In Situ Profiling of Microbial Communities in Full-Scale Aerobic

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In Situ Profiling of Microbial Communities in Full-Scale Aerobic Sequencing Batch Reactors Treating Winery Waste in Australia Simon J. McIlroy, Lachlan B. M. Speirs, Joseph Tucci, and Robert J. Seviour* Biotechnology Research Centre, Department of Pharmacy and Applied Science, La Trobe University, Bendigo, Victoria 3552, Australia ABSTRACT: On-site aerobic sequencing batch reactor (SBR) treatment plants are implemented in many Australian wineries to treat the large volumes of associated wastewater they generate. Yet very little is known about their microbiology. This paper represents the first attempt to analyze the communities of three such systems sampled during both vintage and nonvintage operational periods using molecular methods. Alphaproteobacterial tetrad forming organisms (TFO) related to members of the genus Defluviicoccus and Amaricoccus dominated all three systems in both operational periods. Candidatus ‘Alysiosphaera europaea’ and Zoogloea were codominant in two communities. Production of high levels of exocellular capsular material by Zoogloea and Amaricoccus is thought to explain the poor settleability of solids in one of these plants. The dominance of these organisms is thought to result from the high COD to N/P ratios that characterize winery wastes, and it is suggested that manipulating this ratio with nutrient dosing may help control the problems they cause.

’ INTRODUCTION The wine industry is a major global industry with over 25 billion liters of wine produced in 2008 alone (www.oiv.org). It has been estimated that for each liter of wine 1
2.5 L of wastewater is produced,1,2 although this can be as high as 14 L.3 Consequently, large amounts of wastewater are generated at various stages during the wine production period (vintage) as well as from bottling and cleaning of equipment and the maintenance of cellar hygiene in nonvintage periods.1,4,5 A wide range of treatment technologies has been applied around the world for dealing with this wastewater, involving both aerobic and anaerobic plant configurations, where one of the main selection criteria used is their operating costs (reviewed by refs 4 and 5). Of the aerobic configurations, which are more efficient for treatment, sequencing batch reactors are used in many Australian wineries (J. Constable, personal communication) because of their simplicity and suitability for coping with the high seasonal variations in winery waste composition and generated loads.1,6 The chemical nature of winery wastewater differs substantially from that of domestic sewage. In particular, it possesses high BOD/ COD:N/P ratios, which, while fluctuating considerably over the course of the year, for COD:P ratios are reported to be in excess of 100:1.1,6 Its pH is also generally lower than domestic sewage being below 4.5.1 These marked differences reflect the high levels of sugars and organic acids in winery wastewaters, and in many plants P and N supplementation of the wastewater is undertaken to facilitate its biological treatment (J. Constable, personal communication). The high carbon load poses serious potential environmental threats, and so again driven by cost considerations, many of the larger wineries treat their own wastes on site instead of discharging them into domestic sewerage systems. Molecular rRNA-based methods have allowed us to begin to resolve the biodiversity of the microbial communities in plants of r 2011 American Chemical Society

many different configurations treating wastewater from domestic and industrial sources.7 Furthermore, they have provided the tools to reveal the in situ functions of many of these populations, especially those involved in P and N removal.8
10 It is quite clear that the microbial community that develops in a treatment process reflects the selective pressures imposed by both the plant operational conditions and the nature of the wastewater being treated.11 As mentioned above, winery wastewaters are highly distinctive in their nature, which poses challenges for their successful biological treatment. Despite this, very few attempts have been made to understand the microbiology involved, and these have been restricted largely to recognizing dominant morphotypes from microscopic examination of the biomass (e.g., ref 12) or from studies using culture dependent methods (e.g., refs 2 and 13). Both are restricted in the levels of information they can provide on community biodiversity. This situation is surprising, because many of the aerobic SBR treatment plants in Australia suffer from operational problems like poor sludge settleability, which are likely to be microbiological in origin. Consequently, we applied molecular techniques to profile communities of three aerobic SBR systems treating the waste in different wine-producing regions in Australia. The data presented here show for the first time that these are each dominated by bacterial populations reported elsewhere in habitats largely characterized by high COD:P ratios, and we propose that these bacteria may cause the operational problems encountered there. Received: May 31, 2011 Accepted: August 29, 2011 Revised: August 29, 2011 Published: August 29, 2011 8794

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Table 1. Plant Operating Parameters Plant A item

a

nonvintage

typical crush (Tonne)

N/A

SBR volume (ML)

1.1

Plant B vintage

30 000

nonvintage N/A

Plant C vintage

20 000

4.6

nonvintage N/A

vintage 1000

0.2

daily flow (kL)

30

280

350

550

4

20

total cycle time (h)

4

12

12

12

12

12

aerate (min)a

30

560

500

400

600

565

settle (min) decant (min)

60 10

60 60

60 40

60 70

105 7

60 40

feed (min)

10

60

130

190

8

55

MLSS (g L
1)

5000

9000
11 000

6000

4480

2400

5600

SRT (days)

Not dewatered

20
30

40
60

20
30

15
20

8
10

COD

1000

8000
10 000

5000
7000

5000
7000

10 400

16 000

BOD

500

6000
8000

3000
4000

4000
6000

6700

10 000

DO (end of aeration)

1
2

1
2

3
4

3
4

7.1

5.8

pH, feed pH, SBR

5
7 8

4
5 7.5

4
5 6.7
7.3

4
5 6.8
7.3

5.7 7.1

4.0 8.1

temp. (°C)

12
14

30
35

12
14

28
30

11

24.6

nutrient dosage (kg week
1)

none

urea, 10; DAPb 5

none

urea, 140

urea, 10; DAP,b 2

urea, 20; DAP,b 5

To reduce operating costs, these reactors are not always aerated during the feed steps or periods of the aeration stages. b Diammonium phosphate.

