Characterization of the loosely attached fraction of activated sludge bacteria

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42 (2008) 843 – 854

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Characterization of the loosely attached fraction of activated sludge bacteria Fernando Morgan-Sagastume1, Poul Larsen, Jeppe Lund Nielsen, Per Halkjær Nielsen Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 57, 9000 Aalborg, Denmark

art i cle info

ab st rac t

Article history:

Bacterial biomass was characterised in supernatants from activated sludge from a nutrient

Received 1 March 2007

removal plant after settling before and after applying gentle shear (G600 s1). Free-

Received in revised form

swimming and floc-associated bacteria were quantified by microscopy and their identity

24 August 2007

was determined by fluorescence in-situ hybridization (FISH). Total cell numbers in the

Accepted 27 August 2007

supernatant after settling ranged within 2–9  107 cells/mL. Most cells (60–70%) were

Available online 7 September 2007

associated with microcolonies or small flocs, which made up 5–10% of the total number of

Keywords: Activated sludge Shear Detachment Bacterial cells FISH

particles. The remaining 30–40% of the cells corresponded to free-swimming, single cells. The small flocs in the supernatants (diameter ¼ 2.5–35 mm) accounted only for 1% of the total number of particles; however, they greatly contributed to the total volume of biomass in suspension (57% and 75%). The shear applied (G600 s1) induced some floc detachment and higher cell numbers in the supernatants (10–70  107 cells/mL). The identity of bacteria in suspension was as diverse as that in the settled sludge; however, bacteria belonging to Planctomycetes, Firmicutes and Deltaproteobacteria were in higher abundance in the sludge supernatants and were enriched in the supernatants due to gentle shear. Potentially active bacteria were quantified based on the ratio of the number of cells fluorescing with the EUBmix gene probe targeting most bacteria to the total number of cells stained with DAPI. Lower ratios of EUBmix to total cells were measured in the supernatants (50%) than in the settled sludge (80%), suggesting that cells in the dispersed fraction of the sludge were potentially less active than those in the average settleable floc. In conclusion, the attachment properties of bacteria in activated sludge were different among groups, rendering floc fractions more susceptible to detachment and suspension depending on their abundance and activity level. & 2007 Elsevier Ltd. All rights reserved.

1.

Introduction

High turbidity and biomass discharge problems in activated sludge wastewater treatment plants are associated with a relatively small fraction of the solids biomass (o10%), which tends to remain in suspension and/or to detach easily from average sludge flocs (Morgan-Sagastume and Allen, 2004,

2005; Wile´n et al., 2000a). Dispersed biomass in activated sludge can be measured as supernatant turbidity after sludge settling, and it is always present in the sludge-mixed liquor. Although the degree of biomass dispersion and deflocculation depends on different factors, complete sludge deflocculation or floc disintegration never occurs (Morgan-Sagastume and Allen, 2004). Only a relatively small fraction of the total sludge

Corresponding author. Tel.: +45 96358503; fax: +45 96350558.

E-mail addresses: [email protected] (F. Morgan-Sagastume), [email protected] (P.H. Nielsen). 1 Present address: AnoxKaldnes AB, Klostera¨ngsva¨gen 11A, 22647 Lund, Sweden. 0043-1354/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2007.08.026

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mass deflocculates under shear stress below 600 s1, and an equilibrium concentration of dispersed biomass is generally achieved (Mikkelsen and Keiding, 1999). Our understanding of activated sludge floc structure has improved in terms of floc size and morphology, organic and inorganic content, and presence and identity of filamentous bacteria (Nielsen, 2002); also recently, in terms of overall microbial community structure (Juretschko et al., 2002; Schmid et al., 2003), and in terms of the main physicochemical interactions governing it (Liss, 2002; Nielsen, 2002). Nevertheless, the mechanisms explaining the susceptibility of a fraction of the sludge to disperse and to detach from flocs are poorly understood despite the large impact of this sludge fraction on treatment performance. Although microbial cells compose a relatively small fraction of the total organic content of activated sludge organic matter, 10–20% (Nielsen, 2002), bacterial activity in activated sludge appears to determine floc structural stability besides catalysing the process of biodegradation (Wile´n et al., 2000a). In particular, sludge deflocculation is commonly linked not only to microbial stresses (Morgan-Sagastume and Allen, 2004) but also to specific substrate metabolism and electron donor conditions (Wile´n et al., 2000a, b). Therefore, a recent approach in understanding bioflocculation in activated sludge has been to identify microcolony-forming bacterial cells and their ability to remain flocculated under different physicochemical conditions. Fluorescence in-situ hybridization (FISH) in municipal activated sludge with nutrient removal has revealed that the most abundant microcolony-forming bacteria belong to Beta-, Alpha- and Deltaproteobacteria (Klausen et al., 2004). In general, bacterial microcolonies resist disintegration, but differences in microcolony strength and in the forces controlling it have been observed among FISH-probedefined bacterial groups under extensive shear. Strong microcolonies are formed by many Beta-, Gamma- and Deltaproteobacteria, and Actinobacteria (Klausen et al., 2004). At the genus level, strong microcolonies have been identified for Rhodocyclus-related and Actinobacteria-related polyphosphate-accumulating organisms (Larsen et al., 2006). The bacterial composition of the dispersed biomass and the loosely attached floc matrix is almost unknown. Only recently, the Alphaproteobacteria and Firmicutes were identified to deflocculate preferentially from microcolonies in activated sludge under extensive shear and physico-chemical treatments (Klausen et al., 2004). Lower relative abundance of Alpha- and Betaproteobacteria and Actinobacteria has been observed in unsettled sludge compared with total mixed liquor sludge (Schmid et al., 2003), and bacteria with morphotypes like Spirochaetes and Spirillum are known freeswimming bacteria in activated sludge (Eikelboom, 2000). Thus, a better phylogenetic and physiological characterisation of activated sludge bacteria in flocs and as single cells in turbid effluent from settled sludge can help link settling and bioflocculating properties to microbial community composition, which can eventually lead to an improved understanding of solids discharge problems and the procurement of solutions in full-scale activated sludge systems. The aim of this study was to quantify and characterise the dispersed and easily detachable bacteria in supernatants from settled municipal activated sludge with and without low

