Scum in Activated Sludge Plants: Impact of Non-filamentous and Filamentous Bacteria*

Schaum in Belebungsanlagen: Einfluss nicht-fdiger und fdiger Bakterien H. Lemmer, G. Lind, E. Mller, M. Schade, and B. Ziegelmayer 1
Keywords: Acinetobacter sp., Hydrophobicity, Microthrix parvicella, Nocardioform Actinomycetes, Scum, Wastewater Treatment Summary: Excessive scum production is a widespread phenomenon in present activated sludge wastewater treatment. The question how foaming is initiated and stabilized is still unanswered. Hydrophobic wastewater ingredients and surface active material such as synthetic surfactants are discussed among others as major causative agents for scum production. Focusing on biological impacts non-filamentous bacteria isolated from scum turned out to contribute to flotation by both cell surface hydrophobicity and emulsification activity, depending on the prevailing substrate and milieu conditions. The biological characterization of scum based on microscopic sludge investigation of conspicuous microorganisms resulted in a significant shift of filamentous and non-filamentous organism populations with Gram-positive bacteria prevailing in present nutrient removal plants as compared to the situation ten years ago. Their hydrophobic cell surface is supposed to support adherence and stabilization of interfaces and thus promote sludge flotation. In scum six types of filamentous bacteria turned out to be numerous: Microthrix parvicella and nocardioform actinomycetes, both of them being enriched in the scum fraction, moreover Nostocoida limicola and Eikelboom types 0041/0675, 1851, and 0092. Possible interactive mechanisms between non-filamentous and filamentous scum bacteria and their selection factors are discussed in order to contribute to a better understanding of scum formation and to provide efficient troubleshooting measures. Schlagwrter: Acinetobacter sp., Hydrophobie, Microthrix parvicella, nocardioforme Actinomyceten, Schaumbildung, Belebungsanlage Zusammenfassung: Starke Schaumbildung ist gegenwrtig ein weit verbreitetes Phnomen in Klranlagen nach dem Belebungsverfahren. Die Frage nach Entstehung und Stabilisierung dieser Schume ist nach wie vor ungeklrt. Hydrophobe Abwasserbestandteile und oberflchenaktive Stoffe wie etwa Tenside werden unter anderem als wichtige Ursache der Schaumbildung diskutiert. Die Suche nach biologischen Einflssen ergab, dass aus Schaum isolierte Bakterien ohne Fadenbildung sowohl durch Oberflchenhydrophobierung als auch durch die Bildung stabiler Emulsionen zur Flotation beitragen, abhngig von den vorherrschenden Substrat- und Milieubedingungen. Die biologische Charakterisierung von Schaum, basierend auf mikroskopischen Untersuchungen aufflliger Organismen in belebtem Schlamm, ergab in Anlagen mit Stickstoff- und Phosphorentfernung eine deutliche Verschiebung hin zu Gram-positiven Bakterien Fadenbakterien und solche, die nicht fdig wachsen im Vergleich zu den vor etwa 10 Jahren blichen Anlagen zur reinen Elimination von Kohlenstoffverbindungen. Die hydrophobe Oberflche dieser Organismen scheint der Adhsion an und Stabilisierung von Grenzflchen und damit der Flotation frderlich zu sein. In Schaum wurden sechs wichtige Fadenorganismen festgestellt: Microthrix parvicella und nocardioforme Actinomyceten, die beide in der Schaumfraktion angereichert werden, des Weiteren Nostocoida limicola und die zunchst noch mit Nummern benannten Fadenbakterien Eikelboom Typ 0041/0675, 1851 und 0092. Mgliche Interaktionen zwischen nicht-fdigen und fdigen Schaumbakterien sowie ihre Selektionsfaktoren werden diskutiert. Damit wird angestrebt, dem Praktiker ber ein besseres Verstndnis der Schaumbildung effektive Bekmpfungsmanahmen an die Hand zu geben.

1 Introduction
Excessive scum production is a widespread problem in biological wastewater treatment plants (WWTPs). The phenom* Presented as plenary lecture at Wastewater 99, May 1820 1999, Teplice, Czech Republic. 1 Dr. rer. nat. Hilde Lemmer, Dipl.-Biol. George Lind, Dipl.Biol. Elisabeth Mller, Dipl.-Biol. Margit Schade, Dipl.-Biol. Birgit Ziegelmayer, Bayerisches Landesamt fr Wasserwirtschaft, Kaulbachstr. 37, D-80539 Mnchen, Germany Correspondence to H. Lemmer E-mail: LEMMER@oec.net
34
Acta hydrochim. hydrobiol. 28 (2000) 1

enon is characterized by the formation of stable gas-filled cells. Their boundaries are stabilized by surface active molecules and/or hydrophobic substances at the gas-water interface. As Gochin and Solari [1] pointed out, hydrophobic sites are absolutely necessary because bubble entrapment in hydrophilic flocs would not be able to rise the particles. Large and open flocs require only a few hydrophobic sites to induce attachment of microbubbles. Thus activated sludge flocs being enlarged by excessive filamentous growth of hydrophobic bacteria are extremely susceptible to flotation. Small compact flocs, in contrast, are less sensitive to flotation. Moreover surfactant molecules, be it synthetic or biogenic, adhere to inter-

