The role of Lecane rotifers in activated sludge bulking control

ARTICLE IN PRESS WAT E R R E S E A R C H

42 (2008) 2483 – 2490

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

The role of Lecane rotifers in activated sludge bulking control Edyta Fia"kowska, Agnieszka Pajdak-Sto´s Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387 Krako´w, Poland

art i cle info

ab st rac t

Article history:

Experiments were conducted on Lecane inermis feeding on filamentous bacteria and living in

Received 25 October 2007

activated sludge to determine if the rotifers can control the growth of the bacteria

Received in revised form

responsible for bulking. The experiments showed that Lecane are capable of significantly

4 January 2008

reducing the density of Microthrix parvicella filaments. The rotifers not only survived the

Accepted 2 February 2008

transfer from the culture to the activated sludge, but they multiplied quickly when foraging

Available online 19 February 2008

on filamentous bacteria. By reducing the number of filaments, the rotifers improved

Keywords: Rotifera

settling properties of the sludge. This is apparently the first report on the possibility of using rotifers to control bulking.

Microthrix

& 2008 Elsevier Ltd. All rights reserved.

Bulking Filaments Bacteria Wastewater Settling

1.

Introduction

The activated sludge process is the one most frequently used in wastewater treatment technology. Activated sludge is the complex suspension of biological and nonbiological components. Among the former are bacteria, fungi, protozoans and metazoans, whereas the nonbiological fraction contains organic and inorganic particles. The suspension is constantly aerated and mixed to ensure the appropriate oxygen level for microorganisms and to prevent sedimentation of the suspension. The bacteria, primarily heterotrophic, nitrifying, denitrifying, poly-P and glycogen-accumulating ones, play the most important role in pollutant removal. However, some of these organisms, namely certain filamentous bacteria, may prove a hindrance to proper functioning of a treatment plant. The most frequently occurring are solids separation problems caused by foaming and bulking of the sludge.

Research initiated by Eikelboom in the second half of the last century (cf. Eikelboom (2000) and the literature therein) led to the description of approximately 30 morphotypes of filamentous organisms appearing in domestic wastewater treatment plants. Some of them were found to be responsible for bulking problems. The bacterium most frequently causing severe bulking in Europe, South Africa and Australia is Microthrix parvicella (Blackbeard et al., 1986; Kristensen et al., 1994; Seviour et al., 1994; Eikelboom et al., 1998; Wanner et al., 1998), whereas in North America, the most commonly observed types were 1701 and 021N (Seviour and Blackall, 1999). Over the years, different methods were employed to control foaming and bulking (Jenkins et al., 2004; Tandoi et al., 2006). Some of them were nonspecific and aimed at improving the settling properties of the sludge without eliminating the cause of problem. Among the most frequently used methods were addition of synthetic organic polymers, inorganic coagulants and flocculants like aluminium and iron salts,

Corresponding author. Tel.: +48 12 664 68 74; fax: +48 12 664 69 12.

E-mail addresses: [email protected] (E. Fia"kowska), [email protected] (A. Pajdak-Sto´s). 0043-1354/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2008.02.001

