Novel Actinomycete Isolated from Bulking Industrial Sludge JOHANNA M

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1986, p. 1324-1330 Vol. 52, No. 6 0099-2240/86/121324-07$02.00/0 Copyright © 1986, American Society for Microbiology Novel Actinomycete Isolated from Bulking Industrial Sludge JOHANNA M. WHITE,' DAVID P. LABEDA,2 MARY P. LECHEVALIER,3 JAMES R. OWENS,' DANIEL D. JONES,' AND JOSEPH J. GAUTHIER'* Department of Biology, University ofAlabama at Birmingham, Birmingham, Alabama 352941; U.S. Department of Agriculture, Northern Regional Research Center, Peoria, Illinois 616042; and Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854-07593 Received 14 July 1986/Accepted 5 September 1986

A novel actinomycete was the predominant filamentous microorganism in bulking activated sludge in a bench-scale reactor treating coke plant wastewater. The bacterium was isolated and identified as an actinomycete that is biochemically and morphologically similar to Amycolatopsis orientalis; however, a lack of DNA homology excludes true relatedness. At present, the isolate (NRRL B 16216) cannot be assigned to the recognized taxa of actinomycetes.

Successful operation of the activated sludge process de- previously (13). Briefly, they consisted of water-jacketed pends on separation of the microbial biomass from the aeration basins, each with a volume of 12 liters. Overflow treated water in a clarifier. Sludge bulking, which is the was collected in a clarifier from which settled sludge was inability of the biomass to settle properly, represents a major pumped back into the aeration basin. To simulate full-scale problem associated with this treatment method. Although treatment conditions, the reactors were maintained at a some bulking incidents are the result of the formation of temperature of 35°C, an oxygen concentration of 3 mg/liter, pinpoint floc or dispersed growth of the biomass, it is known a pH of 7.2, and a hydraulic retention time of 42 h. Each that bulking can be associated with the presence of filamen- reactor was inoculated with mixed liquor from an activated tous bacteria (6, 8, 27, 28). In addition, recent quantitative sludge process treating coke plant wastewater. For the first data indicate that the extent of bulking is related to the 30 days, the feed contained an average (per liter) of 50 mg of amount of filamentous growth (15, 35). ammonia and 350 mg of total organic carbon, including 200 Control of bulking has been largely empirical, and studies mg of phenol, 135 mg of thiocyanate, and 2 mg of cyanide. have often lacked a characterization of the organism (or The initial mixed liquor suspended solids concentration was organisms) involved. Conditions suspected to cause bulking about 3,000 mg/liter. Wasting was accomplished by removal include low dissolved-oxygen concentration, high and low of 0.5% of the mixed liquor from the aeration basin once organic loading, industrial inputs, nutrient deficiencies, and each day, resulting in a mean cell residence time of 200 days. flow configuration. A few studies have attempted to corre- After 30 days, the influent loading was gradually raised by late a single condition with the presence of filamentous increasing the proportion of unprocessed ammonia liquor in growth (26, 33). Organisms are usually characterized on the the feed as previously described (13). By 67 days of opera- basis of microscopic observations (6-8, 35). tion, the influent total organic carbon concentration was A rational approach to the control of filamentous bulking increased to 900 mg/liter and the ammonia concentration was depends on understanding the precise physical and nutri- increased to 780 mg/liter. During this time the concentration tional conditions that, taken together, provide a selective of suspended solids in mixed liquor rose to approximately advantage to a particular filamentous organism. It is there- 8,000 mg/liter. A control reactor was operated under the fore essential to know specifically which organism becomes same conditions but without increasing the organic loading. predominant under a given set of conditions. Isolation of the filamentous organism. A 10-ml sample of Most filamentous bacteria isolated from bulking sludge coke plant mixed liquor from the reactor and 90 ml of 0.2 M have been described as gram-negative, sheathed rods (6-8, K2HPO4 were blended for 3 min in a Waring blender, serially 29). Although actinomycetes have been identified in acti- diluted, and spread onto van Veen agar (6). After 7 to 10 vated sludge, they are not thought to cause bulking, but have days of incubation at 35°C, isolated colonies of the filamen- been associated primarily with foaming problems in munic- tous organism were restreaked until pure cultures were ipal treatment plants (6, 20, 27, 35). In this study, a gram- obtained. The