’ EXPERIMENTAL SECTION Samples. Samples were taken from three full-scale sequencing batch reactors (SBR) treating winery waste in New South Wales (plant A) in nonvintage and South Australia (plant B) and Tasmania (plant C) in both vintage (during wine production) (April 2011) and nonvintage (July 2010) operating periods. Operating parameters for each plant are given in Table 1. Typical influent total P values were 5
10 mg/L for plants A and B and 5
30 mg/L for plant C. Fluorescence in Situ Hybridization (FISH). FISH was carried out using PFA (paraformaldehyde) fixed biomass samples as described by Daims et al.14 The FISH probes listed in Table 2 were used and hybridizations performed at 46 °C for 1.5 h unless otherwise stated. All probes were purchased from ProOligo (Sigma-Aldrich, Australia). Slide wells were mounted in Vectashield (Vector Laboratories) and viewed on a Nikon E800 epifluorescence microscope. To increase cell wall permeability to some of the FISH probes, pretreatments with lysozyme, achromopeptidase, and mild acid hydrolysis as detailed in the protocol of Kragelund et al.15 were applied to biomass samples. Appropriate controls for biomass autofluorescence using the non-EUB probes were included in all analyses. Histochemical Staining. Intracellular polyphosphate granules were detected by applying 4,6-diamidino-2-phenylindole (DAPI) solution (50 μg mL
1) for 10 min to air-dried biomass smears as described by Kawaharasaki et al.46 Biomass was also stained for the presence of intracellular polyhydroxyalkanoate (PHA) granules with Nile Blue A (100 mg L
1 in ethanol) at 55 °C for 10 min, based on the method of Ostle and Holt47 as described in Ahn et al.48 The India ink negative capsule and Neisser staining methods were carried out as described by Jenkins et al.49 16S rRNA Gene Clone Library Preparation from Plant A Biomass. Biomass DNA was extracted using three different methods in attempts to minimize possible extraction biases

associated with each: the sodium trichloroacetate-based method of McIlroy et al.,50 the potassium xanthogenate-based method of Tillett and Neilan,51 and the FastDNA SPIN Kit (Qbiogene, Melbourne, Australia). For each of the DNA extracts, 16S rRNA genes were PCR amplified in quadruplicate with the 27f/1492r and 27f/1525r primer sets,52 with annealing temperatures of 42 and 52 °C, respectively, and the resulting products pooled and stored at
70 °C until required. All other PCR, cloning, and sequencing details are those described by Nittami et al.29 Partial sequence reads (>500 bp) were organized into OTUs based on shared 99% sequence similarities and a ‘complete’ 16S rRNA sequence obtained for a representative from each OTU of interest. 16S rRNA Sequencing of Cultured Isolates. Attempts were made to culture some of the dominant morphotypes from plant C by streaking out the biomass onto solid media. Both GS and R2A agar were used as described by Maszenan et al.53 Colonies were screened microscopically, and those with the desired morphotype (see Results) were subcultured repeatedly until Gram staining suggested they were pure. A single colony was then used for 16S rRNA sequencing as above, except the DNA was extracted from cells as described by McIlroy et al.,54 and PCR products were sequenced directly by AGRF (Brisbane, Australia).

’ RESULTS Application of the EUBmix FISH probe set indicated that the majority of the organisms present in all three winery wastewater treatment plant biomass samples taken during both vintage and nonvintage operational periods were Bacteria. Large yeast-like cells, fungal hyphae, and Protozoa were seen too, particularly in the plant C waste sample, but always in low abundance. Almost all bacterial cells stained strongly for PHA inclusions, while a smaller number (approximately 50% of biovolume); +++ = very abundant (approx. 25
50% of biovolume); ++ = common (approx. 5
25% of biovolume); + = some to few (approx. 97% similar to Candidatus ‘A. europaea’ (W-OTU1 and W-OTU2) (Figure 3). These sequences had only a single terminal mismatch to that of

the Noli-644 probe target (Table 4), suggesting this FISH probe is the one binding to this filament in situ. They also contained a potential binding site for the DF988 probe, with 3 terminal mismatches and a missing internal base (Table 4). The positions of the terminal mismatches were similar to those allowing nonspecific binding of the DF988 FISH probe to the same position in the 16S rRNA sequence of cluster III Defluviicoccus members29 as shown in Table 4 and have a lower theoretical ΔΔG°overall (calculated for the perfectly matched sequence with mathFISH software57). The ability of FISH probes to bind to target sites despite there being missing bases in the target site and forming a bulge over these missing bases has been demonstrated previously.58 However improbable the DF988 probe binding to Candidatus ‘A. europaea’ despite four mismatches in its target site might seem, inclusion in the FISH protocol of an unlabeled competitor probe with the perfect match to this putative probe target sequence, as shown in Table 4, eliminated all the above-background level fluorescence (data not shown). FISH data also suggested that cluster I TFO members of the alphaproteobacterial Defluviicoccus vanus-related group were 8797

