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1 Section of Environmental Engineering, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark
2 BIOCLEAR BV, Rozenburglaan 13, 9727 DL Groningen, The Netherlands
3 VERMICON AG, Emmy-Noether-Str. 2, 80992 München, Germany
4 ASIS vof, Deventerweg 38, 7203 AK Zutphen, The Netherlands
5 CNR, Water Research Institute, Via Reno 1, 00198 Rome, Italy
Correspondence
Per Halkjær Nielsen
phn{at}bio.aau.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Eikelboom & van Buijsen, 1983; Jenkins et al., 1993, 2004) and on molecular methods, mainly involving development of specific oligonucleotides for identification by fluorescence in situ hybridization (FISH) (Amann, 1995; Kanagawa et al., 2000; Wagner et al., 1994).
Filamentous Alphaproteobacteria have been shown to be very important in industrial WWTPs where they are often associated with bulking incidents or deteriorating settling properties of the sludge (Levantesi et al., 2004; van der Waarde et al., 2002). The filamentous Alphaproteobacteria have been observed and described in several studies in the past 4 years (Dionisi et al., 2002; Eikelboom & Geurkink, 2002; Levantesi et al., 2004; Thomsen et al., 2006; van der Waarde et al., 2002) and, based on 16S rRNA gene sequences from six uncultured bacteria and one isolated species, they have now been described as constituting seven distantly related phylogenetic clusters: ‘Candidatus Combothrix italica’, ‘Candidatus Catenimonas italica’, ‘Candidatus Sphaeronema italicum’, ‘Candidatus Alysiosphaera europaea’, ‘Candidatus Monilibacter batavus’, ‘Candidatus Alysiomicrobium bavaricum’ (Levantesi et al., 2004) and Meganema perideroedes (Thomsen et al., 2006). Oligonucleotide probes for detection of these species by FISH are also available (Levantesi et al., 2004; Thomsen et al., 2006).
A FISH survey applying species-specific gene probes of the filamentous Alphaproteobacteria has been conducted on samples originating from 86 industrial WWTPs in Denmark, the Netherlands, Germany and Italy (Levantesi et al., 2004). The most common species was ‘Ca. Alysiosphaera europaea’, which was seen in approximately 24 % of the WWTPs investigated, followed by ‘Ca. Alysiomicrobium bavaricum’ (16 %) ‘Ca. Monilibacter batavus' (10 %), ‘Ca. Sphaeronema italicum’ (10 %) and Meganema perideroedes (2 %). ‘Ca. Combothrix italica’ and ‘Ca. Catenimonas italica’ were only rarely observed. In total, the Alphaproteobacteria were found in 66 % of all samples and the above-mentioned species were the dominant filamentous species in approximately 25 % of the samples, clearly emphasizing their importance in industrial WWTP. Unidentified filamentous Alphaproteobacteria in the samples were estimated to constitute 15 % based on hybridization with the group-specific alpha probe (ALF968) (Levantesi et al., 2004).
Several attempts to isolate and grow these filamentous Alphaproteobacteria have largely failed and currently little information on their physiology is available. Such knowledge is of major importance for developing efficient control strategies. At present only isolates of Meganema perideroedes exist in pure culture (Levantesi et al., 2004; Thomsen et al., 2006), but isolates of ‘Ca. Alysiosphaera europaea’ coexisting with a yeast (Debaryomyces hansenii) are available (Snaidr et al., 2002). The physiological properties of Meganema perideroedes have been examined in pure culture (Levantesi et al., 2004; Thomsen et al., 2006) and data on the ecophysiology of Meganema perideroedes have been obtained (Kragelund et al., 2005). This filamentous bacterium is a very versatile consumer of a variety of organic substrates under aerobic conditions and it has a very large storage capacity, measured as polyhydroxyalkanoates (PHAs) formed from different substrates in the presence of different electron acceptors (e-acceptors). Denitrification was not observed in pure culture, although nitrate could be reduced to nitrite by some isolates. However, studies on ecophysiology showed that different substrates were taken up and stored under different redox conditions (Kragelund et al., 2005), demonstrating the use of nitrate and nitrite as e-acceptors. These data indicate that Meganema perideroedes is most probably able to perform denitrification (Kragelund et al., 2005).
