A novel method of enumerating Nocardia amarae in foaming activated sludge

JOURNAL OF FERMENTATIONAND BIOENGINEERING Vol. 77, NO. 6, 674-678. 1994

A Novel Method of Enumerating N o c a r d i a a m a r a e in Foaming Activated Sludge MASANORI FUJITA,* KEISUKE IWAHORI, AND H I R O N O R I TAKI Department of Environmental Engineering, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565, Japan

Received 25 January 1994/Accepted 7 March 1994 Various experimental investigations were carried out in order to develop a novel method of enumerating N o c a r d i a amarae in foaming activated sludge using n-octadecane as a carbon source (OD medium). Colonies of N. amarae isolate appeared about 3 d earlier on OD medium than on MS medium. N. amarae counts on OD medium with 20 m g / l of nalidixic acid, using both synthetic and practical activated sludges, were always

higher than those on MS medium. It is concluded that OD medium with nalidixic acid was more effective for the count of N. amarae in activated sludge than MS medium.

Cultivation media MS medium (peptone, 5.0g; sodium propionate, 4.5 g; yeast extract, 2.5 g; glucose, 1.0 g; agar, 17.0 g; deionized water, 1/) with the pH adjusted to 7.5 was used for subcultivation, precultivation and viable cell count of N. amarae. OD medium using n-octadecane as a carbon source was developed to determine the viable cell count of N. amarae; n-octadecane, 5.93 g; yeast extract, 1.0 g; NaC1, 0.303g; KCI, 0.14g; CaC12, 0.18g; NH4CI, 0.35g; MgSO4, 0.2 g; Tween 80, 0.593 g; agar, 17.0 g, deionized water, 1 l. The pH of the OD medium was adjusted to 7.5. CGY medium was used for the total heterotroph count. The components of the medium were casitone, 5.0g; glycerol, 1.0g; yeast extract, 5.0g and deionized water, 1 1. The pH was adjusted to 7.2. Preparation of OD medium 5.93 g of n-octadecane and 0.593 g of Tween 80 were mixed at 50°C and emulsified with ultrasonic treatment for 2 min. The emulsified n-octadecane solution was then mixed with yeast extract and minerals (except CaCI2). After agar was added to this solution, the pH was adjusted to 7.5. This solution was autoclaved for 20rain at 120°C and then sterile CaCI2 was added. This solution was poured into petri dishes. Droplets of n-octadecane were blotted on the surface of the agar plate. Sterile polytton filters (PF060; Advantec Toyo Co.,Tokyo) made of pure tetrafluoroethylene resin were immediately placed on the agar surface before solidification of the agar and then removed after the agar had completely solidified. This filter could be reused by washing with ethyl alcohol followed by UV radiation for about 1 h. Viable call count methods Viable cell count was measured using both OD and MS media. Each sample was dispersed by ultrasonication for 1 min. This dispersed solution was then diluted in turn by 5 mg/l of sterile sodium tripolyphosphate solution. Three petri dishes at each dilution stage were prepared and 0.1 ml of the diluted sample was spread on the surface of the agar plates. The plates were incubated at 30°C and colonies appearing were counted with the naked eye or a focusing microscope ( < 10 ×).

Recently, many problems associated with abnormal foaming or scum in the activated sludge process have been reported by researchers from several countries, including the United States (1), Japan (2), England (3), South Africa (4) and Australia (5). It has been stated by many (6-9) that these problems are due to actinomycetes (Nocardia amarae and Rhodococcus rhodochrous etc.) which possess strong hydrophobic properties. Moreover, it was reported by Dhaliwal (10) that the occurrence of foaming depends only upon the existing amount of actinomycetes biomass. Therefore, a method for determing actinomycetes in the activated sludge process is important to prevent these problems. At present, the viable cell count method using MS medium (11) or starch-casein agar medium (12) is the most widely adopted biomass determination method for actinomycetes. Nevertheless, various problems arise when using MS medium, while others have been described during practical application, such as the cultivation period is long (10 d or more) and it is very difficult to distinguish and identify the actinomycetes because they are hidden by other bacterial colonies growing on the agar plates. Therefore, an urgent matter in biomass determination is the development of a simple and quick viable cell count method. It was reported by Fujita et al. (13) that N. amarae had the peculiar characteristics of utilizing hydrocarbons, particularly n-octadecane, as substrates. Since few microorganisms which can utilize n-octadecane are present in the activated sludge process, it is thought that the above-mentioned problems for viable cell count can be solved using n-octadecane as a carbon source. In this paper, a novel method for enumerating N, amarae has been developed using n-octadecane as a carbon source in the viable cell count medium. With this method, N. amarae in foaming activated sludge obtained from a sewage treatment plant can be quantified. MATERIALS AND M E T H O D S Microorganism N. amarae (9) isolated from foaming activated sludge was used in this study.

