomplished the goal of 3819

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the culture in late stage 4, after anaerobic acetate uptake was complete (similar to Figure 4A). FISH analyses showed that, in late stage 4 (day 356), cluster 2 Defluviicoccus spp. represented 31 ± 7% of the EUB probe-positive bacteria (similar to Figure 4B) and cluster 1 Defluviicoccus spp. comprised about 3.3 ± 0.4% of the EUB-positive bacteria (Table 2). Competibacter spp. and Accumulibacter spp. were not detected. 16s rRNA genebased 454 pyrosequencing data were consistent with these results (Table 2, Figure 5) and are discussed further below.

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DISCUSSION Acetate Concentration and pH Effects on PAO/GAO Competition. The loss of PAO activity when the pH was decreased in stage 2 was consistent with previous research showing that lower pH values tend to favor GAOs over PAOs under typical laboratory SBR operation (giving high in-reactor acetate concentrations).4,11,14,16,19,34 The recovery of PAOs when the acetate addition rate was decreased in stage 3 confirmed this study’s hypothesis that the competitive advantage of GAOs at lower pH values may only exist at high in-reactor acetate concentrations. To our knowledge, this is the first report of successful EBPR in this lower pH range (although a modeling study suggested PAOs may dominate under low temperature, low pH conditions46). According to ecological theory, “r-strategist” organisms may dominate systems with high per capita food supplies and low population densities, while “K-strategists” are favored in environments with low per capita food supplies and high population densities.47 In the lower pH range studied here, PAOs exhibited the K-strategist phenotype (stage 3), while GAOs exhibited the r-strategist phenotype (stages 2 and 4). In the higher pH range (stage 1), PAOs also exhibited the rstrategist phenotype, suggesting these classifications may be pH-dependent. Energy for Acetate Uptake. These findings provide experimental evidence for the hypothesis that PAOs may rely on energy-consuming active transport for anaerobic acetate uptake while GAOs do not, which could explain the PAO competitive advantage at low acetate concentrations.27,28 Previous calculations of ATP produced by PAOs and GAOs, based on substrate and storage product measurements in EBPR systems, indicated that PAOs produced approximately 1 mol of ATP/mol of acetate uptake in excess of internal metabolic requirements for acetate transformation to PHB, which could be used for an active transport mechanism.27 GAOs had no excess ATP available for anaerobic acetate transport, and so, they may rely on diffusion, a much slower process at low substrate concentrations external to the cell. Burow et al.28 came to similar conclusions, suggesting that active transport contributed more to acetate uptake in a PAO-enriched (Accumulibacter spp.) culture than in a GAO-enriched (Defluviicoccus spp.) culture, based on measurements using chemical inhibitors. They suggested both PAOs and GAOs drive acetate uptake using the cellular chemiosmotic gradient but that PAOs expend more energy to generate this gradient and can therefore utilize acetate permease (ActP), which can scavenge low amounts of acetate.48 Further research is required to elucidate PAO and GAO acetate uptake mechanisms. GAO Competition Was Not the Immediate Cause of PAO Failures. An unexpected finding was that GAO competition for acetate was not the immediate cause of PAO failures in stages 2 and 4. This conclusion is based on both functional and population measurements. First, PAO failure

Figure 4. Typical late stage 2 images: (A) Neisser stain, day 203, showing the GAO phenotype tetrad morphology (blue arrows); (B) FISH for late stage 2 sample (day 205) with probes EUBMIX (TAMRA) targeting most bacteria60 and DF2MIX (Cy5) targeting cluster 2 Defluviicoccus spp. (cells positive for both probes appear orange).