34

40 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000 03234320/00/01010034 $ 17.50+.50/0

faces. They are prone to adsorb molecules from the bulk water at this interface which is then stabilized (adsubble processes [2, 3]). Dispersed gas bubbles are available as air or oxygen bubbles in the aeration tank, as nitrogen or nitrous oxide in denitrification zones, or as methane in digesters. Hydrophobic ingredients such as oil and grease might enter the plant via the primary effluent as is the case in various WWTPs with pronounced industrial influence. However, there are many municipal plants suffering from foam where hardly any grease and oil input can be detected. Hydrophobic ingredients seem also to be available by an abundant growth of organisms with a hydrophobic surface. Stability of foam is then not only dependent on the organisms hydrophobicity but also on solids concentration in the foam [4]. Hydrophobicity is due to lipoproteins, lipopolysaccharides, and phospholipids in many Gramnegative bacteria, or polypeptides in some Gram-positives [5]. It is strongly influenced by the prevailing substrate and milieu conditions, as was shown by pure culture investigations. As an example many microorganisms are known to strongly increase their hydrophobicity by loosing their polysaccharid slime capsule during starvation phases with carbonaceous substrate limitation [6]. Nocardia amarae was shown to form hydrophobic cell surface fimbriae during growth on hydrocarbon substrates [7]. Other evidence of enhanced sludge hydrophobicity came from Frlund and coworkers [8] who compared biopolymer composition in a high load (sludge age c. 8 d) and a low load nutrient removal plant (sludge age c. 36 d). They found a pronounced difference between the amounts of hydrophobic property molecules in the low load sludge making up to 45.1% of chromatogram area as compared to 14.2 % in high load sludge. Comparing scum with the corresponding activated sludge there seems to be a striking difference in the uronic acid content of extracellular polysaccharides [9, 10] with high amounts of uronic acids prevailing in foams: the higher the uronic acid concentration the more hydrophobic the scum layer. Uronic acids in the cell wall of Gram-positives are known to replace the polyol phosphate chains of teichoic acids during phosphorus deficiency. They might thus well be an indicator for such conditions. Surface active material, on the one hand, may enter the WWTP via the primary effluent or else by bacterial production. In particular, synthetic surfactants originating from washing powders and cleansing products have been discussed as a causative agent for scum production. However, many modern synthetic surfactants are readily degradable and not accumulating in the sludge fraction [11]. On the other hand, many microorganisms are known to excrete biosurfactants into the medium at nutrient deficiency conditions, e.g., Pseudomonas aeruginosa producing rhamnolipids at nitrogen deficiency or Acinetobacter calcoaceticus forming emulsifying minicapsules on the cell surface at amino acid deficiency [12]. Other bacteria produce surface active material in order to emulsify and make available hydrophobic substrate: Rhodococcus erythropolis and many other bacteria and yeasts are known to produce phospholipids during growth on hexadecane and other long chain alkanes [13]. Many of the above mentioned bacteria are readily found in activated sludge. Why then not hypothesize that similar effects as shown in pure culture investigations might be true in situ? Our investigations therefore focus on the impact of the dominant scum proliferating activated sludge bacteria community on hydrophobicity and emulsifying activity. Conspicuous scum bacteria were investigated by microscopic sludge investigation and their occurrence compared to the prevailing in situ
Acta hydrochim. hydrobiol. 28 (2000) 1

milieu conditions. Non-filamentous bacteria from scum layers were preselected by adsorption to a hydrophobic carrier and characterized with respect to taxonomy, growth behavior, cell surface hydrophobicity, and emulsification capability at various substrate conditions. Moreover, their fatty acid profiles during growth on different substrates were investigated in order to elucidate whether they might be a possible long chain alkane substrate supply for specialized scum bacteria during lysis processes in stabilized sludge since this type of substrate seems to be crucial for hydrophobicity and emulsification [14, 15]. Possible interactive mechanisms between non-filamentous and filamentous scum bacteria promoting sludge flotation are discussed in order to elucidate efficient troubleshooting measures.

