FEMS Microbiology Reviews 29 (2005) 4964 www.fems-microbiology.

org

Microthrix parvicella, a lamentous bacterium causing bulking and foaming in activated sludge systems: a review of current knowledge
Simona Rossetti
b

a,*
a

, Maria C. Tomei a, Per H. Nielsen b, Valter Tandoi

Water Research Institute, CNR, Via Reno 1, 00198 Rome, Italy Section of Environmental Engineering, Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark Received 12 November 2003; received in revised form 5 July 2004; accepted 13 September 2004 First published online 19 October 2004

Abstract This review summarizes the microbiology and physiology of Microthrix parvicella and the methods of its growth control in activated sludge wastewater treatment plants. This lamentous bacterium is of high interest because of its worldwide involvement in severe bulking and foaming at wastewater treatment plants. We present a critical analysis of physiological and kinetic data on M. parvicella and discuss its growth and storage abilities in various environments with the aim of understanding the strategies of this organism to successfully compete with other bacteria in activated sludge. Additionally, this review elaborates on research needs for dening reliable control strategies of bulking and foaming based on key features of M. parvicella. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Microthrix parvicella; Activated sludge; Bulking; Foaming; Filamentous bacteria; Control strategies

Contents 1. The Microthrix parvicella puzzle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Isolation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Tools for in situ identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Techniques for studying the physiology and growth kinetics of M. parvicella . 2.1. Pure culture studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. In situ activated sludge studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiology of M. parvicella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Basic metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Storage capabilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Growth kinetics of M. parvicella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Aerobic growth rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Growth yield coefficient (Y) and saturation constant (Ks). . . . . . . . . . . . 4.3. pH and temperature effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors affecting growth of M. parvicella in activated sludge plants . . . . . . . 5.1. Growth characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Substrate storage capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
*

2.

3.

4.

5.

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

50 50 51 52 52 53 53 54 54 55 56 56 57 58 58 59 60

Corresponding author. Tel.: +39 06 8841451; fax: +39 06 8417861. E-mail address: rossetti@irsa.rm.cnr.it (S. Rossetti).

0168-6445/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsre.2004.09.005

50

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

6. 7.

Control strategies for M. parvicella bulking and foaming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Research needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

1. The Microthrix parvicella puzzle Microthrix parvicella is a lamentous bacterium commonly occurring in activated sludge wastewater treatment plants (WWTPs) with dierent operating conditions and aeration basin congurations. Several global surveys have shown that this organism is most frequently responsible for the problems of solidliquid separation in bulking and foaming [13] and substantial eorts have been directed towards solving the operational and performance problems it causes in WWTPs. Activated sludge represents a unique ecosystem and this bacterium has been detected only there. Sequences of 16S rRNA that appear in clone libraries from several other environments including marine sediments [4], soil [5,6], and marine environments [5] are only distantly related to Candidatus Microthrix parvicella (<92% 16S rRNA sequence similarity). Microthrix parvicella is a long, thin (diameter of 0.60.8 lm), non-branched and unsheathed lamentous bacterium. Its coiled appearance and characteristic gram-positive reaction make it easy to recognize it by microscopy in activated sludge samples (Fig. 1). The organism was originally described by Pasveer [7] and while several isolates have been cultured [814], only a few of them are maintained in pure culture [1113]. 1.1. Isolation Many attempts have been made to isolate M. parvicella from WWTPs and twenty years elapsed since the rst isolates were obtained in the 1970s, before a taxonomically identied reference strain of M. parvicella became available [11]. The rst pure culture was obtained by van Veen [8], although several earlier studies mention attempts to isolate this organism [7,15,16]. The descriptions of the laments studied by Pasveer [7] are identical to current descriptions of M. parvicella. Pure cultures exhibited lamentous morphology only in an atmosphere of 90% CO2 + 10% air, at a pH below 6 and when glucose was the carbon source. These isolates were then identied as Escherichia coli [7] without giving any details of the identication methods used. Farquhar and Boyle [15,16] stated that their laments resembled lament-forming lactic acid bacteria and they biased their isolation conditions towards favouring this group of microorganisms. None of these rst reports described any diculties in lament isola-

tion, so it is probable that the microorganism isolated was not M. parvicella. The description of M. parvicella laments and their isolation as given by van Veen [8] is detailed, and suggest that isolation is complex and time consuming. Later Eikelboom [9] utilized a medium comprising sludge hydrolysate and a complex vitamin mixture to obtain several isolates of M. parvicella. No indication of any possible diculties in obtaining these cultures was given. The organisms grew to 1 mm colonies on the sludge agar after 10 d subculturing. The same isolation medium was used by Slijkhuis and Deinema [17] but they reported that the sludge hydrolysate contained colloidal particles, which negatively affected cell yield. Slijkhuis [10] subsequently developed a chemically dened medium for studying the pattern of carbon utilization by M. parvicella. This axenic culture study generated the rst physiological data on the presumptive M. parvicella. In contrast to previous work [8], the isolates obtained by Slijkhuis had an unusual metabolism: they did not grow on simple substrates such as sugars and organic acids, and required oleic acid or its polyoxyethylene sorbitan ester (Tween 80) as carbon and energy source. The Slijkhuis isolates were characterized only morphologically [10] and detailed biochemical properties or 16S rRNA analyses are not available to compare them to the recently isolated strains. In the next decade no other pure cultures were obtained. Slijkhuis results had led to a widespread opinion that Tween 80 was essential substrate for growth of M. parvicella, although this substrate did not support the growth of subsequent isolates. Our comprehension of this lamentous bacterium was further complicated by description of an isolate of M. parvicella obtained by micromanipulation on Tween 80-based growth medium by Forster and coworkers [1820]. Their isolate showed morphology that was quite dierent to that published for any pure culture of M. parvicella. Their description of this isolate included an ability to undergo a rod-lament transition, to form spores and possess motility and to sometimes be present as gram-negative rods and then convert to gram-positive extended laments. Most likely their report describes the wrong organism and their results have never been conrmed by any available phylogenetic analysis of the isolate nor by later work. Blackall et al. [11] suggested that this isolate was a Bacillus sp., and lamentous members of this genus are commonly present in activated sludge.

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

51

sional presence of swollen and necrotic cells, which is consistent with the view that M. parvicella pure cultures are probably under environmental stress that may explain their poor growth under axenic conditions [14,22]. Subsequently, additional strains of M. parvicella have been cultured [12,13] with a complex culture medium (R2A medium [23]). None of these isolates could grow on the chemically dened medium of Slijkhuis [10], suggesting that considerable physiological dierences exist among these strains [1113] and the original Slijkhuis strain. The extent of these dierences was assessed by Tandoi et al. [22] who showed that all recently isolated strains did not have a specic requirement for long-chain fatty acids as carbon and energy sources. Table 1 details the media, isolation methods and growth conditions used for culturing M. parvicella axenically. The only common feature to all the studies is the diculty in maintaining this lamentous microorganism in culture, which may explain why early isolates of this bacterium, including those obtained by Slijkhuis and Eikelboom, are no longer viable.