ARTICLE IN PRESS 2484

WAT E R R E S E A R C H

42 (2008) 2483– 2490

talc, chlorine and hydrogen peroxide. Each of these methods, however, has serious drawbacks such as high costs, increasing sludge mass and interference with nitrification (for a review see Jenkins et al., 2004). They have not always been effective. Seka et al. (2001) showed that Type 021N, identified by fluorescent in situ hybridization, originating from two different plants showed different susceptibility to the same chemical—chlorine. In one case, Type 021N turned out to be chlorine-susceptible, whereas in the other, it was chlorineresistant. Other attempts to avoid bulking problems include using specific methods such as addition of macro and micronutrients, or installation of selectors. The selectors are smaller reactors, often compartmentalized, in which raw sewage and sludge are mixed for a short time before being introduced into biological reactors (for review see Tandoi et al., 2006). In many cases, the selectors helped to resolve the problem of bulking, but in numerous others, they were not sufficient, especially if the organism causing the problem was M. parvicella (Jenkins et al., 2004). Relatively little attention was paid to the possibility of eliminating filamentous bacteria by their natural enemies occurring in activated sludge. Inamori et al. (1991) showed that two species of ciliates equipped with a cytopharyngeal basket were able to ingest filaments of the Type 021N and Sphaerotilus natans. They suggested that bulking might be eliminated quite quickly if the density of ciliates was high enough. Also, Drzewicki and Hul (1997), in an experiment conducted in a treatment plant, showed that a ciliate Trithigmostoma cucullulus, when present in high densities, was able to significantly limit the densities of Type 021N. During our microscopic observations of activated sludge, we noticed that at least two other organisms—rotifers and testate amoebae—readily ingested filamentous bacteria. Therefore, we decided to check if among the numerous data we had collected from different treatment plants, there are any relationships between the number of rotifers and/or amoebae and the density of filamentous bacteria. We also designed a set of experiments to determine if rotifers transferred from a culture to activated sludge from different treatment plants would be able to survive, proliferate and eliminate filamentous microorganisms. Our aim was also to check if the elimination of filaments by rotifers can influence the settling ability of sludge.

2.

Materials and methods

Over a period of 3 years, we collected samples of activated sludge from four wastewater treatment plants situated in southern Poland. The type and basic parameters of the plants are given in Table 1. Samples consisting of approximately 200 ml of sludge were taken from aeration tanks and sent to our laboratory. To ensure good aerobic conditions during transport, the vials were only filled halfway. The samples were always analyzed within 24 h of being taken. The analyses were made according to the method of Eikelboom (2000) based on a simplified assessment of organism density that did not require counting them. In this method, the densities of filamentous organisms observed in a sample are compared with reference images contained in the method’s manual (Eikelboom, 2000; Fia"kowska et al., 2005) and expressed as degrees on a six-point scale called the filament index (FI). The densities of proto- and metazoans are expressed as degrees on a scale from 0 to 3 where 0 means none and 3 means numerous cells/colonies per slide. In this study, to attain greater precision, we introduced half-point intervals to the above scales. As the first step in our search for the relationship between the filamentous bacteria density and organisms living in activated sludge, we conducted correlation analyses for all 133 samples collected over 3 years. We tested correlations between the FI and the indices of ciliates, flagellates, naked amoebae, testate amoebae, rotifers and nematodes. Then, all the correlations were repeated for each plant separately. Only in the case of testate amoebae and rotifers were there noticeable correlations, so we chose them for further investigations. The amoebae turned out to be extremely difficult to cultivate; therefore, we used only the rotifers in our experiments. We carried out a laboratory experiment to see if rotifers are able to reduce the number of filamentous bacteria. Direct microscopic observations revealed that rotifers of the genus Lecane feed predominantly on such bacteria, so we decided to use them in our experiment. To obtain a clonal population of Lecane inermis, single individuals were transferred with a micropipette from a sludge sample to separate wells in the tissue culture test plates filled with Z˙ywiec brand mineral

Table 1 – Type and basic parameters of treatment plants Wastewater treatment plant System Pre-clarifier Aerobic reactor Anoxic reactor Anaerobic reactor Fraction of industrial wastes Type of industrial wastes The range of temperatures (1C) Winter Summer

