- Email: [email protected]
Identification of filamentous microorganisms from activated sludge: A compromise between wishes, needs and possibilities
Wat. Res. Vol. 23, No. 7, pp. 883-891, 1989 Printed in Great Britain. All rights reserved
0043-1354/89 $3.00+0.00 Copyright © 1989 Maxwell PergamonMacmillan pie
IDENTIFICATION OF FILAMENTOUS MICROORGANISMS FROM ACTIVATED SLUDGE: A COMPROMISE BETWEEN WISHES, NEEDS AND POSSIBILITIES J. WANNER~ and P. GRAU~) Department of Water Technology and Environmental Engineering, Prague Institute of Chemical Technology, Suchbfitarova 5, CS-166 28 Prague 6, Czechoslovakia (First received April 1988; accepted in revised form February 1989)
Abstract--Different approaches to the identification of filamentous microorganisms have been evaluated in this paper. The needs for the identification and taxonomic location have been discussed. It has been shown that the identification to types according to Eikelboom is suitable for the purpose of an effective activated sludge bulking and foaming control. The Eikelboom types have been divided into groups with similar properties, occurrence and problems they cause. Key words--activated sludge, filamentous microorganisms, bulking, foaming, identification, taxonomy
INTRODUCTION
Investigation into filamentous microorganisms observed in bulking and foaming activated sludge has become a very important task for both researchers and practitioners in recent years, especially after introducing the nutrient removal activated sludge processes often reported to have bulking and foaming problems. At the same time, this problem requires close cooperation between sanitary and chemical engineers and microbiologists, which is impossible without mutual understanding. The aim of this paper is to summarize the present state of possibilities and needs for a correct identification of filamentous microorganisms and its impact on curing filamentous bulking and foaming problems. THE ROLE OF FILAMENTOUS MICROORGANISMS IN ACTIVATED SLUDGE
The role of filamentous microorganisms in the biocenosis of activated sludge can be evaluated from different viewpoints, but the main aspects are as follows: (i) According to Sezgin et al. (1978), the filamentous microorganisms are believed to form a "backbone" of activated sludge flocs on which flocforming bacteria are fixed by means of extracellular polymers. Although this filament backbone model of flocs formation is now widely accepted in the United States and South Africa (e.g. Jenkins et al., 1984; Melmed et al., 1986), the authors of this paper are not convinced that the skeletal structure is absolutely necessary for forming strong and settleable flocs. We observed that the only case when the filaments created the true skeleton of flocs was after suppressing filamentous growth under anoxic conditions (Figs 1-3) (Wanner et al., 1987a, b,c). In contra-
diction to Sezgin's theory the presence of filamentous microorganisms did not prevent the forming of "pinpoint" flocs (Wanner and Proske, 1988). It seems more probable that the glycocalyx matrix rather than the filamentous skeleton is the primary factor in forming flocs with desirable physical properties (Chudoba, 1989). (ii) The deterioration of activated sludge settling properties caused by an excessive occurrence of filamentous microorganisms in the biocenosis is now a serious problem, with which almost every operator is familiar. A comprehensive survey of activated sludge filamentous bulking causes and methods of control was given by Chambers and Tomlinson (1982), Chudoba (1985, 1989), Jenkins et al. (1984), Wanner and Grau (1988), and by many others. A relatively new problem of activated sludge foaming was reviewed by Jenkins et al. (1984). (iii) The increased occurrence of filamentous microorganisms in the biocenosis of activated sludge indicates that the activated sludge system is not designed or operated properly. Jenkins and his co-workers (Richard et al., 1982; Strom and Jenkins, 1984; Jenkins et al., 1984) proposed that the presence of certain filaments indicate the conditions causing activated sludge bulking, such as low DO, low F/M, low pH, increased concentration of sulphides and nutrient deficiency. Unfortunately, at the present state of knowledge, the indicative role of filamentous microorganisms is rather questionable. For instance, Williams and Unz (1985a) observed Type 1701, a "typical" low DO microorganism, in an aeration basin operated at high DO levels. In our previous experiment (Wanner et al., 1987a), Thiothrix caused serious bulking problems in a lab-scale activated sludge system with anaerobic zone. After the reinoculation, however, the activated sludge did not display any excessive growth of Thiothrix filaments 883
884
J. WANNER and P. GRAU
Fig. 1. Micrographs of an activated sludge floc without the "filamentous backbone". (a) Amorphous floc; (b) fingered zoogloeal floc.
although all operational parameters remained the same as during the bulking period. Furthermore, as it will be shown later, there are certain filamentous microorganisms which are connected with both bulking and foaming problems and it is difficult to predict in advance what kind of nuisance they will cause. On the other hand, if we were able to recognize more exactly all growth and nutrition requirement of filamentous microorganisms, then the correlation between their occurrence and bulking causes should be tighter. The conditions causing bulking should be further examined.
THE NEED FOR IDENTIFICATION
A correct finding of the taxonomic position is necessary for the documentation of a microorganism and for its preservation in microbiological collections. Once the microorganism is positioned taxonomically, the identification of isolates from activated sludge will become much faster and easier. The knowledge of the taxonomic position will also help in recognizing growth and nutrition requirements of a given microorganism by comparison to other strains within a genus or family.
Filamentous microorganisms identification
885
Fig. 2. Micrograph of filaments covered with bacteria. Such correct taxonomic location is still very difficult and expensive. Fortunately, the real needs of engineers are more prosaic. A microbiological examination of the bulking and/or foaming activated sludge must result in such a kind of information which enables the engineers: (i) To find out or predict the extent of filamentous bulking or foaming, i.e. not only to quantify the number or length of filaments but also to specify the type of filaments present in a given sludge. It is a recognizable fact that all filamentous microorganisms do not deteriorate the settling properties of activated
sludge in the same manner. According to our experience, much more severe bulking problems are to be expected when Sphaerotilus spp, Type 021N, and Thiothrix are the dominating filaments in comparison with, for example, Microthrix parvicella, Haliscomenobacter hydrosis, and Nostocoida limicola bulking. The foaming problems threaten only when special kinds of filaments are present (see below). (ii) To estimate the causes of the presence of filamentous microorganisms in the biocenosis. By making such an estimate other factors should also be considered, especially wastewater character and composition, aeration basin(s) configuration and all basic
Fig. 3. Micrograph of the floc developed on the "filamentous skeleton" (Wanner et al., 1987b).
