JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 69, No. 3, 143-147. 1990

Isolation of Sphaerotilus natans-Lysing Microorganism from Activated Sludge

RIKIYA TAKAHASHI,* AKIHIKO HATTORI, TOSHIKI INOUE, KOUICHI OGIWARA, MASAHARU SUZUKI, AND HITOSHI YONEYAMA

Department of Brewing and Fermentation, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156, Japan

Received 21 August 1989/Accepted 18 December 1989

Sphaerotilus natans is thought to be a causative agent for bulking of activated sludge. Four micro- organisms lysing S. natans were isolated from sewage activated sludge. One of lhe isolates, K-01, was in- vestigated further. K-01 formed plaques 3-5 d after plating together with S. natans on PYG agar plates. The plaques enlarged gradually into surrounding S. natans cells and finally covered the entire surface of the plates. The isolate could not be filtered through a membrane filter with a pore size of 0.2/tm. Electron microscopic observation revealed that K-01 was long-rod shaped, Gram negative microorganism. K-01 could not be cultured on PYG agar medium without S. natans, but it was able to form small colonies on diluted bouillon agar medium without the host cell. K-01 strongly suppressed the growth of S. natans in liquid culture.

The activated sludge process has been widely used for treatment of various polluted waters. If a wastewater con- tains a large amount of soluble carbonaceous pollutants, filamentous microorganisms proliferate abundantly within the sludge (1-4.) Consequently, the purifying capacity of wastewater in final settling tanks seriously deteriorated with the bulking sludge. Because of this phenomenon the use of the process has a limitation on the amount loaded. Therefore, a great deal of effort has been made to control the growth of the filamentous microorganisms in the sludge (5-7).

Addition of some disinfectants (8-10), coagulants, or metal ions (11) into the sludge have occasionally improved the sedimentation of the bulking sludges. Improvement of the property was also accomplished sometimes by the adjustment of dissolved oxygen or pH in aeration tanks (12, 13).

Biological interactions of the filamentous microbes with other microorganisms were also reported. Takiguchi et al. showed that Streptomyces organonensis produced an antibiotic against S. natans (14). Curds et al. (15) observed that a kind of protozoan, Trithigmostoma cucullulus, preyed on the filamentous bacterium. As to bacteria, Venosa noticed that freely living nonfilamentous cells of S. natans were lysed by a parasitic bacterium, Bdellovibrio bacteriovorus, but the filamentous cells were not (16).

This paper deals with the isolation of microorganisms which lyse filamentous cells of S. natans and the identifica- tion of several characteristics.

MATERIALS A N D METHODS

Source of isolation Activated sludges were collected from sewage treatment facilities of Kase, in Kanagawa prefecture, and used as the sources for isolation. They were cultivated for 3 d by supplying artificial sewage, com- posed of 80mg/l of glucose, 0.03%0 Polypepton (Daigo Nutritive Chemicals Ltd.), 0.02%0 meat extract (Kyokuto), 0.005%0 urea, and 0.005%0 Na2HPO4, in a treatment test

* Corresponding author.

apparatus. The sludges were then treated with a homoge- nizer (Ikemotorika, RKI-1076) at 1,300 rpm for 10 rain just before use.

Host bacterium The host filamentous bacterium, S. natans IFO 13543, was cultured at 25°C for 5 d in PYG liquid medium which consist of 0.05% glucose, 0.2%0 meat extract (Kyokuto), and 0.1% yeast extract (Daigo Nutritive Ltd.) with pH 7.2.

Susceptibility of several microorganisms to K-01 was studied by inoculating K-01 together with these host bacteria on the agar medium.

Isolation procedure of the S. natans-lysing micro- organisms A sample of the culture of S. natans was spread on PYG agar medium that was made by adding 2.0O/oo agar. A small amount of the homogenized sludge was sprayed over the same plates. They were incubated at 25°C for 3-5 d. Plaques appearing on the agar plates were picked up, resuspended in 5 ml of sterile water, and homogenized. Then these homogenates were plated out in the same way and incubated again. The procedure was repeated 3 to 5 times for the purification of the plaque formers. The purified isolates were maintained on an agar slant or in the liquid medium with the host bacterium.

Electron microscopy To prepare specimens of the isolate (K-01), cultured broth of the isolate with the cells of S. natans was treated with 0.5~0 formaldehyde, and negative staining was done by adding the same volume of 1.0% phosphotungstic acid. An electron microscope (JEOL, Ltd., SEM 100CX) was used for the observations.