42 (2008) 843– 854

turbulent shear in order to evaluate whether these were different from the average sludge composition. It was hypothesised that the bacterial average structure of the biomass in suspension is different from that of the average settling flocs, and that the easily detachable biomass also has a different physiological state and bacterial composition from that of the settling biomass, reflecting specific bacterial flocculating properties. Both free-swimming and small-flocassociated cells were quantified and identified by FISH. Furthermore, the levels of potentially active bacterial cells in both the detachable and the settleable fractions were quantified based on the ratio of FISH-detectable cells.

2.

Materials and methods

2.1.

Activated sludge samples

Mixed liquor activated sludge was collected from the aeration tank of the Aalborg East wastewater treatment plant (WWTP) (Aalborg, Denmark). This plant conducts advanced biological N and P removal, as well as some chemical P removal with the addition of FeSO4. Aalborg East WWTP is designed for 100,000 population equivalents and operates with a mean sludge retention time of 25–30 days. All measurements and tests were initiated 45 min after sample collection. The mixed liquor total suspended solids (MLSS) in all the samples were on average 4500 mg/L with a 65% content of volatile suspended solids (VSS). The filament abundance in the original mixed liquor samples was described as ‘‘common’’ or filament index ¼ 3 based on Eikelboom (2000), and the sludge volume index, determined after 30 min settling in a 1000 mL graduated cylinder, averaged 140 mL/g MLSS.

2.2. Collection and preservation of dispersed and loosely attached biomass samples To study both the activated sludge inherent dispersed biomass and the loosely attached floc fraction, activated sludge-mixed liquor samples were allowed to settle for 30 min by gravity in a 1 L graduated cylinder both before and after low shear tests. The biomass present in the supernatant from settled mixed liquor without shear was considered as dispersed biomass inherent to the sludge sample (undisturbed supernatant), whereas the biomass present in the supernatant from settled mixed liquor after undergoing low shear was considered to contain the loosely attached fraction of sludge flocs (shear supernatant). The supernatants were gently decanted, ensuring that settled solids did not mix with the supernatant. Immediately after collection, both supernatants and settled sludge were fixed and stored at 4 1C in 2% formaldehyde for 4,6-diamino-2-phenylindoldihydrochloridedilactate (DAPI) staining. For FISH analyses, the supernatants and the settled sludge were fixed in 4% paraformaldehyde for 3 h at 4 1C for hybridisation of Gram-negative cells. The fixed samples were rinsed with sterile filtered tap water by centrifugation (3  for 8 min at 3400g) and stored at 20 1C in 1:1 PBS/ethanol (Amann, 1995). For hybridisation of Grampositive cells, samples were fixed and stored in 50% ethanol.

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2.3.

Low shear experiments

2.5. DAPI-based particle size distributions in sludge supernatants

The low-level shear experiments were conducted with the same set-up described elsewhere (Klausen et al., 2004; Larsen et al., 2006), but with the following modifications. The experiments were carried out with 1 L of mixed liquor in a Plexiglas cylindrical reactor (diameter ¼ 10.5 cm) with four vertical baffles (each 1  13 cm). A single-bladed paddle placed 4 cm above the bottom was stirred at 660 rpm, corresponding to a relatively gentle average turbulent shear rate (G) of 600 s1 in this type of reactor set-up, as previously determined elsewhere (Mikkelsen et al., 2002). Shear was applied continuously at 2071 1C for 3 h, which corresponded to the lowest time necessary for turbidity to plateau at its maximal value, as illustrated in Fig. 1. The shear rate of 600 s1 was more gentle than the shears previously used in these types of deflocculation tests (G ¼ 2200 s1) (Klausen et al., 2004; Larsen et al., 2006).

2.4.

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The size distribution of cells, microcolonies and small flocs in the supernatants was evaluated based on their specific crosssectional area (biovolume), as observed under epifluorescence microscopy. Such area frequency distributions were assessed from epifluorescent microscopic images of DAPI-stained polycarbonate filters taken under oil immersion at 630  magnification. DAPI staining was conducted as outlined above without homogenisation. For each particle size distribution, images of 25 fields of view were taken randomly, and the total number of particles from all 25 fields of view counted were 22,700–25,300 for shear supernatants and 4200–5900 for undisturbed-sludge supernatants. Particles with an area o0.2 mm2 were considered as background image noise and were not included in the analyses. The particle size distributions are reported based on the assumption that particles are spherical with a diameter, d.