886
J. WANNERand P. GRAU
operational parameters, It is evident that neither a microbiologist nor an engineer, however experienced, can trace the causes himself. A close cooperation between the specialists is of extreme importance. (iii) To rectify the bulking and foaming problems. This is in fact the final object of our effort. The information resulting from the identification of filamentous microorganisms must help in selecting proper measures against their excessive growth. The following ways of filamentous bulking and foaming control are available: --selective "killing" of filaments protruding from the flocs or growing outside the flocs by dosing chlorine, ozone or hydrogen peroxide, ---change of inappropriate wastewater characteristics, e.g. by removing sulphide, neutralizing pH, adding some nutrients, --modification of operation, e.g. by increasing DO concentration or decreasing SRT, --kinetic selection of non-filamentous microorganisms by installing an oxic selector (Chudoba, 1985), --metabolic selection of non-filamentous microorganisms by means of anaerobic and/or anoxic zones/selectors (Tracy et al., 1986; Wanner et al., 1987a, b, c), --mechanical removal of the scum from the surface of basins and clarifiers. To choose the best way, we must know how the bulking and foaming sludge will respond to these measures, which is impossible without knowing what kinds of filaments are present. The standpoints of microbiologists and engineers differ to a certain extent. The determination of the exact taxonomic position of filamentous microorganisms is not inevitable for selecting one of the above-enumerated measures for curing the bulking and foaming problems. For such purposes it is necessary to know not the Latin name of the microorganism but its morphological properties (mainly shape, length and width of filaments), metabolic characteristics (capability of utilizing substrate under oxic, anoxic and anaerobic conditions and of forming reserve materials, such as PHB, PHV, glycogen, polyphosphate or sulphur granules), kinetic and growth parameters (tt .... Ks, Y), nutrition requirements, etc. It is evident that the taxonomic positioning into species, subspecies, and strains often differing only on the genetic code level is too luxurious for our practical needs. In addition, it requires highly specialized and well equipped laboratories. The identification and location of microorganisms into groups with similar properties and behaviour is sufficient in most cases and can be performed in laboratories in many plants. THE POSSIBILITIESOF IDENTIFICATION
Conventional methods of identification The conventional method of identification of bacteria or other microorganisms is based on their
isolation from mixed cultures and on subsequent exhaustive tests of morphological, biochemical and physiological features. The results of these tests are then compared with standard references given in the manual such as Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974). The main advantage of the conventional method seems to be in the unambiguity of taxonomic location obtained in this way. But at present the taxonomic position of a given microorganism without genetic studies can hardly be considered as "definite". For the purposes of isolating the filamentous microorganisms from the diverse biocenosis of activated sludge, the conventional method is too cumbersome, time-consuming, and mostly unnecessary or even unappropriate because of the following reasons: (i) There is a need of at least 30 stable features and of as many as possible variable features (e.g. morphology of cells and cell agglomerates including trichomes) for a formal recognition of nomenclatoric taxon. (ii) There is still a severe lack of pure cultures of filamentous microorganisms originating from activated sludges that isolates can be compared with. (iii) There is a great probability that many morphological, physiological and genetic changes may occur in the filamentous microorganisms isolated from activated sludges in comparison with the pure cultures from bacteriological collections as a result of various undefinable influences on them in wastewater treatment plants (Ottovfi, Personal Communication). (iv) The difficulties in preparing a pure inoculum from the mixed culture of activated sludge, especially due to the removal of accompanying cells of other microorganisms. (v) The low growth rates of most filamentous microorganisms cause the isolation and identification to take weeks or months. Thus, the results obtained do not correspond with the actual state of the biocenosis in the treatment plant the isolates originate from. The conventional identification techniques were also used by Farquhar and Boyle (1971a, b). It was one of the first attempts to systematize filamentous microorganisms occurring in activated sludges. Their detailed determinative key was, however, founded rather on an algological basis and some bacteriological criteria were omitted (Ottovfi, Personal Communication).
The identification to types The identification and classification techniques used by Eikelboom (Eikelboom, 1975; Eikelboom and van Buijsen, 1981) represented a great break in the investigation of activated sludges filamentous bulking and foaming. Eikelboom's Manual was practic~.lly immediately accepted by most of the researchers and practitioners interested in this field throughout the world. The method of Eikelboom
Filamentous microorganisms identification was further modified by Jenkins et al. (1984). The techniques are based on phase contrast microscopic observations of morphology, relationship to other organisms present and staining characteristics (Gram stain, Neisser stain and observation of sulphur and PHB granules). The filamentous microorganisms are classified (with several exceptions) into the so-called types according to the following features: cell shape dimension, presence of sheath, filament, morphology staining characteristics and presence or absence of polyphoshate, sulphur and PHB granules. The taxonomic position of most of the types is uncertain. From 29 categories of filamentous microorganisms only a few represent, undoubtedly, taxonomical species and most of them are not yet fitted into the accepted microbiological classification systems. However, this fact in no case contradicts enormous advantages of this classification system. The identification to types according to Eikelboom and Jenkins is fast and can be performed even by trained nonmicrobiologists. Despite the fact that the types do not represent taxonomical species, their description meets all the practical needs for identification. As will be demonstrated below, all 29 Eikelboom's types can be classified into four groups according to morphological, physiological and metabolic similarity, their occurrence under the same operational conditions and the problems they cause. Group I: Sphaerotilus-like microorganisms. Historically, until the 70s the sheathed bacterium Sphaerotilus natans was considered as the main filamentous microorganism causing bulking problems (for literature review see Eikelboom, 1975). Since the pioneer works of Cyrus and Sladkfi (1970) and Sladk~ and Ottovfi (1973) more than 30 different filamentous microorganisms have been identified in bulking sludges. This can be explained by gradually developing identification techniques in the course of time. But the changes in wastewater composition and the development of the activated sludge process itself have also played an important role. For instance, Sphaerotilus natans has never been found in South African nutrient removal plants with anaerobic and/or anoxic zones and Type 1701 has only been identified with an insignificant frequency of dominance and occurrence (Blackbeard et al., 1988), which is in agreement with our previous findings showing that Sphaerotilus spp are not capable of utilizing substrate under anaerobic or anoxic conditions (Wanner et al., 1987a, b,c). The Sphaerotilus-like filaments are composed of rod- or square-shaped cells contained in a clear sheath. False branching may be observed. Their occurrence in activated sludges is connected with saccharidic or other readily biodegradable wastewaters, higher SRT, and low DO. Besides Sphaerotilus spp, Eikelboom's Types 1701, 0041 and 0675 can be included in this group. Williams and Unz (1985a) have shown that Type 1701 may be related to the genus Sphaerotilus. Group H: Leucothrix, Thiothrix and Eikelboom's WR 23J7-43
887
Type 021N strains. The problem of a proper identification of Leucothrix spp, Thiothrix spp and Type 021N is one of the most exciting in the taxonomy of filamentous microorganisms. The genus Leucothrix was introduced by Cyrus and Sladkfi (1970) for describing filamentous microorganisms resembling colourless blue-green algae. Although the genus Leucothrix was observed in bulking sludges by other authors (Chudoba et al., 1973; Poffe et al., 1979), it was not mentioned in either Eikelboom's or Jenkins's Manual. The primary reason seemed to be that this microorganism was considered to be of marine origin (Brock, 1966). Type 02IN is a very common filamentous microorganism in bulking sludges from conventional activated sludge plants (for survey see Jenkins et al., 1984). But it is quite rare in nutrient removal treatment plants (Blackbeard et aL, 1988), which is also connected with its inability to survive under anaerobic or anoxic conditions (Wanner et al., 1987a, b, c). Filamentous microorganism Thiothrix is also very widespread and was described in detail by Nielsen (1984). Thanks to the exhaustive studies of Williams and Unz (1985a, b), Shimizu (1985) and Williams et al. (1987) we can conclude that Type 02IN is not identical with either the genus Leucothrix or the genus Thiothrix. A final decision on the taxonomic position of Type 021N can be made only after genetic relatedness studies. Both the genus Leucothrix and Type 021N are connected with the filamentous bulking caused by readily biodegradable wastewaters, high SRT and probably by nutrient deficiency. There is no evidence that they have evoked bulking problems in the systems with anaerobic and/or anoxic zones. Thiothrix can cause serious bulking problems whenever it can take advantage of its mixotrophic way of life (Nielsen, 1984; Wanner et al., 1987a, c) (Fig. 4). Group III: Microthrix parvicella and other microorganisms capable of utilizing substrate not only under oxic conditions. Microthrix parvicella was morphologically described by van Veen (1973) and its nutrition requirements were studied by Slijkhuis (1983). According to Williams and Unz (1985a), Type 0803 appears to be similar to M. parvicella. Interest in M. parvicella has increased recently after the finding that this microorganism does not behave as a "typical" filament. It has been found that M. parvicella not only utilizes substrate under anoxic conditions but is also able to accumulate a significant portion of degradable substrate and thus to compete against flock-forming bacteria (Bat6k, 1988; Wanner and Grau, 1988). The ability to accumulate substrate may explain the fact that selectors do not control SVI to very low levels when M. parvicella causes bulking as reported by Jenkins et al. (1984). Rensink and his co-workers (Mulder and Rensink, 1987; Rensink and Donker, 1987) reported the cases when M. parvicella caused serious bulking problems in the systems
888
J, WANNERandP. GRAU
Fig. 4. Micrograph of the activated sludge with an excessive growth' of Thiothrix filaments.