bacterium was deposited in the collection of positive filamentous bacterium proliferated until sludge bulk- the U.S. Department of Agriculture Northern Regional ing occurred in a bench-scale reactor treating coke plant Research Center, Peoria, Ill., as NRRL B 16216. wastewater of gradually increasing strength. The organism Other microorganisms. Nocardia orientalis ATCC 19795 was isolated, but taxonomic studies show that it cannot be (subsequently renamed Amycolatopsis orientalis) and readily accommodated within any of the presently recog- Nocardia amarae ATCC 27808 were obtained from the nized taxa of actinomycetes. American Type Culture Collection, Rockville, Md. Phase-contrast microscopy. Wet mounts of the filamentous MATERIALS AND METHODS organism were observed by using a Nikon model 87697 phase-contrast microscope. Operation of bench-scale reactors. Bench-scale reactors Fluorescence microscopy. Samples of either pure cultures (chemostats) used in these experiments have been described of the organism or mixed liquor from the reactor aeration basin were mixed 1:1 with 0.05% acridine orange, incubated * Corresponding author. at room temperature for 5 min, and viewed with a Leitz- 1324 VOL. 52, 1986 NOVEL ACTINOMYCETE FROM BULKING SLUDGE 1325

Ortholux-2 fluorescence microscope (Leitz/Opto-Metric none peaks was confirmed by collecting the peak fractions Div. of E. Leitz Inc., Rockleigh, N.J.) equipped with a and determining the mass ions by chemionization mass 490-nm excitation filter and 350-nm barrier filter. spectroscopy. Electron microscopy. Pure culture specimens were fixed in Isolation and purification of DNA and determination of glutaraldehyde and veronal acetate buffer and embedded in G+C content. DNA was extracted from the filamentous 2% molten agar noble (Difco Laboratories, Detroit, Mich.); isolate NRRL B 16216 and the type strain A. orientalis ISP after dehydration in acetone and staining with osmium 5040 by phenol extraction by the method of Doi (5) after tetroxide, macerated sections of the specimens were embed- initial cell breakage by sonication or passage through a ded in Epon Polybed 812 (PolyScience Inc., Warrington, French pressure cell. Calf thymus DNA (G+C content, 40%) Pa.); thin sections on copper grids were poststained with obtained from Sigma Chemical Co., St. Louis, Mo., was lead citrate and uranyl acetate and examined with a Zeiss used as the standard. Melting-point determinations were EM 10 A microscope. performed by the methods of Mandel and Marmur (22). Characterization. The isolate was characterized by diag- Determination of base composition by depurination was nostic substrate utilization by the methods of Gordon et al. performed by the method of Huang and Rosenberg (12). (10), Lechevalier and Lechevalier (20), and Goodfellow and Isolation and purification of DNA and DNA homology. Minnikin (9). In addition, to determine whether antimicro- Freeze-dried biomass was thoroughly ground in a mortar and bial agents were produced by the organism, Escherichia coli, pestle, suspended in lx SSC (lx SSC is 0.15 M NaCI plus Staphylococcus aureus, Streptococcus faecalis, Bacillus 0.015 M sodium citrate)-0.5% sodium lauryl sulfate-100 jig megaterium, Pseudomonas aeruginosa, and Serratia mar- of pronase per ml, and placed on a rotary shaker at 100 rpm cescens were streaked in close proximity to a lawn of the in an incubator at 25°C for 24 h to solubilize the DNA. The isolate. DNA was then isolated and purified by the method of Lipid analysis. The cells were grown on Trypticase soy Marmur (24) and further purified by hydroxylapatite chro- agar (BBL Microbiology Systems, Cockeysville, Md.) until matography. The extent of DNA homology between strain confluent colonies appeared in the streak region. One heavy NRRL B 16216 and the type strain A. orientalis NRRL 2450 loopful of bacteria was used for the analysis of whole-cell was determined spectrophotometrically from Coto.5 values fatty acid extracts by gas chromatography in the HP 5898A by using the method of Seidler and Mandel (32) and Seidler Microbial Identification System (Hewlett-Packard Co., Palo et al. (31) as reported by Kurtzman et al. (14). Alto, Calif.). Growth studies. Growth of the isolate on phenol as the sole Phosphatidylethanolamine and phosphatidylcholine were source of carbon and energy and under different conditions determined by the method of Lechevalier et al. (18). of pH and temperature was assessed by the determination of Mycolic acids. Mycolic acids were extracted by the cell dry weight. method of Minnikin et al. (25) and Lechevalier et al. (19) To assess growth under anaerobic conditions, cultures from cultures of N. amarae, Streptomyces fradiae, and the freshly inoculated on van Veen agar or half-strength nutrient filamentous isolate. agar were incubated at 35°C in