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Figure 1. Micrographs of SBR-activated sludge biomass from (a
d) plant A and (e and f) plant B. (a) Neisser stain of the dominant filament in plant A; (b) phase contrast images with corresponding composite FISH image with the EUBmix (Fluos = green) and DF988 (CY3 = red) probe sets (green + red = yellow); (c) phase contrast image with corresponding fluorescence image after Nile Blue A staining (PHA granules fluoresce red); (d) phase contrast image with corresponding composite FISH image with the EUBmix (green) and DF218 (red) probe sets; (e) phase contrast image with corresponding composite FISH image with the ALF968 (green) and DF218 (red) probe sets; (f) phase contrast image with corresponding composite FISH image with the EUBmix (green) and DF988 (red) probe sets. Scale bars indicate 10 μm.

codominant with this filament in the plant A community, being present mainly as tight spherical clustered cells within the flocs (Figure 1d). These populations were detected in the clone library (W-OTU3) (Figure 3). Members of cluster II Defluviicoccus TFO were also present in the flocs but at much lower abundances. Unfortunately, analysis of the corresponding bacterial community

during the vintage period was not possible as the plant was decommissioned at the end of the nonvintage period. FISH Analysis of Plant B. The microbial community composition of the biomass taken from this plant in both vintage and nonvintage was very similar, with both being dominated overwhelmingly by TFO cells responding to the alphaproteobacterial 8798

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Figure 2. Micrographs of SBR-activated sludge biomass from plant C: (a) phase contrast image with the corresponding composite FISH image with the ALF968 (Fluos = green) and AMAR 839 (CY3 = red) probes (green + red = yellow); (b) phase contrast image with the corresponding fluorescence image after Nile Blue A staining (PHA granules fluoresce red); (c) phase contrast image with the corresponding fluorescence image after DAPI staining (PolyP granules fluoresce light yellow); (d) phase contrast image with India ink capsule staining; (e) phase contrast images with the corresponding composite FISH image with the EUBmix (green), ZRA23a (red), and Beta42a (CY5 = blue) probe sets (green + red + blue = white). Scale bars indicate 10 μm.

probe ALF968. The vast majority of these were identified as members of clusters I and II of Defluviicoccus-related organisms. Cluster I members, the more dominant of the two, occurred in large irregular clusters at times exceeding 200 μm in diameter (Figure 1e). Cluster II members were present as smaller clusters of TFO scattered throughout the biomass (Figure 1f). Thin Neisser-negative, PHA-positive filaments with a morphology similar to that of Haliscomenobacter hydrossis but not hybridizing with the HHY probe were seen commonly protruding from the flocs. These hybridized with the Beta42a probe but were too thin to be Sphaerotilus natans, a betaproteobacterial filament implicated in bulking in activated sludge systems, and they also failed to respond to the SNA probe designed to target it. Filamentous Chloroflexi were also present, although in much lower abundances, and some of these responded to the FISH probes targeting the Eikelboom morphotypes type 080341 and type 1851,37 with the former the more abundant of the two. Little

difference in the community composition was observed between samples taken during the nonvintage and vintage periods, except that cluster I Defluviicoccus cell clusters were generally smaller and more loosely associated in the vintage community. FISH Analysis of Plant C Community. This community from the nonvintage period was again dominated by alphaproteobacterial TFO arranged in large sheets (Figure 2a
d). However, these did not respond to any of the Defluviicoccus subgroup FISH probes but instead fluoresced with the AMAR839 probe targeting members of the genus Amaricoccus (Figure 2a). The intensity of the fluorescence signal with this probe varied substantially between individual cells and was improved considerably after a lysozyme prehybridization treatment. These AMAR839-positive cells also stained brightly for PHA inclusions (Figure 2b), and DAPI staining indicated that many possessed small polyP inclusion bodies (Figure 2c). Clusters were surrounded by substantial capsular material (Figure 2d). This community was codominated 8799

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Environmental Science & Technology by betaproteobacterial coccibacilli arranged in large microcolonies. These were identified after FISH with the ZRA23a probe as members of the genus Zoogloea (Figure 2e), and staining showed the cells were again heavily capsulated (Figure 2d). A small number of Chloroflexi filaments, most of which responded to the probe targeting type 0803 (T0803
0654), and H. hydrossis were also detected by FISH, but filamentous bacteria were generally rare. In the plant sample taken from the vintage period, both Amaricoccus TFO and Zoogloea were still present but now in much lower abundance (estimated visually to be about a 50% reduction in both). During the nonvintage period plant C suffered from poor solids settleability or bulking. Yet the biomass possessed insufficient numbers of bacterial filaments to explain this, and interfloc bridging was absent.49 Furthermore, the few fungal filaments seen (Figure 2a) were not associated with the flocs but suspended in the mixed liquor. Instead, the flocs contained heavily capsulated Amaricoccus TFO and Zoogloea (Figure 2d), and it seems much

Figure 3. Maximum likelihood tree of 16S rRNA gene sequences obtained in this study (represented in bold) (all sequences were at least 1200 bp long). The JF957136 sequence represents isolates W-TFO1, W-TFO2, and W-TFO3. Parsimony bootstrap values were calculated as percentages of 1000 analysis and are only indicated for values g 75%: (O) bootstrap value g 75%; (b) bootstrap value g 95%. Scale bar corresponds to 0.1 substitutions per nucleotide position. Brackets define Defluviicoccus-related clusters. AS = Activated sludge. OTU = Operational taxonomic unit.