with anaerobic zones. According to Melmed et al. (1986), even the inclusion of an anoxic selector prior to the anaerobic zone of the Phoredox-type reactor failed to suppress the M. parvicella growth. Therefore, M. parvicella is one of the most common filamentous microorganisms in nutrient removal plants (Blackbeard et al., 1988). It results from the survey prepared by Blackbeard et al. (1988) that Type 0092 is a microorganism with the far highest frequency of dominance and occurrence in activated sludges from nutrient removal plants. But the explanation of this fact still calls for more detailed metabolic studies. This statement is believed to be generally valid for all filamentous microorganisms with a higher occurrence in nutrient removal plants because today we know very little about their behaviour under anaerobic and anoxic conditions. The filamentous microorganisms from this group can be generally characterized as microorganisms which are able to accumulate or utilize substrate under anoxic conditions (anoxic respiration, denitrification) or to utilize substrate under anaerobic conditions, which is connected with the phenomenon of polyphosphate formation and degradation. Group IV:foam-forming microorganisms. The activated sludge foaming caused by certain types of filamentous microorganisms must be clearly distinguished from an "abiotic foaming" resulting from a higher content of surfactants, grease and oil in treated wastewater and from "sludge-rising" in secondary clarifiers resulting from denitrification. The activated sludge foaming was initially reported to be connected with the presence of actinomycete
bacteria of the genus Nocardia. The formation of a chocolate-brown, viscous and stable scum, which is very difficult to deal with, was explained by abundant branched hyphae of actinomycetes which form a net which entraps oil droplets and gas bubbles. The foam formation is supported by excreting surface active agents (Pitman, 1984). The causes of actinomycete growth in activated sludges were described by Jenkins et al. (1984) and Lemmer (1986). The main factor initiating the enhanced occurrence of actinomycetes appears to be a high SRT of activated sludge and of the scum (in which it can be even higher than the mean SRT of mixed liquor). At high SRTs, Nocardia has a metabolic advantage in competing for substrate under low F/M conditions. Therefore, the most successful method for preventing the Nocardia growth is considerably lowering the SRT, which is, however, in contradiction with the requirements of nitrifiers. Thus, the most effective way of suppressing the Nocardia growth cannot be applied to nutrient removal plants where the foaming problems are most frequent. Not only actinomycete bacteria cause foaming problems. Goddard and Forster (1987) reported on the production of a stable foam when Nostocoida limicola and Type 0041 were present in the biocenosis of activated sludge. The authors explained this fact with the ability of N. limicola and Type 0041 to produce biosurfactants, such as lipids, lipopeptides, proteins and carbohydrates and, therefore, to form a foam. Both N. limicola and Type 0041 also caused the first serious case of activated sludge foaming which was registered in Czechoslovakia. These filaments were identified in the activated sludge from the
Filamentous microorganisms identification biological phosphorus removal plant of Kaplice, Southern Bohemia. The .plant was initially designed as a two-stage activated sludge process ("AB System"), but due to continuous operational problems with this type of process, and owing to the fact that the effluent from the plant importantly contributed to eutrophication of a drinking water reservoir, it was decided to rebuild the plant from an AB System to a biological phosphorus removal plant. The aeration basins and the settling tank of the first stage (see Fig. 5) were changed to anaerobic reactors mechanically mixed by submerged pumps. In September 1987, when the phenomenon of biological phosphorus removal was already stabilized and total-P in the influent was reduced from more than 10rag/! to below 0.5 mg/1 POa-P in mixed liquor in oxic zones, the SVI values started to increase and browncoloured scum appeared on the surface of aeration basins and final clarifier. Microscopic examination according to Jenkins et al. (1984) revealed that Nosto. coida limicola II and Type 0041 were the cause of both bulking and foaming. It was interesting to note that while in the foam predominated with Type 0041 (occurrence frequency of Type 0041 and N. limicola filaments was 2:1), the ratio was exactly converse in the mixed liquor. The treatment plant is characterized with high SRT values ( > 10-15 days) because of the problems with the final sludge disposal. The problem of foaming has not been solved yet but it has been considerably reduced by regularly skimming the scum from the surface ((~ech, Personal Communication). According to Blackbeard et al. (1988) the most frequently observed microorganisms in the foam from nutrient removal activated sludge plants in
889
South Africa are as follows: Type 0092, M. parvicella, and Type 0041, while the typical "foam" microorganism Nocardia occurs with much less frequency. Type 0092 is a microorganism about which not a lot of information is known and its taxonomic position is unknown. M. parvicella may belong to actinomycete bacteria. Finally, it can be hypothesized that the filamentous microorganisms connected with activated sludge foaming: (i) have to be able to produce biosurfactants enabling them to froth and create scum, (ii) seem to be able to utilize substrate (or simply to survive) under anaerobic and/or anoxic conditions because the foaming problems are reported chiefly for nutrient removal plants. The ability to froth is of primary importance for foam formation and many of filamentous microorganisms found in activated sludge foams can be only "entrapped" into the foam formed by the "true" foam-forming microorganisms. SUMMARY An excessive appearance of filamentous microorganisms in the biocenosis of activated sludge leads to two nuisance cases: activated sludge bulking and activated sludge foaming. A proper identification of filamentous microorganisms enables: (i) to predict what kind and which extent of problem is to be expected, (ii) to estimate the causes of the presence of the filaments in the biocenosis, (iii) to rectify the bulking and foaming problems.
1
An1
An2
[
An1
1
0x
Ox
Ox
I
I
I al
Ox
T
4
Fig. 5. Schematic outline of the Kaplice wastewater treatment plant ("AB System" retrofitted to biological phosphorus removal) Anl, anaerobic zones rebuilt from the aeration basins of the first stage; An2, anaerobic zone rebuilt from the settling tank of the first stage; Ox, oxic zones; ST, settling tank; 1, raw sewage; 2, final effluent; 3, returned activated sludge; 4, excessive activated sludge.