Determination of cell numbers in sludge supernatants

The numbers of total cells and of free-swimming cells (free cells) in the supernatants were calculated by enumerating DAPI-stained cells filtered onto black polycarbonate filters (0.22 mm, Millipore). Total cells and free cells were enumerated from homogenised and non-homogenised samples, respectively. Only single, free cells or 2 attached cells were enumerated as free cells; filamentous bacteria and bacteria in aggregates of more than 3 cells were not considered as free cells. Homogenisation was conducted on diluted samples using a glass tissue grinder (Thomas Scientifics, USA). DAPI staining was conducted on top of the filter with 5% w/v DAPI for 10 min. Dilutions ensured that 50–200 DAPI-stained cells were counted per field of view. A total of 10 fields of view per sample were enumerated using epifluorescence microscopy and oil immersion at 1000  magnification.

0.008

2.6. FISH and quantification of bacterial abundance in sludge FISH oligonucleotide probing was conducted based on Amann (1995) protocols. The oligonucleotide probes used are listed in Table 1, and further details about the probes are provided at probeBase (Loy et al., 2003). The relative abundance of each probe-defined bacterial group was estimated as the ratio of area fluorescing with each specific probe (CY3 labelled) to the area fluorescing with the EUBmix probe (FLUOS labelled) on images from sludge supernatants and settled sludge. For each sample (settled sludge and supernatants from sludge C, D and E), 20 images of randomly selected fields of view were taken at 630  magnification from 2 different hybridisation wells on Teflon-coated slides (with 6 wells) containing the same sample on the slide. Citifluor was used as antifadent.

G=250s-1

Turbidity (OD650 /g MLSS)

G=400s-1 G=600s-1

0.006

0.004

0.002

0.000 0

50

100 Time (min)

150

200

Fig. 1 – Typical supernatant turbidity increase during a 3 h shear experiment with municipal activated sludge (sample C) at different shear levels (G). Average turbidity values are reported with standard deviations from 3 independent measurements from the same reactor (n ¼ 3).

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2.7. Quantification of potentially active bacteria (EUBmix/ DAPI ratio) The levels of potentially active bacterial cells were investigated based on the ratio of the number of cells fluorescing with the FLUOS-EUBmix gene probe targeting most bacteria to the number of DAPI-stained cells. Quantification was conducted in 10 (n ¼ 10) fields of view, ensuring that a

Table 1 – Oligonucleotide probes used in FISH and their specificity Oligonucleotide probe

Specificity

Reference

ALF968 BET42a

Many Alphaproteobacteria Betaproteobacteria

GAM42a

Gammaproteobacteria

Neef (1997) Manz et al. (1992) Manz et al. (1992) Neef et al. (1998) Manz et al. (1996) Roller et al. (1994) Meier et al. (1999) Amann et al. (1992); Rabus et al. (1996) Bjo¨rnsson et al. (2002); Gich et al. (2001) Daims et al. (2000) Daims et al. (1999)

Pla46 CF319a+b HGC69a LGC354a+b

Planctomycetes Cytophaga– Flavobacterium group of the Bacteroidetes Actinobacteria Many Firmicutes

SRB385+SRB385Db

Deltaproteobacteria and Desulfobacteriaceae

GNSB941+CFX1223

Phylum Chloroflexi

Ntspa712

Phylum Nitrospira/ Leptospirillum All Bacteria

EUBmix (EUB338, EUB338-II and EUB338-III) NONEUB

Negative control

Wallner et al. (1993)

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minimum of a total of 400 cells as FLUOS or DAPI were counted. FISH and DAPI staining were conducted on wells of Teflon-coated glass slides, following the approach outlined above.

2.8.

Analytical techniques, microscopy and image analysis

Total and VSS in the mixed liquor and supernatants were determined as per standard methods (APHA et al., 2005). Supernatant turbidity was assessed as the optical density (OD) at 650 nm after 3 min centrifugation at 426g. Turbidity values were normalised to suspended solids (SS). Microscopic enumerations and microscopic images were obtained using an Axioskope II epifluorescent microscope (MetaVue software 6.4; Universal Imaging Corp., Downington, PA). Image analyses for fluorescing areas were conducted using the imageprocessing software ImageJ 1.34s (http://rsb.info.nih.gov/ij/; National Institutes of Health, US) with macros specifically developed for these analyses.

2.9.

Statistical approach

Fresh activated sludge was collected 5 times in total within a 2.5-month period. In undisturbed supernantants, the numbers of total cells and free cells (Table 2), and total number of particles used in particle size distributions (Fig. 2) were determined in all 5 samples of fresh activated sludge (A–E). Only the later 3 samples (C–E) were subjected to low shear, and were the source of samples for further analyses (Table 2, Figs. 2–5). Mean values are reported with 71 standard deviation, except for the EUBmix/DAPI ratios (Fig. 3), percent specific probe binding (Fig. 4) and cell number (Fig. 5) for which 95% confidence intervals were used, to emphasize the interval where the population mean would lie. The means of EUBmix/ DAPI ratios (Fig. 3), percent specific probe binding (Fig. 4) and cell number (Fig. 5) were calculated by treating each sample average as a single data point (n ¼ 3 from sludge samples C–E), so as to reflect the variability of the mean value from