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more likely that these two heavily capsulated populations were both responsible for this episode of nonfilamentous ‘viscous’ bulking.49 Identification of Isolates from Plant C Biomass. Four of the 100 isolates cultured onto GS medium from plant C were Gramnegative tetrads. The 16S rRNA gene sequence of the first three of these isolates (designated W-TFO1, W-TFO2, and W-TFO3: Acc. No. JF957136) were identical and 97.3% similar to Amaricoccus kaplicensis (Figure 3), also isolated from activated sludge.53 The 16S rRNA sequence of the fourth of these isolates (W-TFO4: Acc. No. JF957137) was 98.8% similarity to the other three and 98.0% similar to A. kaplicensis (Figure 3). All isolates were pleomorphic, with large variation in their cell sizes, typical of Amaricoccus.59,60 These also stained positively for aerobic polyphosphate and PHA storage and produced an extensive capsule. Unlike other Amaricoccus isolates,53 these did not grow on R2A or PYC agar. Their ability to accumulate polyphosphate is also different to that reported for the other Amaricoccus isolates.53

’ DISCUSSION This paper describes for the first time the community composition of winery wastewater treatment plants using cultureindependent rRNA-based methods. It also provides an explanation for the dominance of certain populations and a possible solution to the operational problems they appear to cause. The FISH data from three SBRs treating winery wastewater unexpectedly showed each community was dominated by members of the Alphaproteobacteria. The dominating TFO populations were identified as members of clusters I and II Defluviicoccus-related organisms and possibly new Amaricoccus spp., Candidatus ‘A. europaea’ and Zoogloea were also codominant in the biomass from plants A and C, respectively. Organisms with a similar TFO morphology to those seen in these plants occur commonly in activated sludge communities analyzed by microscopy.61 TFO have also been reported to dominate the biomass treating wastes from wineries,12 although these TFO were not identified nor were they in the study of Liu et al.,62 where they dominated overwhelmingly the community of an EBPR laboratory reactor supplied with a high C:P feed. Bacteria growing as tetrads and representing a wide phylogenetic diversity are known to occur in activated sludge communities.61 However, the TFO of Liu et al.62 are considered likely to be members of the genus Defluviicoccus, since the alphaproteobacterial TFO in the community of a reactor set up to operate in exactly the same way were identified as cluster I members of this genus.58,63 Furthermore, bearing in mind the earlier reports

Table 4. Mismatches in Target Sites Between FISH Probes and Selected 16S rRNA Sequences

*

E. coli position numbering. Base mismatches to the Noli-644 and DF988 probes are highlighted and italicized. 8800

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Environmental Science & Technology concerning TFO distribution, it is probably more than a coincidence that the only cultured member of this genus, Defluviicoccus vanus, was isolated from an activated sludge plant treating high carbohydrate brewery wastes.60 In fact, members of this genus are seen frequently in large numbers in anaerobic:aerobic EBPR plant communities operating with low P removal capacity. They are thought to outcompete the polyphosphate-accumulating organisms (PAO) under P-limiting conditions, producing extensive capsular material and storing instead glycogen, thus earning the description of glycogen-accumulating organisms (GAO).11,64 Yet they are not restricted to plants of this configuration, and have been detected in high abundance in communities of plants dealing with paper mill waste, which is also characterized by its high COD:P ratio.30,65
67 Furthermore, extremely high Defluviicoccus enrichments (up to 95%) have been achieved in laboratoryscale reactor communities fed high COD:P feeds,68
70 adding further evidence to support the view that these conditions, so typical of these winery wastes, support their excessive proliferation. The ecology of Amaricoccus spp. is less clear than that of Defluviicoccus, yet they seem to share several similar ecological traits. Originally isolated from a laboratory-scale EBPR reactor fed glucose and not acetate and showing low EBPR capacity,71 these TFO ‘G-Bacteria’ were identified as members of a new genus Amaricoccus.53 Four species have been described so far with Amaricoccus tamworthensis, like D. vanus, being isolated from an activated sludge plant treating carbohydrate-rich brewery malting wastes. Amaricoccus spp. were also detected by FISH in high abundance in the communities of plants treating paper mill wastes.72 Yet their impact on EBPR capacity is likely to be less detrimental than that of Defluviicoccus. Pure cultures of A. kaplicensis have shown no ability to assimilate substrates under anaerobic conditions,59 meaning that they should not pose a threat to the polyphosphate-accumulating organisms (PAO) in the anaerobic zones of EBPR plants. Why Candidatus ‘A. europaea’ should dominate the community of plant A is unclear, since little is known of its ecophysiology.55 However, Levantesi et al.56 reported its presence in large amounts especially in activated sludge plants treating paper mill and potato wastes, where again the raw plant influent in both would be distinguished by its very high COD:N/P ratio. All the Alphaproteobacteria in these winery wastes appear to store considerable intracellular PHA stores in these winery wastes, and Defluviicoccus, Amaricoccus, and Zoogloea (especially the latter two) also synthesize substantial exocellular capsular material (Figure 2d). These metabolic features are typical responses of bacterial populations to conditions of unbalanced growth73 which would arise from high COD:P/N ratio feeds. Zoogloea populations are commonly seen in activated sludge plants,22 and pure culture studies74 suggest exocellular polysaccharide production by them is encouraged under N-limiting conditions. Therefore, it seems likely that Defluviicoccus, Amaricoccus, ‘Alysiosphaera’, and Zoogloea share a similar ecological niche in that they proliferate under nutrient-limiting high C:N/P feed conditions, and this operational feature probably explains their dominance in these winery wastewater treatment systems. The high relative abundances of floc-associated Amaricoccus and Zoogloea aggregates (Figure 2) may also explain the operational solids separation problems experienced by plant C. The nonfilamentous viscous bulking49 experienced by the plant during the nonvintage period of operation is considered to be caused by them, encouraged to proliferate excessively by the high COD:P/N