890
J. WAN~R and P. GRAU
The available techniques of the identification of filamentous microorganisms have been discussed. It has been concluded that the knowledge of a correct taxonomic position of filamentous microorganisms is helpful but not absolutely necessary for preventing or curing the bulking and foaming problems. Therefore, the identification to types according to Eikelboom is quite sufficient. It has been demonstrated that most of the Eikelboom's types can be classified into four groups of filamentous microorganisms on the basis of their similarity, occurrence under the same operation conditions and causing of the same problems. The groups are as follows: (i) oxic zone growers S (Sphaerotilus-like microorganisms), (ii) oxic zone growers C (Cyanophycae-like microorganisms, i.e. Leucothrix, Thiothrix and Type 021N), (iii) all zone growers A (Microthrix parvicella and other heterotrophic microorganisms capable of utilizing substrate not only under oxic conditions), (iv) foam-forming microorganisms F (Nocardia spp, Nostocoida limicola, Type 0092 and other microorganisms producing biosurfactants). Further metabolic studies of all filamentous microorganisms occurring in bulking or foaming activated sludges are necessary for a more accurate classification into the groups. The classification into four groups reflects the most frequent bulking and foaming problems in wastewater treatment practice. Special cases of bulking have not been considered in the paper.
REFERENCES
Batik J. (1988) A development of wastewater treatment plant with nitrification and denitrification. Ph.D. dissertation, Prague Institute of Chemical Technology, Prague (in Czech). Blackbeard J. R., Gabb D. M. D., Ekama G. A. and Marais G. v. R. (1988) Identification of filamentous organisms in nutrient removal activated sludge plants in South Africa. Water SA 14, 1-18. Brock T. D. (1966) The habitat of Leucothrix mucor, a widespread marine microorganism. Limnol. Ocean ogr. I 1, 303. Buchanan R. E. and Gibbons N. E. (Eds) (1974) Bergey's Manual of Determinative Bacteriology, 8th edition. Williams & Wilkins, Baltimore, Md. Chambers B. and Tomlinson E. J. (Eds) (1982) Bulking of Activated Sludge: Preventative and Remedial Methods. Ellis Horwood, Chichester. Chudoba J. (1985) Control of activated sludge filamentous bulking--VI. Formulation of basic principles. Wat. Res. 19, 1017-1022. Chudoba J. (1989) Control of activated sludge filamentous bulking. Sci. Papers Prague Inst. Chem. Technol. F27. In press. Chudoba J., Ottovfi V. and Madrra V. (1973) Control of activated sludge filamentous bulking--I. Effect of the hydraulic regime or degree of mixing in an aeration tank. Wat. Res. 7, 1163-1182. Cyrus Z. and Sladk~ A. (1970) Several interesting organisms present in activated sludge. Hydrobiologia 35, 383-396.
Eikelboom D. H. (1975) Filamentous organisms observed in activated sludge. Wat. Res. 9, 365-388. Eikelboom D. H. and van Buijsen H. J. (1981) Microscopic Sludge Investigation Manual. IMG-TNO Report A94a, Delft. Farquhar G. J. and Boyle W. C. (1971a) Identification of filamentous micro-organisms in activated sludge. J. Wat. Pollut. Control Fed. 43~ 6044522. Farquhar G. J. and Boyle W. C. (1971b) Occurrence of filamentous micro-organisms in activated sludge. J. Wat. Pollut. Control Fed. 43, 779-798. Goddard A. J. and Forster C. F. (1987) A further examination into the problem of stable foams in activated sludge plants. Microbios 50, 29-42. Jenkins D., Richard M. G. and Daigger G. T. (1984) Manual on the Causes and Control of Activated Sludge Bulking and Foaming. Water Research Commission, Pretoria. Lemmer H. (1986) The ecology of scum causing actinomycetes in sewage treatment plants. Wat. Res. 20, 531-535. Melmed L. H., Hart M. A. Osborn D. W. and Lrtter L. H. (1986) Investigation into filamentous organisms causing scum formation and bulking in nutrient removal activated sludge systems. Paper presented at the Technology Transfer Symposium "Towards a Better Understanding of Biological Phosphorus Removal", Johannesburg. Mulder J. W. and Rensink J. H. (1987) Introduction of biological phosphorus removal to an activated sludge plant with practical limitations. In Biological Phosphate Removal from Wastewaters (Edited by Ramadori R.). Pergamon Press, Oxford. Nielsen P. H. (1984) Oxidation of sulfide and thiosulfate and storage of sulfur granules in Thiothrix from activated sludge. Wat. Sci. Technol. 17, 167-181. Pitman A. R. (1984) Operation of biological nutrient removal plants. In Theory, Design and Operation of Nutrient Removal Activated Sludge Processes (Edited by Wiechers H. N. S. et al.). Water Research Commission, Pretoria. Poffe R., Vanderleyden J. and Verachtert H. (1979) Characterization of a Leucothrix-type bacterium causing sludge bulking during petrochemical waste-water treatment. Eur. J. appL Microbiol. Biotechnol. 8, 229-237. Rensink J. H. and Donker H. J. G. W. (1987) The influence of bulking sludge on enhanced biological phosphorus removal. In Biological Phosphate Removal from Wastewaters (Edited by Ramadori R.). Pergamon Press, Oxford. Richard M. G., Jenkins D., Hao O. and Shimizu G. (1982) The isolation and characterization of filamentous microorganisms from activated sludge bulking. Report No. 81-2, Sanit. Engng and Envir. Health Res. Lab., University of California, Berkeley. Sezgin M., Jenkins D. and Parker D. S. (1978) A unified theory of filamentous activated sludge bulking. J. Wat. Pollut. Control Fed. 50, 362-381. Shimizu G. P. (1985) The growth and nutrient uptake kinetics of Type 02IN, a novel Thiothrix-like bacterium responsible for activated sludge bulking. Ph.D. dissertation, University of California, Berkeley. Sladk~i A. and Ottovfi V. (1973) Filamentous organisms in activated sludge. Hydrobiologia 43, 285-299. Slijkhuis H. (1983) Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium. Appl. envir. Microbiol. 46, 832-839. Strom P. F. and Jenkins D. (1984) Identification and significance of filamentous microorganisms in activated sludge. J. Wat. Pollut. Control Fed. 56, 449-459. Tracy K. D., Adams M. E. and Flammino A. (1986) Control of activated sludge settling characteristics with anaerobic selectors. Paper presented at the 59th Annual Conference Water Pollution Control Federation, Los Angeles.
Filamentous microorganisms identification van Veen W. L. (1973) Bacteriology of activated sludge, in particular the filamentous bacteria. Antonie van Leuwenhoek J. Microbiol. Serol. 39, 189-205. Wanner J., Ottovfi and Grau P. (1987a) Effect of an anaerobic zone on settleability of activated sludge. In Biological Phosphate Removal from Wastewaters (Edited by Ramadori R.). Pergamon Press, Oxford. Wanner J., Chudoba J., Kucman K. and Proske L. (1987b) Control of activated sludge filamentous bulking--VII. Effect of anoxic conditions. Wat. Res. 21, 1447-1451. Wanner J., Kucman K., Ottov~ V. and Grau P. (1987c) Effect of anaerobic conditions on activated sludge filamentous bulking in laboratory systems. War. Res. 21, 1541-1546. Wanner J. and Grau P. (1988) Filamentous bulking in
891
nutrient removal activated sludge systems. )Vat. Sci. Technol. 20, 1 4 . Wanner J. and Proske L. (1988) Unpublished data. Williams T. M. and Unz R. F. (1985a) Isolation and characterization of filamentous bacteria present in bulking activated sludge. Appl. Microbiol. Biotechnol. 22, 273-282. Williams R. M. and Unz R. F. (1985b) Filamentous sulfur bacteria of activated sludge: characterization of Thiothrix, Beggiatoa, and Eikelboom Type 021N strains. Appl. envir. Microbiol. 49, 887-898. Williams T. M., Unz R. F. and Doman J. T. (1987) Ultrastructure of Thiothrix spp and "Type 021N" bacteria. Appl. envir. Microbiol. 53, 1560-1570.