Table 2 – Average numbers7standard deviations (n ¼ 10) of total and free cells in supernatants from undisturbed sludge and sludge exposed to low shear (G ¼ 600 s1) Sample

Total cells (cells/mL)

Free cells (cells/mL)

Free/total cell ratio

0.1970.2  107 1.370.4  107 0.9675.2  107 3.170.7  107 2.270.9  107 1.970.8  107

0.8 0.2 0.2 0.5 0.2 0.470.2

3.372.1  107 2378  107 1674  107 1479.8  107

0.3 0.3 0.3 0.370.0

Supernatant from undisturbed activated sludge A B C D E Average

2.470.7  107 6.571.6  107 4.571.2  107 6.873.8  107 8.971.6  107 5.872.5  107

Supernatant from activated sludge subjected to gentle shear G ¼ 600/s C 1273  107 D 73717  107 E 54710  107 Average 46731  107 A, B, C, D and E refer to samples collected from the same plant on different dates.

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WAT E R R E S E A R C H

35

100

Number (%)

Undisturbed Shear Cumulative - undisturbed Cumulative - shear

25

80 70 60

20

50 15

40

10

30 20

5

Cumulative number (%)

90

30

10

≥8 .1

8 2. 6-

2. 5 1. 6-

1. 6 1. 2-

.1 -1 0.

81

-0 63 0.

0. 5-

.8

0 0. 62

0

Diameter (μm)

100 Undisturbed Shear Cumulative - undisturbed Cumulative - shear

80

60

80 70 60

50

50

40

40

30

30

.1 ≥8

8 62.

2. 61.

1. 21.

-1 0.

81

-0 63 0.

0. 50.

5

0 6

10

0 .1

20

10 .8

20

62

Biovolume (%)

70

90

Cumulative biovolume (%)

90

Diameter (μm) Fig. 2 – Number (A) and area (B) distributions in supernatants from undisturbed sludge and sludge exposed to gentle shear (G ¼ 600 s1). For each area interval, an average7standard deviation was calculated from 5 (n ¼ 5 for undisturbed) and 3 (n ¼ 3 for shear) independent size distributions, corresponding to different sludge samples collected on different dates. For the area distributions (B), the middle area for each area interval was used for estimating the total area per interval based on the number of particles within each interval. For the last interval (X50 lm2), an area of 100 lm2 was used.

Settled sludge

Supernatant undisturbed sludge

Supernatant after shear

0.90 EUB mix/DAPI ratio

0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 C

D E Aalborg East activated sludge sample

Average∗

Fig. 3 – Average EUBmix/DAPI ratios (n ¼ 10) evaluated in settled sludge and supernatants from undisturbed sludge and sludge exposed to low shear levels (G ¼ 600 s1). The error bars represent 95% confidence limits. *Average of all the data (n ¼ 30) from the three samples.

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Settled sludge

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Supernatant undisturbed sludge

Supernatant sheared sludge

os lp ha pi ra pr ot eo ba B et ct ap er ro ia te G ob am ac m te ap ria ro te ob D ac el ta te pr ria ot eo ba ct er ia Fi rm ic ut A es ct in ob ac Pl te an ria ct om yc et B es ac te ro id et es

itr A

N

hl or of le

xi

40 35 30 25 20 15 10 5 0

C

Percent specific probe binding (specific probe/EUBmix∗100)

WA T E R R E S E A R C H

Fig. 4 – Bacterial population structure of settled activated sludge and supernatants from undisturbed and sheared sludge based on probe-defined FISH quantification. The relative abundance for each group corresponds to the average795% confidence limits from the quantification of three (n ¼ 3) independent samples (C, D and E).

Supernatant sheared sludge

es et id ro

ac B

ct Pl

an

te

om

ob in ct A

et yc

te ac

ic rm Fi

es

ria

es ut

er ct

eo ot pr

D

el

ta

m am G

ba

ob te ro ap

ro ap et B

ia

. t.. ac

te ac ob te

eo ot pr

ha lp A

ria

ia er ct ba

os itr N

C

hl

or

of

pi

le

ra

xi

Cell number (cells/g SS)

Supernatant undisturbed sludge

1.6E+12 1.4E+12 1.2E+12 1.0E+12 8.0E+11 6.0E+11 4.0E+11 2.0E+11 0.0E+00

Fig. 5 – Estimated concentrations of cells hybridized with group-specific probes in supernatants from undisturbed and sheared sludge. The error bars correspond to 95% confidence limits from n ¼ 3 independent quantifications (C, D and E) (see Fig. 4). these 3 independent samples. The statistical significance of differences between means before and after low shear was assessed by paired Student’s t hypothesis tests at the 95% confidence level. The level of significance of the tests (p) are reported for those cases where a significant difference was found.

3.

Results

3.1.