2 Methods
Hydrophobic bacteria were preselected from Microthrix parvicella-dominated scum layers by adsorption to octyl sepharose beads (hydrophobic interaction chromatography, HIC) as described previously [16]. Preselected isolates were taxonomically characterized by fatty acid analysis of whole cell hydrolysates (MIS, MIDI, Newark, Delaware), and hybridization with rRNA-targeted oligonucleotides according to Wagner et al. [17]. Impact of substrate conditions on hydrophobicity and emulsifying activity was tested in batch cultures for several strains of Acinetobacter calcoaceticus, Hydrogenophaga pseudoflava, and Rhodococcus fascians. Emphasis was laid on growth phases and substrate conditions relevant in situ, such as different load conditions (F/M ratios) with respect to readily degradable substrate prevailing in present municipal scum WWTPs. High load conditions were simulated with trypticase soy broth (BOD 16850 mg L1), low load conditions with R2A-medium at different concentrations (100% with a BOD of 2055 mg L1, 50%, 25%). Tween 20 served as long chain hydrolysable substrate, acetate and ethanol as readily degradable short chain substrate. Lysed sludge bacteria as found in stabilization plants or else in floated sludge fractions were simulated by adding autoclaved Gram-negative (Enterobacter agglomerans) and Gram-positive bacteria (Micrococcus varians). Hydrophobicity of the cell surface was determined by a microbial adhesion to hydrocarbons (MATH)test with hexadecane. Emulsification capability was tested by emulsifying a water/hexadecane system as described earlier [16]. Fatty acid composition at different substrate conditions was determined by the above mentioned MIDI procedure as percent of total fatty acids in order to screen for high amounts of unsaturated C16 and C18 fatty acids being discussed as substrate favored by scum bacteria.

3 Results and Discussion

Microbiological characterization of scum bacteria communities is still in its infancy. Albeit in situ fluorescent staining techniques with gene probes being commonly adopted, unfortunately we are still far from an overall description of activated sludges. Therefore we started to characterize scum by microscopic investigations of foaming sludges and determining the conspicuous, predominantly filamentous bacteria most of which still resist cultivation and thus thorough description. For characterization of non-filamentous scum bacteria we investigated preselected hydrophobic bacteria from Microthrix parvicella-dominated sludges despite the known imponderabilities of cultivation methodes in describing bacteria communities [17]. The hitherto maintained distinction between filaments and non-filaments might be overcome as soon as we will know more about activated sludge bacteria growth behavior because it is generally accepted that morphological variability at different substrate and milieu conditions is widespread.
35

34

40

3.1 Microscopic Sludge Investigation of Conspicuous Microorganisms

Our investigations resulted in a significant shift from Gram-negative filamentous and non-filamentous organisms to Gram-positive bacteria prevailing in present nutrient removal plants as compared to high load plants common ten years ago [18]. Their hydrophobic cell surface is supposed to support adherence and stabilization of interfaces and thus promote sludge flotation. Six types of filamentous bacteria turned out to be numerous in scum: Microthrix parvicella and nocardioform actinomycetes, both of them being enriched in the scum fraction, moreover Nostocoida limicola and, to some extent, Eikelboom types 0041/0675, 1851, and 0092. This is true of many other countries such as South Africa [19], Australia [20], Sweden [21], The Netherlands [22], or Czech Republic [23]. Nocardioform scum actinomycetes with genera such as Rhodococcus, Gordona and Nocardia [2426] turned out to be versatile with respect to carbon, nitrogen, and phosphorus sources which is in agreement with earlier findings. This makes this group competitive at rich supply with hydrolysable, in particular hydrophobic substrate [27] available in WWTPs without primary clarifier or in nutrient-rich scum layers where they can utilize lysed cells constituents. On the other hand, some rhodococci tested here are also competitive in carbondeficient environment prevailing in low load nitrification stages by the ability of using CO2 as a carbon source [16]. In contrast to other bacteria, hydrophobicity of actinomycetes in general is strong at many growth conditions, most pronounced at hydrophobic substrate supply [28]. Excretion of surface active material is pronounced during post-logarithmic growth [16], their biosurfactants being known to produce very stable foams [4]. Sodium nitrate as a nitrogen source provided higher amounts of biosurfactants as compared to ammonia salts [29]. Nocardioforms, even if not growing as hyphae and being overlooked during microscopic sludge investigation, are prone to contribute to flotation in all stages of WWT, in particular with high amounts of long chain alkane substrate prevailing, and to stabilization of old sludge blankets by both hydrophobicity and emulsifying activity, in particular at long mean cell residence times [16]. Microthrix parvicella scum problems are at present the most severe because they comprise scum in aeration tanks as well as in digesters. Unfortunately, no proper description of the organism is available yet. Thus, for instance, it is still not clear whether the Microthrix parvicella strains of Australia are the same as those in Europe. There is evidence M. parvicella to be a deep branching member of actinomycetes [30]. It is generally accepted that M. parvicella proliferates at low load conditions and temperatures below 15C making the organism competitive in nitrifying systems in moderate climate. The Microthrix strain described by Slijkhuis [31], however, has been shown to prefer reduced nitrogen and sulfur sources over oxidized substrate favoring the bacterium at ammonia shock load conditions (e.g. by uncontrolled digester supernatant input) or in reduced environments such as decaying scum layers or anaerobic and anoxic zones comprised in nutrient removal plants. Promising physiological data are now available by the elegant technique of in situ microautoradiography introduced by Andreasen and Nielsen [32, 33]. They found M. parvicella to thrive on the long chain fatty acids oleic and palmitic acid, and on trioleic acid with oleic acid being utilized at aerobic, anoxic, and anaerobic conditions. Interestingly, this was the only type of substrate utilized by the Microthrix strain described by
36