maintaining very low acetate concentrations during the anaerobic phase, with acetate concentrations less than the detection limit throughout stage 3 (Figures 1B and 2B). During the first 60 days of stage 3, Pns/TSS and Prel/Ac ratios increased, suggesting the PAOs returned to dominate the reactor (Figure 2). Pns/TSS increased from 0.02 at the beginning of stage 3 to an average value of 0.16 ± 0.01 mg/mg from day 266 to the end of stage 3. Similarly, the Prel/Ac ratio increased steadily from 0.02 at the beginning of stage 3 to an average value of 0.55 ± 0.04 mol/C-mol from day 266 until the end of stage 3 (Table 1). Consistent with these results, early stage 3 Neisser staining was similar to that of late stage 2, with few Neisser-positive cells and many tetrad-forming bacteria (image not shown, but similar to Figure 4A), but in late stage 3, the culture was once again dominated by Neisser-positive cells (similar to Figure 3A). FISH analyses indicated 77 ± 11% of the EUB-positive bacteria were Accumulibacter spp. (PAOmix probes, day 283), while Competibacter and Defluviicoccus spp. were not detected or were very low (Table 2). 16s rRNA genebased 454 pyrosequencing data were consistent with these results (Table 2, Figure 5) and are discussed further below. Stage 4: Low pH, Rapid Acetate Addition. In stage 4, the reactor was returned to stage 2 conditions by increasing the acetate addition rate (Table 1). EBPR failed once again, as indicated by rapidly decreased biomass P content and Prel/Ac uptake ratios (Table 1, Figure 2). The relatively low Prel/Ac uptake ratio coupled with complete anaerobic acetate uptake after day 344 was consistent with GAO dominance of the culture. Consistent with these performance data, Neisserpositive cocci declined rapidly in early stage 4, indicating few PAOs, and tetrad-forming bacteria were not initially present either. Bacteria with tetrad morphology only began to dominate 3820

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Figure 5. Percentage of OTUs determined by 454 pyrosequencing of samples from stages 3 and 4 at genus-level taxonomy. Classes are in parentheses (abbreviated) in legend. Only genuses comprising at least 1% of the total OTUs in at least one sample are listed; others are included in the “other” category, which also includes some OTUs that could not be classified at the genus level (see the Materials and Methods section). See the Supporting Information for class-level groupings.

was more or less immediate at the beginning of stages 2 and 4, with rapid decreases in Pns/TSS and anaerobic P release/ acetate uptake values (Figure 2). Simultaneously, end-anaerobic acetate concentrations increased rapidly, indicating that acetate was plentiful during the anaerobic phase while PAO activity deteriorated, demonstrating that GAO competition for acetate was not the cause of PAO failure. Furthermore, FISH analyses demonstrated that Accumulibacter decreased in population to nondetectable levels in early stages 2 and 4 before GAOs (Defluvicoccus) began to dominate the cultures (Table 2, Figure 4). Finally, the pyrosequencing results also indicated an initial decrease in Accumulibacter population in early stage 4, preceding the increase in Defluvicoccus (Figure 5, discussed further below). If GAOs were not the immediate causes for the PAO failures, other factor(s) related to environmental changes must have been responsible. In going from stages 1 to 2, a high pH, high in-reactor acetate concentration-acclimated PAO culture failed when the pH was decreased. This could be attributed to a failure of the PAOs to quickly adapt to the lower pH value, but this does not apply to the failure that occurred in stage 4, as the stage 3 PAOs were acclimated and thriving at the lower pH value. In this case, the environmental change was an increase in the rate of acetate addition, resulting in higher reactor acetate concentrations. The PAO failures in both stages 2 and 4 suggest that it was the combination of high in-reactor acetate concentrations and low pH that led to these PAO declines. It