0043-1354/89 $3.00+0.00 Copyright © 1989 Maxwell PergamonMacmillan pie
IDENTIFICATION OF FILAMENTOUS MICROORGANISMS FROM ACTIVATED SLUDGE: A COMPROMISE BETWEEN WISHES, NEEDS AND POSSIBILITIES J. WANNER~ and P. GRAU~) Department of Water Technology and Environmental Engineering, Prague Institute of Chemical Technology, Suchbfitarova 5, CS-166 28 Prague 6, Czechoslovakia (First received April 1988; accepted in revised form February 1989)
Abstract--Different approaches to the identification of filamentous microorganisms have been evaluated in this paper. The needs for the identification and taxonomic location have been discussed. It has been shown that the identification to types according to Eikelboom is suitable for the purpose of an effective activated sludge bulking and foaming control. The Eikelboom types have been divided into groups with similar properties, occurrence and problems they cause. Key words--activated sludge, filamentous microorganisms, bulking, foaming, identification, taxonomy
INTRODUCTION
Investigation into filamentous microorganisms observed in bulking and foaming activated sludge has become a very important task for both researchers and practitioners in recent years, especially after introducing the nutrient removal activated sludge processes often reported to have bulking and foaming problems. At the same time, this problem requires close cooperation between sanitary and chemical engineers and microbiologists, which is impossible without mutual understanding. The aim of this paper is to summarize the present state of possibilities and needs for a correct identification of filamentous microorganisms and its impact on curing filamentous bulking and foaming problems. THE ROLE OF FILAMENTOUS MICROORGANISMS IN ACTIVATED SLUDGE
The role of filamentous microorganisms in the biocenosis of activated sludge can be evaluated from different viewpoints, but the main aspects are as follows: (i) According to Sezgin et al. (1978), the filamentous microorganisms are believed to form a "backbone" of activated sludge flocs on which flocforming bacteria are fixed by means of extracellular polymers. Although this filament backbone model of flocs formation is now widely accepted in the United States and South Africa (e.g. Jenkins et al., 1984; Melmed et al., 1986), the authors of this paper are not convinced that the skeletal structure is absolutely necessary for forming strong and settleable flocs. We observed that the only case when the filaments created the true skeleton of flocs was after suppressing filamentous growth under anoxic conditions (Figs 1-3) (Wanner et al., 1987a, b,c). In contra-
diction to Sezgin's theory the presence of filamentous microorganisms did not prevent the forming of "pinpoint" flocs (Wanner and Proske, 1988). It seems more probable that the glycocalyx matrix rather than the filamentous skeleton is the primary factor in forming flocs with desirable physical properties (Chudoba, 1989). (ii) The deterioration of activated sludge settling properties caused by an excessive occurrence of filamentous microorganisms in the biocenosis is now a serious problem, with which almost every operator is familiar. A comprehensive survey of activated sludge filamentous bulking causes and methods of control was given by Chambers and Tomlinson (1982), Chudoba (1985, 1989), Jenkins et al. (1984), Wanner and Grau (1988), and by many others. A relatively new problem of activated sludge foaming was reviewed by Jenkins et al. (1984). (iii) The increased occurrence of filamentous microorganisms in the biocenosis of activated sludge indicates that the activated sludge system is not designed or operated properly. Jenkins and his co-workers (Richard et al., 1982; Strom and Jenkins, 1984; Jenkins et al., 1984) proposed that the presence of certain filaments indicate the conditions causing activated sludge bulking, such as low DO, low F/M, low pH, increased concentration of sulphides and nutrient deficiency. Unfortunately, at the present state of knowledge, the indicative role of filamentous microorganisms is rather questionable. For instance, Williams and Unz (1985a) observed Type 1701, a "typical" low DO microorganism, in an aeration basin operated at high DO levels. In our previous experiment (Wanner et al., 1987a), Thiothrix caused serious bulking problems in a lab-scale activated sludge system with anaerobic zone. After the reinoculation, however, the activated sludge did not display any excessive growth of Thiothrix filaments 883
884
J. WANNER and P. GRAU
Fig. 1. Micrographs of an activated sludge floc without the "filamentous backbone". (a) Amorphous floc; (b) fingered zoogloeal floc.
although all operational parameters remained the same as during the bulking period. Furthermore, as it will be shown later, there are certain filamentous microorganisms which are connected with both bulking and foaming problems and it is difficult to predict in advance what kind of nuisance they will cause. On the other hand, if we were able to recognize more exactly all growth and nutrition requirement of filamentous microorganisms, then the correlation between their occurrence and bulking causes should be tighter. The conditions causing bulking should be further examined.
THE NEED FOR IDENTIFICATION
A correct finding of the taxonomic position is necessary for the documentation of a microorganism and for its preservation in microbiological collections. Once the microorganism is positioned taxonomically, the identification of isolates from activated sludge will become much faster and easier. The knowledge of the taxonomic position will also help in recognizing growth and nutrition requirements of a given microorganism by comparison to other strains within a genus or family.
Filamentous microorganisms identification
885
Fig. 2. Micrograph of filaments covered with bacteria. Such correct taxonomic location is still very difficult and expensive. Fortunately, the real needs of engineers are more prosaic. A microbiological examination of the bulking and/or foaming activated sludge must result in such a kind of information which enables the engineers: (i) To find out or predict the extent of filamentous bulking or foaming, i.e. not only to quantify the number or length of filaments but also to specify the type of filaments present in a given sludge. It is a recognizable fact that all filamentous microorganisms do not deteriorate the settling properties of activated
sludge in the same manner. According to our experience, much more severe bulking problems are to be expected when Sphaerotilus spp, Type 021N, and Thiothrix are the dominating filaments in comparison with, for example, Microthrix parvicella, Haliscomenobacter hydrosis, and Nostocoida limicola bulking. The foaming problems threaten only when special kinds of filaments are present (see below). (ii) To estimate the causes of the presence of filamentous microorganisms in the biocenosis. By making such an estimate other factors should also be considered, especially wastewater character and composition, aeration basin(s) configuration and all basic
Fig. 3. Micrograph of the floc developed on the "filamentous skeleton" (Wanner et al., 1987b).