Slijkhuis [31]. However, for Microthrix scum control we have to keep in mind that the author repeatedly pointed out that fatty acids being toxic at high concentrations are apt as a substrate only at very low substrate: dry weight ratios, i.e. below 0.025 mg oleic acid per milligram dry weight (in comparison, rhodococci do not mind two orders of magnitude higher ratios). That means, in order to utilize high amounts of fatty acids, M. parvicella must already have constituted a high biomass concentration. The ground of some Microthrix problems might therefore well be prepared by other scum bacteria, be it filaments or not, and furtheron stabilized by M. parvicella. This is in agreement with the observations of Hart [34], who describes a scum problem in South Africa due to a nonfilamentous bacterium tentatively determined as Acinetobacter species, and with own observations of floating sludge problems being initiated by excessive coryneform growth. Nostocoida limicola is found in moderately loaded scumming WWTPs with F/M ratios around 0.2 kg kg1 d1 (BOD) (see [18]; or 0.33 kg kg1 d1 (COD), see [35]). It has been described to produce much less stable foam layers as compared to actinomycetes or Microthrix parvicella. This is the reason why Khan and coworkers [5] think Nostocoida limicola not to be a causative species. The physiological properties of this organism are not well investigated. As is true of M. parvicella also Nostocoida limicola is not yet a well defined organism and it might well be that many different strains, species or even genera are hiding behind this name. Nevertheless, Andreasen and Nielsen [32] provide interesting data on the in situ uptake of different substrates. In contrast to M. parvicella which turned out to be restricted to the uptake of lipid substrate Nostocoida limicola utilizes besides oleic acid glucose, glycine, and leucine, i.e. readily degradable substrate. This might explain why the organism is found in WWTPs where soluble substrate might still be available, i.e. at a higher F/M ratio than is favorable for M. parvicella. Eikelboom types 0041/0675, 1851, and 0092 are not yet well understood. Neither their physiological properties nor their taxonomic home are yet defined. As mentioned above, type 0041 is supposed to produce a lipid/protein complex and thus contribute to foam formation by surface-active material [35].

3.2 Characterization of Non-filamentous Scum Bacteria

The Acinetobacter strains tested belonged to A. calcoaceticus and A. johnsonii. They turned out versatile in utilizing long chain and short chain substrate, however, showed weak growth on lysed bacteria and on organic phosphorus sources. In the Acinetobacter calcoaceticus strains tested, the major constituents of the fatty acid profile turned out to be 16:0 saturated palmitic acid making up 10 to 20%, 16:1 7c unsaturated palmitoleic acid making up 25 to 40%, and 18:1 oleic acid making up 20 to 30% of the total fatty acids in both log and stationary growth phase (see Table 1). A significant change in fatty acid profile during growth on TSA and R2A was not observed. The amount of the unsaturated fatty acids was somewhat higher with TSA, though. This is in agreement with the fatty acid profiles of ethanol-grown Acinetobacter calcoaceticus described by Loffhagen et al. [36] who compared its growth on various alcohols. Hydrophobicity was low during growth on TSA with 0 to 5% of cells being adsorbed to hexadecane. It got strikingly high at growth on 100%, 50%, and 25% R2A with adsorption from 50 to 90% and was moderate
Acta hydrochim. hydrobiol. 28 (2000) 1

34

40

Table 1: Fatty acid profiles of selected scum bacteria at various substrate conditions. Fettsureprofile ausgewhlter Schaumbakterien bei verschiedenen Substratbedingungen. Organism Acinetobacter spp. TSA high load WWT R2A low load WWT both log and stationary phase 16:0 16:1 18:1 16:0 16:1 18:1 52...60% 20...23% 10...20% 25...40% 20...30% 20...30% 48...56% 5...13% 20...30% 5% stationary phase 30%

Hydrogenophaga spp.

Rhodococcus spp.

iso/anteiso branched fatty acids 15:0 anteiso 15:0 iso log phase 16:0 10...20% 16:1 < 5%

with acetate and ethanol. On the other hand, emulsification activity of both cells and cell-free supernatant was pronounced on all media, in particular with ethanol. Cell suspension showed strong emulsifying activity during lag and log growth phase. In contrast, cell-free supernatant showed high activity during log and stationary growth phase. The ability of Acinetobacter strains to excrete surface active material such as emulsan in order to emulsify hydrophobic substrate such as crude oil, triglycerides, and middle-length alkanes is well documented [37]. Khan et al. [5] hypothesize Acinetobacter spp. to produce biosurfactants with a high proteinaceous and lipid content. The contribution to flotation in WWT might be by cell surface hydrophobicity, especially at low F/M conditions with readily degradable substrate and, at special growth conditions with hydrophobic substrate being available or else by emulsification during post-log conditions (see Table 2). The Hydrogenophaga pseudoflava strains tested showed weak growth on long chain and short chain substrate as well as on lysed cells. They were able to use inorganic and organic phosphorus sources. They showed similar major constituents