Deflocculation at low turbulent shear rates

Turbulent shear rates lower than 600 s1 (i.e., G ¼ 250 and 400 s1) had little impact on particle detachment from flocs and on increasing sludge supernatant turbidity (Fig. 1). A G ¼ 600 s1 consistently yielded saturation turbidity profiles that plateaued at significantly higher values (0.008, 0.012 and 0.017 OD650/g MLSS) than the original mixed liquor (0.002, 0.001 and 0.006 OD650/g MLSS) in 3 independent shear tests. In addition, this highest shear rate (G ¼ 600 s1) yielded a higher

supernatant SS level (3472 mg SS/L). In contrast, the lower shear rates (G ¼ 250 and 400 s1) produced comparable supernatant turbidity values (o0.004 OD650/g MLSS) to the original mixed liquor and only slightly elevated supernatant SS levels (12 and 15 mg SS/L) compared with the initial levels in the untreated sludge (8 mg SS/L). Therefore, a G ¼ 600 s1 was used to study the detachment of the loosely bound portion of sludge flocs since it consistently yielded higher levels of effluent SS (420 mg/L) and distinctive saturation profiles of supernatant turbidity. Higher shear rates (G ¼ 800–1100 s1), previously demonstrated to cause floc erosion (Mikkelsen and Keiding, 2002), are even much higher than the shear rates typically present in an activated sludge plant (G90–220 s1) (Das et al., 1993), and therefore were not used in this study.

3.2. Size distribution of cells, microcolonies and small flocs in supernatants DAPI was used to stain not only free cells and cells in microcolonies but also cells in flocs, thus revealing some

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structure of flocs in suspension. Microcolonies are considered as aggregates formed by the same types of cells, whereas flocs have a diverse composition (Nielsen, 2002). DAPI-based particle size distributions (all free cells, microcolonies and small flocs, Fig. 2A) from the supernatant of undisturbed sludge indicate that most particles (ca. 95%) inherently present in the supernatant exhibited a cross-sectional area p2 mm2 (equivalent diameter d ¼ 1.6 mm). These small particles contributed to only 21% of the total particle biovolume (Fig. 2B), and they were mostly single cells, small microcolonies and protozoan debris stained by DAPI. In contrast, the few particles (5%) with larger cross-sectional areas (dX1.6 mm), which mainly corresponded to small flocs and filaments, contributed to a very large part of the total particle biovolume (80%). As expected, shear forces resulted in a greater number of larger particles in the supernatant, such as small flocs and longer filaments with higher cross-sectional areas (10% with a dX1.6 mm; Fig. 2A). These particles contributed to 70% of the total particle biovolume (Fig. 2B). Overall, the size distributions were very similar in the two types of supernatants with the majority of particles (99%) being small particles with a diameter below 2.5 mm and contributing to 26–44% of the total biovolume. The larger particles with a diameter between 2.5 and 35 mm represented only 1% of the total number of particles stained by DAPI (Fig. 2), but made up a large fraction of the total biovolume in the supernatants (74% and 56% without and with shear, respectively).

3.3.

Number of cells in supernatants

The total number of cells (including free cells, cells in small flocs and filaments) in the supernatants from different samples of undisturbed sludge ranged within 2–9  107 cells/mL with an average of 5.872.5  107 cells/mL (Table 2). In supernatants from sheared sludge, the total number of cells was higher ranging within 10–70  107 cells/mL (Table 2). These higher cell numbers indicate that approximately 2–10-fold more non-settleable cells were dispersed into the supernatant due to the applied shear (G ¼ 600 s1). The number of free cells in the supernatant from sheared sludge also tended to be 3–10fold higher than those from the undisturbed sludge supernatants. A comparison between free cells and total cells shows that in both supernatants ca. 30–40% of the cells were freeswimming, single cells and that 60–70% were associated with microcolonies, small flocs and filaments accounting for 5–10% of the total number of particles, as outlined above (Table 2 and Fig. 2).

3.4.

Potentially active bacterial cells in supernatants

Potentially active bacteria were quantified based on the ratio of the number of cells fluorescing with the EUBmix gene probe targeting most bacteria to the total number of cells stained by DAPI. Overall, lower bacterial EUBmix/DAPI ratios were determined in the supernatants than in the settled sludge (Fig. 3). The ratios in the supernatants from sludge subjected to shear were consistently and statistically sig-

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849

nificantly lower than those from settled sludge (undisturbed: po0.07; sheared: po0.006; paired t-test). The ratios in the settled sludge were on average 7675%, whereas in the supernatants from sheared sludge these ratios were around 5171% (Fig. 3). Although the EUBmix/DAPI ratios also tended to be lower in supernatants from undisturbed sludge than in the settled sludge, some of these differences were not statistically significant. No consistent pattern was observed when comparing EUBmix/DAPI ratios between both supernatants. These results suggest that more of the potentially less active cells are present in the supernatant than in the settled sludge, and that the deflocculated fraction of the sludge was enriched by less active cells from the sludge.