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ratio of the feed. Heavily encapsulated TFO bacteria were also thought to be responsible for nonfilamentous bulking in a plant treating winery waste in Hungary.12 This solids separation problem observed in plant C did not occur in vintage, where the Amaricoccus population decreased substantially. The reason for this decline in relative abundance of Amaricoccus is unclear. Clearly more studies of this kind are needed with winery plants to see if the microbiological trends recorded here occur globally. The data from this study suggest that winery wastewater treatment plants and others treating carbohydrate-rich waste may contain a rich reservoir of further biodiversity of Defluviicoccus and Amaricoccus members. They also suggest that episodes of nonfilamentous viscous bulking, invariably linked in the past to Zoogloea, may in fact be caused by other bacterial populations also favored by N- and/or P-limiting conditions like Amaricoccus. If so, bulking control in these plants may be controlled readily by careful P and N supplementation of the influent wastes to allow balanced growth of the communities and to discourage the excessive proliferation of these potentially troublesome bacterial populations.

’ AUTHOR INFORMATION Corresponding Author

*Phone: +61 3 5444 7459. Fax: +61 3 5444 7476. E-mail: r.seviour@ latrobe.edu.au.

’ ACKNOWLEDGMENT The authors would like to thank the plant operators, Robert Morris, Andrew Johns, Grant Kohlhagen, and Nichola Seymour, for supplying samples and plant operation information. Thanks also go to John Constable (JJC Engineering Pty. Ltd) for his helpful advice and information. S.M. and L.S. were supported by a Post Graduate Writing Up Award from La Trobe University and APA Ph.D. scholarship, respectively. Photograph of a stream used in the TOC art was taken by Kristy McIlroy and used with permission. ’ REFERENCES (1) Brito, A. G.; Peixoto, J.; Oliveira, J. M.; Oliveira, J. A.; Costa, C.; Nogueira, R.; Rodrigues, A. Brewery and winery wastewater treatment: some focal points of design and operation. In Utilization of by-products and treatment of waste in the food industry; Oreopoulou, V., Russ, W., Eds.; Springer: New York, 2007; Vol. 3, pp 109
131. (2) Eus
ebio, A.; Petruccioli, M.; Lageiro, M.; Federici, F.; Duarte, J. C. Microbial characterisation of activated sludge in jet-loop bioreactors treating winery wastewaters. J. Ind. Microbiol. Biotechnol. 2004, 31 (1), 29–34. (3) Eusebi, A. L.; Nardelli, P.; Gatti, G.; Battistoni, P.; Cecchi, F. From conventional activated sludge to alternate oxic/anoxic process: the optimization of winery wastewater treatment. Water Sci. Technol. 2009, 60 (4), 1041–1048. (4) Arvanitoyannis, I. S.; Ladas, D.; Mavromatis, A. Wine waste treatment methodology. Int. J. Food Sci. Technol. 2006, 41 (10), 1117–1151. (5) Frost, P.; Kumar, A.; Correll, R.; Quayle, W.; Kookana, R.; Christen, E.; Oemcke, D. Current practices for winery wastewater management and its reuse: an Australian industry survey. Wine Ind. J. 2007, 22 (1), 40–46. (6) Torrijos, M.; Moletta, R. Winery wastewater depollution by sequencing batch reactor. Water Sci. Technol. 1997, 35 (1), 249–257. (7) Seviour, R. J.; Nielsen, P. H. Microbial Ecology of Activated Sludge; IWA Publishing: London, 2010. 8801