in their fatty acid profiles as were found for Acinetobacter strains with 20 to 30% 16:0 palmitic acid, 48 to 56% 16:1 7c palmitoleic acid, and 5 to 13% 18:1 oleic acid in both log and stationary growth phase (see Table 1). This is in agreement with results of Wilkinson [38] who described 19% 16:0, 58% 16:1, and 8% 18:1 in Pseudomonas pseudoflava. Cell surface hydrophobicity with Hydrogenophaga strains turned out to be strain-dependent. Emulsification was pronounced with mixed substrates such as R2A or else with acetate. As Knallgas bacteria, i.e. hydrogen-oxidizing bacteria, Hydrogenophaga spp. grow with hydrogen as a source of reduction equivalents. This corresponds to these organisms being competitive and therefore readily found in low load stages of nutrient removal plants. Cell suspensions showed strong emulsifying activity on all media during lag, log, and stationary growth phase, cellfree supernatants on TSA during log, and on all media during their stationary growth phase. Hydrogenophaga species might thus contribute to flotation at low load conditions with high sludge ages by both hydrophobicity and emulsification capability (see Table 2).

Table 2: Hydrophobicity (H) and emulsification capacity (EC) of selected scum bacteria at different WWT conditions. Hydrophobie (H) und Emulgierungsfhigkeit (EC) ausgewhlter Schaumbakterien unter verschiedenen Betriebs- und Substratbedingungen in einer Klranlage. Organism Acinetobacter spp. Hydrogenophaga pseudoflava Rhodococcus fascians + other nocardioform actinomycetes Cytophaga-Flavobacterium-group Microthrix parvicella Sludge age (F/M) low (high) high (low) H1 EC H1 EC H 1) EC EC H ? stat. phase grazing?
34 40

Type of substrate hydrophilic hydrophobic EC

N/P limitation H EC

H ? C16/18 in situ?
37

strain dependent

Acta hydrochim. hydrobiol. 28 (2000) 1

The nocardioform actinomycetes being selected belonged to the Rhodococcus species R. rhodnii, R. fascians, R. globerulus, and R. rhodochrous and turned out to be the most versatile utilizing all substrates offered. Growth on Tween 20, however, was surprisingly weak. Rhodococcus fascians strains showed, as expected, strikingly different fatty acid profiles as compared to Acinetobacter and Hydrogenophaga species. The prevailing constituents were, as described typical for Grampositives by OLeary and Wilkinson [39], high amounts of branched iso/anteiso fatty acids with 15:0 anteiso pentadecanoic acid making up 52% to 60% and 15:0 iso 20% to 23% of the total fatty acids during log and stationary phase at growth on TSA. Growth on R2A yielded clearly smaller amounts of branched acids, however, it yielded 10 to 20% 16:0 palmitic acid and less than 5% 16:1 7t palmitoleic acid in the log growth phase with 16:1 increasing up to 30% during the stationary growth phase (see Table 1). Hydrophobicity tests with Rhodococcus fascians strains turned out to be inconclusive, as was shown in previous work with hydrophobicity being strongly dependent on strain and substrate and milieu conditions. This coincides with observations of Khan and Forster [4] who found a maximum of cell hydrophobicity-dependent foam height with Rhodococcus strains after 24 h growth followed by a strong decrease. Excretion of surface active material, in contrast, was found to be strong during stationary growth phase in some of our strains. For R. rubra Khan et al. [5] describe a carbohydrate/protein polymer to be involved. The results clearly demonstrate that for an understanding of scum formation in WWT it is not only conspicuous filamentous bacteria to be considered. Also non-filamentous bacteria are able to support flotation by changing cell surface hydrophobicity and emulsifying activity under defined substrate and milieu conditions (Table 2). It is worth mentioning that the nocardioforms tested here were not growing as hyphae in the scum layer they were isolated from, so they would not have been recognized as a culprit during microscopic sludge investigation. Scum initiation or later stabilization may thus not be a mere filament problem.

thrix parvicella (as mentioned above [31, 44]). In general, long chain alkylic substrates are prone to play an important role in flotation events as was shown above with respect to substrate-dependent hydrophobicity and emulsifying activity of different scum isolates. As examples remember Acinetobacter spp. to be emulsifying and Rhodococcus spp. to be strongly hydrophobic at hydrophobic substrate conditions prevailing. Another aspect which is not yet well understood is the availability and utilization of lysed cells as a nutrient source in very low loaded sludge stabilization systems and in scum layers being cut off from the substrate supply of the mixed liquor. Hydrophobic substrate might well be available from lysed cell walls at these conditions, e.g., in our case both saturated and unsaturated C16 fatty acids readily found in the Gram-negatives tested. Moreover phospholipids from lysed cell walls may be utilized as organic phosphorus sources in particular by organisms with a high cell surface hydrophobicity producing extracellular, non-cell-bound phosphatases [45]. Interestingly, the authors found the growth rates of two Acinetobacter baumannii isolates with hydrophobic phosphatidic acid as phosphorus source to exceed the ones with hydrophilic inorganic phosphorus or -glycerophosphate. Synthetic surfactants have also been discussed as a causative agent for scum formation. However, in situ surfactant concentrations in primary effluents in the range of 5 to 10 mg L1 are far beyond critical micelle concentrations [46]. Moreover modern anionic, nonionic, and cationic surfactants have been shown to be readily degradable and not to be accumulated in the sludge fraction to a great extent. However, we have to keep in mind that production of biogenic surfactants is widespread among bacteria and is favored by defined milieu conditions, in particular by long chain alkanes and/or nutrient deficiency, as was shown above for scum isolates. Thus present WWT is prone to enhance biogenic surfactant formation. Goddard and Forster [35] hypothesized Eikelboom type 0041 to produce a saturated lipid/protein complex surfactant, Rhodococcus and Nocardia strains turned out to produce foam-stabilizing biosurfactants which at least in part are cellbound [4, 47]. Are these organisms together with their non-filamentous colleagues such as Acinetobacter and Hydrogenophaga strains to be accused for scum stabilizing surfactant production?