CZ

WW

ZPM

PK

Complete mix  + + + o1% Fish, fruit, vegetable processing

Complete mix + + +  o1% Meat processing

SBR Flotator + +  100% Meat processing

Complete mix  + + + o1% Vegetable processing

8–9 18–20

0–3 16–19

5–7 20–23

7–9 18–20

ARTICLE IN PRESS WAT E R R E S E A R C H

42 (2008) 2483 – 2490

water. A rice grain, sterilized earlier in boiling water, was added to each well. The rotifers fed on bacteria proliferating on the rice. The cultures were kept at the light intensity of 70 mm photon m2 s1, under a 12-light:12-dark regime at 20 1C. For the experiments, we chose the clone that reached the highest density. The rotifer species were determined according to Segers (1995). We used activated sludge from three different treatment plants: two (CZ, WW) were chosen from among the four used in preliminary analyses, and the third (SU) was one constantly suffering from bulking. Before the experiment, we identified filamentous bacteria and assessed their density in each sample according to the method of Eikelboom (2000). In the sludge from the CZ plant, the FI was 3.5 with distinct dominance of M. parvicella, in the WW, the index was 4.5 and the only filamentous bacterium present was M. parvicella, and in the SU, the index was 4.0 and M. parvicella and Actinomycetes were present in equal proportions. To confirm our identification of M. parvicella, we applied the FISH technique using the Vermicon KIT-Microthrix to the sample from the WW treatment plant. The experiments were carried out in Cell WellsTM (Corning). Into each of 16 wells we transferred 1 ml of thoroughly mixed activated sludge. The wells were divided into two 8-well groups, one of which served as a control. One hundred rotifers, picked up individually with a pipette, were transferred into each well of the experimental group. During the transfer, approximately 100 ml of medium was moved as well, so an equal volume of Z˙ywiec brand mineral water was added to the control wells. The experiment was repeated with the sludge from each treatment plant. The cell plates were left for 1 week in darkness at 20 1C. After the week, we took 25 ml subsamples out of each well, mixed each with 10 ml of acridine orange on a microscope slide and covered them with 22  22 mm2 cover slips. The use of acridine orange made all live filaments clearly visible. We took pictures of 10 randomly chosen fields of view for each slide. Each picture was then analyzed with the help of the Lucia image analysis system. We counted the number of filaments crossing the measurement frame of 28 mm  28 mm. We called the value ‘‘density factor’’ (DF). The mean value of DF was then calculated for each experiment and control well. For the observation, we used a Nikon Eclipse 80i fluorescence microscope equipped with a B-2A filter (excitation 450–490 nm) working at a total magnification of 1000  . In the next step, we repeated the experiment on a larger scale. For this purpose, we chose activated sludge WW because M. parvicella was the only filamentous bacterium present in this sludge, and its index was relatively high (FI ¼ 4.5). In addition, there were no L. inermis naturally occurring in this sludge. The experiment was carried out in eight 2 l beakers. Each beaker contained 1 l of activated sludge. From the numerous sludge analyses we had made, we estimated that the most frequently occurring rotifer density in activated sludge ranged from 100 to 300 ind/ml. To get the density of rotifers within this range, 50 ml of Lecane sp. suspension containing approximately 3200 ind/ml was added to four experimental beakers. In this way, the approximate density of 150 ind/ml in each beaker was reached. Into the remaining four beakers, serving as a control, 50 ml of Z˙ywiec brand mineral water was added. Activated

2485

sludge was aerated with an aquarium pump and kept at 20 1C. To avoid differences in aeration between beakers, diffusers were moved daily between beakers in a random pattern. Using the method described above, we calculated the DF 24 h and 7 days after the release of the rotifers. From each beaker, two subsamples were taken and the mean DF was calculated for the experiment and control. On the fifth and seventh days, we also counted the number of Lecane sp. individuals in eight 25 ml subsamples taken from each beaker. Then, the mean number of rotifers in 1 ml of sludge was calculated. On the eighth day the sludge in each beaker was gently but thoroughly mixed, and 100 ml was poured into each of eight cylinders and left for 30 min. Then, the volume of settled sludge was measured and the pictures were taken.

3.