dx.doi.org/10.1021/es2018576 |Environ. Sci. Technol. 2011, 45, 8794–8803

Environmental Science & Technology (8) He, S.; McMahon, K. D. Microbiology of Candidatus Accumulibacter in activated sludge. Microb. Biotechnol. In press, doi:10.1111/ j.1751-7915.2011.00248.x. (9) McMahon, K. D.; He, S.; Oehmen, A. The microbiology of phosphorus removal. In Microbial ecology of activated sludge; Seviour, R. J., Nielsen, P. H., Eds.; IWA Publishing: London, 2010; pp 281
319. (10) Daims, H.; Wagner, M. The microbiology of nitrogen removal. In Microbial ecology of activated sludge; Seviour, R. J., Nielsen, P. H., Eds.; IWA Publishing: London, 2010; pp 259
280. (11) Oehmen, A.; Lemos, P. C.; Carvalho, G.; Yuan, Z.; Keller, J.; Blackall, L. L.; Reis, M. A. M. Advances in enhanced biological phosphorus removal: from micro to macro scale. Water Res. 2007, 41 (11), 2271–2300. (12) Jobb
agy, A.; Liter
athy, B.; Tardy, G. Implementation of glycogen accumulating bacteria in treating nutrient-deficient wastewater. Water Sci. Technol. 2002, 46 (1
2), 185–190. (13) Gonzalez, J. M.; Jurado, V.; Laiz, L.; Zimmermann, J.; Hermosin, B.; Saiz-Jimenez, C. Pectinatus portalensis non. sp., a relatively fastgrowing, coccoidal, novel Pectinatus species isolated from a wastewater treatment plant. Antonie van Leeuwenhoek 2004, 86 (3), 241–248. (14) Daims, H.; Stoecker, K.; Wagner, M. Fluorescence in situ hybridization for the detection of prokaryotes. In Molecular Microbial Ecology; Osborn, A. M., Smith, C. J., Eds.; Taylor & Francis: New York, 2005; pp 213
239. (15) Kragelund, C.; Remesova, Z.; Nielsen, J.; Thomsen, T.; Eales, K.; Seviour, R.; Wanner, J.; Nielsen, P. Ecophysiology of mycolic acidcontaining Actinobacteria (Mycolata) in activated sludge foams. FEMS Microbiol. Ecol. 2007, 61 (1), 174–184. (16) Amann, R. I.; Binder, B. J.; Olson, R. J.; Chisolm, S. W.; Devereux, R.; Stahl, D. A. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 1990, 56 (6), 1919–1925. (17) Daims, H.; Br€uhl, A.; Amann, R.; Schleifer, K.; Wagner, M. The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 1999, 22 (3), 434–444. (18) Wallner, G.; Amann, R.; Beisker, W. Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 1993, 14 (2), 136–143. (19) Manz, W.; Amann, R.; Ludwig, W.; Wagner, M.; Scheifer, K.-H. Phylogenetic oligodeoxynucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst. Appl. Microbiol. 1992, 15 (4), 593–600. (20) Zilles, J.; Peccia, J.; Kim, M.; Hung, C.; Noguera, D. Involvement of Rhodocyclus-related organisms in phosphorus removal in fullscale wastewater treatment plants. Appl. Environ. Microbiol. 2002, 68 (6), 2763–2769. (21) Crocetti, G. R.; Hugenholtz, P.; Bond, P. L.; Schuler, A.; Keller, J.; Jenkins, D.; Blackall, L. L. Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation. Appl. Environ. Microbiol. 2000, 66 (3), 1175–1182. (22) Rossello-Mora, R. A.; Wagner, M. The abundance of Zoogloea ramigera in sewage treatment plants. Appl. Environ. Microbiol. 1995, 61 (2), 702–707. (23) Hess, A.; Zarda, B.; Hahn, D.; Haner, A.; Stax, D.; Hohener, P.; Zeyer, J. In situ analysis of denitrifying toluene- and m-xylene-degrading bacteria in a diesel fuel-contaminated laboratory aquifer column. Appl. Environ. Microbiol. 1997, 63 (6), 2136–41. (24) Wagner, M.; Amann, R.; K€ampfer, P.; Assmus, B.; Hartmann, A.; Hutzler, P.; Springer, N.; Schleifer, K.-H. Identification and in situ detection of gram-negative filamentous bacteria in activated sludge. Syst. Appl. Microbiol. 1994, 17 (3), 405–417. (25) Kong, Y.; Ong, S. L.; Ng, W. J.; Liu, W.-T. Diversity and distribution of a deeply branched novel proteobacterial group found in anaerobic-aerobic activated sludge processes. Environ. Microbiol. 2002, 4 (11), 753–757. (26) Neef, A.; Witzenberger, R.; K€ampfer, P. Detection of sphingomonads and in situ identification in activated sludge using 16S rRNA-

ARTICLE

targeted oligonucleotide probes. J. Ind. Microbiol. Biotechnol. 1999, 23 (4
5), 261–267. (27) Wong, M.-T.; Tan, F. M.; Ng, W. J.; Liu, W.-T. Identification and occurrence of tetrad-forming Alphaproteobacteria in anaerobic-aerobic activated sludge processes. Microbiology 2004, 150 (11), 3741–3748. (28) Meyer, R. L.; Saunders, A. M.; Blackall, L. L. Putative glycogenaccumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing. Microbiology 2006, 152 (2), 419–429. (29) Nittami, T.; McIlroy, S.; Seviour, E. M.; Schroeder, S.; Seviour, R. J. Candidatus Monilibacter spp., common bulking filaments in activated sludge, are members of Cluster III Defluviicoccus. Syst. Appl. Microbiol. 2009, 32 (7), 480–489. (30) McIlroy, S.; Seviour, R. J. Elucidating further phylogenetic diversity among the Defluviicoccus-related glycogen-accumulating organisms in activated sludge. Environ. Microbiol. Rep. 2009, 1 (6), 563–568. (31) Maszenan, A.-M.; Seviour, R. J.; Patel, B. K.; Wanner, J. A fluorescently-labelled r-RNA targeted oligonucleotide probe for the in situ detection of G-bacteria of the genus Amaricoccus in activated sludge. J. Appl. Microbiol. 2000, 88 (5), 826–835. (32) Snaidr, I.; Beimfohr, C.; Levantesi, C.; Rossetti, S.; van der Waarde, J.; Geurkink, B.; Eikelboom, D.; Lemaitre, M.; Tandoi, V. Phylogenetic analysis and in situ identification of “Nostocoida limicola”like filamentous bacteria in activated sludge from industrial wastewater treatment plants. Water Sci. Technol. 2002, 46 (1
2), 99–104. (33) Roller, C.; Wagner, M.; Amann, R.; Ludwig, W.; Schleifer, K.-H. In situ probing of Gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides. Microbiology 1994, 140 (10), 2849–2858. (34) Meier, H.; Amann, R.; Ludwig, W.; Schleifer, K. Specific oligonucleotide probes for in situ detection of a major group of Grampositive bacteria with low DNA G + C content. Syst. Appl. Microbiol. 1999, 22 (3), 186–196. (35) Bj€ornsson, L.; Hugenholtz, P.; Tyson, G. W.; Blackall, L. L. Filamentous Chloroflexi (green non-sulfur bacteria) are abundant in wastewater treatment processes with biological nutrient removal. Microbiology 2002, 148 (8), 2309–2318. (36) Gich, F.; Garcia-Gil, J.; Overmann, J. Previously unknown and phylogenetically diverse members of the green nonsulfur bacteria are indigenous to freshwater lakes. Arch. Microbiol. 2001, 177 (1), 1–10. (37) Beer, M.; Seviour, E. M.; Kong, Y.; Cunningham, M.; Blackall, L. L.; Seviour, R. J. Phylogeny of the filamentous bacterium Eikelboom Type 1851, and design and application of a 16S rRNA targeted oligonucleotide probe for its fluorescence in situ identification in activated sludge. FEMS Microbiol. Lett. 2002, 207 (2), 179–183. (38) Kragelund, C.; Levantesi, C.; Borger, A.; Thelen, K.; Eikelboom, D.; Tandoi, V.; Kong, Y.; van der Waarde, J.; Krooneman, J.; Rossetti, S.; Thomsen, T.; Nielsen, P. Identity, abundance and ecophysiology of filamentous Chloroflexi species present in activated sludge treatment plants. FEMS Microbiol Ecol 2007, 59 (3), 671–682. (39) Speirs, L.; Nittami, T.; McIlroy, S.; Schroeder, S.; Seviour, R. J. Filamentous bacterium Eikelboom type 0092 in activated sludge plants in Australia is a member of the phylum Chloroflexi. Appl. Environ. Microbiol. 2009, 75 (8), 2446–52. (40) Speirs, L. B. M.; McIlroy, S. J.; Petrovski, S.; Seviour, R. J. The activated sludge bulking filament Eikelboom morphotype 0914 is a member of the Chloroflexi. Environ. Microbiol. Rep. 2011, 3 (2), 159–165. (41) Kragelund, C.; Thomsen, T. R.; Mielczarek, A. T.; Nielsen, P. H. Eikelboom’s morphotype 0803 in activated sludge belongs to the genus Caldilinea in the phylum Chloroflexi. FEMS Microbiol. Ecol. 2011, 76 (3), 451–62. (42) Neef, A.; Amann, R.; Schlesner, H.; Schleifer, K.-H. Monitoring a widespread bacterial group: in situ detection of Planctomycetes with 16S rRNA-targeted probes. Microbiology 1998, 144 (12), 3257–3266. (43) Manz, W.; Amann, R.; Ludwig, W.; Vancanneyt, M.; Schleifer, K. Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum Cytophaga-Flavobacter-Bacteroides in the natural environment. Microbiology 1996, 142 (5), 1097–1106. 8802