The purpose of this study was to carry out a comprehensive ecophysiological investigation of the five most important filamentous alphaproteobacterial species known to date. Ecophysiological data on Meganema perideroedes have been included (Kragelund et al., 2005) and the different alphaproteobacterial species have been compared to find common physiological traits within this group with the aim of developing potential control strategies.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. In general, most of the industrial sites only carried out organic carbon removal, nitrification and chemical phosphorus removal. The temperature range in the plants was from 20 to 40 °C and the sludge age was between 12 and 37 days.
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Morphological description.
The filamentous bacteria present in the sludge were morphologically identified using the Eikelboom classification system for municipal and industrial WWTPs (Eikelboom, 2002; Eikelboom & Geurkink, 2002; Eikelboom & van Buijsen, 1983). Morphological identification involved the use of phase-contrast microscopy, and Gram-, Neisser- and PHA-staining properties (Eikelboom & van Buijsen, 1983; Ostle & Holt, 1982).
FISH and gene probe design.
FISH was applied using group- and species-specific 16S rRNA-targeted nucleic acid probes; their details are described in probeBase (Loy et al., 2003) and are depicted in Table 2. The probes were labelled with 5(6)-carboxyfluorescein-N-hydroxy-succinimide ester (FLUOS) or with the sulfoindocyanine dyes (Cy3 and Cy5) (Thermohybaid Interactive). The FISH procedure was conducted as described elsewhere (Amann, 1995). A confocal laser scanning microscope (LSM 510; Carl Zeiss) equipped with a UV laser (351 and 364 nm), an Ar ion laser (458 and 488 nm) and two HeNe lasers (543 and 633 nm) was used to record fluorescent signals from the gene probes. Light microscopy was used to assess silver grain density from the microautoradiography (MAR) procedure. FISH was combined with other in situ methods as listed below and modification of procedures can be found elsewhere (Kragelund et al., 2005). An overview of theoretical perfect-match organisms targeted by the different probes is shown in Fig. 1. New more specific probes were constructed for ‘Ca. Alysiomicrobium bavaricum’ and for ‘Ca. Sphaeronema italicum’ as the ecophysiological investigation suggested that the existing probes for these two species were unspecific and hybridized with more than one species. Therefore, the specificity of the two probes was investigated using Probe Match (http://rdp.cme.msu.edu/index.jsp) and this confirmed that more sequences were targeted than just ‘Ca. Alysiomicrobium bavaricum’ and ‘Ca. Sphaeronema italicum’ sequences. Probe PPx3-1428 was also found to target sequences within other phyla, e.g. Deltaproteobacteria (1), Firmicutes (15) and Actinobacteria (3). Probe Sita-649 targeted unclassified sequences within the Proteobacteria (1) and Firmicutes (2). A hierarchical approach was therefore used, where the group-specific probe (ALF968) was applied together with more specific probes, which ensured that only filamentous Alphaproteobacteria were investigated. These theoretical probe match searches, however, indicated that it is very likely that two probes, Sita-649 and PPX3-1428, could hybridize with more than one species of filamentous Alphaproteobacteria and therefore additional probes were designed.
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). To evaluate the formamide concentration for optimum stringency, the designed probes were analysed on formamide-fixed activated sludge from the plant where the filaments were originally observed, by applying hybridization buffer containing 0–60 % formamide (in 5 % increments) using image analysis software with a designed macro (ImageJ 1.33s, Rasband W; National Institutes of Health, USA; http://rsb.info.nih.gov/ij/).
MAR.
MAR experiments were performed with 3H- or 14C-labelled tracers. The procedure is described briefly here, but further details are available in Lee et al. (1999) and Kragelund et al. (2005). MAR-positive and -negative filaments were assessed by comparing silver grains on top of the filaments to the background. A minimum of 30 gene-probe-defined filaments were investigated for each incubation. Aerobic uptake of short- and long-chained fatty acids, sugars, alcohol, amino acids and bicarbonate with thiosulfate for autotrophic/mixotrophic behaviour was tested for the five Alphaproteobacteria species. Diluted sludge (1 g SS l–1) was added to a glass serum vial and mixed with tracer (10 µCi=3.7x105 Bq) and the unlabelled organic substrate to a final concentration of 2 mM (except oleic acid, final concn 0.5 mM), and the vial was closed with an airtight rubber stopper. The use of other potential e-acceptors apart from oxygen was tested with selected substrates, including nitrate (2 mM) or nitrite (0.5 mM) as potential e-acceptors, or under anaerobic conditions (without oxygen, nitrate or nitrite present). Nitrate and nitrite concentrations were measured in the sludge water using sticks. If either nitrate or nitrite was present, incubation of sludge was performed to remove these prior to MAR experiments. This was done by adding acetate and degassing the samples, thus allowing the consumption of nitrate or nitrite using these as e-acceptors. Moreover, a preincubation step of 2 h with unlabelled organic substrate (with nitrate or nitrite present as e-acceptor) was included before labelled substrate was added. This was done to ensure that only bacteria able to take up large amounts of substrates (for growth or storage) would be MAR-positive (Andreasen & Nielsen, 2000). Pasteurized sludge was used for chemography. A 3 h incubation period was used in all experiments except 5 h for bicarbonate and thiosulfate. After the incubation period, the sludge was prepared and processed as described by Lee et al. (1999). The exposure time was 4–6 days. Details on the radiochemicals used are listed elsewhere (Kragelund et al., 2005).