Results

Analysis of all the data collected throughout 3 years from all four plants combined, showed that the index of filamentous bacteria (FI) correlates negatively with the density of rotifers (r ¼ 0.3182, po0.001) and testate amoebae (r ¼ 0.3805, po0.001), whereas correlations with the density of other organisms such as ciliates, flagellates, naked amoebae and nematodes were not significant. Correlation analysis performed for each plant separately showed that the correlation between the rotifer index and the FI was negative and statistically significant in the treatment plants PK (Fig. 1a, r ¼ 0.4968, p ¼ 0.001) and ZPM (Fig. 1b, r ¼ 0.5476, p ¼ 0.0056). In the CZ treatment plant (Fig. 1c, p40.5) there was no correlation, whereas in the WW the correlation was marginally significant (Fig. 1d, r ¼ 0.313, p ¼ 0.08). In the case of the CZ plant, there was a significant correlation between the density of testate amoebae and FI (Fig. 2, r ¼ 0.41, p ¼ 0.01). In the ZPM, the correlation was marginally significant (r ¼ 0.3817, p ¼ 0.06), whereas in the WW and the PK plants, the correlation was not significant. Figs. 3a–c show the results of a laboratory-scale experiment in which rotifers were placed in activated sludge from three different plants: CZ, WW and SU. In two cases, CZ (Fig. 3a) and WW (Fig. 3b), there were significant differences between the experiment and the control. The t-test for independent samples confirmed that rotifers significantly reduced the density of filamentous bacteria in the CZ sludge (t ¼ 3.411, df ¼ 158, po0.001) and the WW sludge (t ¼ 5.484, df ¼ 158, po0.001), although the effect was less pronounced in the case of the former. There was no difference between the treatment and control in the experiment with SU activated sludge (Fig. 3c). The experiment carried out in beakers confirmed the ability of Lecane to reduce the number of filamentous bacteria. At the beginning, the mean density of filaments in the experimental and control beakers was similar. After a week, the density of filaments in beakers with rotifers decreased significantly (t ¼ 3.392, df ¼ 157, po0.001), whereas in the control, it remained at the same level. At the end of the experiment, the difference between the DF in experimental and control beakers was highly significant (Fig. 4, t ¼ 5.9437, df ¼ 157, po0.001).

ARTICLE IN PRESS 2486

WAT E R R E S E A R C H

42 (2008) 2483– 2490

N=40 r = -0,4968 p=0,0011

N=24 r =-0,5476 p=0,0056 5

5 PK

4

3 FI

FI

3 2

2 1

1

0

0 0.0

0.5

1.0 1.5 2.0 rotifer index

2.5

3.0

3.5

0.0

N=37 r=-0,1016 p=0,5497

0.5

1.0

1.5 2.0 2.5 rotifer index

3.0 3.5

N=32 r=-0,3130 p=0,0841

5

5 CZ

4

WW

4

3

3 FI

FI

ZPM

4

2 1

2 1

0

0 0.0

0.5

1.0 1.5 2.0 2.5 rotifer index

3.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 rotifer index

3.5

Fig. 1 – Correlation between rotifer density (rotifer index) and filaments index (FI) in PK treatment plant (a), ZPM treatment plant (b), CZ treatment plant (c) and in WW treatment plant (d).

N=37 r= -0,4147 p=0,0107 5

where t is the day of experiment, nt is the number of rotifers on the day t of the experiment and n0 is the initial number of rotifers. During the first 5 days, the growth rate varied between 0.2 and 0.3 ind/day, and during the last 2 days it reached 0.5–0.8 ind/day. Fig. 6 shows the volume of settled sludge after 30 min. The level of sludge was visibly lower in cylinders with the sludge containing Lecane than in the control. The volume of sludge ranged from 14 to 18 ml in the experimental cylinders, and from 21 to 44 ml in the control cylinders.

4

FI

3 2 1 0 0.0

0.5

1.0 1.5 2.5 2.0 testate amoebae index

3.0

3.5

Fig. 2 – Correlation between testate amoebae density (testate index) and filaments index (FI) in CZ treatment plant.