anaerobic jars (GasPak; Analysis of cell wall components. Cells were grown in BBL). To determine whether cells had been killed by half-strength nutrient broth, shaken at 30°C for 1 week, and anaerobiosis, the jars were opened sequentially at 24-h harvested by centrifugation at 10,000 x g for 10 min. The intervals and the plates were then incubated aerobically. cells were washed twice with deionized water, suspended in 50 ml of 1% sodium dodecyl sulfate in 1 mM EDTA, and kept RESULTS in a water bath (100°C) for 30 min. The suspension was then Description of the filamentous organism in mixed liquor. sonified with a Branson Cell Disruptor 350 (Branson Sonic When the bench-scale reactors were first inoculated with Power Co., Danbury, Conn.). for 4 min at 50% cycle biomass from an actual coke waste biological-treatment (continuous operation; output, 4), dialyzed against two plant, filaments were barely detectable. A gradual increase changes of distilled water at 40°C for 12 h, and centrifuged at was observed both in the reactors receiving increased levels 15,000 x g for 30 min. of feed and in the control reactor receiving the constant level For the determination of diaminopimelic acid (DAP), 5-mg of feed normally maintained in the treatment plant. How- samples were hydrolyzed in 1 ml of 6 N HCl in sealed tubes ever, the filaments appeared earlier and became more abun- at 100°C and then lyophilized. The method of Becker et al. dant under conditions of increased organic loading than in (2) was followed for DAP analysis by descending paper the control reactor (13). They first appeared extending from chromatography. In addition to standards of purified meso- the edges of floc particles (Fig. 1) and eventually formed a and L-DAP, E. coli cell hydrolysate was used as a reference sparsely branching mesh that often bridged adjacent floc for meso-DAP. particles. Throughout the period of increase in organic For the analysis of sugar components, samples were loading, the filaments formed few inclusions and thickenings hydrolyzed by the method of Becker et al. (3) and analyzed in the mixed liquor. Individual filaments measured about 1.0 by the methods of Staneck and Roberts (34). to 1.5 ,um in width and eventually reached a length of 250 to Isolation and purification of menaquinones. Menaquinones 500 ,um. were extracted and purified by the method of Collins et al. Isolation. Isolation of the most abundant filamentous or- (4). The menaquinone extracts were analyzed by high- ganism was achieved on a medium in which a comparatively performance liquid chromatography by the method of low concentration of nutrients limited the size of competing Tamaoka et al. (36) by using a Zorbax ODS reverse-phase bacterial colonies. The organism appeared as grey-white column (4.5 by 250 mm; E. I. du Pont de Nemours & Co., pinpoint colonies on van Veen medium after 4 to 5 days of Wilmington, Del.) with an isopropyl ether:methanol (1:3) incubation at 35°C. The colonies were partially embedded in solvent system at a flow rate of 1 ml/min. Menaquinones the agar and difficult to dislodge and became increasingly dry were detected by using a model SP 8440 variable wavelength and leathery during growth. UV/VIS monitor (Spectra Physics, Autolab Division, San Morphology in liquid media. In shaken cultures of van Jose, Calif.) at 270 nm. The identity of the major menaqui- Veen medium, the organism grew in discrete aggregates 2 to 1326 WHITE ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 1. Initial appearance of filaments in the mixed liquor. Bar, 30 ,um. 3 mm in diameter which settled quickly, leaving a clear In half-strength nutrient broth, the organism formed larger supernatant solution. Viewed microscopically, a particle aggregates (4 to 8 ,um) with short filaments (typically less consisted of a tighly packed mass of filaments surrounded by than 25 pm long) extending from the edges. After 3 to 5 days, a halo of relatively short (less than 50 ,Lm), sparsely branch- refractile and nonrefractile inclusions and thickenings ap- ing filaments (Fig. 2). After 48 h, a ring of organisms adhered peared terminally and subterminally (Fig. 3). to the glass at the air-liquid interface. Inclusion granules The isolate grown in nutrient-poor medium and the bulk- were not detected in fresh cultures but appeared in cultures ing organism in the mixed liquor appeared identical in older than 10 days. morphology, cellular inclusions, and filament width. Al-

FIG. 2. Microcolony of isolate B 16216 growing in a nutrient-poor liquid medium. Bar, 10 ,.m. VOL. 52, 1986 NOVEL ACTINOMYCETE FROM BULKING SLUDGE 1327