1.2. Taxonomy The identity and the widespread occurrence of M. parvicella in WWTPs suering from severe bulking have been conrmed by molecular methods. Blackall et al. [11] were the rst to report the denitive phylogenetic aliation of M. parvicella. These workers placed M. parvicella (strain DAN1-3) as a novel, deep-branching member of the Actinobacteria based on sequencing of its 16S rRNA gene. An identical phylogenetic position was shown for the Italian M. parvicella isolate, strain RN1 [12], which represents the sole strain unequivocally identied on a molecular basis, used for the physiological and kinetic studies. Fig. 2 shows the phylogenetic tree of all publicly available sequences of M. parvicella. In 1996, DNA from a non-viable culture of the original isolate obtained by van Veen [8], kept at 4 C, was extracted and the nearly complete 16S rRNA gene was subsequently amplied and cloned [24]. Sequence analysis showed that this strain had 98% similarity to the 16S rRNA of M. parvicella strain DAN1-3 [24]. However, this information was never published in a scientic journal and these sequence data are not available in any public sequence database. While M. parvicella is not a taxonomically valid name because of lack of any detailed phenotypic characterization, it was proposed that the name be retained, and the organism be elevated to Candidatus status because M. parvicella is the vernacular name for this lament used throughout the wastewater industry [14]. However, as this review shows, we think that the requisites for a name validation can be satised based on the

Fig. 1. Light micrograph of the microscopic appearance of Microthrix parvicella in an activated sludge sample taken from the aeration basin of a conventional plant: (a) dark, coiled laments present in the bulk solution; (b) typical gram-positive laments. Bar is 10 lm.

In 1994 a new strain (strain DAN1-3) was isolated [11] from a conventional activated sludge plant in Dandenong (Australia) using culture medium (modied NTM medium) that did not contain any long-chain fatty acids. Ultrastructure studies of strain DAN1-3 conrmed that it had a typical gram-positive wall [14,21] and the characteristic uneven appearance of the laments was from the presence of spherical swollen cells along their length. Transmission electron microscopy (TEM) showed that these cells contained no lipid deposits or any other accumulated intracellular storage material, and were not spores or cysts, but possessed some features consistent with resting structures [21]. Filaments in activated sludge usually appear much more even with only an occa-

52 Reference

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

physiological and kinetic data that are now available for this organism.
[8] [9] [17] [11] [13] [12]

1.3. Tools for in situ identication

Slant cultures at 4 C Slant cultures at 4 C NR Slant cultures at 4 C NR (storage at 80 C in glycerol failed) Slant cultures at 4 C

Table 1 Growth media and methods used for the isolation and maintenance of M. parvicella

NR, not reported. It contains: yeast extract, proteose peptone, casamino acids, glucose, soluble starch and sodium pyruvate.

Ammonium sulphate NR NR Ammonium sulfate and organic nitrogen Organic nitrogen

Nitrogen source

Organic nitrogen

Specic 16S rRNA-targeted probes were developed by Erhart et al. [25] (Table 2) for the uorescence in situ hybridization (FISH), which could successfully highlight M. parvicella laments in activated sludge biomass samples. These workers also designed MPA650 probe that requires competitor probes to allow for its relative non-specicity (CompMPA650.1 and CompMPA650.2 in Table 2). M. parvicella, like some other grampositive bacteria, needs a pre-treatment with mutanolysin to enhance cell permeability prior to whole-cell probing. However, despite this necessary step, FISH has been successfully applied to detect M. parvicella in WWTPs in several countries [25,27]. As for other lamentous bacteria in wastewater systems [28,29], a suite of antibodies has also been developed for detection and quantication of M. parvicella in activated sludge [30]. The antibodies were produced against strain RN1 and showed varying reactivity against putative M. parvicella laments in activated sludge samples [30]. Dierent from the FISH probing, routine application of this technique for in situ detection of M. parvicella has not become popular. Although some of the questions concerning the in situ growth of M. parvicella have been addressed, more knowledge base is needed before ecient control measures against this bacterium can be dened and implemented. Its slow growth rate, diculty in storing isolates and in generating phenotypic information have hindered attempts to elucidate the reasons for the proliferation of M. parvicella in WWTPs. These diculties have inuenced research eorts, at times causing these to be misdirected. Therefore, it is proper time to summarize the current state of our understanding of the socalled M. parvicella puzzle [31] and to speculate on what still needs to be accomplished to better control the bulking and foaming problems it causes.

Maintenance

Dilution and plating Dilution and plating NR Micromanipulation

Micromanipulation Contained in yeast extract 7.2

Isolation

B12 and thiamine complex complex B12 and thiamine

Growth factor added

pH

NRa NR NR 8.0

Vitamin Vitamin Vitamin Vitamin

Sulphur source

Sulphate NR NR Organic sulfur

Organic sulfur and/ or sulfate Organic sulfur and/ or sulfate

7.2

Contained in yeast extract

Micromanipulation

2. Techniques for studying the physiology and growth kinetics of M. parvicella Both pure culture studies and culture-independent techniques have been used to study the key phenotypic features of this lamentous bacterium. Most eorts have attempted to address the problems arising from its slow growth rate in culture. The major diculties in studying M. parvicella emerge from the number of novel techniques that have been tested and applied. However, this work has now allowed us to reach satisfactory conclusions on many aspects of its physiology and its growth

Glucose Sludge hydrolysate Sludge hydrolysate Succinate and peptone

Complex mediumb R2A

Media

I H H NTM

R2A

Complex mediumb

Carbon source

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

53

Fig. 2. Phylogenetic tree showing all publicly available sequences of Candidatus Microthrix parvicella.

Table 2 Sequences, target sites and hybridization condition for rRNA-targeted probes for Microthrix parvicella [25] Probe MPA60 MPA223 MPA645 MPA650 CompMPA650.1 CompMPA650.2
a

Probe sequence (5 0 3 0 ) GGATGGCCGCGTTCGACT GCCGCGAGACCCTCCTAG CCGGACTCTAGTCAGAGC CCCTACCGGACTCTAGTC CCCTACCGCACTCTAGTC CCCTACCGAACTCTAGCC

Target site (16S rRNA position)a 6077 223240 645661 650666 650666 650666

Stringency: formamide concentration (%) 20 20 20 20 20 20

From Escherichia coli numbering of Brosius et al. [26].

kinetics, and to validate them by comparing the results from dierent experimental approaches. 2.1. Pure culture studies Only two studies with pure cultures of M. parvicella have been reported. The rst, performed in batch and in continuous culture [10], is the only one in which steady state growth conditions were used. In all other studies, the paucity of the biomass has been the major drawback in investigating the growth kinetics. In the second study, total extended lament length (TEFL) measurement [1] was used for estimating growth rates in batch pure culture of M. parvicella in both liquid and solid media [22,32]. This technique is particularly useful for evaluating the physiology and growth kinetics of this and other slow-growing bacteria, without altering the growth environment during the experiment, because only a very small amount of biomass (corresponding to a total lament length of 100500 lm) is required for analysis. The TEFL measurement method has also been tested on faster-growing laments such as Thiothrix sp. and no dierence between growth parameter values obtained with the TEFL-approach and those estimated

with more traditional methods such as changes in ATP content and culture turbidity was found [33]. 2.2. In situ activated sludge studies Microautoradiography (MAR) has been used in several studies to investigate the physiology of M. parvicella under in situ conditions. This method is described in detail in several publications (e.g., [3437]). Activated sludge samples containing high numbers of M. parvicella are incubated under dened conditions with appropriate radiolabelled substrates. After cell xation and photographic processing, the bacterial cells able to take up the labelled substrate (MAR-positive bacteria) can be visualized by light microscopy because they are covered with silver grains. Microautoradiography combined with FISH and uorescence microscopy, has several advantageous features for studying the in situ physiology of M. parvicella. Investigations can be performed directly on the bacteria present in the sludge without any enrichment or pre-cultivation and under conditions identical to those experienced by bacteria in the treatment plant. Among other methods applied for studying the physiological characteristics of