The number of rotifers increased significantly during the experiment (Fig. 5). The increase was slower at the beginning—the number of rotifers increased 4-fold over 5 days, then at the end of the experiment it increased 3.5-fold over only 2 days. Because exponential growth should follow the equation: Nt ¼ n0 ert , the growth rate was calculated as 1 r ¼ ðln nt  ln n0 Þ, t

4.

Discussion

Although numerous studies have been conducted in search of methods that would help to control the bulking of sludge, no universal method has been found (Lemmer, 1998; Seviour and Blackall, 1999; Eikelboom, 2000; Tandoi et al., 2006). Thus, it seemed reasonable to investigate whether organisms naturally occurring in activated sludge might be used as a tool to control the growth of filamentous bacteria. The results of first attempts at using ciliates were promising (Inamori et al., 1991; Drzewicki and Hul, 1997), but when we collected data from treatment plants in Poland, it turned out that the ciliates used in those studies appeared in sludge only rarely. However, we found a negative correlation between the number of rotifers and/or amoebae and the density of filamentous bacteria, which suggested that rotifers and amoebae may be potential grazers of filaments.

ARTICLE IN PRESS WAT E R R E S E A R C H

7

Mean Mean±SE

CZ 6

∗

Mean± 1,96 SE

5

5

4

4

DF

DF

7

Mean Mean±SE

6

2487

42 (2008) 2483 – 2490

3

3

2

2

1

1

0

WW ∗

Mean± 1,96 SE

0 control treatment

control treatment 7

Mean Mean±SE

6

SU ∗

Mean± 1,96 SE

DF

5 4 3 2 1 0 control treatment Fig. 3 – Mean filament density factor (DF) 7 days after inoculation of rotifers in CZ sludge (a), WW sludge (b) and SU sludge (c).

7

5 4 3 2 1

Mean umber of rotifers/ml

Mean±SE Mean±1,96∗SE

6

DF

2500

Mean

2000 1500 1000 500 0 0

0 control treatment

5

7

Time (days)

Fig. 4 – Mean filament density factor (DF) 7 days after inoculation of rotifers in WW sludge laboratory experiment in beakers.

Fig. 5 – Mean number of rotifers per milliliter counted in beakers with WW sludge at the start, in 5th and 7th day of experiment.

The first experiment, in which Lecane rotifers were placed in activated sludge from three different treatment plants, showed that rotifers are able to decrease the density of filaments, but not in every case. They turned out to be ineffective in the SU sludge. There may have been two reasons for the lack of rotifers’ effect in SU. One is that in this sludge, Actinomycetes constituted approximately half of the

filamentous organisms present. Although Actinomycetes are classified as filamentous organisms, their morphology differs from that of other filamentous bacteria. Their filaments are shorter and, what might be even more important in terms of being ingested, they are branched. This might make them a less accessible source of food for rotifers. To determine if this was really the case, we also made direct microscopic

ARTICLE IN PRESS 2488

WAT E R R E S E A R C H

42 (2008) 2483– 2490

Fig. 6 – The volume of activated sludge taken from control (1–4) and experimental beakers (5–8) after 30 min of sedimentation in cylinders. The dark stripes at the top of liquid in control cylinders consist of floating sludge.