FIG. 3. Sporelike swellings and inclusions in mature cultures of B 16216 in nutrient-rich liquid medium. Bar, 10 ,um though inclusions and thickenings were rare in the filaments hyphae. The spores were of irregular size and shape and in mixed liquor, they appeared identical to those in pure varied in number from 3 to about 50. Chains of spores were culture. However, in the mixed liquor, filament length was also observed on the vegetative hyphae. Older spores could often more than 500 p.m, whereas in pure culture, it rarely swell up to 5 ,um in diameter. exceeded 100 ,um. Electron microscopy. Transmission electron micrographs Morphology on solid media. Growing on agar plates, the showed the isolate to be a true-branching, procaryotic fila- isolate produced chains of smooth spores on the aerial mentous organism (Fig. 4). Each filament was composed of

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FIG. 4. Electron micrograph showing true branching and cell structure. Bar, 10 p.m. 1328 WHITE ET AL. APPL. ENVIRON. MICROBIOL. rod-shaped cells separated by a partially completed cell wall. TABLE 1. Comparison of NRRL B 16216 and A. orientalis The outer surface of the cell wall consisted of a light- and a ISP5040 dark-staining layer, similar in appearance to the cell wall of Characteristic B 16216 A. orientalis Nocardia spp. (1, 20). The outer dark layer, which formed a continuous cover on the surface of the filament, was not a Decomposition part of the septum. Adenine Casein + Fluorescence microscopy. During early exponential Esculin most of the filaments a fluores- + + growth, yielded greenish Gelatin + cence, whereas filaments in the stationary phase contained Hippurate + + areas of green, yellow, orange, and red. In older cultures, Starch + + thickened cells appeared green fluorescing in a mass of Tyrosine + + red-fluorescing cells, indicating a possible difference in via- Xanthine + + bility or metabolic activity (11). Cell wall analysis. Cell wall analysis was performed to Nitrate reduction + + determine the presence of either L-DAP or meso-DAP, which, in combination with sugar patterns, determines the Growth 10°C + identification of actinomycetes (3, 16). The data showed the 450C presence of meso-DAP, arabinose, and galactose, indicating Lysozyme a type IV cell wall and a type A sugar pattern. Lipid analysis. Phospholipid analysis was performed to Acid production detect the compounds of diagnostic value in the identifica- Adonitol tion of actinomycetes (18). Phosphatidylethanolamine was Arabinose + detected in extracts from the isolate, but phosphatidyl- Cellobiose + Dulcitol choline and glucosamine-containing phospholipids were not. + acid revealed a of Galactose + Fatty analysis spectrum typical Glucose + The 14 to 20 + actinomycetes. fatty acids, containing carbons, Glycerol + + were of the iso and anteiso types; tuberculostearic acid was Inositol + present in small amounts. No mycolic acids were detected. Lactose + Analysis of menaquinones by high-performance liquid Maltose + + chromatography revealed the presence of only one major Mannitol + peak which was determined to be MK-8 (H4) based on its Mannose + retention time and the mass of its molecular ion (z, 720). A Melezitose + second, much smaller peak was determined to be MK-8 (H2) Melibiose + a-Methyl-D-glucoside + (z, 718). Raffinose Biochemical characterization. The was + organism gram pos- Rhamnose + itive, variably acid fast, strongly catalase positive, and Sorbitol weakly oxidase positive. Trehalose + Tests of biochemical and physiological traits by the Xylose + schemes of Goodfellow and Minnikin (9) and Lechevalier et al. (16, 19, 20) showed that the isolate resembled A. Utilization orientalis ISP5040 (Table 1). However, the G+C content of Acetate + + the isolate was determined to be 73% (versus 67% for A. Benzoate + orientalis), and DNA homology studies indicated only 11.5% Citrate + + between A. orientalis and B 16216. Lactate + + homology Malate + + Growth studies. The organism was able to grow on all of Oxalate the common amino acids except cysteine and glutamic acid Propionate + + in liquid culture. Growth in full-strength nutrient broth was Pyruvate + + slow compared with growth in half-strength nutrient broth. Succinate + + Phenol, the predominant carbon source in coke plant wastewater, supported growth at a concentration of 0.025%, whereas an increase to 0.05% caused the severe reduction of anaerobically. Although the cultures remained viable even growth, and an increase to 0.10% caused complete cessation after an exposure of several days to anaerobic conditions, no of growth. After an initial lag phase of 8 to 10 days, growth growth occurred anaerobically. The longer anaerobic condi- proceeded at the normal rate in half-strength nutrient broth tions were maintained, the longer it took for growth to occur amended with 20 pLg of cyanide per ml. A. orientalis did not after reexposure of the cultures to oxygen. survive incubation in similar concentrations of phenol or Antibiotic activity. No antibiotic