is perhaps relevant that the acetic acid (CH3COOH) form of the acetic acid/acetate acid−base pair is a known uncoupler of the proton motive force and is toxic to some bacteria,49 and its concentration increases as pH decreases (its relative concentration increases approximately 10-fold as pH decreases from 7.5 to 6.5). The higher concentrations of CH3COOH at lower pH values, when combined with the relatively high concentrations occurring when it is added rapidly in an SBR, may be inhibitory to PAOs. According to this hypothesis, the eventual dominance of Defluvicoccus spp. during these stages suggested that this GAO was more tolerant of the low pH, high CH3COOH condition than was Accumulibacter. Further research is required to test this hypothesis and to determine relevance to other causes of PAO failures, such as high temperature and long SRT. Population Changes Determined by Pyrosequencing. 454 pyrosequencing was conducted on several samples from late stage 3 and throughout stage 4. Because pyrosequencing, like other PCR-based methods, is subject to biases such as variable DNA extraction efficiencies, gene copy numbers per cell, and rates of amplification, its application as a semiquantitative tool should be viewed with caution. Nevertheless, pyrosequencing results were comparable to quantitative FISH results (Table 2), supporting its utility for comparing samples and even absolute quantification in this case. At the class level (Supporting Information, Figure S1), the Betaproteobacteria OTUs were dominant in late stage 3 (76− 3821

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on day 365). Lysobacter is in the same family (Xanthomonadaceae) as Pseudoxanthomonas. Dokdonella (also Xanthomonadaceae) increased to 2% of the total OTU in late stage 4, suggesting the potential importance of this family in failed EBPR systems. Tetrasphaera also transiently increased in early stage 4 (Figure 5). Tetrasphaera have been previously suggested to be PAOs,53−55 and so, it is possible these bacteria briefly occupied the PAO niche vacated by Accumulibacter in early stage 4. The most common OTU in this group was 99% similar to Tetrasphaera elongata (GenBank NR_024735; ref 55), which was previously proposed as a PAO. Tetrasphaera decreased later in stage 4 (Figure 5), possibly due to competition from the GAO Defluvicoccus for acetate in the anaerobic phase. Tetrasphaera may have also contributed to PAO activity during the PAO-dominant stage 3, as they were 1−2% of the OTUs in these samples. Phycicoccus, which is in the Intrasporangiaceae family with Tetrasphaera, represented 9% of the OTUs on day 334 but like Tetrasphaera quickly decreased to less than 1% of the population in the later samples. Further research is required to determine whether Phycicoccus may also include PAOs that behaved similarly to Tetrasphaera in this study. Practical Implications. These findings are potentially of practical importance because full-scale systems are likely to have lower acetate concentrations than laboratory SBRs. Fullscale systems are typically designed as completely mixed reactors, in which influent is immediately diluted upon addition to the reactors, leading to much lower substrate concentrations than SBRs. Even systems designed as plug flow reactors have complete-mix characteristics due to nonideal hydraulics. Furthermore, acetate in full-scale systems is typically generated in situ through fermentation, which is also likely to lead to low in-reactor concentrations. Influent acetate concentrations in laboratory EBPR research studies have typically been much higher than domestic wastewater to help enrich for PAO populations, and the SBR configuration further increases inreactor concentrations. The observations reported here showing that higher inreactor acetate concentrations may favor GAOs suggest that some previous laboratory studies may have overstated the practical importance of GAO competition, and consequent inhibition of EBPR, at least at lower pH values, which improves our understanding of the role of GAOs in full-scale systems. Indeed, this may be one reason why GAOs have been commonly reported in SBRs with deteriorated EBPR,5,7,9,56−58 while similar reports in full-scale systems are more rare.59 In light of results described above suggesting GAOs are opportunists, increasing only after EBPR had already failed, the simple presence of GAOs in failed systems does not necessarily indicate that GAOs caused the failure. From a design perspective, these results suggest that maintaining low in-reactor acetate concentrations (but not decreasing the amount of acetate) may be beneficial to EBPR. This could be achieved in full-scale SBR systems using a strategy similar to that used in this study, by slowing rates of influent acetate addition during anaerobic phases. Systems with plug flow or complete mix anaerobic reactors in series could benefit from addition of influent (or supplemental acetate, when it is used) at multiple points along the flow stream (stepfeed configuration), rather than only at the upstream end, to reduce in-reactor concentrations.

