Growth rate of aspidiscidae isolated from activated sludge

Water Research Pergamon Press 1972. Vol. 6, pp. 137-144. Printed in Great Britain

GROWTH RATE OF ASPIDISCIDAE ISOLATED FROM ACTIVATED S L U D G E RYUICHI SUDO and SHUICHI AIBA Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan (Received 9 September 1971)

Abstract--Isolation, stock and in addition, monoxenic culture of specific species of Aspidiscidae, Aspidisca costata and Aspidisca lynceus were attempted successfully. Aspidiscidae are the typical protozoa found frequently in the activated sludge from municipal sewage treatment. Using a heterogeneous bacterial population isolated from the activated sludge as the protozoan food, the monoxenic growth rate (logarithmic growth phase) of Aspidiscidae and the effect of temperature on the growth rate were measured. The specific growth rate was 1'2-1"3 day -1 at 20°C, about one-half of that observed with Vorticellidae at the same temperature. Aspidiscidae examined were "stenophagic" and "horozoic". The optimum temperature for growth was about 30°C and the value of activation energy for growth was AE = 13,800 cal mole -1. INTRODUCTION IT IS WELL known that activated sludge in the biological treatment of waste waters is a typical ecosystem, composed of bacteria, fungi, protozoa and smaller metazoa. A sequence of reactions--soluble organic and inorganic substances in the raw waste are metabolized first by bacteria and/or fungi, secondly, these microbes are taken up by protozoa and finally, the protozoa serve as food of smaller metazoa--is expected in the biological oxidation process. Activated sludge functions as "food chain" in reducing BOD values of an original waste. Though the BOD reduction kinetics is a basis for designing and operating the biological treatment plant, some indices such as transparency, concentration of coliform bacteria with respect to effluent from the plant are important, besides the value o f BOD, especially when excellent quality of effluent is of primary interest. Contrary to the wealth of information concerning the kinetics, factors contributing to the quality of effluent have yet to be determined. PILLAI and SUBRAHMANYAN(1942, 1944) emphasized the significance of protozoa in the activated sludge. HARDIN (1943), WATSON (1945) and CURDS (1963) urged that some species of protozoa excrete specific materials to cause flocculation of bacteria and/or suspended solids in the waste water, suggesting the protozoan role to improve the effluent quality. CURDS and VANDYKE (1966) isolated some protozoa from the activated sludge to study the protozoan growth rate, not mentioning, however, of Aspidiscidae at all. CURDS et al. 0968) confirmed experimentally that the quality of effluent improved if the original sludge free from protozoa was impregnated artificially with specific protozoa belonging to Ciliata. They attributed this improvement to the predatory fimction of the protozoa and attempted a monoxenic culture of Tetrahymenapyriformis to reveal more quantitatively the prey-predator relationship between protozoa and bacteria (CURDS and COCKBURN, 1968). In addition, CURDS and FEY (1969) observed that E. coli was markedly removed f r o m effluent when protozoa were found in the activated sludge. Recently, SUDO and AIBA (1970) pointed out that the effluent quality WATER 6/2--A

137

138

RYU1CHI Stroo and SHUICHIAIBA

w a s e n h a n c e d w h e n t h e a c t i v a t e d s l u d g e w h e r e i n e i t h e r " f i x i n g " o r " c r a w l i n g " species o f p r o t o z o a d o m i n a t i n g was settled u n d e r g r a v i t y . TABLE 1 s u p p l e m e n t s t h e a b o v e a r g u m e n t . By a n d large, it is n o t e d f r o m the t a b l e t h a t t h e s l u d g e r i c h in e i t h e r V o r t i c e l l i d a e o r A s p i d i s c i d a e p r o d u c e d effluent in g o o d q u a l i t y . A s p i d i s c i d a e w e r e d o m i n a n t in t h e A c t i v a t e d S l u d g e P l a n t s in T o k y o a r e a f r o m early summer (June) to late autumn (October) when the water temperature exceeded 20°C. TABLE 1. MICROFAUNA OF ACTIVATED SLUDGE AS RELATED TO QUALITY OF EFFLUENT (EXAMPLES)