54

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

M. parvicella, detection of surface-associated exoenzymes with uorogenic substrates and uorescent hydrophobic beads to characterize surface properties have also been used [38]. 3. Physiology of M. parvicella 3.1. Basic metabolism Pure-culture studies show that M. parvicella is an aerobic chemoorganotroph. Its presumed ability to denitrify (because of its proliferation in anoxic/aerobic systems, i.e., the Carrousel plants) [2] was resolved by pure-culture studies [22], which showed that isolates of M. parvicella are aerobic, non-fermentative and can reduce nitrate not further than to nitrite. Cultures of M. parvicella can grow over a wide range of oxygen partial pressures, and good growth is obtained under microaerophilic conditions. At low dissolved oxygen (DO) concentration ($0.4 mg l1) it produces long and regular laments with none of the empty or deformed cells observed at higher values of dissolved oxygen (Fig. 3). Because limiting dissolved oxygen conditions seem to be benecial for the growth of this microorganism, low DO may represent a metabolic advantage for M. parvicella proliferation in activated sludge. It has been reported that high DO concentrations (>6 mg l1) may be toxic to it and so M. parvicella might be considered a microaerophile [10]. Andreasen and Nielsen [36] observed that M. parvicella assimilated [3H] oleic acid in the presence of various electron acceptors after starvation periods from 4 h to 7 d. M. parvicella remained longer viable under anoxic and anaerobic conditions than in oxic conditions, an observation that agrees with the assumption that it is a microaerophile [10,22]. The eect of prolonged anaerobic incubation times was studied with pure cultures [32]. M. parvicella, grown aerobically, was incubated under anaerobic con-

Fig. 3. Light micrograph of the microscopic appearance of M. parvicella, strain RN1 grown in liquid media under (a) fully aerobic (DO > 2 mg l1) and (b) microaerophilic conditions (DO < 0.4 mg l1). Bar is 10 lm.

dition at 20 C for up to three months without losing its capability to grow at rates comparable to those before anaerobic incubation, when aerobic conditions were restored. Similar behaviour was observed with strains incubated under anoxic conditions [32]. There is considerably more controversy regarding the organic substrates used by the various isolates of M. parvicella in pure culture and in situ (Table 3). For the Dutch isolate, both Tween 80 and oleic acid could serve as a carbon and energy source [10]. Growth also occurred on polyoxyethylene sorbitan derivates of stearic acid (Tween 60), palmitic acid (Tween 40) and lauric acid (Tween 20). The free long-chain fatty acids as well as saturated fatty acids with medium chain length (i.e., caproic, caprylic) and volatile fatty acids (acetic and butyric acids) were barely used by M. parvicella. Fructose, glucose, citric acid, succinic acid and lactic acid were not utilized by the Dutch isolate. Moreover, M. parvicella required L -methionine as a source of reduced sulphur, and this could be replaced by other reduced-sulphur compounds such as L -cysteine, sodium sulphide and sodium thiosulphate [10]. This stringent growth requirement has not been investigated further, and all subsequent pure culture studies have utilized growth media containing these S compounds. The only other isolate that has been extensively characterized in pure culture is M. parvicella strain RN1 [22]. This strain utilized a wide range of carbon sources, including organic acids, complex substrates and fatty acids as sole carbon and energy sources. In contrast to the Slijkhuis isolate, neither oleic acid nor its sorbitan esters supported growth of this strain (Table 3). Blackall et al. [11,14] showed that neither pure cultures of their M. parvicella nor activated sludge isolates from several countries could grow on oleic acid or its sorbitan esters. In situ microautoradiography studies [34,35,38] showed that the long-chain fatty acids (LCFA), oleic and palmitic acids, and to a lesser extent trioleic acid, were taken up by M. parvicella growing in activated sludge, but simple substrates such as acetate were not (Table 3). Dierences in substrate utilization may be explained in terms of the dierent culture environments. In pure cultures, isolates, grown in a non-competitive environment are metabolically diverse and can use a range of simple carbon sources. In activated sludge plants, where the competition for easily biodegradable substrates is more intense, M. parvicella seems to grow on more complex compounds that are preferentially accessible to it. While pure culture experiments show the physiological potential of this organism, activated sludge experiments demonstrate its functional physiology. Finally, it is worth noting that dierences between the recent [11,12,14] and Slijkhuis [10] M. parvicella isolates cannot be easily addressed, mainly because of the lack of information about the phylogenetic alia-

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964 Table 3 Summary of the main physiological properties of Microthrix parvicella in pure culture and in situ studies Parameter Pure cultures Candidatus M. parvicella [22] Aerobic growth on: Acetic acid Pyruvic acid Propionic acid Butyric acid Citric acid Lactic acid Glucose Fructose Yeast extract Casamino acids Glycine Leucine Sodium oleate Proteose peptone Oleic acid Palmitic acid Trioleic acid Tween 80-oleic acid Tween 60-stearic acid Tween 40-palmitic acid Tween 20-lauric acid Ethanol Autotrophic growth Denitrication: Reduction of nitrate to N2 Reduction of nitrate to nitrite Microaerophilic growth Storage: Aerobic Anaerobic Anoxic
a b

55

In situ [35,38] Slijkhuis isolate [10] nda nd nd nd nd nd nd nd nd + nd nd + + + + nd nd +(lipid) nd nd nd nd nd nd nd nd nd nd + + + nd nd nd nd nd nd nd +(lipid) +(lipid) nd

+ + nd nd nd nd nd + + nd nd + nd nd nd nd nd nd + + +(PHAb, polyphosphate) +(PHA) +(PHA)

nd, not determined. PHA, poly b-hydroxyalkanoates.

tion of the latter. Future studies and phylogenetic comparison of new pure cultures to existing Candidatus M. parvicella [1113], may help to resolve these metabolic discrepancies. 3.2. Storage capabilities The substrate storage capabilities of M. parvicella have been reported in a number of pure culture studies under aerobic [10,12,22,32] and anoxic/anaerobic conditions [32]. Anoxic/anaerobic storage was conrmed by incubation of aerobically-grown laments under anoxic or anaerobic conditions, and then observing the intracellular storage of poly b-hydroxyalkanoates (PHA)-like inclusions by epiuorescence microscopy after Nile Blue A staining (Fig. 4(a)). Similar results have been obtained in experiments on mixed biomass from a conventional treatment plant by combining FISH and Nile Blue A staining (Fig. 4(b)) [32]. Formation of lipid storage granules in M. parvicella has also been observed during in situ studies of

activated sludge samples from nutrient removal plants [38]. Added [14C] oleic acid in activated sludge containing an excessive amount of M. parvicella was completely and rapidly removed (within 1020 min) under both aerobic and anaerobic conditions. All soluble oleic acid was either taken up by the bacteria or adsorbed to the oc surfaces (Fig. 4(c)). FISH-MAR analysis using a FISH probe for M. parvicella conrmed that uptake occurred under both aerobic and anaerobic conditions. Extraction and identication of the radiolabelled lipids by gas chromatography showed that the storage compound was most probably a triglyceride. Cell surface hydrophobicity is important in LCFA uptake: in situ studies with hydrophobic microspheres showed that M. parvicella laments in treatment plants (with or without foaming) are more hydrophobic than most other activated sludge bacteria [38]. A hydrophobic surface can attract lipids, LCFA and other nonpolar substrates, and so will be advantageous in any competition for this type of substrate. In pure culture M. parvicella can produce extracellular esterases

56

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

Fig. 4. Fluorescence micrograph of anaerobic storage products in strain RN1 after Nile Blue A staining (a) and in laments of M. parvicella present in activated sludge (b). Some PHA granules are arrowed. Bar is 10 lm in (a) and 5 lm in (b). (c) In situ anaerobic uptake of radiolabelled oleic acids detected by microautoradiography. The bacteria were stained with DAPI and the silver grains can be seen as small black dots. Epiuorescence and bright-eld microscopy are combined.