3.3 Substrate Availability in Scum WWTPs and Impact on Microorganism Selection

There are some substrates repeatedly discussed to be favoring scum production. Hydrophobic substrates such as oil and grease originating from the primary effluent of WWTPs are widely accused as causative agents for scum formation. However, in most cases it is not the hydrophobic substrate as such that is promoting sludge flotation. A simple calculation shows that it would take 5.9 g of grease to lift 1 g of activated sludge dry matter, that means 86% fat in the dry matter [40]. So in most cases it is trapped gas that causes flotation. However, hydrophobic substrate can act as an indirect promoter for excessive growth of organisms adapted to degradation of this kind of substrate. It is well documented that nocardioform actinomycetes such as Rhodococcus or Nocardia species proliferate at high loads of grease and oil because of their affinity to long chain alkanes and fatty acids [27, 4042]. In pure culture studies N. amarae was shown to have a stronger affinity for long chain fatty acids than Pseudomonas aeruginosa and thus was more competitive in activated sludge processes that receive sewage containing a high proportion of long chain fatty acids, oils, and fats [43]. Proliferation of filamentous bacteria is dependent on load conditions. High load of grease and oil will promote nocardioforms, low load will select for Micro38

4 Conclusions
Stable foam formation is due to the presence of hydrophobic substances and surface-active material in present WWTPs. Low load conditions with nutrient deficiencies are prone to select for both filamentous and non-filamentous sludge bacteria with pronounced capability to form hydrophobic cell surfaces and to stabilize emulsions by cellbound or excreted substances. Long chain fatty acids and other long chain alkane substrates turned out to play a crucial role for enhancing hydrophobicity and/or emulsifying activity in various activated sludge bacteria, and thus for proliferating flotation processes. Therefore an important control tool for, in particular, actinomycete- and Microhrix'-dominated scum is avoiding high concentrations of these substrates by all means. This includes primary effluent control as well as preventing long scum residence times with concomitant cell lysis and decay processes.
Acta hydrochim. hydrobiol. 28 (2000) 1

34

40

 High concentrations of mixed liquor suspended solids are prone to provide high amounts of surface active material. Lowering MLSS content as such (coming along with a higher F/M ratio) might thus be beneficial, in particular for Microthrix control. Stability of foams is not only dependent on hydrophobicity but also on solids concentration in the foam. This in turn is organism-dependent. Effective control measures can therefore either focus on decreasing the biomass of the scum proliferating species or else on preventing floated sludge fractions from the very beginning by preventing quiescent zones where floating sludge accumulates and by promptly skimming off any flotate.

5 Outlook
Finally we should keep in mind that together with the changes of load and aeration regime in present WWT the concomitant increase of protozoa and metazoa might contribute to the observed population shift by changing competition within the activated sludge biocenosis. Bacteria are able to cope with grazing pressure by increasing the hydrophobicity of their cell wall [48]. This might well be another reason why today activated sludge biocenoses are dominated by hydrophobic bacteria. Another strategy to meet the grazing pressure is to produce filaments [49]. In the present investigation morphological variability was most pronounced within the rhodococci, the phenomenon is known, however, for many other filaments. Therefore future investigations into the microbial ecology of WWT should include these neglected aspects of biological interactions in wastewater biocenoses. Moreover much more emphasis is to be laid on determining community structure and substrate availability in situ as a key for understanding foaming and flotation processes.

Acknowledgements
We gratefully acknowledge financial support by grant A 5.12 to HL from the Lnderarbeitsgemeinschaft Wasser/ Abwasser LAWA via the Kuratorium fr Wasserwirtschaft KfW and the Abwassertechnische Vereinigung ATV , moreover financing by the grants for the projects Waschmittel and Florenanalyse by the Bavarian State Ministry for State Development and Environmental Affairs.