Effluent quality Water temp. (°C) (aeration unit) Transparency (cm) COD (mg l - l ) * BOD (mg 1-')

Plant site, Date Mikawashima, Sunamachi, Shibaura, July 25, 1969 Aug. 15, 1969 July 22, 1970 24-7

27.0

52 16 10

35 16 12

24.0 100 12 8

Microfauna in sludge and number concentration per ml of mixed liquor Vorticella convallaria -60 2120 Vorticella alba -40 -Vortieella sp. -40 -Vorticella mierostoma -140 -Epistylis sp. 360 140 400 Carchesium polypinum --280 A~pidisca costata -21,020 3180 Aspidisca spp. 2800 --Loxophyllum spp. --160 Litonotus sp. 500 120 180 Amoeba spp. 100 100 -Arcella vulgaris 1020 80 -Astasia sp. 20 -20 Bodo edax -160 1O0 Pleuromonas sp. 200 140 -Oikomonas terma 20 --Peranema sp. --120 Tokophrya spp. 100 20 -Podophrya sp. -20 -Philodina roseola --20 * K M n O , method. A s s u m i n g Aspidisca costata a n d Aspidisca lynceus as r e p r e s e n t a t i v e s o f A s p i d i s c i d a e in t h e a c t i v a t e d s l u d g e (HORASAWA, 1945 ; BROWN, 1965 ; SUDO a n d OHGOSHI, 1964), t h e i s o l a t i o n , s t o c k a n d m o n o x e n i c c u l t u r e o f the A s p i d i s c i d a e will b e d i s c u s s e d in this w o r k ; m o n o x e n i c c u l t u r e o f V o r t i c e l l i d a e has b e e n discussed e l s e w h e r e (SUDO a n d AIBA, 1971). MATERIALS

AND METHODS

The media used for isolating and stocking both Aspidiscidae and heterogeneous b a c t e r i a l p o p u l a t i o n as t h e p r o t o z o a n f o o d w e r e n e a r l y t h e s a m e as t h o s e in t h e p r e v i o u s s t u d y o n V o r t i c e l l i d a e (SUDO a n d AIBA, 1971).

Growth Rate of Aspidiscidae Isolated from Activated Sludge

139

Media LE-medium (LEVINE,1959; SUDO and AIBA, 1971) prepared from tender leaves o f lettuce and boiled egg yolk was used to isolate and/or stock the protozoa. The medium was sterilized at 121°C for 15 rain (pH = 6.8-7.0). SE-medium (PRAKASAMand DONDERO, 1967; SCDO and AIBA, 1971) obtained from the sludge extract (via boiling mixed liquor suspended solid taken from a sewage treatment plant at 121°C for 15 rain, followed by filtration of the mixed liquor) was used to stock heterogeneous bacterial population as protozoan food, while SE-agar medium (agar = 1 per cent) was for isolating the bacterial population from the sludge.

Isolation of heterogeneous bacterial population from the sludge and its stock procedure A loop from the activated sludge was streaked on the SE-agar surface or a dilute aqueous suspension of the original sludge (dilution ratio = 104-105) was added (0-2 ml) on to the agar. Incubation was at 20°C for 5 days. Colonies taken from the agar surface were inoculated into the SE-medium for stocking at 5°C. These colonies, if inoculated into the LE-medium, served as food for the protozoan isolation and/or its stock. With a specific sludge wherein Flagellata and Amoebina were dominant, dilution was advisable to reduce these protozoa in the bacterial isolation process. Transinoculation was made once every 2 weeks for the stock at 5°C. If the bacteria were to be used as food for Aspidiscidae in the growth rate measurement, a preculture from the stock in a fresh SE-medium at 20°C for 48 h was required. The preculture period was twice as long as that needed for Vorticellidae (SLrDOand AIBA, 1971). The longer period was to secure the bacterial floc; the floes were deemed necessary for Aspidiscidae due to their crawling habit.