* Corresponding author. 674

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RESULTS AND DISCUSSION

Viable cell count using N . a m a r a e isolates Timeserial variations of N . a m a r a e colonies using both OD and MS media are shown in Fig. 1. For OD medium, N. a m a r a e colonies were macroscopically recognized after 3 d of incubation. The number of colonies became constant after 5 d. Colonies were about 1 mm in diameter on average and had a uniform size (Fig. 2). On the other hand, the colonies for MS medium appeared after 6 d and their number became constant after 8 d. Colonies on MS medium had very peculiar shapes and the irregular diameters ranging from 1 to 3 mm (Fig. 3). The sizes of the colonies increased after 8 d, while those for OD medium did not. The viable cell counts on the plateau were 1.77 × 107 CFU/ml and 1.35 x 106 CFU/ml for OD and MS media, respectively. Colonies appeared about 3 d earlier on OD medium than on MS medium, and the viable cell count on OD medium was about one order greater than that for MS medium. The earlier appearance of colonies on OD medium than on MS medium may be due to the higher specific growth rate of N . a m a r a e during the logarithmic phase since its specific growth rate has been found to be about 3 times higher than that of MS medium for n-octadecane (13). The approximate one order of magnitude more viable cell count on OD medium than on MS medium may be attributable to the fact that less than one to two orders of counts may be obtained using the plating method than the direct counting method with a microscope when counting heterotroph viable cells. It has also been suggested that many strains uncountable by visual or focusing microscope observations exist on the medium. Therefore, the early-growing colonies may have covered up nearby N . a m a r a e colonies. Small and uniform colonies on OD medium formed rapidly. This permitted the measurement of colonies uncountable on MS medium to be measured on OD medium. Therefore, it is concluded that the viable cell count on OD medium using N. a m a r a e broth is more effective than on MS medium. Additional effects of antibiotics It has been thought that n-octadecane is a substrate that is not readily used by activated sludge microorganisms. However, a large number of small colonies appeared on the OD medium and covered the N . a m a r a e colonies making it

FIG. 2. Colonies after 13 d on OD medium using N. amarae isolates. difficult to count them. Yeast extract could not be omitted from the OD medium because of its colony formation factor. The inhibitory effects of antibiotics (nalidixic acid and polymixin B sulfate) on Gram-negative bacteria were investigated becuase most activated sludge microorganisms are Gram-negative. The viable cell count values for N. a m a r a e and activated sludge bacteria with various concentrations of nalidixic acid are shown in Fig. 4. Nalidixic acid had no inhibitory effects on N . a m a r a e and the sizes of the colonies were almost unchanged compared with the control up to the added concentration of 40 m g / l . Meanwhile, the relative ratio of the N . a m a r a e colonies decreased rapidly and colonies became considerably smaller when the concentration of nalidixic acid exceeded 40 m g / l . For activated sludge, the ratio decreased rapidly to 72.2% and 49.8% at concentrations of 10mg/l and 20mg/l, respectively, but was about 50% when concentrations were over 40 m g / l . These results are

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FIG. 3. Colonies after 13 d on MS medium using N. amarae isolates.

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FIG. 4. Effects of nalidixic acid addition to viable cell counts on OD medium. Symbols: 0, N. amarae; O, activated sludge. in agreement with a study on P s e u d o m o n a s sp. (14). N . amarae was not affected by the addition o f polymixin B sulfate concentrations up to 2 m g / l . However, the growth o f activated sludge was sometimes inhibited by about 20% and sometimes totally by the addition o f 2 m g / l (data not shown). This is a result o f the unstable nature o f polymixin B sulfate in alkali environments and its extremely slow dispersion into agar. Therefore, this antibiotic was considered to lack sufficient reproducibility and to be unsuitable for addition to n-octadecane. Hence, OD m e d i u m with 20 m g / l o f nalidixic acid was selected as the most be suitable for counting N . amarae in activated sludge.