Chloroform treatment of the isolate (K-01) Three drops of chloroform was added to 5 ml of isolate suspen- sion, and left for 10 rain. Chloroform was blown out by air. One ml of cultured broth of S. natans was mixed with 3 ml of the isolate suspension and agitated vigorously. The mixture was use for plaque counts.

Counting of the plaque former Ten ml of the isolate suspension was mixed with 1 ml of the precultured broth of S. natans. Three-tenths of a ml of the mixture was spread on the PYG agar plate. After 3-7 d of cultivation, the numbers of plaques were counted. The density of the plaque former in the suspension was expressed as plaque

143

144 TAKAHASHI ET AL. J. FEkMENI. BIOEN~.,

forming units (PFU/ml ) . Viable counts of S. na tans A mixed culture of S.

natans and K-01 was homogenized and diluted and, 0.3 ml was plated out on the PYG agar medium. The colonies ap- pearing were counted after 2 d of cult ivation and the viable counts were expressed as colony forming units (CFU/ml) .

Culture media The following seven culture media (A-G) were used for the growth test of K-01. They were A, PYG agar medium; B, diluted malt agar medium con- sisting of 0.2% malt extract (Difco) and 2% agar at pH 7.0; C, bouil lon agar composed of l ° t meat extract (Kyokuto), 1% Polypepton (Nihonseiyaku), 0.5% NaC1, and 204 agar at pH 7.0; D, diluted bouil lon agar medium composed of 0.2%0 meat extract (Kyokuto) , 0.20/00 Polypep- ton (Nihonseiyaku), 0.1°/oo NaC1, and 2% agar at pH 7.0; E, diluted Standard medium (JIS K-0102) (17); F, activated sludge extract agar medium (pH 7.0); and G, diluted ac- tivated sludge extract agar medium (pH 7.0). To prepare activated sludge extract, 1 / of sewage activated sludge, which contained 10 g of dry matter , was washed twice with distilled water by centr ifugat ion at 3,000 rpm for 10 rain. It was boiled for 2 h in 1 l of distilled water and centri- fuged at 3,000 rpm for 10 rain to remove cell debris. The supernatant was used for the culture media.

Escherichia coli was cultured aerobical ly in a liquid medium consisting of 1.0% Polypepton , 1.0% lactose, 0.5% NaC1, 0.2o/00 K2HPO~ and 0.2% ammonium ferric citrate at pH 7.4, or the agar medium supplemented with 2.0%o agar.

X a n t h o m o n a s oryzae was cultured in a liquid medium consisting of 1.0% Polypepton , 0.2%0 yeast extract, 0 .1% MgSO4.7H20, and plated out on the agar medium sup- plemented with 1.5% agar at pH 7.0. Yeasts were cultured in a liquid medium consisted of 1.0% malt extract (Difco), and solid medium with 2.00/00 agar at pH 7 .0±0 .1 .

Growth of K-01 on S. natans cells S. natans cells was precultured in PYG liquid medium for 5 d and washed with sterile water by centr ifugation. Precultured K-01 cells were also harvested by centr ifugat ion at 10,000rpm for 10 rain and suspended in sterile water. A por t ion of the suspension was spread on washed living cells or autoclaved cells of S. natans solidified by 2% agar at a density of 1.5 to 2 . 0 m g / m l . After 4-5 d of cult ivation, plaques were counted. In parallel with this, the p laque-forming abil i ty of the suspension was examined on PYG agar medium by mixed plating with S. natans.

Growth suppression test of S. na tans Eleven ml of mixed culture of K-01 and S. natans in PYG medium at 25°C for 6 d, was supplemented with 1 ml of S. natans, which was separately cultured in PYG medium at 25°C for 5 d. One ml of the freshly mixed culture was inoculated into 10 ml of PYG medium and cult ivation was started at 25°C. S. natans was also cultured without K-01 as a con- trol. The viable counts, both of S. natans and K-01, were done for 10-15 d.

RESULTS

Isolation of lytic microorganisms for S. na tans Homogenized sludges were inoculated on the PYG agar medium together with S. natans and incubated for 3-5 d. Several clear zones appeared on the lawn of S. natans. The lytic microorganisms were obtained from the plaques and they were classified into four types according to their grow- ing characteristics on PYG agar medium (data not shown). One of them, K-01, was studied further in this paper.

o b

c d FIG. 1. Photographs of plaques produced by K-01 on the lawn

of S. natans. (a) S. natans without K-01, after 6 d of cultivation; (b) 5 d; (c) 6 d; (d) 7 d of cultivation.