Isolation of Aspidiscidae ; its purification, identification and stock preparation A small amount of activated sludge sampled from several plant sites in Tokyo was placed in a Petri dish (6 cm dia.); using a micro pipette (0.1-0.2 mm dia.) and needles under a stereomicroscope, Aspidiscidae could be transferred from the sludge to a LE-medium reservoir of a ring placed concentrically on another Petri dish (9 cm dia.). The ring (2 cm dia.) was fixed on the dish by a thin layer of the SE-agar medium. The reservoir circumscribed by the inner wall of the ring and the gelatinized SE-agar medium on the dish was provided with LE-medium to isolate the protozoa. The LEmedium (about 1 ml) was impregnated with the heterogeneous bacteria from the sludge. The Petri dish with the ring was incubated at 20°C for several days and from time to time the proliferation of Aspidiscidae was checked under the stereomicroscope and in particular, the presence and/or absence of foreign protozoa other than Aspidiscidae was examined for carefully under the microscope. The isolation procedure was repeated until the monoxenic culture of Aspidiscidae could be established. An effective isolation was difficult, if Flagellata, Amoebina and smaller metazoa were present. An aqueous solution of methyl cellulose was added to the monoxenic culture of Aspidiscidae to arrest motility. Another aqueous solution of methylene blue (100 mg in 1-1. of tap water) was used to stain the protozoa to facilitate the morphological identification under a microscope (Ktroo, 1954; JAPANESESEWAGEWORKSASSOCIATION, 1964).

140

Rvl.rtCHl SUDOand SHUICHIAIBA

Aspidiscidae isolated thus were stocked in test tubes, containing 5 ml of the LEmedium impregnated with the bacterial food, each at 5°C and 10°C. Transinoculation, once every month when stocked at 5°C and once every 2 weeks at 10°C was repeated. M e a s u r e m e n t o f growth rate

A clonal cultivation from the stock to obtain the test material was at 20°C for 48 h. The number of cells transferred from the clonal culture onto a slide glass was N = 2-4. The center of the slide glass was slightly concaved. Before the transfer the clonal culture was rinsed fully with sterile tap water. A definite amount of the bacterial suspension (0.1 ml) was also added into the concave. The slide glass was placed on small stays which were fixed inside a Petri dish (9 cm dia.). Each dish contained 10 ml of sterile water to minimize the evaporation of culture medium. The cultivation temperature extended from 2 ° to 37°C, but most of the experiments were at 20°C. The cell growth was observed from time to time by exposing the slide glass under the stereomicroscope. It was confirmed from the preliminary experiments that the growth was logarithmic during 24-48 h after the initiation of each run. Accordingly, the specific growth rate tz of the cells was assessed from the numbers of cells No and N observed at t ----0 and t = 24 h. The/z values appearing later are the averages of experimental data taken repeatedly 5-22 times.

TABLE 2. SPECIFICATIONS OF ASPIDISCIDAE ISOLATED

Size (t~m) Protozoa Aspidisea costata Dujardin strain S Aspidisca lynceus Ehrenberg strain M

Plant s i t e

Length

Width

Sunamachi

31

22

Mikawashima

40

30

TABLE 3. SPECIFIC GROWTH RATE OF ASPIDISCIDAE IN COMPARISON WITH THAT OF OTHER PROTOZOA AT 2 0 ° C .

Growth rate Species Aspidisca costata strain S Aspidisea lynceus strain M Vorticella microstoma strain S Vortieella eonvallaria strain M Paramecium eaudatum strain MO

t~ (day-l) *

tq (h)t

1'2 1.3 3.3 2.2 1.4

13"6 12"4 5"0 7"6 12"0

* Specific growth rate based on number -- d I n N dt 0.693 t Generation time --

Growth Rate of Aspidiscidae Isolated from Activated Sludge RESULTS

141

AND DISCUSSION

TABLE 2 shows the species of Aspidiscidae isolated in this work from sewage plant sites in Tokyo area; the two species were identified as Aspidisca costata and Aspidisca lynceus. Their specific growth rates in comparison with those of protozoa other than Aspidiscidae at 20°C are shown in TABLE 3. The values of ~ for Vorticellidae and Parameciidae are cited from the previous work (SUDO and AIBA, 1971). The data in each row of the table are averages (deviation of less than 15 per cen0 of repeated observations and it may be said that the value of/~ for Aspidiscidae was nearly one-half of those observed with VorticeUidae. Aspidisca lynceus did not proliferate on the slide glass due to unknown reasons and so, t h e / , value in TABLE 3 was estimated from the cells which proliferated inside the ring reservoir fixed on the SE-agar medium. Since it was difficult to count accurately the cell number in the specific space, the growth rate of Aspidisca costata which could grow on the slide glass was studied in details as will be shown later. The bacterial food

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~S~__ rnb°t

Microbial Food originating

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~n+o ]

/~

i Shibaura Sewage~Treot.