(possibly lipases) when growing on Tween substrates [10]. In situ studies in activated sludge using a uorogenic substrate showed that M. parvicella possesses lipase activity on its cell surface [38]. The presence of this lipase should improve its ability to take up lysis products and LCFA from lipids associated with the cell surface. It is not clear how M. parvicella obtains the energy required for LCFA uptake and storage product formation under anaerobic conditions because the organism does not appear to have an active uptakerelease of orthophosphate as do the polyphosphate accumulating organisms (PAOs) [36]. It is possible that other as yet unidentied intracellular reserves such as glycogen provide energy for uptake and storage to occur under anaerobic conditions. When oxygen (or maybe nitrate) is available as electron acceptor, the storage material is used for growth. M. parvicella is the rst activated sludge organism that has a physiological ability to accumulate lipids, so it can be described as a lipidaccumulating organism (LAO) [38]. 4. Growth kinetics of M. parvicella Quantitative kinetic data are required to predict the development of individual microbial populations in mixed cultures found in WWTPs. For example, control of lamentous bulking of activated sludge will become more feasible when the known kinetic characteristics of lamentous and oc-former bacteria allow the construction of reliable process models. Filamentous bacteria have so far mainly been characterized on a qualitative basis, as research eorts have

mainly been directed toward dening the taxonomic position of any new isolates. Only limited quantitative data on their physiology and kinetics are available. Kinetic characterization of M. parvicella is hindered because the organism is dicult to isolate and cultivate, so in spite of its importance in bulking and foaming, very limited quantitative kinetic and stoichiometric data exist for it. 4.1. Aerobic growth rate Slijkhuis studied the physiological and kinetic characteristics of this bacterium under a range of environmental conditions and with dierent carbon and energy sources [10]. Its maximum growth rate on Tween 80 was determined in batch and continuous culture at 25 C. Batch growth was assessed by measuring its protein content rather than dry weight because of the large contribution to dry weight of the lipids stored intracellulary, especially during the early phases of batch growth. This probably occurred because of a high initial substrate/biomass ratio, and the dynamic conditions of batch growth encouraged substrate storage [39]. The maximum batch growth rate (lmax) was 0.38 d1. A typical batch kinetic curve is shown in Fig. 5. The steady state growth of this strain was studied in a chemostat at dilution rate in the range of 0.0110.069 h1. In these experiments it was possible to use dry weight for biomass concentration measurements because lipid storage was not substantial. The calculated lmax was 1.44 d1, which is higher than in batch cultures. Besides the dierences between batch and chemostat conditions, some of the discrepancies

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

57

14

12

10

ln (X)

(a) (b)
0 2 4 6 8 10 12 14

ria prevented these from continuing. TEFL increases were also followed on isolates obtained by micromanipulation from the same sludge sample. The same mean lmax value (0.66 d1) was found for both pure and mixed cultures, showing that the presence of other activated sludge organisms had no signicant eect on growth kinetics of M. parvicella. Table 4 provides an overview of the available literature data on M. parvicella lmax values. It is worth noting that the lmax values determined in batch culture cover a quite narrow range and are generally much lower than those observed in continuous systems. This trend needs to be conrmed with data from other strains, but there is evidence that higher growth rates do exist in WWTPs, and that continuous systems like WWTPs can be better simulated in a chemostat [40] rather than in batch culture systems. 4.2. Growth yield coecient (Y) and saturation constant (Ks) The growth yield coecient (Y), dened as mass of biomass formed per unit of utilized substrate), was evaluated by Slijkhuis [10] from continuous culture data at various dilution rates (D) in the range of 0.010.05 h1 by the Pirt equation [41]; values of 0.34 (g cell dry weight g Tween 801) or 0.17 (mg VSS mg COD1) 1 have been published. From TEFL measurements on the RN1 strain grown on R2A medium, a value of 1.2 108 lm mg TOC1 (corresponding to %0.1 mg VSS mg COD1) 2 was determined. These low Y values indicate that M. parvicella has a high energy requirement for growth. M. parvicella has a very high substrate anity (low Ks values) [1,2]. Evaluation of Ks intrinsic values is dicult because of the complications introduced by substrate diusion rates in occulent cultures, so the estimated values are usually dened as apparent values. Intrinsic values have been reported for only a few lamentous bacterial species in pure culture. The apparent Ks value was estimated for the Slijkhuis isolate by respirometric batch experiments using Tween 80 and oleic acid as growth limiting substrates, and values of 15 and 8 mg l1 (corresponding to COD values of 30 and 23 mg l1) were obtained for Tween 80 and oleic acid, respectively. The Ks value for strains RN1 and 4B, was estimated with the same approach as used for determining their lmax. The Ks value of 3.9 mg COD l1 was obtained for both strains. The apparatus used in these experiments allowed the growth of a single lament to be

time (d)
Fig. 5. Growth curves of M. parvicella obtained by batch tests in liquid pure cultures: (a) TEFL (lm ml1) vs. time (data from [22]) and (b) protein content (mg l1) vs. time (data from [10]).

in lmax values could come from using dierent biomass measurement parameters [10]. A quantitative determination of lmax by TEFL measurement was carried out on isolates of M. parvicella strain RN1 grown in liquid R2A medium [22]. The lmax of this strain ranged between 0.3 and 0.5 d1. Fig. 5 shows a typical TEFL prole obtained during a batch test. A comparison of the batch growth curves given in Fig. 5 shows that the lmax values, estimated for dierent strains grown on dierent substrates, are very similar as indicated by their slopes. Growth kinetics for strain RN1 and another isolate, strain 4B [32], were also studied on solid R2A medium with TEFL measurements. At 20 C and for substrate concentrations in the range of 503000 mg COD l1, lmax values were 0.46 0.04 and 0.37 0.05 d1 for strains 4B and RN1, respectively. The substrate concentration in the range mentioned above had no eect on the organisms lmax. These lmax values conrm the characteristic slow growth of M. parvicella. Results from pure culture studies, while essential for physiological and kinetic characterizations, cannot be used directly to predict the behaviour of a microorganism in a WWTP since possible interactions with other microorganisms and/or availability of specic substrates cannot be precisely reproduced in pure culture experiments. To better simulate in situ growth conditions for M. parvicella, kinetic tests have been conducted in activated sludge [32]. The lmax was estimated at 20 C by measuring the extended lament length in activated sludge samples spread onto agar plates; TEFL measurements were performed until overgrowth by other bacte-

COD/Tween 80 ratio = 2. Conversion factor of TOC to COD of 0.35 and assuming a lament average diameter of 1 lm.
2

58

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

Table 4 Summary of maximum growth rate values of M. parvicella estimated in pure culture and in mixed biomass Conditions Batch (liquid medium) Batch (liquid medium) Batch (agar plate) Batch (agar plate) Chemostat Culture Pure Pure Pure Mixed Pure Limiting substrate Tween 80 Complex carbon sources R2A R2A Tween 80 Growth indicator Protein TEFL TEFL TEFL Dry weight lmax (d1) 0.38 0.30.5 0.370.66 0.66 1.44 Reference [10] [22] [32] [32] [10]

followed, so that any interference from substrate diusion was reduced in comparison to the situation in an activated sludge oc. Consequently, the Ks value determined here should be quite close to the intrinsic value and would apply directly to a lament growing in the bulk liquid. 4.3. pH and temperature eects Slijkhuis studied the eect of pH on M. parvicella growth over a pH range from 7.1 to 8.0 [10]. During growth, the medium pH decreased from 8 to 7.6 due to acid production. Growth rate decreased with pH and was zero at an initial pH value 67.1. Strain RN1 seems less sensitive to pH: kinetics tests carried out at 20 C on R2A medium, under non-limiting substrate conditions, showed that pH had no signicant eect on the maximum growth rate over the range of 6.78.0, and only a slight decrease in lmax was detected at pH 8.4 [22]. The eect of temperature on the growth of M parvicella was investigated using Tween80/peptone as substrates [10]. Optimum growth occurred at about 25 C. Some growth occurred at 8 C while at 35 C very poor biomass development was observed up to 70 h and no growth was seen at higher temperatures. The temperature dependence of lmax for strains 4B and RN1, evaluated by kinetic tests carried out under non-limiting carbon and nutrient conditions, is shown in Fig. 6. The optimal growth rate occurred at @22 C; no growth was detected at 30 C but at 7 C the maximum growth rate was still appreciable (lmax 0.103 0.032 d1 for 4B and 0.085 0.019 d1 for RN1). Growth rate data over the temperature range of 720 C (where lmax increases with temperature) have been correlated with the simplied form of the Arrhenius equation. 3 Estimated temperature coecient values (h) for strains 4B and RN1 were 1.114 and 1.105. These coecients indicate marked temperature dependence and are comparable to the values reported for nitrifying bacteria (h = 1.123) [42].
max (d-1)