83% of total OTUs), they decreased dramatically after the acetate addition rate was increased at the beginning of stage 4, and they were less than 1% of the sequences by late stage 4. Alphaproteobacteria OTUs increased in early stage 4 (with PAO failure), and they dominated the sequences by late stage 4 (60− 71% of total OTUs). Several groups increased in early stage 4 and decreased in late stage 4, including the Gammaproteobacteria, which were the largest component of the total OTUs in early stage 4 at 45% on day 334 and decreased to 14% by day 365: the Actinobacteria and the Sphingobacteria. At the genus level, Accumulibacter was 60−80% of the OTUs, in the late stage 3 samples, accounting for most of the Betaproteobacteria. Accumulibacter was represented by a single OTU that was 99% similar to a Candidatus Accumulibacter sp. clone (Genbank accession JN679116), and it was 98% similar to the Candidatus Accumulibacter phosphatis clade IIA str. UW1 genome (Genbank accession CP001715). Dechloromonus, which is in the same family as Accumulibacter (Rhodocyclaceae), comprised 16% of the OTU’s on day 308 (stage 3). The role of Dechloromonas in EBPR is not clear: they have been previously suggested to include PAOs50 as well as GAOs.51,52 Their large presence on day 308 and the observation that PAO activity was higher on this day than on day 286 (Figure 2) suggest that Dechloromonas spp. may have behaved as PAOs in this study, but further research is necessarily to clarify their role. After the acetate addition rate was increased to begin stage 4 on day 326, the Accumulibacter and Dechloromonas populations rapidly decreased to less than 0.2% of the OTUs on day 334 and later (Figure 5). The GAO genus Defluviicoccus was less than 2% of the early stage 4 sample OTUs (days 334 and 336), when PAO failure had already occurred. Consistent with FISH results (Table 2) and increased tetrad-forming bacteria (image not shown), Defluvicoccus OTUs dominated the system in late stage 4 (33% and 46% of total OTUs on days 356 and 365, respectively; Figure 5), when anaerobic acetate uptake was once again complete (Figure 2). As discussed above, the delayed increase in Defluvicoccus until after PAO populations and activity suggested these GAOs did not cause PAO failure but rather opportunistically increased in number only after EBPR had already failed and anaerobic acetate was available. There were a large number of other Rhodospirillaceae, the family in which Defluvicoccus resides, in late stage 4 as well (6−7% of total OTUs). These organisms were less than 92% similar to any known species, but their similarity to Defluvicoccus and their large increase in late stage 4, when the GAO phenotype was evident, suggest their potential as GAOs. Similarly, the genus Methylocystis (also Alphaproteobacteria) steadily increased during stage 4 (to 11% of the OTUs on days 356 and 365), suggesting they may also be of interest as potential GAOs. Early stage 4, a transitional period between PAO and GAO domination, exhibited several population shifts. Ottowia and Pseudoxanthomonas increased during early stage 4 but were negligible later. Because the primary carbon source (acetate) was transiently available aerobically during early stage 4, these organisms may have been heterotrophs with an ordinary aerobic metabolism, and when acetate was once again unavailable in the aerobic phases of late stage 4 due to uptake by GAOs, their presence decreased. Pseudoxanthomonas was previously found to increase in a laboratory system after an allaerobic EBPR system failed for unknown reasons.51 Lysobacter also increased in early stage 4 (16−18% of the total OTUs), but this genus was more resilient in later stage 4 (decreasing to 10% 3822

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ASSOCIATED CONTENT

S Supporting Information *

Pyrosequencing results by class. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation, grant no. 0852469. The authors thank Drs. Cristina TakacsVesbach and Dan Colman (UNM Biology Department), and Meghan Preut (UNM Civil Engineering Department) for help with pyrosequencing data processing and analyses.

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