886
J. WANNERand P. GRAU
operational parameters, It is evident that neither a microbiologist nor an engineer, however experienced, can trace the causes himself. A close cooperation between the specialists is of extreme importance. (iii) To rectify the bulking and foaming problems. This is in fact the final object of our effort. The information resulting from the identification of filamentous microorganisms must help in selecting proper measures against their excessive growth. The following ways of filamentous bulking and foaming control are available: --selective "killing" of filaments protruding from the flocs or growing outside the flocs by dosing chlorine, ozone or hydrogen peroxide, ---change of inappropriate wastewater characteristics, e.g. by removing sulphide, neutralizing pH, adding some nutrients, --modification of operation, e.g. by increasing DO concentration or decreasing SRT, --kinetic selection of non-filamentous microorganisms by installing an oxic selector (Chudoba, 1985), --metabolic selection of non-filamentous microorganisms by means of anaerobic and/or anoxic zones/selectors (Tracy et al., 1986; Wanner et al., 1987a, b, c), --mechanical removal of the scum from the surface of basins and clarifiers. To choose the best way, we must know how the bulking and foaming sludge will respond to these measures, which is impossible without knowing what kinds of filaments are present. The standpoints of microbiologists and engineers differ to a certain extent. The determination of the exact taxonomic position of filamentous microorganisms is not inevitable for selecting one of the above-enumerated measures for curing the bulking and foaming problems. For such purposes it is necessary to know not the Latin name of the microorganism but its morphological properties (mainly shape, length and width of filaments), metabolic characteristics (capability of utilizing substrate under oxic, anoxic and anaerobic conditions and of forming reserve materials, such as PHB, PHV, glycogen, polyphosphate or sulphur granules), kinetic and growth parameters (tt .... Ks, Y), nutrition requirements, etc. It is evident that the taxonomic positioning into species, subspecies, and strains often differing only on the genetic code level is too luxurious for our practical needs. In addition, it requires highly specialized and well equipped laboratories. The identification and location of microorganisms into groups with similar properties and behaviour is sufficient in most cases and can be performed in laboratories in many plants. THE POSSIBILITIESOF IDENTIFICATION
Conventional methods of identification The conventional method of identification of bacteria or other microorganisms is based on their
isolation from mixed cultures and on subsequent exhaustive tests of morphological, biochemical and physiological features. The results of these tests are then compared with standard references given in the manual such as Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974). The main advantage of the conventional method seems to be in the unambiguity of taxonomic location obtained in this way. But at present the taxonomic position of a given microorganism without genetic studies can hardly be considered as "definite". For the purposes of isolating the filamentous microorganisms from the diverse biocenosis of activated sludge, the conventional method is too cumbersome, time-consuming, and mostly unnecessary or even unappropriate because of the following reasons: (i) There is a need of at least 30 stable features and of as many as possible variable features (e.g. morphology of cells and cell agglomerates including trichomes) for a formal recognition of nomenclatoric taxon. (ii) There is still a severe lack of pure cultures of filamentous microorganisms originating from activated sludges that isolates can be compared with. (iii) There is a great probability that many morphological, physiological and genetic changes may occur in the filamentous microorganisms isolated from activated sludges in comparison with the pure cultures from bacteriological collections as a result of various undefinable influences on them in wastewater treatment plants (Ottovfi, Personal Communication). (iv) The difficulties in preparing a pure inoculum from the mixed culture of activated sludge, especially due to the removal of accompanying cells of other microorganisms. (v) The low growth rates of most filamentous microorganisms cause the isolation and identification to take weeks or months. Thus, the results obtained do not correspond with the actual state of the biocenosis in the treatment plant the isolates originate from. The conventional identification techniques were also used by Farquhar and Boyle (1971a, b). It was one of the first attempts to systematize filamentous microorganisms occurring in activated sludges. Their detailed determinative key was, however, founded rather on an algological basis and some bacteriological criteria were omitted (Ottovfi, Personal Communication).
The identification to types The identification and classification techniques used by Eikelboom (Eikelboom, 1975; Eikelboom and van Buijsen, 1981) represented a great break in the investigation of activated sludges filamentous bulking and foaming. Eikelboom's Manual was practic~.lly immediately accepted by most of the researchers and practitioners interested in this field throughout the world. The method of Eikelboom
Filamentous microorganisms identification was further modified by Jenkins et al. (1984). The techniques are based on phase contrast microscopic observations of morphology, relationship to other organisms present and staining characteristics (Gram stain, Neisser stain and observation of sulphur and PHB granules). The filamentous microorganisms are classified (with several exceptions) into the so-called types according to the following features: cell shape dimension, presence of sheath, filament, morphology staining characteristics and presence or absence of polyphoshate, sulphur and PHB granules. The taxonomic position of most of the types is uncertain. From 29 categories of filamentous microorganisms only a few represent, undoubtedly, taxonomical species and most of them are not yet fitted into the accepted microbiological classification systems. However, this fact in no case contradicts enormous advantages of this classification system. The identification to types according to Eikelboom and Jenkins is fast and can be performed even by trained nonmicrobiologists. Despite the fact that the types do not represent taxonomical species, their description meets all the practical needs for identification. As will be demonstrated below, all 29 Eikelboom's types can be classified into four groups according to morphological, physiological and metabolic similarity, their occurrence under the same operational conditions and the problems they cause. Group I: Sphaerotilus-like microorganisms. Historically, until the 70s the sheathed bacterium Sphaerotilus natans was considered as the main filamentous microorganism causing bulking problems (for literature review see Eikelboom, 1975). Since the pioneer works of Cyrus and Sladkfi (1970) and Sladk~ and Ottovfi (1973) more than 30 different filamentous microorganisms have been identified in bulking sludges. This can be explained by gradually developing identification techniques in the course of time. But the changes in wastewater composition and the development of the activated sludge process itself have also played an important role. For instance, Sphaerotilus natans has never been found in South African nutrient removal plants with anaerobic and/or anoxic zones and Type 1701 has only been identified with an insignificant frequency of dominance and occurrence (Blackbeard et al., 1988), which is in agreement with our previous findings showing that Sphaerotilus spp are not capable of utilizing substrate under anaerobic or anoxic conditions (Wanner et al., 1987a, b,c). The Sphaerotilus-like filaments are composed of rod- or square-shaped cells contained in a clear sheath. False branching may be observed. Their occurrence in activated sludges is connected with saccharidic or other readily biodegradable wastewaters, higher SRT, and low DO. Besides Sphaerotilus spp, Eikelboom's Types 1701, 0041 and 0675 can be included in this group. Williams and Unz (1985a) have shown that Type 1701 may be related to the genus Sphaerotilus. Group H: Leucothrix, Thiothrix and Eikelboom's WR 23J7-43
887
Type 021N strains. The problem of a proper identification of Leucothrix spp, Thiothrix spp and Type 021N is one of the most exciting in the taxonomy of filamentous microorganisms. The genus Leucothrix was introduced by Cyrus and Sladkfi (1970) for describing filamentous microorganisms resembling colourless blue-green algae. Although the genus Leucothrix was observed in bulking sludges by other authors (Chudoba et al., 1973; Poffe et al., 1979), it was not mentioned in either Eikelboom's or Jenkins's Manual. The primary reason seemed to be that this microorganism was considered to be of marine origin (Brock, 1966). Type 02IN is a very common filamentous microorganism in bulking sludges from conventional activated sludge plants (for survey see Jenkins et al., 1984). But it is quite rare in nutrient removal treatment plants (Blackbeard et aL, 1988), which is also connected with its inability to survive under anaerobic or anoxic conditions (Wanner et al., 1987a, b, c). Filamentous microorganism Thiothrix is also very widespread and was described in detail by Nielsen (1984). Thanks to the exhaustive studies of Williams and Unz (1985a, b), Shimizu (1985) and Williams et al. (1987) we can conclude that Type 02IN is not identical with either the genus Leucothrix or the genus Thiothrix. A final decision on the taxonomic position of Type 021N can be made only after genetic relatedness studies. Both the genus Leucothrix and Type 021N are connected with the filamentous bulking caused by readily biodegradable wastewaters, high SRT and probably by nutrient deficiency. There is no evidence that they have evoked bulking problems in the systems with anaerobic and/or anoxic zones. Thiothrix can cause serious bulking problems whenever it can take advantage of its mixotrophic way of life (Nielsen, 1984; Wanner et al., 1987a, c) (Fig. 4). Group III: Microthrix parvicella and other microorganisms capable of utilizing substrate not only under oxic conditions. Microthrix parvicella was morphologically described by van Veen (1973) and its nutrition requirements were studied by Slijkhuis (1983). According to Williams and Unz (1985a), Type 0803 appears to be similar to M. parvicella. Interest in M. parvicella has increased recently after the finding that this microorganism does not behave as a "typical" filament. It has been found that M. parvicella not only utilizes substrate under anoxic conditions but is also able to accumulate a significant portion of degradable substrate and thus to compete against flock-forming bacteria (Bat6k, 1988; Wanner and Grau, 1988). The ability to accumulate substrate may explain the fact that selectors do not control SVI to very low levels when M. parvicella causes bulking as reported by Jenkins et al. (1984). Rensink and his co-workers (Mulder and Rensink, 1987; Rensink and Donker, 1987) reported the cases when M. parvicella caused serious bulking problems in the systems
888
J, WANNERandP. GRAU
Fig. 4. Micrograph of the activated sludge with an excessive growth' of Thiothrix filaments.