observations on activated sludge in which M. parvicella and Actinomycetes were present in equal proportions. In 75% of all observed situations, when rotifers attacked the filaments, M. parvicella were eaten whole, while in 16% of cases, the rotifers tried to ingest Actinomycetes but abandoned the attempts and in only 8% of cases did they succeed in ingesting very small fragments of Actinomycetes colonies. The other possible reason for rotifers’ low effectiveness in SU might be the chemical composition of the sludge. Practically each sludge is unique and its chemical as well as biological composition is so complex that it is not possible to control all parameters. So far, relatively little attention has been paid to factors determining rotifer presence in sludge. They are reported to appear in systems with low organic loads and/or higher sludge age (Eikelboom, 2000; Jenkins et al., 2004; Drzewicki et al., 2007). Rotifers are also considered to be, along with ciliates, the most sensitive to toxins introduced into the system with wastewater, and thus can be used as indicators of toxicants (Jenkins et al., 2004). The sludge we used might have suffered from a toxic compound coming to the plant that made the rotifers less active. Their condition may have also been affected by chemicals used in this plant to precipitate phosphorus. The results show that the effectiveness of rotifers may depend on the sludge composition. Kaewpipat and Grady (2002) showed that the microbial composition of sludge, taken from the same plant and kept under identical conditions in laboratory-scale sequencing batch reactors, was dynamic and diverged significantly. Thus, if rotifers were to be used on a technological scale, it would be advisable to check their effectiveness on a smaller laboratory scale prior to the treatment. As our experiment showed, it is possible to note the increasing rotifers’ growth rate even during the first 5 days after inoculation. The second experiment confirmed that L. inermis are effective in removing filamentous bacteria from activated sludge. It also showed that rotifers significantly improve settling properties of activated sludge. After 30 min, the volume of sludge with rotifers was noticeably lower than the volume of sludge without them (Fig. 6). The variability in the level of the sludge among experimental cylinders was smaller than that among control cylinders. What is more, in the control cylinders there was a thin layer of sludge floating

at the surface. Such a layer was absent in experimental cylinders (Fig. 6). It seems that rotifers can affect the volume of sludge in two ways. One of them is by simply consuming filaments in large quantities. It was found that rotifers can consume several times their body weight per day (Clement and Wurdak, 1991), so they can be really effective in removing filaments from the environment. Moreover, by reducing the number of filaments, they remove the so-called ‘‘bridges’’ between flocs, which are responsible for poor settling of the sludge. The experiment also showed that rotifers transferred from a culture to activated sludge in which they had not been present earlier not only survived, but in fact thrived. Nogrady et al. (1993) stated that the amount of available food is the main factor limiting rotifer population growth. Apparently, activated sludge provided rotifers with the quantities of food that enabled them to multiply at the rate of even 0.8 ind/per day, which created really high densities in a short time. An additional advantage of rotifer foraging activity is their impact on sludge volume. All over the world, the disposal of excess sludge is considered a bottleneck of wastewater treatment. Pe´rez-Elvira et al. (2006) presented a review of current techniques aimed at reducing sludge production. Among them, predation on bacteria is reported to decrease biomass production by up to 43% in the case of predatory ciliates, and up to 80% when protists and metazoans were employed. Also, Lapinski and Tunnacliffe (2003) showed that bdelloid rotifers are effective in removing suspended solids by consumption and improving settleability. Their experiments suggested that rotifers can reduce biomass production and improve effluent clarity. These papers demonstrated the importance of the role that rotifers play in activated sludge. However, research to date has concentrated on the impact of rotifers on dispersed unicellular bacteria. Our experiments proved that filamentous bacteria can also be limited by rotifers. It turned out that by feeding on filamentous bacteria, rotifers simultaneously decrease sludge production, which in fact should be considered a positive side effect of their activity. Such reduction is probably less efficient than the reduction in anaerobic digesters. However, the latter method, although seen as cost-effective and environmentally friendly, in fact requires considerable investments to work properly