0.6

0.5

4B RN1

0.4

0.3

0.2

0.1

0.0 6 8 10 12 14 16 18 20 22 24 26

T (C)
Fig. 6. Maximum growth rate of M. parvicella as function of temperature for the two strains, 4B and RN1. Error bar is the standard deviation (data from [32]).

5. Factors aecting growth of M. parvicella in activated sludge plants According to Wanner [2], the distribution of ocforming and lamentous bacteria in the biomass of activated sludge plants is determined by operating conditions causing the establishment of kinetic and metabolic selection mechanisms in the reaction environment. In the rst case the growth kinetics is aected while in the second the metabolic pathways of substrate utilization can be enhanced or hindered. In the case of M. parvicella, the experimental evidence obtained from pure culture and in situ studies suggests that its proliferation in activated sludge might be caused by several factors. Both kinetic and metabolic selection could be operative: kinetic selection mechanisms derive from the organisms growth characteristics that allow it to grow at appreciable rates under critical conditions like low temperatures and dissolved oxygen levels. At the same time its marked storage capacity in all reaction environments represents a strong metabolic peculiarity able to induce metabolic selection mechanisms. Generally, in real activated sludge plants more

lmax(T) = lmax(20) h(T20), where T = temperature (C), lmax(20) and lmax(T) = maximum growth rates (d1) at 20 C and T, respectively, and h = temperature coecient.
3

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

59

causes for kinetic and metabolic selection mechanisms would exist and currently available full-scale plant data suggest that this lamentous organism is highly versatile. It thus possesses several metabolic strategies necessary to grow and survive under a wide range of operating conditions. Because of this M. parvicella cannot be used to indicate what the operational cause of bulking might be, and neither can its detection in mixed liquor allow a simple diagnosis for bulking in terms of kinetic and metabolic selection. 5.1. Growth characteristics Reported maximum growth rate values for M. parvicella in the range of 0.31.4 d1 indicate that fairly high sludge ages are required for its survival in WWTPs. This observation is consistent with the occurrence of M. parvicella in nutrient removal plants and nitrifying conventional plants [1,2]. From a survey of Colorado (USA) plants treating domestic wastewater, a sludge age hc P 10 d was required for M. parvicella to cause operational problems [43]. This is a quite common value adopted in plant design (even if nitrogen removal is not required) [44,45] in order to maintain safe operational conditions in the presence of temporary inuent overloads, or if an increase in the population served by the plant is expected. Furthermore, scum layers or physical barriers, which in conventional plants cause the establishment of regions with sludge retention times higher than the nominal sludge age, could represent a potential seeding source for this lament. The practice of recycling biological scum through the system must therefore be avoided. Another factor, reported to promote M. parvicella proliferation in the nutrient removal plants, is the availability of nitrogen compounds. It has been hypothesized that lamentous bulking in nitrication-denitrication systems may occur as a result of competition for substrates between the lamentous and oc-forming organisms [46]. In particular, if denitrication is incomplete, the intermediate nitric oxide was hypothesized to accumulate in the oc-forming organisms and exert a toxic eect by preventing them from utilizing the slowly biodegradable COD under aerobic conditions. However, as lamentous organisms have been thought not to accumulate nitric oxide, they are not inhibited and so they may use available substrates in the aerobic zone, thus out-competing the oc-formers. With regard to M. parvicella this hypothesis is consistent with experimental evidence that pure cultures are able to perform only the rst step of denitrication. It has been shown that M. parvicella requires ammonia for growth [10] and it has been suggested that ammonia, available under aerobic conditions in incompletely nitrifying biological nutrient removal (BNR) plants, is a preferential nitrogen source for M. parvi-

cella [47]. The positive eect of nitrogen sources such as nitrate and ammonia (arising from a failure of complete denitrication and nitrication, respectively) on M. parvicella growth has also been observed in many full-scale biological nutrient removal plants [4854]. Dissolved oxygen (DO) concentration strongly aects growth of M. parvicella in activated sludge. Because this organism appears to be a microaerophile, it proliferates in conventional WWTPs with spatial or temporal low DO concentrations and in nutrient removal plants operating with anoxic - aerobic zones. Experimental evidence to support this behaviour was rst reported in 1988 [55], and later Ekama et al. [56] found, in an investigation of bulking occurrence in South African WWTPs, that continuous aeration of activated sludge eliminated M. parvicella when DO concentration of 23 mg l1 has been reached. Madoni and Davoli [57] suppressed M. parvicella by mixing inuent wastewater with return activated sludge (RAS) under oxic conditions in an activated sludge plant operated with an intermittently aerated initial zone. M. parvicella bulking at the UOSA plant, VA, USA, was controlled by installing an aerobic selector (DO > 2 mg l1) in this single sludge nitrication facility, and by replacing mechanical aeration with ne-bubble diused aeration [58]. The ability of M. parvicella to grow at appreciable rates at temperatures as low as 7 C, which has been normally observed in both pure cultures and full-scale plants, provides it with a signicant competitive advantage during the cold season. In fact, proliferation of M. parvicella in full-scale activated sludge plants typically has a distinctive seasonal pattern. In the Czech Republic a system with a compartmentalized pre-denitrication zone suered from bulking caused by M. parvicella only when the mixed liquor temperature dropped below 1215 C for an extended period [2]. Similar seasonal patterns have been reported for a plant treating domestic and industrial wastewater in Italy [59] and for the Johannesburg Northern wastewater treatment plant where M. parvicella occurred in the winter and Type 0092 dominated in the summer [60]. Kruit et al. [54] found that for four BNR plants in The Netherlands (with fully biological nutrient removal), all of which suffered from M. parvicella bulking, there was a distinct pattern of high sludge volume index (SVI) in the winter and spring, lowest values in the summer and gradually increasing values in the autumn. In a survey of 38 Danish plants, (all with biological nitrogen removal or biological nitrogen and phosphorus removal) the most dominant lament type was M. parvicella, which was most abundant in the winter [61]. The preferential growth of M. parvicella at low temperature was suggested to be attributed to its eect in reducing the solubility of lipids that then concentrated on the activated sludge basin surface, thus becoming