Characteristics of K-01 Small and circular plaques appeared after 3-5 d of cultivation on a plate seeded with S. natans. They enlarged gradually and overlapped after 6-8 d, finally covering the entire surface of the plates (Fig. 1 ).

To distinguish K-01 from bacteriophages, the suspen- sion of plaque former was put on a membrane filter (DISMIC, Toyoroshi Co. Ltd.) with a pore size of 0 .2pro, but it did not go through. Moreover, the growth of S. natans was not affected at all when the filtrate was added to the agar plates.

A small part of a plaque on the plate was resuspended in 5 ml of distilled water, and treated with chloroform as described in Materials and Methods. The plaque-forming abili ty of the suspension was measured. But no plaques were formed, suggesting that the plaque former is suscepti- ble to chloroform.

Morphology Electron microscopic observations showed that K-01 was a long-rod shaped bacterium-like microorganism (Fig. 2). Pleomorphic cells of K-01 was also observed (Fig. 2a, c). The cells often have relatively high density regions near both ends (Fig. 2c).

The sizes of K-01 and S. natans were estimated from the electron micrographs and are shown in Table I. The cells

TABLE 1. Comparisons of cell size

Width Length Microorganisms (~m) (,um)

K-01 0.25--0.4 3.5--13.8 S. natans 0.7 --0.8 2.7-- 4.0 B. bacteriovorus (20) 0.25--0.4 0.8-- 1.2

Voi.. 69, 1990

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a

Sh

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Sph

b

KOI'

S h

C

FIG. 2. Electron micrographs of K-01. K-01 was cultivated for 14 20 d in PYG liquid medium in the presence of S. natans as described in Materials and Methods. (a) x 2000; (b) x 10000; (c) x 10000. Sph: S. natans; K01: K-01; Sh: Sheath of S. natans.

o f K-01 was o f t e n longer t h a n those o f S. na tans , bu t the w i d t h was a b o u t the h a l f o f S. n a t a n s cell. So the cells o f K-01 were easily d i s t i n g u i s h a b l e f r o m the cells o f S. n a t a n s in the i r sizes.

Host specificity of K-01 K-01 was i n o c u l a t e d t o g e t h e r wi th E. col i I F O 3301, X . oryzae I F O 3995, a n d S a c c h a r o m y c e s cerevis iae I FO 2042, I F O 2000, or K7 o n the aga r m e d i a desc r ibed in M a t e r i a l s a n d M e t h o d s . K-01 was ab le to p r o d u c e p l aques on ly o n X . o r y z a e b u t no t o n E. col i a n d the yeast cells. T h e c u l t i v a t i o n o f K-01 in l iqu id

SPHAEROTILUS NA TANS-LYSING MICROORGANISM

TABLE 2. Growth test of K-01 and S. natans on various agar medium

145

Media K-01 S. natans

PYG agar No Yes Diluted malt extract agar a No Yes Bouillon agar Yes No Diluted bouillon agar a Yes Yes Diluted Standard agar b (JIS K-0102) No Yes Activated sludge extract agar No Yes Diluted activated sludge extract agar ~ No Yes

Yes, Colonies were formed; No, growth was not detected. Diluted to 1/5.

h Diluted to 1/100. Diluted to 1/2.

TABLE 3. Colony and plaque forming activities of K-01

Numbers a of Numbers b of colonies the plaques

After 14 d cultivation 65 61

~, b Number of colonies and the plaques were represented as average numbers on PYG plates.

m e d i u m wi th X . oryzae has no t succeeded yet. C u l t u r e m e d i u m fo r K-01 a n d S. n a t a n s A cu l tu re

m e d i u m t h a t a l lows K-01 to g row i n d e p e n d e n t l y in the absence o f S. na tans a n d X . oryzae was f o u n d . As s h o w n in T a b l e 2, b o u i l l o n aga r a n d d i lu ted b o u i l l o n aga r m e d i u m s u p p o r t e d the i n d e p e n d e n t g r o w t h . A b o u t 1 m m in d i a m e t e r , s m o o t h , t r a n s l u c e n t , pa le yel low co lon ies o f K - 01 were f o u n d o n the d i lu ted b o u i l l o n aga r p la te a f t e r 9 d o f cu l t i va t i on . S. n a t a n s also p r o d u c e d whi t e co lon ies o n the b o u i l l o n aga r p la tes . E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n o f the co lon i zed K-01 was also d o n e (Fig. 3). T h e cells s h o w e d s imi la r m o r p h o l o g y as was o b s e r v e d in Fig. 2, a n d p l a q u e f o r m a t i o n was o b s e r v e d aga in w h e n a p o r t i o n o f the smal l yel low c o l o n y was p la t ed ou t o n the P Y G agar m e d i u m t o g e t h e r w i th S. natans . W h e n the s ame a m o u n t o f a cell s u s p e n s i o n o f K-01 was p l a t ed ou t , b o t h o n the d i lu ted b o u i l l o n a n d S. na tans seeded P Y G aga r med ia , two p la tes gave the same n u m b e r s o f co lon ies a n d p l aques (Tab le 3). K-01 f o r m e d p l aques o n l iving cells o f S. na tans