References
[1] Gochin, R. J., Solari, J.: The role of hydrophobicity in dissolved air flotation. Water Res. 17, 651657 (1983). [2] Lemlich, R.: Adsubble processes: Foam fractionation and bubble fractionation. J. Geophys. Res. 77, 52045210 (1972). [3] Sutton, R.: Use of biosurfactants produced by Nocardia amarae for removal and recovery of non-ionic organics from aqueous solutions. Water Sci. Technol. 26 (911), 23932396 (1992). [4] Khan, A. R., Forster, C. F .: An investigation into the stability of foams related to the activated sludge process. Enzyme Microbial Technol. 12, 788793 (1990). [5] Khan, A. R., Kocianova, E., Forster, C. F .: Activated sludge characteristics in relation to stable foam formation. J. Chem. Technol. Biotechnol. 52, 383392 (1991). [6] Kjelleberg, S., Hermansson, H.: Starvation-induced effects on bacterial surface characteristics. Appl. Environ. Microbiol. 48, 497503 (1984).
Acta hydrochim. hydrobiol. 28 (2000) 1

[7] Cairns, W. L., Cooper, D. G., Zajic, I. E., Wood, J. M., Kosaric, N.: Characterization of Nocardia amarae as a potent biological coalescing agent of water-oil emulsions. Appl. Environ. Microbiol. 43, 362366 (1982). [8] Frlund, B., Keiding, K., Nielsen, P . H.: A comparative study of biopolymers from a conventional and an advanced activated sludge treatment plant. Water Sci. Technol. 29 (7), 137 141 (1994). [9] Kerley, S., Forster, C. F .: Extracellular polymers in activated sludge and stable foams. J. Chem. Technol. Biotechnol. 62, 401404 (1995). [10] Baxter-Plant, V . S., Hayes, E., Forster, D. F .: The examination of activated sludge from three plants in relation to the problem of stable foam formation. Process Saf. Environ. Prot. 761, 161165 (1998). [11] Schade, M., Lemmer, H.: Scum in activated sludge wastewater treatment plants: a synthetic surfactant or a biogenic problem? In preparation. [12] Rosenberg, E.: Microbial surfactants. CRC Crit. Rev. Microbiol. 3, 109132 (1986). [13] Kosaric, N., Choi, H. Y., Blaszczyk, R.: Biosurfactant production from Nocardia SFC-D. Tenside Surfactants Deterg. 27, 294296 (1990). [14] Stratton, H. M., Webb, R., Seviour, E. M., Blackall, L. L., Seviour, R. J.: Ultrastructure of Microthrix parvicella from activated sludge. Lett. Appl. Microbiol. 23, 8588 (1996). [15] Sunairi, M., Iwabuchi, N., Yoshizawa, Y., Murooka, H., Morisaki, H., Nakajima, M.: Cell-surface hydrophobicity and scum formation of Rhodococcus rhodochrous strains with different colonial morphologies. J. Appl. Microbiol. 82, 204210 (1997). [16] Lemmer, H., Lind, G., Schade, M., Ziegelmayer, B.: Autecology of scum producing bacteria. Water Sci. Technol. 37 (45), 527530 (1998). [17] Wagner, M., Amann, R., Lemmer, H., Schleifer, K.-H.: Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ. Microbiol. 59, 15201525 (1993). [18] Lind, G., Lemmer, H.: Biologische Charakterisierung von Schumen in Belebungsanlagen. Teil I. Bedeutung fdiger Belebtschlammbakterien. GWF Gas Wasserfach: Wasser Abwasser 139, 16 (1998). [19] Blackbeard, J. R., Ekama, G. A., Marais, G. V . R.: A survey of filamentous bulking and foaming in activated sludge plants in South Africa. J. Water Pollut. Control Fed. 85, 90100 (1986). [20] Seviour, E. M., Williams, C. J., Seviour, R. J., Soddell, J. A., Lindrea, K. C.: A survey of filamentous bacterial populations from foaming activated sludge plants in Eastern States of Australia. Water Res. 24, 493498 (1990). [21] Dillner Westlund, A., Hagland, E., Rothman, M.: Bulking and foaming caused by Microthrix parvicella at three large sewage treatment plants in the greater Stockholm area. Water Sci. Technol., 34 (56), 281287 (1996). [22] Eikelboom, D. H., Andreadakis, A., Andreasen, K.: Survey of filamentous populations in nutrient removal plants in four European countries. Water Sci. Technol. 37 (45), 281289 (1998). [23] Wanner, J., Ruzickova, I., Jetmarova, P ., Krhutkova, O., Paraniakova, J.: A national survey of activated sludge separation problems in the Czech Republic: Filaments, floc characteristics and activated sludge metabolic properties. Water Sci. Technol. 37 (45), 271279 (1998). [24] Lemmer, H., Kroppenstedt, R. M.: Chemotaxonomy and physiology of some actinomycetes isolated from scumming activated sludge. Syst. Appl. Microbiol. 5, 124135 (1984). [25] Blackall, L. L., Harbers, A. E., Greenfield, P .F ., Hayward, A. C.: Foaming in activated sludge plants: A survey in Queensland, Australia and an evaluation of some control strategies. Water Res. 25, 313318 (1991). [26] Goodfellow, M., Stainsby, F . M., Davenport, R., Chun, J. S., Curtis, T.: Activated sludge foaming: The true extent of actinomycete diversity. Water Sci. Technol. 37 (45), 511519 (1998).
39