60

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

more easily available for M. parvicella present in the scum layers [43] but this hypothesis has not yet been veried experimentally. Temperature eects were also investigated in bench scale studies [62]. High temperatures decreased the growth rate of M. parvicella, even when the substrate concentrations (oleate and acetate) were not limiting. The microorganism was completely eliminated at 29 C, indicating that high temperatures not only decrease the availability of lipids and fats to it, but also allow other bacteria to out-compete M. parvicella. 5.2. Substrate storage capacity M. parvicella isolates can take up and store a variety of dierent carbon substrates under aerobic, anoxic and anaerobic conditions. Anaerobic storage allows the microorganism to survive stress conditions imposed by long anaerobic periods and especially provides a strong competitive advantage over most oc-forming bacteria that do not take up and store substrates anaerobically. Moreover, the availability of storage compounds is a key factor in enhancing resistance to starvation that often occurs under the typical and unavoidable dynamic conditions in domestic wastewater treatment plants [32]. No kinetic data on the anaerobic substrate uptake and storage by M. parvicella are available. However, since this organism commonly occurs in enhanced biological phosphorus removal (EBPR) activated sludge plants, its anaerobic substrate uptake and storage rates should be comparable to those of the PAOs and/or GAOs (glycogen accumulating organisms) favoured in EBPR systems. While most isolates of M. parvicella

grow on simple, soluble organic substrates [22], such data do not necessarily mean that this type of substrate is preferred when growing in a mixed biomass such as activated sludge. Indeed, in situ substrate uptake and storage capacity in M. parvicella seems to be limited specically to LCFA. Nielsen et al. [38] hypothesized that this capability gives M. parvicella the ability to out-compete other bacteria in activated sludge systems operating with alternating anaerobic-aerobic zones (Fig. 7). In an initial anaerobic phase the dissolved LCFA are preferentially available to, and directly stored by M. parvicella, while the particulate LCFA fraction is hydrolysed by the oc-formers and provides an additional source of dissolved lipids for M. parvicella. The anaerobic storage products are then utilized for its growth in the subsequent aerobic phase. On the contrary, in an initial aerobic environment, the oc-formers can compete with M. parvicella for both LCFA fractions. It has been shown [32] that the stored substrate cannot be used for growth under anaerobic conditions, but it is still unknown whether M. parvicella can metabolize the substrate under anoxic conditions. In order to conrm this hypothesis, the mechanisms of anaerobic storage and possible fate of stored lipids under anoxic conditions need to be investigated further. The lipid content of municipal wastewater organic carbon varies from 14% [63] to 2535% [64,65]. LCFA could also be produced during the activated sludge process. Lemmer et al. [66] thus suggested that LCFA could be generated from synthetic surfactant degradation and from the lysis of cell material. Only a few quantitative data on these additional LCFA contributions are available and further analysis is required to establish their potential eects on M. parvicella-mediated bulking.

Fig. 7. Hypothesis explaining LCFA utilization by M. parvicella in alternating anaerobicaerobic activated sludge plants (modied from [38]).

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964

61

6. Control strategies for M. parvicella bulking and foaming Filamentous bulking and foaming can be controlled by specic strategies that address the causes of lament proliferation, or by non-specic control methods, which treat the bulking and foaming symptoms. Specic methods are preferable as they are selective for the target microorganism and do not damage the remaining biomass, while non-specic methods (such as chlorine or hydrogen peroxide) are only temporary solutions, potentially detrimental for all the biomass. No reliable control method specic for M. parvicella exists for activated sludge plants, although manipulation of some operating parameters can reduce its abundance. If the sludge retention time (SRT) can be reduced sufciently, growth of M. parvicella can be minimised. This control method is not suitable for plants that must nitrify because the SRT required for M. parvicella control also eliminates nitrifying bacteria from activated sludge [2]. This control measure also has to include all parts of the plants where sludge retention times can be higher than the nominal value (i.e., some reactor zones delimited by physical barriers). Another important M. parvicella control strategy is to maintain a DO concentration of >2 mg l1 at all places in the aerobic zones. This action has already proved eective in some full-scale plants [1,57,67]. Because lipids can provide an advantage to M. parvicella, a control measure for wastewaters of high lipid content would be to remove some of these compounds by a pretreatment such as otation. The oated material could then be introduced into the aerobic zone of the activated sludge system where other bacteria would compete with M. parvicella for the uptake of LCFA [38]. This method, however, would be costly because the lipid fraction of wastewater cannot be readily separated. The high storage capability of M. parvicella under all environmental conditions (aerobic, anoxic and anaerobic conditions) suggests that the use of selectors would be ineective [1,2]. In spite of this (and complicating the M. parvicella puzzle) there are a few reports of the successful application of aerobic contact zones for controlling the growth of M. parvicella [68,69]. The most common non-specic method for bulking control is the use of oxidizing chemicals such as chlorine to destroy laments at the oc surfaces and extending into the bulk liquid. This treatment is best carried out together with a microscopic examination of the sludge to monitor the general eects of the chlorine chemicals on the biomass. Detailed descriptions of the protocols for chlorine dosing and operating conditions are given by Jenkins et al. [1] and Wanner [2]. Recently it has been reported that the addition of polyaluminium chloride (e.g. PAX-14) is an eective

method of controlling M. parvicella in WWTPs. The eect could be specic for M. parvicella because PAX addition seems to be ineective against other lamentous bacteria [70,71]. The recommended PAX-14 dosage is 23 g Al kg MLSS1 d1 in the recycled activated sludge stream for at least three weeks. Nitrication and COD removal eciencies appear to be unaected [70]. PAX-14 dosing appears to change the morphological characteristics of the M. parvicella in parts of the laments [70,71]. The mechanism of PAX14 in controlling M. parvicella in activated sludge is still unknown. Some hypotheses relate to possible changes in cell surface characteristics and to toxic eects [70]. The polyaluminium could aect the hydrophobic nature of the cell wall of M. parvicella and thereby reduce its selective/competitive advantage in the mixed sludge biomass or prevent it from metabolising the hydrophobic substrates. PAX-14 could also remove lipids from the aqueous phase by their occulation followed by entrapment in ocs.

7. Research needs This review has critically examined the current state of knowledge of the microbiology and physiology of M. parvicella and the available methods for controlling its proliferation in activated sludge plants. The review has revealed several crucial aspects of M. parvicella growth and physiology that need to be investigated to provide the basis for ecient and reliable control strategies for this nuisance organism. It would be valuable to obtain additional pure cultures of M. parvicella particularly from systems such as industrial WWTPs, where it has not been extensively monitored and studied. This would permit verication of the phylogeny of industrial representatives of M. parvicella and improve the specicity of oligonucleotide FISH probes for its in situ detection. Additional pure cultures studies on a wider range of isolates are needed to provide a better understanding of the physiological and kinetic properties that still need clarifying. One clear priority is a better understanding of its energy sources, lipid storage products, and biochemical mechanisms for carbon uptake under anaerobic conditions, because anaerobic storage of lipids is a key property of this lamentous organism that may be shared by only a few other bacteria in activated sludge. Conrmation of Ks and Y values would be useful, but considering the diculties in predicting organisms behavior in full-scale plants from pure culture studies, a major challenge would be to use in situ growth studies for quantitative determination of kinetic and stoichiometric parameters. This is now possible because of the elucidation of the taxonomic position of M. parvicella