FIG. 3. Electron Micrographs of K-01. K-01 was cultivated with the diluted bouillon agar medium without the host bacterium for 14 d. x 2,600. The cells were withdrawn into 0.5%0 formaldehyde solu- tion and was used as a specimen of the electron microscopic observa- tion.

146 TAKAHASHI ET AL.

TABLE 4. Comparison of the growth of K-01 on living and dead S. natans cells

Numbers of Cultivation Culture media plaques time

(102/ml) (d)

S. natans cell agar 71 10 Autoclaved S. natans cell agar 0 10 PYG agar 139 4

TABLE 5. Characteristics of K-01

Morphological characteristics Irregular long rod 0.25-0.35 by 3.5-13.8 ~m Nonmotile Gram negative Pleomorphism

Cultural characteristics Grown on bouillon and diluted bouillon agar aerobically Lyse S. natans and X. oryzae but E. coli and yeasts Suppress growth of S. natans in liquid medium

Feature of colony 1 to 3 mm in diameter with a halo ranged 7 to 10 mm in

diameter Pale yellow Translucent Circular Convex Smooth

solidified by 2% agar, but plaques were not observed on the dead cells (Table 4).

M i x e d cul ture o f K - 0 1 and S. natans in l iquid m e d i u m Effects of K-01 on the growth of S. natans in the PYG liquid medium were investigated. The viable counts of S. natans and K-01 were measured in the course of the cultiva- tion. The results are shown in Fig. 4. It was obvious that the growth of S. natans was greatly suppressed by the presence of K-01 and that the viable counts of K-01 in- creased rapidly and then decreased gradually.

Characteristics of K-01 were summarized in Table 5.

DISCUSSION

It was suggested that K-01 was a kind of ectoparasitic microorganism for S. natans, because K-01 produced plaques on the lawn of S. natans and on the washed cells, and production of growth-interfering substances against S. natans was not recognized in the culture broth. It was supported again by the finding that K-01 grew in PYG liquid in presence of S. natans but did not without the host bacterium.

Electron microscopic observation showed that K-01 is a 0.25-0.4 by 3.5-13.8 ~m long rod-shaped bacterium. K-01 is not an obligately parasitic organism because it was able to grow on bouil lon agar medium without the host cells.

The characteristics of K-01, that enlarging of the plaques stopped in a refrigerator around 8°C, and the plaques were colored slightly yellow after prolonged cultivation, were similar to that of Bdel lovibr io , a predator on E. coli (18, 19). Unexpectedly, K-01 was distinguished from B. bacter iovorus by the shape of electron micrograph and their size (20). Recently Hasuo et al. isolated a Gram- positive, yeast-lysing bacterium from activated sludge and named it Rarobac ter faec i tab idus (21), but it could not lyse S. natans (22). Therefore, K-01 was also evidently

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~ / 0 . . . . . . . . . ~ < , . . . . . . . . . . . ~=~

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Cultivation time (d) FIG. 4. Inhibition of the growth of S. natans by a parasite, K-01.

Viable counts of S. natans ( • ) and numbers of the parasite (A) were estimated in the course of cultivation. As a control, growth of S. natans without the parasite was also presented (O).

distinguishable from R. faec i tab idus by Gram-staining, cell size, and host specificity.

The presence of the plaque formers in the activated sludge was assumed to be very frequent, because they were obtained easily by isolation, but the ecological significance of their presence in activated sludge is not apparent.

We observed here that K-01 in the mixed culture with S. natans strongly suppressed the growth of S. natans. This predator and prey relationship probably works even in ac- tivated sludge and controls the numbers of S. natans. In other words, the numbers or predatory activity of K-01 in activated sludge is critical to prevent bulking of activated sludge caused by S. natans.

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