Viable cell count of N . araarae in synthetic activated sludge Viable cell counts were carried out using OD medium with 20 m g / l o f nalidixic acid. Three samples were prepared as follows; N . amarae broth (421 rag//), activated sludge solution (1,629 m g / / ) and a mixture o f equal amounts o f N . amarae and activated sludge with a suspended solids (SS) concentration o f 1,025 m g / l . Representative time-serial variations o f the viable cell count for the mixed sample are shown in Fig. 5. The

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FIG. 5. Time-serial variations of viable cell count of synthetic activated sludge. Symbols: o , N. amarae count on OD medium; O, total count on OD medium; zx, total count on CGY medium. total cell count on O D m e d i u m was 3.95 x 107 C F U / m l after 5 d, while that on CG Y medium was 6.63 x l0 s C F U / m l after 7 d. Cell counts identified as N . amarae on O D m e d i u m became constant at 1.30× 107 C F U / m l after 5 d. These results are summarized in Table 1. Viable cell numbers o f the mixture were also measured on O D m e d i u m without nalidixic acid and MS medium (Table 1). F o r O D medium, the count (total viable cell count) o f the N . a m a r a e broth was about twice that o f the mixture. Fo r CG Y medium, the total viable cell count o f the mixture was about half that o f activated sludge solution. Therefore, it was confirmed that highly reproducible measurements were obtained. N . a m a r a e counts on O D media with and without nalidixic acid were the same. Moreover, approximately 60% suppression o f activated sludge microorganism growth was obtained by adding nalidixic acid, added for the easy discernment o f N.

TABLE 1. Viable cell counts of N. amarae broth, activated sludge and mixture iV. amarae broth

Medium

N. amarae count

OD medium with nalidixic acid without nalidixic acid MS medium CGY medium

Activated sludge Total count (CFU/ml) (CFU/ml)

(CFU/ml)

Total count (CFU/ml)

N. arnarae count

2.97 x 107 (6 d) . . --

-. . . . 1.03 × 107 (7 d )

N.D.

6.33 × 107 (6 d)

--

1.35 x 109 (7 d)

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Mixture Total count (CFU/ml) (CFU/ml)

N. amarae count

1.25 x 107 (5 d) 3.95 x 107 (5 d) 1.30 x 107 (5 d) 1.01x 10s (5 d) 3.47 x 104 (7 d) 4.43 × 107 (7 d) -6.63 x 108 (7 d)

The numbers in parentheses are the cultivation periods (in days) needed to reach the steady state. N.D., Not detected. TABLE 2.

Suspended solids (SS) concentration, pH and N. amarae viable cell count of samples from a sewage treatment plant

Sample Raw sewage Primary effluent Activated sludge Scum Return sludge Secondary effluent

SS concentration (mg//)

pH

258 83 2206 95.4a 8480 6

7.55 7.64 6.76 6.52 6.52 6.63

OD medium with nalidixic acid Count b (CFU/ml) Ratioc (%) 5.00 × 103 (1.94 × 104) 4.00 x 103 (4.80 x 104) 9.00 x 105 (4.08 × 105) - - (8.67x 106) 7.67 × 104 (9.04 × 105) 1.33 x 103 (2.22 x 105)

a Water content (%). b Unit in parenthesis is CFU/mg SS. c Ratio of N. amarae count to total viable count by CGY medium in each sample.

0.014 0.033 0.097 3.042 0.219 0.014

MS medium Count b (CFU/ml) Ratio c (%) N.D. N.D. 6.67 × 104 (3.02 x 104) - - (8.60x 105) < 104 (--) N.D.

0 0 0.007 0.302 -0

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FIG. 8. Colonies after 7 d on OD medium using scum. FIG. 6. Colonies after 12d on OD medium with 20mg/l of nalidixic acid using mixture. a m a r a e colonies since the proportion of the N . a m a r a e

count to total cell count increased from 13% to 34%. The N . a m a r a e count on OD medium was about 4 times higher than that on MS medium, with the total count on the 2 media being almost identical. It is obvious from Figs. 6 and 7 that the number of colonies of heterotrophs on OD medium is less than on MS medium. Colonies identified as N . a m a r a e were transferred to MS medium by replica culture. All colonies appearing after 5 d' cultivation were identified as N . a m a r a e , like those of Fig. 3. Hence, the effectiveness of OD medium was confirmed using synthetic activated sludge. Viable cell count of N . a m a r a e in actual activated sludge Various samples for the viable cell count of N . a m a r a e were prepared as shown in Table 2. The presence of Nocardia type actinomycetes was observed in the scum using an optical microscope. As shown in Figs. 8 and 9, some colonies on OD medium were shaped like donuts with holes in the center, while their peripheries were split into 3 or 4 yellowish-cream colored segments. Thus, they were identified as N . a m a r a e since these characteristics were those of the type culture.

FIG. 7. Colonies after 12 d on MS medium using mixture.