Storage capabilities.
The PHA storage capabilities of the gene-probe-defined filamentous Alphaproteobacteria species were investigated using MAR incubations with different carbon substrates with different e-acceptors. Samples were spread out on gelatin-coated slides and stained according to the protocol for Nile blue (Ostle & Holt, 1982); details are described by Kragelund et al. (2005). The intensity was measured using image analysis software (ImageJ 1.33s, Rasband W). For each filament, a segment of at least 15 µm was analysed by recording the maximum intensity in each horizontal line perpendicular to the filament image. The mean value of these measurements (min. 100) was calculated for each analysed filament in a given incubation. At least 22 filaments were examined for each substrate incubation and mean fluorescence intensity±SD was calculated. Student's t-test was used as a statistical tool to evaluate significant PHA formation compared to the corresponding control. To determine the background level of PHA in the different species, both pasteurized samples and formate incubations were used. No PHA accumulation was ever observed after the addition of formate.
Enzyme-labelled fluorescence (ELF) FISH.
The presence of exoenzyme activity was determined using ELF (ELF-97; Molecular Probes), where substrates form a fluorescent precipitate on the surfaces of bacteria upon enzymic cleavage (Kragelund et al., 2005). The following enzymes were evaluated: ELF-97 esterase substrate (ELF-97 acetate), ELF-97 lipase substrate (ELF-97 palmitate), ELF-97 
-D-galactosidase substrate (elf-97 -D-galactopyranoside), ELF-97 -D-glucuronidase substrate (elf-97 -D-glucuronide), ELF-97 chitinase/N-acetylglucosaminidase substrate (ELF-97 N-acetylglucosaminide; ELF-97 NAG) and the ELF-97 Endogenous Phosphatase detection kit. Sludge (100 µl) was mixed with 0.2 mM of either substrate and was placed in the dark for 2–3 h. Five microlitres of this mix was spread out on a gelatin-coated glass slide and images were taken. The slide was washed with dH2O prior to FISH. The enzyme reaction was subsequently fixed in paraformaldehyde for 1 h and washed in tap water prior to the normal FISH procedure. The remaining enzyme reaction was resuspended in PBS and stored at 4 °C.
Microsphere adhesion to cells (MAC) FISH.
To determine the surface properties of the filamentous bacteria, sulfate-modified microspheres (Molecular Probes) were applied (Nielsen et al., 2001; Kragelund et al., 2005). A 0.002 % solution of microspheres with a diameter of 0.02 µm was sonicated for 100 min at 60 W. Activated sludge (10 µl, 4–5 g SS l–1), 100 µl sterile, deionized water and 0.5 µl microsphere solution were mixed. Subsamples were spread out on gelatin-coated glass slides, images of filamentous bacteria with and without microspheres were recorded using a confocal laser scanning microscope and their positions were recorded. The identity of the filamentous bacteria was subsequently determined by FISH.
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RESULTS |
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). The diameter varied between 1 and 2 µm, without a visible sheath. Epiphytic growth was rarely observed. Other granules apart from sulfur were frequently seen. All species contained PHA (see below). They were usually Gram-negative, but occasionally filaments or parts of filaments were Gram-positive. No uniform Neisser staining was found. As evident in Table 3, any clear distinction between these species was impossible using only morphological methods, confirming the requirements of species-specific gene probes for their identification. In one WWTP (AAU12), an additional very thin morphotype that showed a positive reaction with oligonucleotide probe PPx3-1428 was observed. However, it was present in small amounts only in this treatment plant and was not targeted by the newly developed probe PPx1002 (see below); therefore, it was not investigated further.
