Novel Actinomycete Isolated from Bulking Industrial Sludge JOHANNA M
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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.