ARTICLE IN PRESS WAT E R R E S E A R C H

42 (2008) 2483 – 2490

(for review see Mata-Alvarez et al., 2000). There still are numerous small- and medium-scale treatment plants that are not equipped with anaerobic digesters. For such plants especially, but also for those with anaerobic digesters, it is better to produce the least amount of excessive sludge. As rotifers can control bulking and improve settling properties of the sludge, it certainly is worth trying to use them on a technological scale in treatment plants. Such a method would require high densities of rotifers, but there are already methods of culturing them on a commercial scale. In the review by Lubzens et al. (2001), there are a few different methods described, such as batch cultures, semi-continuous and continuous cultures. For example, Brachionus plicatilis and Brachionus rotundiformis, used as food for larval stages of marine fish, were fed green algae or yeast cells. L. inermis used in our experiments were cultured on bacteria proliferating on rice grains, and they reached high densities, suggesting that the costs of culturing might be acceptable. An additional advantage is that, as our experiment showed, rotifers can multiply at a high rate when they feed on filamentous bacteria in the sludge. Thanks to a fact that the initial inoculum for a treatment plant would not have to contain extremely high densities of rotifers. Our experiments proved that Lecane rotifers may be a useful, natural tool to control growth of M. parvicella in activated sludge. One may wonder if the rotifers would be equally effective in controlling filaments which form the foam on reactors’ surfaces. The foam creates a different type of medium, much denser than activated sludge itself, so it certainly is less beneficial for rotifers. However, as the foam is usually formed as a consequence of excessive growth of filamentous bacteria in the sludge, it is likely that if the rotifers are applied early enough to limit the growth of filaments, the foam will not appear at all. It has also yet to be tested whether the rotifers are similarly effective against other troublesome filaments. It would also require further studies to find the most effective and applicable method of rearing rotifers in sufficient quantities. Nevertheless, if one takes into account all the advantages of using rotifers to limit the growth of filaments and improve the settling properties of sludge, this is certainly worth the effort.

5.

Conclusions

1. Lecane rotifers are capable of significantly reducing the number of filamentous bacteria in activated sludge. 2. This activity significantly improves settling properties of activated sludge. 3. Rotifer effectiveness differs depending on the type of filaments. The rotifers feed more readily on Microthrix parvicella than Actinomycetes. 4. The rotifers are able to survive and proliferate when transferred to a sludge in which they did not occur earlier.

Acknowledgments The authors thank Dr. Irena Bielan´ska-Grajner for identification of rotifer species used in the research and Msc.

2489

Ma"gorzata P"awecka for technological data and comments concerning the treatment process. They also thank Prof. J. Koz"owski and Dr. W. Fia"kowski and anonymous reviewers for many valuable comments made on earlier versions of this paper. This work was supported by the European Community project Centre of Excellence IBAES No. EVK2-CT-2002-80009. R E F E R E N C E S