-~.--~

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I Mikawashima

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1

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t l , , ' 0

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96

t, h FIG. 1. Growth pattern of Aspidisca costata at 20°C. The microbial food came from separate cultures of heterogeneous bacteria in SE-medium. Originally, the bacteria were isolated from the sludge of sewage treatment plants in Tokyo area. Dominant species of protozoa in the respective sludge were: * Aspidisca costata; ~ Vorticella microstoma; ~, Vorticella convallaria.

142

RYUI CHI SUDO a n d SHUICHI AIBA

given in each measurement of the growth rate was prepared, unless otherwise noted, from the activated sludge wherein the protozoa in question was dominant. The growth pattern shown in FIG. 1 for Aspidisca costata changed appreciably depending on the bacterial food. It is evident from the figure that growth was most favorable (/z = 1"2 day - I , cf. TABLE3) when the food from the specific sludge, wherein the same species (Aspidisca costata) dominating, was given. If bacterial food from another sludge, wherein Vorticellidae dominating, was used, the growth rate of Aspidisca costata tapered off and/or the protozoa died ultimately. It is surmised from the figure that Aspidisca costata was "stenophagic".

i

J

5:0

o

1.0

0.5

0.1 3.1

32

/._.........~ ~ 3"3 3.4

ix T

3-5

3-6

3"7

~03

FIo. 2. Effect o f t e m p e r a t u r e o n growth rate o f Aspidisca costata.

In addition, the protozoa species was considered "horozoic", judging from the fact that no growth was observed when the bacterial food was inactivated by either penicillin (5000 U ml -~) or streptomycin (100/~g ml -~); i.e., axenic culture of Aspidisca costata was unsuccessful. This characteristic has also been observed with VorticeUidae (Suoo and AreA, 1971). The effect of temperature on the growth rate is shown in FIG. 2. The range of temperature studied was from 2 ° to 37°C; no growth was observed at 2 ° and 35°C, while at 37°C the protozoa became non-viable. It is noted from the figure that the optimum temperature was around 30°C. The activation energy for growth was estimated from FIG. 2 as AE = 13,800 cal mole -1. There have been few papers published on the effect of temperature on the growth rate of Ciliata. Some of the data on AE will be cited.

Growth Rate of Aspidiscidae Isolated from Activated Sludge

143

F o r Paramecium aurelia, A E ----23,000 cal m o l e -~ (12°-25°C) (HALL, 1953). F o r Tetrahymena pyriformis, A E = 9000-18,000 cal m o l e -1 (10.5°-33.5°C). T h e range o f A E d e p e n d e d on the p r e c u l t u r e t e m p e r a t u r e (TRoRMAR, 1962). F o r Vorticella microstoma, A E = 18,300 cal m o l e -1 (3°-25°C) (SODO a n d AIBA, 1971). T h e value o f A E assessed in this w o r k f o r Aspidisca costata was a p p a r e n t l y in the lower range o f those r e p o r t e d p r e v i o u s l y b y o t h e r w o r k e r s f o r Ciliata. T h e e m p i r i c a l finding t h a t A s p i d i s c i d a e have rarely been identified f r o m the sludge o f Sewage T r e a t m e n t P l a n t s in T o k y o when the o p e r a t i o n t e m p e r a t u r e was lower t h a n 20°C, i.e. 10°-15°C, VortieeUidae a n d O p e r c u l a r i i d a e being p r e d o m i n a n t however, m i g h t have resulted f r o m the l o w e r value o f / ~ a t these temperatures. A c c o r d i n g to ROGOVSKAJA et al. (1968), Aspidisca costata d i s a p p e a r e d f r o m a sludge higher t h a n 30°C in t e m p e r a t u r e . I f the o p t i m u m t e m p e r a t u r e at 30°C f o r the g r o w t h o f Aspidisca costata s t u d i e d here is acceptable, the d i s a p p e a r a n c e d e p e n d i n g on specific t e m p e r a t u r e s higher t h a n 30°C c o u l d be envisaged f r o m FIG. 2. Acknowledgement--The authors are indebted to Dr. I. HORASAWA,Director, Research Institute of Sanitary Engineering, Japan for his constant guidance and valuable suggestions; technical assistance from K. ISHmA, S. CmDA, Mrs. T. YOSHINO and Mrs. J. HAGIWARA(Sewerage Bureau, Tokyo Metropolitan Government) is also appreciated.