34

40

[27] Lemmer, H., Baumann, M.: Scum actinomycetes in sewage treatment plants: Part 2. The effect of hydrophobic substrate. Water Res. 22, 761764 (1988). [28] Stratton, H., Seviour, B., Brooks, P .: Activated sludge foaming: What causes hydrophobicity and can it be manipulated to control foaming? Water Sci. Technol. 37 (45), 503509 (1998). [29] Ramsey, B., McCarthy, J., GuerraSantos, L., Kppeli, O., Fiechter, A.: Biosurfactant production and diauxic growth of Rhodococcus aurantiacus when using n-alkanes as the carbon source. Can. J. Microbiol. 34, 12091212 (1988). [30] Blackall, L. L., Seviour, E. M., Cunningham, M. A., Seviour, R. J., Hugenholtz, P .: Microthrix parvicella is a novel, deep branching member of the Actinomycetes subphylum. Syst. Appl. Microbiol. 17, 513518 (1994). [31] Slijkhuis, H.: The physiology of the filamentous bacterium Microthrix parvicella. PhD Thesis, Landbouwhogeschool Wageningen, The Netherlands, 1983. [32] Andreasen, K., Nielsen, P . H.: Application of microautoradiography to the study of substrate uptake by filamentous microorganisms in activated sludge. Appl. Environ. Microbiol. 63, 36623668 (1997). [33] Andreasen, K., Nielsen, P . H.: In situ characterization of substrate uptake by Microthrix parvicella using microautoradiography. Water Sci. Technol. 37 (45), 1926 (1998). [34] Hart, M. A.: Scum formation in a nutrient removing activated sludge plant. Water SA 11, 171178 (1985). [35] Goddard, A. J., Forster, C. F .: A further examination into the problem of stable foams in activated sludge plants. Microbios 50, 2942 (1987). [36] Loffhagen, N., Hrtig, C., Babel, W.: Fatty acid patterns of Acinetobacter calcoaceticus 69-V indicate sensitivity against xenobiotics. Appl. Microbiol. Biotechnol. 44, 526531 (1995). [37] Towner, K.: The genus Acinetobacter. In: Balows, A., Trper, H. G., Dworkin, M., Harder, W., Schleifer, K.-H. (Eds.): The Prokaryotes, Chapt. 164. Springer, New York, 1992, pp. 31373143. [38] Wilkinson, S. G.: Gram-negative Bacteria. In: Ratledge, C., Wilkinson, S. G. (Eds.): Microbial Lipids, Vol.1, Chapt. 7. Academic Press, London, 1988, pp. 299488.

[39] OLeary, W. M., Wilkinson, S. G.: Gram-positive Bacteria. In: Ratledge, C., Wilkinson, S. G. (Eds.): Microbial Lipids, Vol.1, Chapt. 5. Academic Press, London, 1988, pp. 117202. [40] Lemmer, H.: The ecology of scum causing actinomycetes in sewage treatment plants. Water Res. 20, 531535 (1986). [41] Jenkins, D., Richard, M. G., Daigger, G. T.: Manual on the causes and control of activated sludge bulking and foaming. Lewis, Boca Raton, 1993. [42] Kappeler, J., Brodmann, R.: Low F/M bulking and scumming: Towards a better understanding by modelling. Water Sci. Technol. 31 (2), 225234 (1995). [43] Chua, H., Tan, K. N., Cheung, M. W. L.: Filamentous growth in activated sludge. Appl. Biochem. Biotechnol. 5758, 851856 (1996). [44] Knoop, S., Kunst, S.: Influence of temperature and sludge loading on activated sludge settling, especially on Microthrix parvicella. Water Sci. Technol. 37 (45), 2735 (1998). [45] Lemke, M. J., Churchill, P .F ., Wetzel, R. G.: Effect of substrate and cell surface hydrophobicity on phosphate utilization in bacteria. Appl. Environ. Microbiol. 61, 913919 (1995). [46] Zhang, C., Valsaraj, K. T., Constant, W. D., Roy, D.: Aerobic biodegradation kinetics of four anionic and nonionic surfactants at sub- and supra-critical micelle concentrations (CMCs). Water Res. 33, 115124 (1999). [47] Blackall, L. L., Marshall, K. C.: The mechanism of stabilization of actinomycete foams and the prevention of foaming under laboratory conditions. J. Ind. Microbiol. 4, 181188 (1989). [48] Gurijala, K. R., Alexander, M.: Effect of growth rate and hydrophobicity on bacteria surviving protozoan grazing. Appl. Environ. Microbiol. 56, 16311635 (1990). [49] Jrgens, K., Gde, H.: The potential importance of grazingresistant bacteria in planktonic systems. Mar. Ecol. Prog. Ser. 112, 169188 (1994).

received 31 May 1999 accepted 22 November 1999

40

Acta hydrochim. hydrobiol. 28 (2000) 1

34

40