3.5. Population structure in supernatants and settled sludge Group-specific oligonucleotide probes were used to quantify the relative abundance of different target bacterial groups in the supernatants and settled sludge (Fig. 4). In the settled sludge, the most dominant bacterial groups were Betaproteobacteria (3673% of all Bacteria targeted by EUBmix), Chloroflexi filamentous bacteria (3374%) and Alphaproteobacteria (177 2%). Bacteroidetes (1371%) and Gammaproteobacteria (1072%) were present in medium abundance. Both supernatants from undisturbed and sheared sludge had a bacterial composition as diverse as that of the settled sludge, but differences (po0.009) in bacterial relative abundance between the supernatants and the settled sludge were observed between 6 out of the 10 probe-defined groups (Fig. 4 and Table 3). Planctomycetes, Deltaproteobacteria and Firmicutes were more abundant in the supernatants, and Betaproteobacteria, Bacteroidetes and Chloroflexi were less abundant. Both supernatants showed a similar tendency in their differences compared with the settled sludge (Fig. 4). No significant differences in bacterial abundance were detected for Alphaproteobacteria, Gammaproteobacteria, Actinobacteria and Nitrospira between the supernatants and the settled sludge. Significant differences in the cell numbers (cells/g SS) of several specific bacterial groups were observed between the supernatants from undisturbed and sheared sludge (Fig. 5 and Table 3). The number of cells in the supernatants was estimated based on the average DAPI cell numbers (Table 2), the average EUBmix/DAPI ratios (Fig. 3), the group-specific probe/EUBmix ratios (Fig. 4) and the average SS in both supernatants (undisturbed ¼ 14 mg SS/L; shear ¼ 50 mg SS/L). Shear increased the number of cells of Planctomycetes, Betaproteobacteria, Bacteroidetes, Firmicutes and Chloroflexi in the supernatant with respect to the supernatant from undisturbed sludge. The cell numbers of Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Deltaproteobacteria and Nitrospira in the supernatant were not significantly affected by gentle shear (Fig. 5). The use of absolute numbers of FISH-positive cells (like Fig. 5) together with relative estimates (Fig. 4) is essential in this type of analysis. For instance, the number of Deltaproteobacteria in the supernatant (Fig. 4) might be wrongly interpreted to decrease as a consequence of increased shear levels. However, as the total number of bacteria increased due to the release of Betaproteobacteria, the number of Deltaproteobacteria

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Table 3 – Summary of observations for each specific bacterial group with respect to hybridisation targets in activated sludge as microcolonies, single free-swimming cells or cells within flocs, and filamentous bacteria, and with respect to changes in relative abundance and cell numbers in the supernatants Bacteria group

Chloroflexi Nitrospira Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Deltaproteobacteria Firmicutes Actinobacteria Planctomycetes Bacteroidetes

Colony

Single cells in/ outside flocs

Filamentous bacteria

Abundance in supernatanta

Shear effect on amount in supernatantb

– +++ ++ +++ +++ ++ – ++ – +

– – + ++ ++ + +++ + +++ ++

+++ – + ++ + ++ ‘‘Chains ++’’ + – ++

. – – . – m m – m .

m – – m – – m – m m

Hybridisation targets were inferred from microscopic observations, and differences in relative abundance and cell numbers are a summary from Figs. 4 and 5, respectively. m, Increase in abundance; ., decrease in abundance; –, no hybridised cells or no change in abundance; +, few hybridised cells; ++, some hybridised cells; +++, many hybridised cells. a Comparison between relative abundances in settled sludge and unsettleable biomass in supernatants (Fig. 4). b Comparison between cell concentrations in supernatants from undisturbed and sheared sludge (Fig. 5).

in the supernatant was actually unchanged, as reflected in Fig. 5.

4.

Discussion

4.1. Physical characterisation of dispersed biomass in sludge supernatants In activated sludge, a bimodal particle size distribution is usually reported, consisting of a small-size group in the 0.5–5 mm range and few particles in the 5–25 mm range (Ekama et al., 1997). The small-size group settles slowly and a fraction of them are present in the effluent from clarifiers, and therefore such particles were expected to be abundant in the supernatants from settled sludge in this study. The DAPIbased number distributions (Fig. 2A) from the sludge supernatants agreed well with the small-size group of this bimodal particle size distribution. The minimal area quantified was 0.2 mm2, which corresponds to a circle diameter of 0.5 mm, a reasonable lower limit for bacteria targeted in this study. The cross-sectional area distributions of cells stained with DAPI were used to infer biovolume characteristics of cells, microcolonies and cell-containing flocs in sludge supernatants after settling. The measurement by microscopy and image analysis represents a means of assessing the presence of the smallest particles (do10 mm) in activated sludge, which are challenging to detect and quantify by other techniques for determining particle size distributions in activated sludge (Govoreanu et al., 2004). The particle size distributions in the supernatants based on DAPI staining (Fig. 2) suggest that detachment of the loosely attached floc fraction largely increases the total volume of suspended biomass via relatively few small flocs with a larger volume. The relatively largest particles in the supernatants,

i.e., small flocs, microcolonies and filaments, were few (5–10% of total number), but contributed to a large fraction of the suspended biomass volume, e.g., the largest flocs and filaments (d ¼ 2.5–35 mm) accounted only for 1% of the total number of particles, but they greatly contributed to the total biomass volume (57% and 75%). This biovolume contribution was smaller (57%) in the supernatant from sheared sludge than from the undisturbed sludge since more single cells and microcolonies of smaller size (d ¼ 0.8–2.5 mm) were also sheared off. The differences in particle size distributions between the supernatants from undisturbed and sheared sludge were small (Fig. 2). Nevertheless, the gentle shear (G600 s1) yielded consistently higher turbidity levels and larger cell numbers in the supernatants than without shear. This shear level is slightly above values reported to induce flocculation (G70 s1) and close to those experienced by sludge in fullscale plants (G90–220 s1) (Das et al., 1993). Given the relatively low shear rates that sludge may experience in full-scale plants (Go300 s1), elevated levels of effluent SS in full-scale plants most likely originate from chemical or physiological changes in the sludge as indicated by the low levels of turbidity measured below 400 s1 (Fig. 1).