62

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964 isolate of Microthrix parvicella. J. Appl. Microbiol. 82, 405 410. Seviour, E.M., Williams, C., DeGrey, B., Soddell, J.A., Seviour, R.J. and Lindrea, K.C. (1994) Studies on lamentous bacteria from Australian activated sludge plants. Water Res. 28, 2335 2342. Blackall, L.L., Stratton, H., Bradford, D., Del Dot, T., Sjorup, C., Seviour, E.M. and Seviour, R.J. (1996) Candidatus Microthrix parvicella, a lamentous bacterium from activated sludge sewage treatment plants. Int. J. Syst. Bacteriol. 45, 186187. Farquhar, G.J. and Boyle, W.C. (1971) Identication of lamentous microorganisms in activated sludge. J. Water Pollut. Cont. Fed. 43, 604622. Farquhar, G.J. and Boyle, W.C. (1971) Occurrence of lamentous microorganisms in activated sludge. J. Water Pollut. Cont. Fed. 43, 779798. Slijkhuis, H. and Deinema, M.H. (1982) The physiology of Microthrix parvicella, a lamentous organism isolated from activated sludge In: Bulking in Activated Sludge: Preventatives and Remedial Methods (Chambers, B. and Tomlinson, E.J., Eds.), pp. 7583. Ellis Horwood Ltd., Chichester. Chacin, E., Kocianova, E. and Forster, C.F. (1994) Foam formation, anaerobiosis and Microthrix parvicella. J. Inst. Water Environ. Manage. 8, 534537. Kerley, S., Kocianova, E. and Forster, C.F. (1994) A biochemical characterization of Microthrix parvicella. Process Biochem. 29, 581586. Kocianova, E., Forster, C.F. and Foot, R.J. (1994) What is Microthrix parvicella?. J. Appl. Bacteriol. 76, 301306. Stratton, H.M., Webb, R., Seviour, E.M., Blackall, L.L. and Seviour, R.J. (1996) Ultrastructure of Microthrix parvicella from activated sludge. Lett. Appl. Microbiol. 23, 8588. Tandoi, V., Rossetti, S., Blackall, L.L. and Majone, M. (1998) Some physiological properties of an Italian isolate of Microthrix parvicella. Water Sci. Technol. 37, 18. Reasoner, D.J. and Geldreich, E.E. (1985) A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microbiol. 49, 17. Munthali, M.T., Coolen, M.J.L., Felske, A., Mirza, M.S., Eikelboom, D.H. and Akkermans, A.D.L. (1996). Microthrix parvicella, lamentous bacteria causing foaming and bulking in activated sludge treatment plants. In: Proceedings of the Symposium on Bacterial Genetics and Microbial Ecology, Nafplion, Greece, p. 19. Erhart, R., Bradford, D., Seviour, R.J., Amman, R.I. and Blackall, L.L. (1997) Development and use of uorescent in situ hybridization probes for the detection and identication of Microthrix parvicella in activated sludge. Syst. Appl. Microbiol. 20, 310318. Brosius, J., Dull, T.J., Sleeter, D.D. and Noller, H.F. (1981) Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J. Mol. Biol. 148, 107127. de los Reyes, F.L, Rothauszky, D. and Raskin, L. (2002) Microbial community structures in foaming and nonfoaming full-scale wastewater treatment plants. Water Environ. Res. 74, 437449. Howgrave-Graham, A.R. and Steyn, P.R. (1988) Application of the uorescent-antibody technique for the detection of Sphaerotilus natans in activated sludge. Appl. Environ. Microbiol. 54, 799 802. Raskin, L., De Los Reyes, M.F., Oerther, D.B. and De Los Reyes, F.L. (1998) Characterization of lamentous foaming in activated sludge systems using oligonucleotide hybridization probes and antibody probes. Water Sci. Technol. 37, 485493. Connery, N., Thompson, A.S., Patrick, S. and Larkin, M.J. (2002) Studies of Microthrix parvicella in situ and in laboratory

and the availability of FISH probes for its in situ identication. Some full-scale plant control strategies need to be validated under controlled conditions. These strategies include the value of aerobic selectors (for which conicting results exist), the eect of nitrogen compounds (ammonium as N source and nitrate as electron acceptor) on M. parvicella growth and the eectiveness of lipid removal (i.e., by otation) in the inuent wastewater. The mechanism by which PAX controls M. parvicella is important to understand so that doses of this high-cost chemical and the additional sludge production can be reduced. Successful completion of these studies will require close collaboration among microbiologists, engineers, and wastewater treatment plant operators, who must apply the results from fundamental research in controlled, real-world conditions. Such a combined approach may eventually solve the severe solidliquid separation problems caused by this microorganism and insert the last pieces into the M. parvicella puzzle.

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

References
[21] [1] Jenkins, D., Richard, M.G. and Daigger, G.T. (2004) Manual on the Causes and Control of Activated sludge Bulking, Foaming and other Solids Separation Problems. Lewis Publishers, Washington DC, 190 pp. [2] Wanner, J. (1994) Activated Sludge Bulking and Foaming Control. Technomic Publishing, Pennsylvania, 327 pp. [3] Martins, A.M.P., Pagilla, K., Heijnen, J.J. and van Loosdrecht, M.C.M. (2004) Filamentous bulking sludge a critical review. Water Res. 38, 793817. [4] Ravenschlag, K., Sahm, K., Pernthaler, J. and Amann, R. (1999) High bacterial diversity in permanently cold marine sediments. Appl. Environ. Microbiol. 65, 39823989. [5] Rheims, H., Sproer, C., Rainey, F.A and Stackebrandt, E. (1996) Molecular biological evidence for the occurrence of uncultured members of the actinomycete line of descent in dierent environments and geographical locations. Microbiology (UK) 142, 2863 2870. [6] Joseph, S.J., Hugenholtz, P., Sangwan, P., Osborne, C.A. and Janssen, P.H. (2003) Laboratory cultivation of widespread and previously uncultured soil bacteria. Appl. Environ. Microbiol. 69, 72107215. [7] Pasveer, A. (1969) A case of lamentous activated sludge. J. Water Pollut. Cont. Fed. 41, 13401352. [8] Van Veen, W.L. (1973) Bacteriology of activated sludge, in particular the lamentous bacteria. Antonie van Leeuwenhoek 39, 189205. [9] Eikelboom, D.H. (1975) Filamentous organisms observed in activated sludge. Water Res. 9, 365388. [10] Slijkhuis, H. (1983). The physiology of the lamentous bacterium M. parvicella. Ph.D. thesis, Agriculture College of Wageningen, The Netherlands. [11] Blackall, L.L., Seviour, E.M., Cunningham, M.A., Seviour, R.J. and Hugenholtz, P. (1994) Microthrix parvicella is a novel, deep branching member of the actinomycetes subphylum. Syst. Appl. Microbiol. 17, 513518. [12] Rossetti, S., Christensson, C., Blackall, L.L. and Tandoi, V. (1997) Phenotypic and phylogenetic description of an Italian