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). When the probes Nost993 and Helper1010 where applied at an optimal formamide concentration (20 %), filamentous ‘Ca. Sphaeronema italicum’ gave a clear fluorescent signal in CNR1 but not in TNO18, also suggesting the presence of two different genotypes in these plants.
Ecophysiology
Uptake of organic substrates under aerobic conditions.
Substrate uptake of the different filamentous species under aerobic conditions was determined in various treatment plants by MAR-FISH and the results are listed in Table 4. Data obtained from the four different alphaproteobacterial species were compared to that of Meganema perideroedes reported previously (Kragelund et al., 2005). All the probe-defined species consumed acetate and most also consumed other short-chain fatty acids (propionate and pyruvate), but not formate and butyrate. The results also show that the five species could be divided into two major physiological groups: Group 1 that primarily consumed short-chain acids (‘Ca. Monilibacter batavus' and ‘Ca. Sphaeronema italicum’) and Group 2 that, besides the short-chain acids, were able to consume various carbohydrates, amino acids and ethanol (‘Ca. Alysiomicrobium bavaricum’, ‘Ca. Alysiosphaera europaea’ and Meganema perideroedes).
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Substrate uptake under denitrifying conditions.
The ability of the filamentous Alphaproteobacteria to take up substrates under anaerobic conditions or denitrifying conditions (with nitrate or nitrite added) is shown in Table 5.
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When nitrite acted as potential e-acceptor, all Group 1 species and one Group 2 species (‘Ca. Alysiomicrobium bavaricum’) took up the same substrates as when nitrate was e-acceptor (Table 5
). No substrate uptake was observed for ‘Ca. Alysiosphaera europaea’, while Meganema perideroedes utilized only glucose and acetate.
Under anaerobic conditions, none of the alphaproteobacterial species investigated showed any uptake of substrates, indicating a lack of anaerobic growth or storage capabilities (data not shown).
Storage capacity
In fresh activated sludge samples, all gene-probe-defined filamentous Alphaproteobacteria species had small PHA granules as visualized by Nile blue staining. A number of substrates were tested for uptake and potential conversion into PHA under different e-acceptor conditions by measuring the increase in fluorescence intensity during a 3 h incubation with different substrates (Table 6 and Table 7). The substrates tested for PHA accumulation were those resulting in a positive signal using MAR for the different species (Tables 4 and 5). For some substrates, substantial uptake and subsequent storage was observed, where the individual cells in the filaments were almost completely filled with PHA granules. There was some variation between filaments, but with similar PHA content in all cells within each filament.
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). Group 2 species also converted almost all substrates tested into PHA, e.g. short-chain fatty acids, sugars and amino acids.
When nitrate was present as e-acceptor, PHA formation was observed for all substrates taken up, as determined by MAR (Table 7). PHA formation with nitrite as e-acceptor showed the same pattern as with nitrate for all species except ‘Ca. Alysiosphaera europaea’.
Exoenzyme activity and surface properties
The investigated species present in the bulk liquid showed no surface-associated exoenzymic activity with any of the ELF enzymes tested, with the exception of some ‘Ca. Monilibacter batavus' filaments which exhibited lipase activity. Also Meganema perideroedes showed lipase activity on one occasion after a factory shutdown (Kragelund et al., 2005). Filamentous bacteria in the different samples exhibited some enzyme activity, observed as fluorescent precipitates along the filament, but these bacteria were not identified. Enzyme activity at the surface of microcolonies and filamentous organisms in the flocs was always present. In general, very high glucuronidase and esterase activity was observed in the flocs. No attempts were made to study possible exoenzyme activity inside sludge flocs.
The surface properties of the filamentous alphaproteobacterial species were investigated using MAC-FISH. The adherence of surface-modified microspheres was investigated for the filaments and for the flocs. Both hydrophobic and more hydrophilic areas were observed in the flocs.