activity was detected cyanide. against the organisms tested. Effects of temperature. No growth occurred in van Veen medium or in half-strength nutrient broth at temperatures DISCUSSION below 10°C. Growth was markedly reduced above 40°C. To determine a possible resistance of the sporelike inclusions to We previously reported the occurrence of an unidentified high temperatures, cultures were immersed in a water bath filamentous bacterium (now designated NRRL B 16216) in at 80°C for 10 to 60 min. The cells were no longer viable after bench-scale reactors used to study the effects of increased 10 min. organic loading of coke plant wastewater (13). The study had Effects of anaerobic conditions. Plate cultures on van Veen to be terminated when sludge bulking became an intractable medium and half-strength nutrient agar were incubated problem. VOL. 52, 1986 NOVEL ACTINOMYCETE FROM BULKING SLUDGE 1329

The organism was difficult to isolate because of slow Amycolatopsis species (67% for A. orientalis) and 73% for B growth and small colony size. Even though it appeared to be 16216. An examination of the DNA homology of A. the predominant organism in microscopic fields of the mixed orientalis and B 16216 showed it to be only 11.5%, indicating liquor, it was easily overgrown by other organisms on agar that there is little or no relationship between the strains. The plates. Attempts to isolate the organism by the use of various only other actinomycetes commonly found in sewage are enrichment media yielded two additional filamentous bacte- rhodococci, mycobacteria, and strains of N. amarae (20). As ria as well as several species of fungi. None of these were previously pointed out, members of these taxa contain judged to be significant contributors to the bulking problem mycolates, whereas B 16216 does not. Likewise, the isolate because they formed few colonies on plates and were rarely differs from typical members of the other actinomycete seen microscopically in mixed-liquor samples. genera having the same cell wall composition; B 16216 A comparison of the morphologies of the pure cultures differs from Saccharomonospora species because in B 16216 obtained showed a clear distinction between the single the spores are in chains, not borne singly on the aerial species identified as the most abundant and the other fila- mycelium, and it differs from Pseudonocardia, Faenia mentous species also isolated. The bulking organism had a (Micropolyspora), Saccharopolyspora, Amycolata, and characteristic morphology in coke plant activated sludge, Actinopolyspora species because it has phospholipid type P i.e., long, sparsely branched filaments. Inclusions and thick- II containing phosphatidylethanolamine as the major nitrog- enings, though rare, were present and were useful for enous phospholipid, rather than type P III, which contains identification. When the organism was cultured on labora- phosphatidylcholine. Thus, we conclude that our isolate is a tory media under low-nutrient conditions, the long, sparsely novel actinomycete not presently assignable to a recogniz- branching filaments appeared identical to those in the mixed able actinomycete taxon. liquor. Filament morphology varied, however, depending on Earlier observations made during the operation of the the nature and amount of nutrients supplied in the growth bench-scale reactors (13) were consistent with the results medium. For identification purposes it is therefore important obtained in the characterization of the isolate. Growth of B to culture an isolate under different conditions. The same 16216 in pure culture was greatly reduced at 40°C. When the organism may display different morphological characteris- temperature of the reactor was raised to 42°C, the numbers tics in a mixed liquor fed wastewater of a different compo- of filaments decreased and the settleability of the sludge sition and hence might be considered a novel strain if improved. When the temperature was returned to 35°C after microscopic observation were used as the major criterion for 35 days, filaments again became predominant and settle- identification. ability was lost. Likewise, a reduction in the concentration Evidence that the isolate was the predominant filamentous of dissolved oxygen, which has been suggested as a means to organism in the mixed liquor rests on the similarity of control sludge bulking (26, 31, 33), would be ineffective microscopic appearance, colony frequency on dilution against B 16216 since the organism is able to survive plates, and the ability of the isolate to utilize phenol and prolonged periods of anaerobiosis. tolerate high concentrations of cyanide. Biological treatment of industrial wastewaters favors the Cell wall analysis showed isolate B 16216 to possess type selection of specialized microbial communities (30). Gaudy