with anaerobic zones. According to Melmed et al. (1986), even the inclusion of an anoxic selector prior to the anaerobic zone of the Phoredox-type reactor failed to suppress the M. parvicella growth. Therefore, M. parvicella is one of the most common filamentous microorganisms in nutrient removal plants (Blackbeard et al., 1988). It results from the survey prepared by Blackbeard et al. (1988) that Type 0092 is a microorganism with the far highest frequency of dominance and occurrence in activated sludges from nutrient removal plants. But the explanation of this fact still calls for more detailed metabolic studies. This statement is believed to be generally valid for all filamentous microorganisms with a higher occurrence in nutrient removal plants because today we know very little about their behaviour under anaerobic and anoxic conditions. The filamentous microorganisms from this group can be generally characterized as microorganisms which are able to accumulate or utilize substrate under anoxic conditions (anoxic respiration, denitrification) or to utilize substrate under anaerobic conditions, which is connected with the phenomenon of polyphosphate formation and degradation. Group IV:foam-forming microorganisms. The activated sludge foaming caused by certain types of filamentous microorganisms must be clearly distinguished from an "abiotic foaming" resulting from a higher content of surfactants, grease and oil in treated wastewater and from "sludge-rising" in secondary clarifiers resulting from denitrification. The activated sludge foaming was initially reported to be connected with the presence of actinomycete
bacteria of the genus Nocardia. The formation of a chocolate-brown, viscous and stable scum, which is very difficult to deal with, was explained by abundant branched hyphae of actinomycetes which form a net which entraps oil droplets and gas bubbles. The foam formation is supported by excreting surface active agents (Pitman, 1984). The causes of actinomycete growth in activated sludges were described by Jenkins et al. (1984) and Lemmer (1986). The main factor initiating the enhanced occurrence of actinomycetes appears to be a high SRT of activated sludge and of the scum (in which it can be even higher than the mean SRT of mixed liquor). At high SRTs, Nocardia has a metabolic advantage in competing for substrate under low F/M conditions. Therefore, the most successful method for preventing the Nocardia growth is considerably lowering the SRT, which is, however, in contradiction with the requirements of nitrifiers. Thus, the most effective way of suppressing the Nocardia growth cannot be applied to nutrient removal plants where the foaming problems are most frequent. Not only actinomycete bacteria cause foaming problems. Goddard and Forster (1987) reported on the production of a stable foam when Nostocoida limicola and Type 0041 were present in the biocenosis of activated sludge. The authors explained this fact with the ability of N. limicola and Type 0041 to produce biosurfactants, such as lipids, lipopeptides, proteins and carbohydrates and, therefore, to form a foam. Both N. limicola and Type 0041 also caused the first serious case of activated sludge foaming which was registered in Czechoslovakia. These filaments were identified in the activated sludge from the
Filamentous microorganisms identification biological phosphorus removal plant of Kaplice, Southern Bohemia. The .plant was initially designed as a two-stage activated sludge process ("AB System"), but due to continuous operational problems with this type of process, and owing to the fact that the effluent from the plant importantly contributed to eutrophication of a drinking water reservoir, it was decided to rebuild the plant from an AB System to a biological phosphorus removal plant. The aeration basins and the settling tank of the first stage (see Fig. 5) were changed to anaerobic reactors mechanically mixed by submerged pumps. In September 1987, when the phenomenon of biological phosphorus removal was already stabilized and total-P in the influent was reduced from more than 10rag/! to below 0.5 mg/1 POa-P in mixed liquor in oxic zones, the SVI values started to increase and browncoloured scum appeared on the surface of aeration basins and final clarifier. Microscopic examination according to Jenkins et al. (1984) revealed that Nosto. coida limicola II and Type 0041 were the cause of both bulking and foaming. It was interesting to note that while in the foam predominated with Type 0041 (occurrence frequency of Type 0041 and N. limicola filaments was 2:1), the ratio was exactly converse in the mixed liquor. The treatment plant is characterized with high SRT values ( > 10-15 days) because of the problems with the final sludge disposal. The problem of foaming has not been solved yet but it has been considerably reduced by regularly skimming the scum from the surface ((~ech, Personal Communication). According to Blackbeard et al. (1988) the most frequently observed microorganisms in the foam from nutrient removal activated sludge plants in
889
South Africa are as follows: Type 0092, M. parvicella, and Type 0041, while the typical "foam" microorganism Nocardia occurs with much less frequency. Type 0092 is a microorganism about which not a lot of information is known and its taxonomic position is unknown. M. parvicella may belong to actinomycete bacteria. Finally, it can be hypothesized that the filamentous microorganisms connected with activated sludge foaming: (i) have to be able to produce biosurfactants enabling them to froth and create scum, (ii) seem to be able to utilize substrate (or simply to survive) under anaerobic and/or anoxic conditions because the foaming problems are reported chiefly for nutrient removal plants. The ability to froth is of primary importance for foam formation and many of filamentous microorganisms found in activated sludge foams can be only "entrapped" into the foam formed by the "true" foam-forming microorganisms. SUMMARY An excessive appearance of filamentous microorganisms in the biocenosis of activated sludge leads to two nuisance cases: activated sludge bulking and activated sludge foaming. A proper identification of filamentous microorganisms enables: (i) to predict what kind and which extent of problem is to be expected, (ii) to estimate the causes of the presence of the filaments in the biocenosis, (iii) to rectify the bulking and foaming problems.
1
An1
An2
[
An1
1
0x
Ox
Ox
I
I
I al
Ox
T
4
Fig. 5. Schematic outline of the Kaplice wastewater treatment plant ("AB System" retrofitted to biological phosphorus removal) Anl, anaerobic zones rebuilt from the aeration basins of the first stage; An2, anaerobic zone rebuilt from the settling tank of the first stage; Ox, oxic zones; ST, settling tank; 1, raw sewage; 2, final effluent; 3, returned activated sludge; 4, excessive activated sludge.
890
J. WAN~R and P. GRAU
The available techniques of the identification of filamentous microorganisms have been discussed. It has been concluded that the knowledge of a correct taxonomic position of filamentous microorganisms is helpful but not absolutely necessary for preventing or curing the bulking and foaming problems. Therefore, the identification to types according to Eikelboom is quite sufficient. It has been demonstrated that most of the Eikelboom's types can be classified into four groups of filamentous microorganisms on the basis of their similarity, occurrence under the same operation conditions and causing of the same problems. The groups are as follows: (i) oxic zone growers S (Sphaerotilus-like microorganisms), (ii) oxic zone growers C (Cyanophycae-like microorganisms, i.e. Leucothrix, Thiothrix and Type 021N), (iii) all zone growers A (Microthrix parvicella and other heterotrophic microorganisms capable of utilizing substrate not only under oxic conditions), (iv) foam-forming microorganisms F (Nocardia spp, Nostocoida limicola, Type 0092 and other microorganisms producing biosurfactants). Further metabolic studies of all filamentous microorganisms occurring in bulking or foaming activated sludges are necessary for a more accurate classification into the groups. The classification into four groups reflects the most frequent bulking and foaming problems in wastewater treatment practice. Special cases of bulking have not been considered in the paper.