The three species belonging to Group 2 (see above) were all completely covered with microspheres and had relatively more microspheres than most other organisms in the sludges. This indicates the presence of a hydrophobic sheath. Group 1 ‘Ca. Monilibacter batavus' filaments appeared much less hydrophobic as only very few microspheres attached to the surfaces. ‘Ca. Sphaeronema italicum’ filaments did not show the same surface properties in the two different plants investigated, reflecting the two different genotypes. The gene-probe-defined population in CNR1 targeted by Nost993 had no microspheres attached to the surface, whereas the filaments in TNO18 targeted by Sita-649 were completely covered.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Levantesi et al., 2004; van der Waarde et al., 2002). Of the five species hitherto described, only the ecophysiology of Meganema perideroedes has been investigated (Kragelund et al., 2005), so the present study provides a more comprehensive study of almost all filamentous Alphaproteobacteria encountered in industrial WWTPs. All species had a morphology resembling that of Type 021N, or Nostocoida limicola, and could only be correctly identified to species level when FISH was applied. Hardly anything is known about the physiology of the different species, so specific control strategies have not yet been developed to combat this group of filamentous organisms. The substrate uptake data suggest that the five species can be divided into two groups with a very similar physiology. Group 1 consists of ‘Ca. Monilibacter batavus' and ‘Ca. Sphaeronema italicum’ and they took up mainly short-chain fatty acids. Group 2 (‘Ca. Alysiomicrobium bavaricum’, ‘Ca. Alysiosphaera europaea’ and Meganema perideroedes) was more versatile and able to take up carbohydrates, amino acids and ethanol in addition to the short-chain fatty acids. Furthermore, the capability to form the storage product PHA was generally large for all species in Group 1 and Group 2 (except for ‘Ca. Alysiosphaera europaea’). Interestingly, when we compare this grouping with the phylogenetic analysis of the species, it appears that the species in Group 1 cluster together, as do two of the Group 2 species. Meganema perideroedes does not cluster with any of the other four species. Thus, it seems that the physiological properties to some extent follow the phylogenetic affiliation of the species.
It is not clear whether the five species were able to perform full denitrification to gaseous nitrogen, but under conditions where nitrate served as potential e-acceptor they were definitely physiologically very active, and able to take up substrates and form PHA from all substrates. When nitrite served as potential e-acceptor all but ‘Ca. Alysiosphaera europaea’ were active and took up largely the same substrates, although none of the species took up substrates under strict anaerobic conditions. This suggests that denitrification to gaseous nitrogen probably took place with some substrates and reduction of nitrate to nitrite occurred only in the presence of a few substrates (e.g. propionate for Meganema perideroedes). It was interesting to note that for all species the number of substrates that could be taken up with nitrate or nitrite as e-acceptor was almost identical, but clearly reduced compared to uptake under aerobic conditions. A similar observation has also been reported for growth of isolates of the denitrifying genus Thauera (Foss & Harder, 1998) and it reinforces the idea that it is virtually impossible to predict the substrate uptake pattern of these bacteria under denitrifying conditions on the basis of only aerobic investigations.
PHA granules were present in all five species. Extensive PHA formation has been reported for Meganema perideroedes (Kragelund et al., 2005; Levantesi et al., 2004) and for an unidentified alphaproteobacterial Nostocoida limicola-like organism (Dionisi et al., 2002). With the results presented here, this seems to be a common trait in filamentous Alphaproteobacteria investigated so far. The ability to form PHA is an obvious advantage, since the storage material can be utilized as energy and/or as a reserve of carbon during periods of unbalanced growth due to unfavourable conditions (Dawes, 1991). ‘Ca. Alysiomicrobium bavaricum’ (AAU12) had a very large storage capacity, as described for Meganema perideroedes (Kragelund et al., 2005), and the variation in storage of PHA in each species may reflect physiological differences or differences in actual physiological status.
Comparing MAR results and PHA quantification, information regarding consumption and transformation of various substrates was obtained. With oxygen as e-acceptor, most short-chain fatty acids were taken up by the species; the Group 2 species (‘Ca. Alysiomicrobium bavaricum’, ‘Ca. Alysiosphaera europaea’ and Meganema perideroedes) in particular transformed most substrates into PHA. In two cases PHA formation was observed after incubation with glucose, which is in agreement with studies on other bacteria (Dawes, 1991; Haywood et al., 1990, 1991). However, this was not observed for Meganema perideroedes under aerobic conditions (Kragelund et al., 2005). This could potentially illustrate differences in the metabolic pathways amongst these species, since Meganema perideroedes only formed PHA from glucose when nitrate or nitrite served as e-acceptor. At present, it is not possible to study glycogen formation at the single-cell level, so differentiation between the storage of PHA and glycogen remains to be investigated.