IV cell wall composition and a type A whole-cell sugar and colleagues (23, 30) have demonstrated that their model pattern, a combination shared by members of the actinomy- for the growth kinetics of the sludge biomass cannot be used cete genera Mycobacterium, Rhodococcus, Nocardia, to predict events during transient states. Chemostat studies Actinopolyspora, Amycolata, Amycolatopsis, Faenia with phenol-containing synthetic wastewater indicated that (Micropolyspora), Saccharomonospora, Pseudonocardia, sudden increases in concentration of the feed result in a and Saccharopolyspora (8a, 16, 21). lower than predicted specific growth rate for the total Lipid analysis indicated the presence of iso and anteiso biomass and an increase in levels of filamentous organisms. fatty acids and tuberculostearic acid, a pattern which is They speculate that the initial response of most organisms to typical of all actinomycete genera having this cell wall type nutrient upshift is the oxidative assimilation of the substrate except Mycobacterium, Rhodococcus, and Nocardia (8a, for conversion into storage products. The subsequent break- 17, 21). The finding that B 16216 lacks mycolates further down of these products may then alter the substrate compo- underscores its differences from the members of these three sition in the reactor and may favor the selective growth of genera, which all contain mycolates. filamentous organisms that are either stimulated by or resist- Phospholipid composition has proved useful in character- ant to these compounds. izing actinomycetes (16, 18). The presence of phos- Once established in the sludge, filamentous morphology phatidylethanolamine, acylated with both hydroxy and nor- favors the survival of a microorganism in this environment. mal fatty acids, and the absence of phosphatidylcholine and Filaments extending from the periphery of floc particles are the glucosamine-containing phospholipids show our isolate surrounded by nutrients in solution in much the same way as to belong to phospholipid type P II. free-floating bacteria. However, whereas unattached bacte- The recently described genus Amycolatopsis (21) was ria are subject to washout from the aeration basin, filaments created to accommodate a number of strains which have a anchored to floc particles are not. The inability of protozoa type IV cell wall composition but lack mycolates and have a and other eucaryotic predators to ingest intact filaments type P II phospholipid cotnposition. However, despite the further favors their selective survival. Even dead filaments similarity in the chemistry of the lipids of the isolate to that are inimical to the treatment process since they remain intact of members of Amycolatopsis and the great resemblance in for many days and appear to cause the same bulking prob- the physiological tests (Table 1) of B 16216 to A. orientalis lems as live filaments. (the type species of the genus), additional studies showed An observation made during this study suggests an addi- that the isolate is probably not related to this group. tional explanation for the proliferation of filaments. In- The principal menaquinones present in Amycolatopsis creases in the levels of B 16216 paralleled dramatic increases species are MK-9 (H2, H4); in our isolate they are MK-8 in the population of a bdelloid rotifer of the genus Philodina. (H2, H4). The G+C percentages are 66 to 69% for These rotifers feed on free bacteria but are unable to ingest 1330 WHITE ET AL. APPL. ENVIRON. MICROBIOL. filaments, thereby providing a selective advantage to fila- growth of floc-forming bacteria: a model for activated sludge mentous organisms. Therefore, this case of bulking may bulking. J. Water Pollut. Control Fed. 56:52-61. to have been caused indirectly by a factor that stimulates 16. Lechevalier, H., and M. P. Lechevalier. 1981. Introduction the order actinomycetales, p. 1915-2027. In M. P. Starr (ed.), The rotifer growth. prokaryotes. Springer-Verlag, New York. 17. Lechevalier, M. P. 1977. Lipids in bacterial taxonomy. Crit. ACKNOWLEDGMENTS Rev. Microbiol. 5:109-211. 18. Lechevalier, M. P., C. de Bievre, and H. A. Lechevalier. 1977. We thank Myron Sasser, University of Delaware, for the gas Chemotaxonomy of aerobic actinomycetes: phospholipid com- chromatography analysis of the fatty acids, Ronald Plattner for the position. Biochem. Syst. Ecol. 5:249-260. mass spectra of the menaquinones, Edward Hoekstra for assisting in 19. Lechevalier, M. P., H. Lechevalier, and A. C. Horan. 1973. preparation of the DNA, A. J. Lyons for running the DNA hybrid- Chemical characteristics and classification of nocardiae. Can. J. ization study, and Magda Gagliardi for cell chemical analyses. Microbiol. 19:965-972. 20. Lechevalier, M. P., and H. A. Lechevalier. 1974. 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