4.2. Free-swimming cells and floc-associated cells in dispersed activated sludge The number of cells in the sludge supernatants determined by direct microscopic counts was in the order of 107 cells/mL and thus 100 times higher than those determined with conventional plate-counting techniques. Plate counts of aerobic microorganisms in effluents from different Danish-activated sludge plants are reported to range from 103 to 105 CFU/mL (Jensen, 2004). Although the biases of cultivation-dependent

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techniques are widely recognised when quantifying microorganisms and assessing genotypic and phenotypic characteristics of complex microbial communities (Amann et al., 1995), plate counts are still commonly used to assess bacterial concentration in effluents from WWTPs (APHA et al., 2005). Direct microscopic counts with a specific stain as DAPI for assessing bacterial concentrations in effluents from treatment plants should be strongly considered. The particle distributions and the cell numbers in the supernatants indicate that not single cells but small flocs and microcolonies contribute largely to the volume of suspended biomass, containing a large number of bacterial cells (60–70%). The similar fractions of free cells from the total cells in both the supernatants from undisturbed and sheared sludge suggest that the relative composition of free cells and microcolony-associated cells in the loosely attached fraction of sludge flocs is similar to that of the inherently dispersed biomass. Therefore, the gentle shear does not enrich significantly for free-swimming cells in the supernatant. This deflocculation process is in agreement with the aggregation– detachment dynamic equilibrium described for activated sludge flocs (Biggs and Lant, 2000; Chaignon et al., 2002; Mikkelsen and Keiding, 1999), implying that the inherently suspended biomass is in a dynamic equilibrium with a weakly flocculated sludge fraction. Part of the inherently suspended biomass may include bacteria from the incoming wastewater to the plant that tend to remain in suspension, to attach weakly to flocs and/or grow in suspension.

4.3. Bacterial population characteristics in settled and unsettled activated sludge The relative abundances of individual bacterial groups assessed by FISH in the activated sludge of this study were at least 3%. These values, including their variability, agree with results from previous studies where these bacterial groups were quantified in different municipal activated sludge samples (Juretschko et al., 2002; Klausen et al., 2004; Schmid et al., 2003; Wagner and Loy, 2002). Betaproteobacteria were the most abundant bacteria in the activated sludge studied, similar to what was reported in 3 different German municipal activated sludge plants (Schmid et al., 2003). Several important functional groups belong to this bacterial group and include ammonium-oxidizing nitrosomonads, denitrifying Azoarcus, Thauera, and Aquaspirillum-related bacteria and polyphosphate-accumulating organisms belonging to Rhodocyclus (Juretschko et al., 2002; Thomsen et al., 2007, 2004). The abundances of the various group-specific probes summed up to slightly above 100% of all Bacteria targeted by the EUBmix probe, suggesting that the most important groups were targeted by the probes applied. An overestimation of the abundance for some groups is possible, but many filamentous bacteria belonging to Chloroflexi were not targeted by the EUBmix, but gave a clear signal with the group-specific probes GNSB941 and CFX1223. This yielded an artificially higher percentage of the relative abundance of Chloroflexi, which, however, still permitted a relative comparison of the abundance of Chloroflexi among settled sludge and supernatants.

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851

Differences in the relative abundance of some of the bacterial groups between the settled and the unsettled sludge fraction support the idea that the microbial community structure of the biomass in suspension and of that potentially present in the effluent from the activated sludge process is somehow different from that of the average activated sludge remaining in the plant. Thus, the lack of settleability of suspended biomass could be explained based on a relatively different microbial composition with potentially different aggregative properties than the settleable sludge. Although microscopic, metabolic and phylogenetic differences have been reported between aggregated and free-living bacteria in marine environments (DeLong et al., 1993) and in micro- and macroflocs from pin-point activated sludge treating paper mill wastewater (Mu¨ller et al., 2002), no equivalent and detailed information has been available for municipal activated sludge. Overall, the bacterial composition in the sludge supernatants is in agreement with some of the previously reported floc-forming properties of bacteria from the different groups. From all the bacterial groups analysed, Planctomycetes and Firmicutes were in a relatively higher abundance in suspended biomass than in settled sludge (Fig. 4). The tendency of Planctomycetes and Firmicutes to grow as single free-swimming cells and/or single cells within flocs appears to make them more likely to remain in suspension and to be susceptible to shear and detachment from flocs. For instance, Alphaproteobacteria and Firmicutes had already been identified to deflocculate easily from flocs under higher shear levels (Klausen et al., 2004). In addition, bacterial groups in which filamentous bacteria were present also showed a tendency to be enriched in the supernatants after gentle shear. Alphaproteobacteria Chloroflexi, Bacteroidetes and Betaproteobacteria holding most filaments in the sludge were enriched in the supernatant after shear as assessed by higher cell concentrations per g SS. Most of these filaments were embedded into the floc material and they could apparently be released to the supernatant by moderate shear. No enrichment of Deltaproteobacteria, Actinobacteria and Nitrospira was observed in the supernatant after shear in agreement with the properties of some bacteria from these groups to form strong microcolonies (Klausen et al., 2004). Despite the fact that some Betaproteobacteria form strong microcolonies, bacteria from this group were enriched in the supernatant after shear. This is reasonable, however, since Betaprotebacteria encompasses many genera of bacteria, some of which are filaments and some of which tend to grow as single cells and may have different properties. The use of more specific gene probes is required to differentiate within this broad group. Finally, the bacterial groups for which abundant differences were observed in the supernatants compared with the settled sludge also showed a tendency to be enriched in the supernatants in response to shear (Table 3), except for Deltaproteobacteria.