The ecophysiology of ‘Ca. Sphaeronema italicum’ (CNR1) and ‘Ca. Alysiomicrobium bavaricum’ was not identical in the two plants investigated for each species. This is different from most other ecophysiological investigations we have conducted on other filamentous bacteria (Nielsen et al., 2002), glycogen-accumulating organisms (Kong et al., 2006) and polyphosphate-accumulating bacteria (Kong et al., 2004), where the physiology for a certain gene-probe-defined species appears very similar in different plants. Therefore, additional gene probes were designed and revealed that different populations were probably present in the different WWTPs, explaining the observed differences in ecophysiology. This seems plausible as there are a number of differences in growth conditions in the plants in terms of temperature and wastewater type. The existence of several closely related species belonging to the two candidate species is not unexpected and a more comprehensive phylogenetic overview must await the determination of more sequences of these filamentous Alphaproteobacteria.
Surface-associated exoenzymic activity was barely observed in these species. ‘Ca. Monilibacter batavus' showed some lipase activity, but was not able to consume the only offered long-chain fatty acid (oleic acid). However, other long-chain fatty acids that were not tested could potentially be taken up, explaining the observed lipase activity. Also, Meganema perideroedes showed lipase activity on one occasion after a factory shutdown, indicating an ability to turn on enzyme activity under periods of stress and/or starvation (Kragelund et al., 2005). The ecophysiology results suggest that all five species utilize mainly low-molecular-mass dissolved organic compounds present in the wastewater rather than particulate organic matter requiring an enzymic step before uptake.
The surface of most of the filamentous Alphaproteobacteria was very hydrophobic, particularly amongst the Group 2 members. It is not known how they benefit from this as they mainly consume hydrophilic soluble substrates and not hydrophobic substrate as does Microthrix parvicella, for example (Andreasen & Nielsen, 2000; Nielsen et al., 2002).
Another factor is the ability of these species to form flocs. It is essential for the filaments to stay attached to the floc material in order to settle in the final clarifier and not to be washed out with the effluent. Previous studies have revealed that most single cells in bulk water in WWTPs are hydrophilic, while many filamentous bacteria protruding from flocs are often hydrophobic (Zita & Hermansson, 1997a, b). Most of the Alphaproteobacteria were observed to be incorporated into the floc material as long as they did not dominate the population. Thus, the primary function of the relatively hydrophobic surface of these bacteria may be attachment to the floc. In several cases we have observed filamentous Alphaproteobacteria in foam in various WWTPs, also reflecting their hydrophobic nature (data not shown).
Possible control measures
The filamentous Alphaproteobacteria are responsible for many bulking incidents, especially in industrial WWTPs. Evidence from this and others studies emphasizes that molecular identification is necessary to distinguish between the different filamentous Alphaproteobacteria. From a physiological point of view, all five of the filamentous Alphaproteobacteria species discussed in this study can be divided into two groups. However, from the point of view of control, they can be considered as a single group as they are all very well adapted to industrial plants with a high content of soluble, readily consumable organic compounds. The survey conducted by Levantesi et al. (2004), and unpublished data (C. Kragelund & P.H. Nielsen), show that the filamentous Alphaproteobacteria in the versatile Group 2 are twice as abundant as Group 1 species in WWTPs.
Currently, the most efficient control strategy for most filamentous bacteria is the introduction of a selector in the plant configuration (Eikelboom, 2002; Wanner, 1994). The advantage of small compartmentalized selectors with a short residence time is the creation of a substrate gradient when raw influent is mixed with sludge. Here, easily degradable substrates are taken up by bacteria with high substrate uptake rates and storage capacities. These properties are generally associated with floc-forming bacteria rather than filamentous bacteria and should thus select for floc-forming bacteria (Shao & Jenkins, 1989; Wanner, 1994). However, both groups of the filamentous Alphaproteobacteria seem to have high substrate uptake rates and storage capacities, probably comparable to floc-formers, so a selector may only work under conditions where the substrate uptake is more restricted. An anaerobic selector would probably work very well, but this is usually difficult to install except in plants with biological phosphate removal. This study showed that nitrate, and in particular nitrite, significantly reduced the number of substrates that can be consumed by these filamentous Alphaproteobacteria, so anoxic selectors using nitrate, or even better nitrite, should be tested. Although the costs of adding nitrate, and particularly nitrite, can be high, it may in some cases be worth consideration. The selector must be designed properly to optimize the amount of soluble substrate to be removed (Martins et al., 2004; Wanner, 1994). For Group 1 members it may also be possible to look at the wastewater stream and see whether it is possible to reduce the amount of short-chain fatty acids.
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ACKNOWLEDGEMENTS |
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Received 1 November 2005;
revised 26 June 2006;
accepted 13 July 2006.
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