The counting results are summarized in Table 2. N. a m a r a e counts on OD medium were about one order of

magnitude larger than those on MS medium, findings similar to that for synthetic activated sludge. In raw sewage, primary effluent and secondary effluent, N . a m a r a e was not detected on MS medium, while 103CFU/ml order of N . a m a r a e was detected on OD medium. It was made clear that the 103 CFU/ml order of N . a m a r a e on OD medium was entering into the aeration tank. This suggests that the number of N . a m a r a e entering the aeration tank has been underestimated in past studies using MS medium. It was also observed that N . a m a r a e in the scum is condensed about 20 times in the aeration tank and that approximately 3% of the total viable cells in the scum is accounted for by N . a m a r a e . Colonies of N . a m a r a e and other bacteria were readily distinguishable with the focusing microscope during the earlier portions of the cultivation period and then macroscopically in the later portions, because of the peculiar colony formation characteristics of N . a m a r a e . N . a m a r a e colonies can be easily identified once the observer becomes experienced. In conclusion, OD medium with nalidixic acid is more effective than MS medium for enumerating N . a m a r a e in an activated sludge process. Although the mechanism of scum generation by actinomycetes is not yet clear, their profiles and behaviours in the activated sludge process are considered to be the first steo needed to solve foam-

FIG. 9. Peculiar colony of iV. amarae on OD medium using scum.

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ing p r o b l e m s . T h e use o f O D m e d i u m , as p r o p o s e d in this study, m a y be effective for o v e r c o m i n g these problems. ACKNOWLEDGMENTS The authors would like to thank to Dr. Y. Sakai of the Japan Sewage Works Agency for providing the pure culture of N . a m a r a e and Mr. S. Kuribayashi of the Kawasaki Municipal Government for providing the activated sludge samples. REFERENCES 1. Pipes, W.O.: Actinomycete scum production in activated sludge processes. J. WPCF, 50, 628-634 (1978). 2. Hiraoka, H. and Tsumura, K.: Suppression of actinomycete scum production--A case study at Senboku Wastewater Treatment Plant. Wat. Sci. Technol., 16, 83-90 (1984). 3. Goddard, A.J. and Forster, C.F.: Stable foams in activated sludge plants. Enzyme Microbiol. Technol., 9, 164-168 (1987). 4. Pretoilus, W.A. and Laubscher, C.J.P.: Control of biology scum in activated sludge plants by means of selective floatation. Wat. Sci. Technol., 19, 1003-1011 (1987). 5. Linda, L. B., Harbers, A.E., Greenfield, P.F., and Hayward, A. C.: Foaming in activated sludge plants: a survey in Queensland, Australia and evaluation of some control strategies. Water Res., 25, 313-317 (1991). 6. Lechevalier, M.P. and Lechevalier, H . A . : N o c a r d i a a m a r a e sp. nov., an Actinomycete common in foaming activated

sludge. Int. J. Syst. Bacteriol., 24, 278-288 (1974). 7. Goodfellow, M., Minnikin, D.E., Tood, C., Alderson, G., MAnnikin, S.M., and Collins, M.D.: Numerical and chemical classification of N o c a r d i a a m a r a e . J. Gen. Microbiol., 128, 1283-1297 (1982). 8. Lemmer, H. and Kroppenstedt, R.M.: Chemotaxonomy and physiology of some Actinomycetes isolated from scumming activated sludge. Syst. Appl. Microbiol., $, 124--135 (1984). 9. Sakai, Y., Moil, T., lida, M., Honda, K., and Matsnmoto, T.: Scum formation by Actinomycetes ( N o c a r d i a sp.) in final clarifier of activated sludge process. J. Jpn. Sew. Works Assoc., 19(214), 56-65 (1982). (in Japanese) 10. Dhaliwal, B.S.: N o c a r d i a a m a r a e and activated sludge foaming. J. WPCF, 51, 344-350 (1979). 11. Moil, T., Sakal, Y., Honda, K., Yano, I., and Hashimoto, S.: Stable abnormal foam in activated sludge process produced by R h o d o c o c c u s sp. with strong hydrophobic property. Environ. Technol. Lett., 9, 1041-1048 (1988). 12. Safferman, R.S.: Standard methods for the examination of water and wastewater, 16th ed. American Public Health Assoc., 993 (1985). 13. Fujita, M., lwahoil, K., Tanigaki, F., lwasaki, D., and Hashimoto, S.: Studies on the utilization of various fatty acids and hydrocarbons of N o c a r d i a a m a r a e from foaming activated sludge. Proc. Environ. Sani. Eng. Res., 27, 75-85 (1991). (in Japanese) 14. Kogure, K., Shimizu, U., and Taga, N.: A tentative direct microscopic method for counting living marine bacteria. Can. J. Microbiol., 26, 318-323 (1979).