dx.doi.org/10.1021/es2018576 |Environ. Sci. Technol. 2011, 45, 8794–8803

Environmental Science & Technology (44) Weller, R.; Gl€ockner, F.; Amann, R. 16S rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum Cytophaga-Flavobacterium-Bacteroides. Syst. Appl. Microbiol. 2000, 23 (1), 107–114. (45) Hugenholtz, P.; Tyson, G. W.; Webb, R. I.; Wagner, A. M.; Blackall, L. L. Investigation of candidate division TM7, a recently recognized major lineage of the domain bacteria with no known pureculture representatives. Appl. Environ. Microbiol. 2001, 67 (1), 411–419. (46) Kawaharasakia, M.; Tanakab, H.; Kanagawaa, T.; Nakamuraa, K. In situ identification of polyphosphate-accumulating bacteria in activated sludge by dual staining with rRNA-targeted oligonucleotide probes and 40 ,6-diamidino-2-phenylindol (DAPI) at a polyphosphateprobing concentration. Water Res. 1999, 33 (1), 257–265. (47) Ostle, A.; Holt, J. Nile blue A as a fluorescent stain for poly-betahydroxybutyrate. Appl. Environ. Microbiol. 1982, 44 (1), 238–241. (48) Ahn, J.; Schroeder, S.; Beer, M.; McIlroy, S.; Bayly, R. C.; May, J. W.; Vasiliadis, G.; Seviour, R. J. Ecology of the microbial community removing phosphate from wastewater under continuously aerobic conditions in a sequencing batch reactor. Appl. Environ. Microbiol. 2007, 73 (7), 2257–2270. (49) Jenkins, D.; Richard, M. G.; Daigger, G. T. Manual on the causes and control of activated sludge bulking, foaming and other solids separation problems, 3rd ed.; CRC Press LLC: London, England, 2004. (50) McIlroy, S.; Porter, K.; Seviour, R. J.; Tillett, D. Simple and safe method for simultaneous isolation of microbial RNA and DNA from problematic populations. Appl. Environ. Microbiol. 2008, 74 (21), 6806–6807. (51) Tillett, D.; Neilan, B. A. Xanthogenate nucleic acid isolation from cultured and environmental Cyanobacteria. J. Phycol. 2000, 36 (1), 251–258. (52) Lane, D. 16S/23S rRNA sequencing. In Modern microbial methods: Nucleic acid techniques in bacterial systematics; Stackebrandt, E., Goodfellow, M., Eds.; John Wiley & Sons: England, 1991; pp 115
175. (53) Maszenan, A.; Seviour, R.; Patel, B.; Rees, G.; McDougall, B. Amaricoccus gen. nov., a Gram-negative coccus occurring in regular packages or tetrads, isolated from activated sludge biomass, and descriptions of Amaricoccus veronensis sp. nov., Amaricoccus tamworthensis sp. nov., Amaricoccus macauensis sp. nov., and Amaricoccus kaplicensis sp. nov. Int. J. Syst. Bacteriol. 1997, 47 (3), 727–734. (54) McIlroy, S.; Hoefel, D.; Schroeder, S.; Ahn, J.; Tillett, D.; Saint, C.; Seviour, R. FACS enrichment and identification of floc-associated alphaproteobacterial tetrad-forming organisms in an activated sludge community. FEMS Microbiol. Lett. 2008, 285 (1), 130–135. (55) Kragelund, C.; Kong, Y.; van der Waarde, J.; Thelen, K.; Eikelboom, D.; Tandoi, V.; Thomsen, T.; Nielsen, P. Ecophysiology of different filamentous Alphaproteobacteria in industrial wastewater treatment plants. Microbiology 2006, 152 (10), 3003–3012. (56) Levantesi, C.; Beimfohr, C.; Geurkink, B.; Rossetti, S.; Thelen, K.; Krooneman, J.; Snaidr, J.; van der Waarde, J.; Tandoi, V. Filamentous Alphaproteobacteria associated with bulking in industrial wastewater treatment plants. Syst. Appl. Microbiol. 2004, 27 (6), 716–727. (57) Yilmaz, L. S.; Parnerkar, S.; Noguera, D. R. mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization. Appl. Environ. Microbiol. 2011, 77 (3), 1118–22. (58) McIlroy, S. J.; Tillett, D.; Petrovski, S.; Seviour, R. J. Non-target sites with single nucleotide insertions or deletions are frequently found in 16S rRNA sequences and can lead to false positives in fluorescence in situ hybridization (FISH). Environ. Microbiol. 2011, 13 (1), 38–47. (59) Falvo, A.; Levantesi, C.; Rossetti, S.; Seviour, R.; Tandoi, V. Synthesis of intracellular storage polymers by Amaricoccus kaplicensis, a tetrad forming bacterium present in activated sludge. J. Appl. Microbiol. 2001, 91 (2), 299–305. (60) Maszenan, A.; Seviour, R.; Patel, B.; Janssen, P.; Wanner, J. Defluvicoccus vanus gen. nov., sp. nov., a novel Gram-negative coccus/ coccobacillus in the Alphaproteobacteria from activated sludge. Int. J. Syst. Evol. Microbiol. 2005, 55 (5), 2105–2111. (61) Seviour, R. J.; Maszenan, A. M.; Soddell, J. A.; Tandoi, V.; Patel, B. K. C.; Kong, Y.; Schumann, P. Microbiology of the ’G-bacteria’ in activated sludge. Environ. Microbiol. 2000, 2 (6), 581–593.