4.4.

Physiology of the dispersed biomass

An indication of the overall microbial activity in the supernatants was obtained by evaluating the number of cells with a relatively high rRNA content as to be hybridised with the EUBmix probe targeting most bacteria with respect to the

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number of cells stained by DAPI. High rRNA levels generally correlate to high bacterial growth rates and levels of protein synthesis (Molin and Givskov, 1999), and although varying from species to species, the intensity of the FISH signal has been used to indicate cell activity in complex environments (Karner and Fuhrman, 1997; Nielsen et al., 2003). The EUBmix/DAPI ratios close to 80% measured in the settled sludge range within values reported for other activated sludge samples (Daims et al., 1999; Nielsen and Nielsen, 2002), indicating that most cells were active. The remaining 20% of DAPI-stained cells may contain too low levels of rRNA to be detected by FISH (dead or inactive), cannot be penetrated by the probes or are not targeted by the EUBmix (as some Chloroflexi). The levels of Archaea (not detected by EUBmix) in this activated sludge were insignificant with respect to the total bacterial levels (data not shown). The relatively lower EUBmix/DAPI ratios in the sludge supernatants suggest that potentially less active cells were present in dispersed, unsettleable biomass than in settled sludge. Chloroflexi filaments enriched in the sheared sludge supernatant could have decreased the EUBmix/DAPI ratios since these cells were detected by DAPI staining, but not all of them with the EUBmix probe, as discussed before. Although the difference in average EUBmix/DAPI ratios between the supernatants from undisturbed and sheared sludge is not statistically significant, it may suggest a decrease in the ratio due to shear (Fig. 3) that is slightly higher than a 2% increase in the fraction of Chloroflexi cells with respect to the total number of cells quantified in the supernatants before and after shear (from 23% to 25%). In addition, since the relative abundance of Chloroflexi was lower in the supernatants than in the settled sludge, the lower EUBmix/DAPI ratios in the supernatants were probably not only due to the enrichment of Chloroflexi cells in the supernatants. These results support the role of microbial activity in determining the flocculating ability of bacterial biomass in activated sludge, and agree with observations of activated sludge deflocculation due to microbial activity inhibition under different conditions (Morgan-Sagastume and Allen, 2005; Wile´n et al., 2000b). Furthermore, stimulation of aerobic heterotrophic activity by specific substrates (e.g., ethanol and glucose) has been shown to promote reflocculation (Wile´n et al., 2000b); however, addition of other substrates (acetate and propionate) decreased floc strength. The higher level of inactive cells in the deflocculated fraction of this work might also explain why several studies have shown that complete reflocculation is impossible. Preferential substrate utilisation by specific bacterial groups or preferential activity-specific grazing by protozoan in the dispersed sludge fraction or flocculating sludge may explain these physiological traits, as also suggested by differences in substrate-utilisation patterns by cells in settleable and nonsettleable sludge flocs (Mu¨ller et al., 2002). Interestingly, no effect on increase in turbidity was measured when inhibition of some flagellates and small ciliates was achieved with the addition of cycloheximide and nystatin in batch tests (results not shown). However, these previously reported inhibitors failed to inactivate most protozoa, rotifers and nematodes even at extremely high concentrations (50–200 mg/L) and after long exposure (6 and 24 h) in contrast to what is

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assumed in the literature, but has not been systematically quantified in activated sludge (Lee and Welander, 1994; Lee and Oleszkiewicz, 2003).

5.

Conclusions

Bacterial biomass characterisation in the settled and unsettled fractions of activated sludge indicates that significant differences exist in the relative abundance of some bacterial groups (Planctomycetes, Firmicutes and Deltaproteobacteria) present in dispersed and settleable sludge. These differences support the hypothesis that inherently dispersed biomass in activated sludge has a different microbial community composition with potentially different aggregative properties than the average settleable sludge. Nevertheless, the biomass in suspension is as diverse as that of the settled sludge. A large number of bacterial cells (60–70%) inherently in suspension in activated sludge are not free-swimming, single cells, but are associated with microcolonies and small flocs in suspension, which contribute to a large fraction of the volume of suspended biomass. Cells in the dispersed and loosely attached fraction of activated sludge appeared potentially less active than cells constituting the average settled sludge flocs, which suggests the importance of microbial activity in regulating flocculating properties. Direct microscopic counts with a specific stain as DAPI for assessing bacterial concentrations in effluents from treatment plants should be strongly considered. The expected numbers of total cells in the supernatant (and effluent) from activated sludge determined by DAPI staining and microscopy were in the order of 107 cells/mL, which is 100 times higher than those determined with conventional plate-counting techniques.

Acknowledgments This study was supported in part by a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada (NSERC) to F.M.-S. and by the Danish Technical Research Council under the framework program ‘‘Activity and Diversity in Complex Microbial Systems’’. R E F E R E N C E S

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