REFERENCES
Batik J. (1988) A development of wastewater treatment plant with nitrification and denitrification. Ph.D. dissertation, Prague Institute of Chemical Technology, Prague (in Czech). Blackbeard J. R., Gabb D. M. D., Ekama G. A. and Marais G. v. R. (1988) Identification of filamentous organisms in nutrient removal activated sludge plants in South Africa. Water SA 14, 1-18. Brock T. D. (1966) The habitat of Leucothrix mucor, a widespread marine microorganism. Limnol. Ocean ogr. I 1, 303. Buchanan R. E. and Gibbons N. E. (Eds) (1974) Bergey's Manual of Determinative Bacteriology, 8th edition. Williams & Wilkins, Baltimore, Md. Chambers B. and Tomlinson E. J. (Eds) (1982) Bulking of Activated Sludge: Preventative and Remedial Methods. Ellis Horwood, Chichester. Chudoba J. (1985) Control of activated sludge filamentous bulking--VI. Formulation of basic principles. Wat. Res. 19, 1017-1022. Chudoba J. (1989) Control of activated sludge filamentous bulking. Sci. Papers Prague Inst. Chem. Technol. F27. In press. Chudoba J., Ottovfi V. and Madrra V. (1973) Control of activated sludge filamentous bulking--I. Effect of the hydraulic regime or degree of mixing in an aeration tank. Wat. Res. 7, 1163-1182. Cyrus Z. and Sladk~ A. (1970) Several interesting organisms present in activated sludge. Hydrobiologia 35, 383-396.
Eikelboom D. H. (1975) Filamentous organisms observed in activated sludge. Wat. Res. 9, 365-388. Eikelboom D. H. and van Buijsen H. J. (1981) Microscopic Sludge Investigation Manual. IMG-TNO Report A94a, Delft. Farquhar G. J. and Boyle W. C. (1971a) Identification of filamentous micro-organisms in activated sludge. J. Wat. Pollut. Control Fed. 43~ 6044522. Farquhar G. J. and Boyle W. C. (1971b) Occurrence of filamentous micro-organisms in activated sludge. J. Wat. Pollut. Control Fed. 43, 779-798. Goddard A. J. and Forster C. F. (1987) A further examination into the problem of stable foams in activated sludge plants. Microbios 50, 29-42. Jenkins D., Richard M. G. and Daigger G. T. (1984) Manual on the Causes and Control of Activated Sludge Bulking and Foaming. Water Research Commission, Pretoria. Lemmer H. (1986) The ecology of scum causing actinomycetes in sewage treatment plants. Wat. Res. 20, 531-535. Melmed L. H., Hart M. A. Osborn D. W. and Lrtter L. H. (1986) Investigation into filamentous organisms causing scum formation and bulking in nutrient removal activated sludge systems. Paper presented at the Technology Transfer Symposium "Towards a Better Understanding of Biological Phosphorus Removal", Johannesburg. Mulder J. W. and Rensink J. H. (1987) Introduction of biological phosphorus removal to an activated sludge plant with practical limitations. In Biological Phosphate Removal from Wastewaters (Edited by Ramadori R.). Pergamon Press, Oxford. Nielsen P. H. (1984) Oxidation of sulfide and thiosulfate and storage of sulfur granules in Thiothrix from activated sludge. Wat. Sci. Technol. 17, 167-181. Pitman A. R. (1984) Operation of biological nutrient removal plants. In Theory, Design and Operation of Nutrient Removal Activated Sludge Processes (Edited by Wiechers H. N. S. et al.). Water Research Commission, Pretoria. Poffe R., Vanderleyden J. and Verachtert H. (1979) Characterization of a Leucothrix-type bacterium causing sludge bulking during petrochemical waste-water treatment. Eur. J. appL Microbiol. Biotechnol. 8, 229-237. Rensink J. H. and Donker H. J. G. W. (1987) The influence of bulking sludge on enhanced biological phosphorus removal. In Biological Phosphate Removal from Wastewaters (Edited by Ramadori R.). Pergamon Press, Oxford. Richard M. G., Jenkins D., Hao O. and Shimizu G. (1982) The isolation and characterization of filamentous microorganisms from activated sludge bulking. Report No. 81-2, Sanit. Engng and Envir. Health Res. Lab., University of California, Berkeley. Sezgin M., Jenkins D. and Parker D. S. (1978) A unified theory of filamentous activated sludge bulking. J. Wat. Pollut. Control Fed. 50, 362-381. Shimizu G. P. (1985) The growth and nutrient uptake kinetics of Type 02IN, a novel Thiothrix-like bacterium responsible for activated sludge bulking. Ph.D. dissertation, University of California, Berkeley. Sladk~i A. and Ottovfi V. (1973) Filamentous organisms in activated sludge. Hydrobiologia 43, 285-299. Slijkhuis H. (1983) Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium. Appl. envir. Microbiol. 46, 832-839. Strom P. F. and Jenkins D. (1984) Identification and significance of filamentous microorganisms in activated sludge. J. Wat. Pollut. Control Fed. 56, 449-459. Tracy K. D., Adams M. E. and Flammino A. (1986) Control of activated sludge settling characteristics with anaerobic selectors. Paper presented at the 59th Annual Conference Water Pollution Control Federation, Los Angeles.
Filamentous microorganisms identification van Veen W. L. (1973) Bacteriology of activated sludge, in particular the filamentous bacteria. Antonie van Leuwenhoek J. Microbiol. Serol. 39, 189-205. Wanner J., Ottovfi and Grau P. (1987a) Effect of an anaerobic zone on settleability of activated sludge. In Biological Phosphate Removal from Wastewaters (Edited by Ramadori R.). Pergamon Press, Oxford. Wanner J., Chudoba J., Kucman K. and Proske L. (1987b) Control of activated sludge filamentous bulking--VII. Effect of anoxic conditions. Wat. Res. 21, 1447-1451. Wanner J., Kucman K., Ottov~ V. and Grau P. (1987c) Effect of anaerobic conditions on activated sludge filamentous bulking in laboratory systems. War. Res. 21, 1541-1546. Wanner J. and Grau P. (1988) Filamentous bulking in
891
nutrient removal activated sludge systems. )Vat. Sci. Technol. 20, 1 4 . Wanner J. and Proske L. (1988) Unpublished data. Williams T. M. and Unz R. F. (1985a) Isolation and characterization of filamentous bacteria present in bulking activated sludge. Appl. Microbiol. Biotechnol. 22, 273-282. Williams R. M. and Unz R. F. (1985b) Filamentous sulfur bacteria of activated sludge: characterization of Thiothrix, Beggiatoa, and Eikelboom Type 021N strains. Appl. envir. Microbiol. 49, 887-898. Williams T. M., Unz R. F. and Doman J. T. (1987) Ultrastructure of Thiothrix spp and "Type 021N" bacteria. Appl. envir. Microbiol. 53, 1560-1570.