Blackbeard, J.R., Ekama, G.A., Marais, G.v.R., 1986. A survey of filamentous bulking and foaming in activated sludge plants in South Africa. Water Pollut. Control 1, 90–100. Clement, P., Wurdak, E., 1991. In: Harrison, F.W., Ruppert, E.E. (Eds.), Rotifera in Microscopic Anatomy of Invertebrates, vol. 4, Aschelminthes. Wiley-Liss & Sons, Chichester, pp. 219–289. Drzewicki, A., Hul, M., 1997. Experimental trial of using a ciliate Trithigmostoma cucullulus to reduce number of filamentous bacteria in activated sludge on the technical scale. (Pro´ba wykorzystania orzeska ˛ Trithigmostoma ccullulus do zwalczania bakterii nitkowatych w osadzie czynnym w skali technicznej). In: Proceedings of 17th Congress of Polish Hyrobiologists, Poznan´, pp. 74–75. Drzewicki, A., Klimiuk, E., Kulikowska, D., 2007. The influence of hydraulic retention time and sludge age on activated sludge biocenosis formation in SBRs treating landfill leachates. Pol. J. Nat. Sci. 22 (3), 425–446. Eikelboom, D.H., 2000. Process Control of Activated Sludge Plants by Microscopic Investigation. IWA Publishing, London. Eikelboom, D.H., Andreadakis, A., Andreasen, K., 1998. Survey of filamentous populations in nutrient removal plants in four European countries. Water Sci. Technol. 37 (4–5), 281–289. Fia"kowska, E., Fyda, J., Pajdak-Sto´s, A., Wia˛ckowski, K., 2005. Activated Sludge—Biology and Microscopic Analysis (Osad czynny – biologia i analiza mikroskopowa). Oficyna Wydawnicza ‘‘Impuls,’’ Krako´w. Inamori, Y., Kuniyasu, Y., Sudo, R., Koga, M., 1991. Control of the growth of filamentous microorganisms using predacious ciliated protozoa. Water Sci. Technol. 23, 963–971. Jenkins, D., Richard, M.G., Daigger, G.T., 2004. Manual on the Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems. Lewis Publishers, Boca Raton, FL. Kaewpipat, K., Grady Jr., C.P.L., 2002. Microbial population dynamics in laboratory-scale activated sludge reactors. Water Sci. Technol. 46 (1–2), 19–27. Kristensen, G.H., Jørgensen, P.E., Nielsen, P.H., 1994. Settling characteristics of activated sludge in Danish treatment plants with biological nutrient removal. Water Sci. Technol. 29 (7), 157–165. Lapinski, J., Tunnacliffe, A., 2003. Reduction of suspended biomass in municipal wastewater using bdelloid rotifers. Water Res. 37, 2027–2034. Lemmer, H., 1998. Bla¨hschlamm, Schwimmschlamm und Schaum in Belebungsanlagen-Ursachen und Beka¨mpfung. Gesellschaft zur Fo¨rderung der Abwassertechnik e. V., Hennef. Lubzens, E., Zmora, O., Barr, Y., 2001. Biotechnology and aquaculture of rotifers. Hydrobiologia 446/447, 337–353. Mata-Alvarez, J., Mace, S., Llabres, P., 2000. Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresourc. Technol. 74, 3–16. Nogrady, T., Wallace, R.L., Snell, T.W., 1993. In: Nogrady, T. (Ed.), Dunomt, H.J. (Coordinating editor), Guides to Identification of Microinvertebrates of the Continental Waters of the World, Rotifera, vol. 1: Biology, Ecology and Systematics. SPB Academic Publishing, Hague.

ARTICLE IN PRESS 2490

WAT E R R E S E A R C H

42 (2008) 2483– 2490

Pe´rez-Elvira, S.I., Nieto Diez, P., Fdz-Polaco, F., 2006. Sludge minimisation technologies. Rev. Environ. Sci. Bio/Technol. 5, 375–398. Segers, H., 1995. The Lecanidae (Monogononta), Rotifera. In: Dumont, H.J.F. (Ed.), Guides to the Identification of the Microinvertebrates of the Continental Waters of the World. SPB Academic Publishing, p. 226. Seka, M.A., Kalogo, S., Hammer, F., Kielemoes, J., Verstraete, W., 2001. Chlorine-susceptible and chlorine-resistant type 021N bacteria occurring in bulking activated sludges. Appl. Environ. Microbiol. 67 (11), 5303–5307. Seviour, R.J., Blackall, L.L. (Eds.), 1999. The Microbiology of Activated Sludge. Kluwer Academic Publishers, Dordrecht.

Seviour, E.M., Williams, C.J., DeGrey, B., Soddell, J.A., Seviour, R.J., Lindrea, K.C., 1994. Studies on filamentous bacteria from Australian activated sludge plants. Water Res. 28, 2335–2342. Tandoi, V., Jenkins, D., Wanner, J. (Eds.), 2006. Activated Sludge Separation Problems. Theory, Control Measures, Practical Experience. IWA Publishing. Wanner, J., Ruzickova, I., Jetmarowa, P., Krhutkova, O., Paranikova, J., 1998. A national survey of activated sludge separation problems in Czech Republik: filaments, flocs characteristics and activated sludge metabolic properties. Water Sci. Technol. 37 (4–5), 271–279.