ARTICLE

(62) Liu, W. T.; Mino, T.; Nakamura, K.; Matsuo, T. Glycogen accumulating population and its anaerobic substrate uptake in anaerobic-aerobic activated sludge without biological phosphorus removal. Water Res. 1996, 30 (1), 75–82. (63) Kong, Y. H.; Beer, M.; Rees, G. N.; Seviour, R. J. Functional analysis of microbial communities in aerobic-anaerobic sequencing batch reactors fed with different phosphorus/carbon (P/C) ratios. Microbiology 2002, 148 (8), 2299–2307. (64) Mino, T.; Liu, W.-T.; Kurisu, F.; Matsuo, T. Modelling glycogen storage and denitrification capability of microorganisms in enhanced biological phosphate removal processes. Water Sci. Technol. 1995, 31 (2), 25–34. (65) McIlroy, S. J.; Nittami, T.; Seviour, E. M.; Seviour, R. J. Filamentous members of cluster III Defluviicoccus have the in situ phenotype expected of a glycogen accumulating organism in activated sludge. FEMS Microbiol. Ecol. 2010, 74 (1), 248–256. (66) Pisco, A. R.; Bengtsson, S.; Werker, A.; Reis, M. A. M.; Lemos, P. C. Community structure evolution and enrichment of glycogenaccumulating organisms producing polyhydroxyalkanoates from fermented molasses. Appl. Environ. Microbiol. 2009, 75 (14), 4676–86. (67) Bengtsson, S.; Werker, A.; Welander, T. Production of polyhydroxyalkanoates by glycogen accumulating organisms treating a paper mill wastewater. Water Sci. Technol. 2008, 58 (2), 323–330. (68) Burow, L. C.; Mabbett, A. N.; Borras, L.; Blackall, L. L. Anaerobic central metabolic pathways active during polyhydroxyalkanoate production in uncultured cluster 1 Defluviicoccus enriched in activated sludge communities. FEMS Microbiol. Lett. 2009, 298 (1), 79–84. (69) Oehmen, A.; Zeng, R.; Saunders, A.; Blackall, L.; Keller, J.; Yuan, Z. Anaerobic and aerobic metabolism of glycogen-accumulating organisms selected with propionate as the sole carbon source. Microbiology 2006, 152 (9), 2767–2778. (70) Dai, Y.; Yuan, Z.; Wang, X.; Oehmen, A.; Keller, J. Anaerobic metabolism of Defluviicoccus vanus related glycogen accumulating organisms (GAOs) with acetate and propionate as carbon sources. Water Res. 2007, 41 (9), 1885–1896. (71) Cech, J. S.; Hartman, P. Competition between polyphosphate and polysaccharide accumulating bacteria in enhanced biological phosphate removal systems. Water Res. 1993, 27 (7), 1219–1225. (72) McIlroy, S. J. Phylogenetic diversity and ecophysiology of alphaproteobacterial glycogen accumulating organisms in enhanced biological phosphorus removal activated sludge systems. Ph.D. Thesis, La Trobe University, Bendigo, 2010. (73) Kessler, B.; Witholt, B. Factors involved in the regulatory network of polyhydroxyalkanoate metabolism. J. Biotechnol. 2001, 86 (2), 97–104. (74) Norberg, A. B.; Enfors, S.-O. Production of extracellular polysaccharide by Zoogloea ramigera. Appl. Environ. Microbiol. 1982, 44 (5), 1231–1237.

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dx.doi.org/10.1021/es2018576 |Environ. Sci. Technol. 2011, 45, 8794–8803