REFERENCES BROWNT. J. (1965) A study of the protozoa in a diffused-air activated sludge plant. J. Proc. Inst. Sew. Purif. 64, 375-378. CURDS C. R. (1963) The flocculation of suspended matter by Paramecium caudatum. J. gen. MicrobioL 33, 357-363. COROS C. R. and VANDYKEJ. M. (1966) The feeding habits and growth rates of some fresh water ciliates found in activated sludge. J. appL Ecology 3, 127-137. CORDS C. R., COCKBUrtN A. and VANDYKEJ. M. (1968) An experimental study of the role of the ciliated protozoa in the activated sludge process. J. Wat. Pollut. Control 67, 312-329. CURDS C. R. and COCKnURNA. (1968) Studies on the growth and feeding of Tetrahymena pyriformis in axenic and monoxenic culture. J. gen. Microbiol. 549 343-358. CURDS C. R. and FEV G. J. (1969) The effect of ciliated protozoa on the fate of Escherichia coli in the activated sludge process. Water Research 3, 853-867. HALL R. P. (1953) Protozoology, 428 pp. Prentice-Hall, Engelwood Cliffs, N.J. HARDIN G. (1943) Flocculation of Bacteria by Protozoa. Nature, Lond. 151, 642. HORSAWAI. (1945) The biological studies on activated sludge in the purification of sewage, 5 pp. Sohgensha, Tokyo. JAPAN SEWAGEWORKSASSOCIATION(1964) Standard Methods for the Examination of Sewage, 218 pp. KUDO R. R. (1954) Protozoology, 839 pp. Thomas, Springfield. LEVINEL. (1959) Axenizing Vorticella convallaria. J. Protozool. 6, 169-171. PILLAIS. A. and SUPBRAHMANYANV. (1942) Role of protozoa in the activated sludge process. Nature, Lond. 150, 525. PILLAI S. C. and SUBRAH~ANVANV. (1944) Role of protozoa in the aerobic purification of sewage. Nature, Lond. 154, 179-180. PRAKASAMT. B. S. and DONDERON. C. (1967) Aerobic heterotrophic bacterial population of sewage and activated sludge. I. Enumeration. Appl. Microbiol. 15, 461-467. ROGOVSKAJAC., LAZAREVEM. and KOSTINAL. (1968) The influence of increased temperatures (30°39°C) on the biocoenosis of activated sludge and the intensity of decomposition of organic compounds. Proc. 4th Int. Conf. Water Pollut. Res. II-9.

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SUDO R. and OHGOSttIY. (1964) The seasonal changes of activated sludge organisms. Water Purif. Liquid Wastes (Japan) 5, 43-49. SUDOR. and AreA S. (1970) A relationship between the sedimentation rate of activated sludge and the quality of effluent. J. Ferm. TeehnoL 48, 342-349. StrDO R. and AreA S. (1971) Growth rate of Vorticellidae isolated from activated sludge. Jap. J. Ecology (in press). THORMARH. (1962) Effect of Temperature on the Reproduction Rate of Tetrahymena pyriformis. Exp. Cell Res. 28, 269-279. WATSONJ. M. (1945) Mechanism of bacterial flocculation caused by protozoa. Nature, Lond. 155, 271-272.