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964 culture: production and use of specic antibodies. Water Sci. Technol. 46, 115118. Eikelboom, D.H. (1994) The Microthrix parvicella puzzle. Water Sci. Technol. 29, 271279. Rossetti, S., Tomei, M.C., Levantesi, C., Ramadori, R. and Tandoi, V. (2002) Microthrix parvicella: a new approach for kinetic and physiological characterization. Water Sci. Technol. 46, 6572. Tomei, M.C., Levantesi, C., Rossetti, S. and Tandoi, V. (1999) Microbiological characterization of pure cultures and its relevance to modelling and control of bulking phenomena. Water Sci. Technol. 39, 2129. Andreasen, K. and Nielsen, P.H. (1997) Application of microautoradiography to study substrate uptake by lamentous microorganisms in activated sludge. Appl. Environ. Microbiol. 63, 36623668. Andreasen, K. and Nielsen, P.H. (1998) In situ characterization of substrate uptake by Microthrix parvicella using microautoradiography. Water Sci. Technol. 37, 1926. Andreasen, K. and Nielsen, P.H. (2000) Growth of Microthrix parvicella in nutrient removal activated sludge plants: Studies of in situ physiology. Water Res. 34, 15591569. Lee, N., Nielsen, P.H., Andreasen, K., Juretschko, S., Nielsen, J.L., Schleifer, K.-H. and Wagner, M. (1999) Combination of uorescent in situ hybidization and microautoradiography a new tool for structure-function analysis in microbial ecology. Appl. Environ. Microbiol. 65, 12891297. Nielsen, P.H., Roslev, P., Dueholm, T. and Nielsen, J.L. (2002) Microthrix parvicella, a specialized lipid consumer in anaerobic aerobic activated sludge plants. Water Sci. Technol. 46, 7380. Daigger, G.T. and Grady Jr., C.P.L. (1982) The dynamics of microbial growth on soluble substrates. A unifying theory. Water Res. 16, 365382. Grady Jr., L.G. and Lim, H.C. (1980) Biological Wastewater Treatment. Theory and Applications. Pollution Engineering and Technology. Marcel Dekker, New York and Basel, 963 pp. Pirt, S.J. (1985) Principles of Microbe and Cell Cultivation. Blackwell Scientic Publications, London, 274 pp. Ekama, G.A. and Marais, G.v.R. (1984) Theory, Design and Operation of Nutrient Removal Activated Sludge Processes, pp. 5.15.18. Water Research Commission, Pretoria. Richard, M.G. (1989). Activated sludge microbiology, J. Water Pollut. Cont. Fed., Alexandria, VA, 73 pp. Tchobanoglous, G., Burton, F.L., Stensel, H.D. and Metcalf & Eddy, Inc. (2002) Wastewater Engineering: Treatment Disposal Reuse, fourth ed. McGraw-Hill, 1830 pp.. German Standard ATV-DVWK-A 131E (2000) Dimensioning of single stage activated sludge plants. German Association for Water, Wastewater and Waste, 57 pp. Casey, T.G., Wentzel, M.C. and Ekama, G.A. (1999). Filamentous organisms bulking in nutrient removal activated sludge systems. Paper 11: a biochemical/microbiological model for proliferation of anoxic-aerobic (AA) lamentous organisms. Water SA 25, 425442. Tsai, M.W, Wentzel, M.C. and Ekama, G.A. (2003) The eect of residual ammonia concentration under aerobic conditions on the growth of Microthrix parvicella in biological nutrient removal plants. Water Res. 37, 30093015. Pilson, R.A., Ekama, G.A., Wentzel, M.C. and Casey, T.G. (1995). The eect of temperature on denitrication kinetics and biological excess phosphorus removal in nutrient removal activated sludge systems in temperate climate (1220 C). Research Report W86, Department of Civil Engineering, University of Cape Town, Rondebosch, 7707 Cape Town, RSA. Ubisi, M.F., Wentzel, M.C. and Ekama, G.A. (1997). Organic and inorganic components of activated sludge mixed liquor.

63

[31] [32]

[50]

[51]

[33]

[34]

[52]

[35]

[53]

[36]

[37]

[54] [55]

[38]

[56]

[39]

[57]

[40]

[58]

[41] [42]

[59]

[60] [61]

[43] [44]

[45]

[62]

[46]

[63]

[64] [65]

[47]

[48]

[66]

[67]

[49]

Research Report W94, Department of Civil Engineering, University of Cape Town, Rondebosch, 7707 Cape Town, RSA. Sneyders, M.J., Wentzel, M.C. and Ekama, G.A. (1998). The eect of dosing unstabilised landll leachate into a nutrient removal activated sludge system. Research Report W95, Department of Civil Engineering, University of Cape Town, Rondebosch, 7707 Cape Town, RSA. Cronje, G.C., Wentzel, M.C. and Ekama, G.A. (2000). Measurement of active heterotrophic organism concentration in nitrication-denitrication activated sludge systems. Research Report W102, Department of Civil Engineering, University of Cape Town, Rondebosch, 7707 Cape Town, RSA. Beeharry, A.O., Wentzel, M.C. and Ekama, G.A. (2001). Evaluation of batch test for measurement of active biomass in activated sludge mixed liquor. Research Report W112, Department of Civil Engineering, University of Cape Town, Rondebosch, 7707 Cape Town, RSA. Casey, T.G. and Alexander, W.V. (2001). Design and operating strategies to minimize bulking by anoxic-aerobic lamentous organisms in nutrient removal activated sludge plants. WRC Report N. 775/1/01, Water Research Commission, Private Bag X03, Gezina 0031, RSA. Kruit, J., Hulsbeek, J. and Visser, A. (2002) Bulking sludge solved. Water Sci. Technol. 46, 457464. Slijkhuis, H. and Deinema, M.H. (1988) Eect of environmental conditions on the occurrence of Microthrix parvicella in activated sludge. Water Res. 22, 825828. Ekama, G.A., Wentzel, M.C., Casey, T.G. and Marais, G.v.R. (1996). Filamentous organisms bulking in nutrient removal activated sludge systems. Paper 6: review, evaluation and consolidation of results. Water SA 22, pp. 147152. Madoni, P. and Davoli, D. (1993) Control of Microthrix parvicella growth in activated sludge. FEMS Microbiol. Ecol. 12, 277284. Daigger, G.T. and Nicholson, G.A. (1990) Performance of four full scale nitrifying wastewater treatment plants incorporating selectors. Res. J. Water Pollut. Cont. Fed. 62, 676683. Miana, P., Grando, L., Caravello, G. and Fabris, M. (2002) Microthrix parvicella foaming at the Fusina WWTP. Water Sci. Technol. 46, 499502. Wentzel, M.C. (1992) Sludge bulking in nutrient removal systems. IAWQ Nutrient Removal Newslett. 2, 713. Kristensen, G.H., Jrgensen, P.E. and Nielsen, P.H. (1994) Settling characteristics for activated sludge in Danish treatment plants with biological nutrient removal. Water Sci. Technol. 29, 157165. Mamais, D., Andreadakis, A., Noutsopoulos, C. and Kalergis, C. (1998) Causes of, and control strategies for Microthrix parvicella bulking and foaming in nutrient removal activated sludge systems. Water Sci. Technol. 37, 917. Henze, M., Kristensen, G.H. and Strube, R. (1994) Rate capacity characterization of wastewater for nutrient removal processes. Water Sci. Technol. 29, 101107. Quemeneur, M. and Marty, Y. (1994) Fatty acids and sterols in domestic wastewater. Water Res. 28, 12171226. Raunkjr, K., Nielsen, P.H. and Hvitved-Jacobsen, T. (1994) Measurement of pools of protein carbohydrate and lipid in domestic wastewater. Water Res. 28, 251262. Lemmer, H., Muller, E. and Schade, M. (2001). Microthrix scum in low load nutrient removal plants: what do these laments feed on? In: Proceedings of the 3rd IWA International Specialized Conference on Microorganisms in activated sludge and biolms processes, Poster no.74, Rome, Italy. Gabb, D.M.D., Still, D.A., Ekama, G.A., Jenkins, D., Wentzel, M.C. and Marais, G.v.R. (1988). Development and full scale evaluation of preventative and remedial methods for control of activated sludge bulking. Research Report W62, Deparment. of

64

S. Rossetti et al. / FEMS Microbiology Reviews 29 (2005) 4964 [70] Eikelboom, D.H. (1997). Control of Microthrix parvicella by addition of PAX-14, 35 pp. TNO-MEP R97/305, TNO Institute of Environmental Sciences, Energy Research and Process Innovation, Appeldoorn, The Netherlands. [71] Roels, T., Dauwe, F., Van Damme, S., De Wilde, K. and Roelandt, F. (2002) The inuence of PAX-14 on activated sludge systems and in particular on Microthrix parvicella.. Water Sci. Technol. 46, 487490.

Civil Engineering, University of Cape Town, Rondebosch, 7707 Cape Town, RSA. [68] Pujol, R. and Canler, J.P. (1994) Contact zone: French practice with low F/M bulking control. Water Sci. Technol. 29, 221 228. [69] Daigger, G.T, Robbins Jr., N.H. and Marshall, B.R. (1985) The design of the selector to control low F/M lamentous bulking. J. Water Pollut. Cont. Fed. 57, 220226.