APPLIED MICROBIOLOGY, Jan., 1967, p. 17-21Copyright ( 1967 American Society for Microbiology

Vol. 15, No. 1Printed in U.S.A.

Occurrence of Zoogloea Colonies and Protozoans at

Different Stages of Sewage Purification1P. M. AMIN AND S. V. GANAPATI

Section of Sanitation, Department of Biochemistry, Maharaja Sayajirao University, Baroda, India

Received for publication 23 May 1966

ABSTRACT

The presence of fingered branch-bearing Zoogloea has been noted on a numberof occasions in the Baroda Sewage Disposal Works. Samples of raw sewage, theeffluent from the continuous flow settling basin, the raw sludge, the floating scum inthe settling basin, the final secondary digested sludge, and the supernatant liquidfrom the secondary digester were kept without any disturbance in 1-liter Pyrexglass beakers, which were loosely covered with petri dishes. Scum was formed on thesurface within 48 hr in all the samples, and fingered Zoogloea colonies resemblingthe pure culture of Zoogloea ramigera reported by Crabtree et al. (5) were found inall except the final secondary digested sludge and the supernatant liquid from thesecondary digester. It is not known whether the Zoogloea colonies discovered in theabove cases are the same as or different from the typical Zoogloea ramigera of acti-vated sludge, and whether they are slime-forming or flocculent types of bacteria. Inany case, they seem to be different in their ecological status and in the nature of theaccompanying protozoans from the typical Zoogloea ramigera. The reasons forthe absence of zoogloeas in two of the samples are unknown.

Extensive studies carried out on the microbialflora and fauna of sewage purified by the activatedsludge process have indicated the presence ofZoogloea ramigera (4-7,1, 24; Unz and Dondero,Bacteriol. Proc., p. 37, 1964) along with cer-tain types of free-swimming or stalked ciliates.The importance of the latter in determining thecondition of activated sludge has also been re-ported by a number of workers (2, 3, 9, 10, 12, 14-21). The special importance of Vorticella in decid-ing the degree of purification of sewage has beenstressed by Pillai and Subrahmanian (14, 16),Reynoldson (22), Hartman (9), and Hawkes (10).During the course of our studies on purification

of Baroda sewage, an attempt was made to detectthe presence of these organisms at different stagesof purification at the Baroda Sewage DisposalWorks. The data thus obtained are reported inthis paper.

MATERIALS AND METHODS

At Baroda, a part of the 8 million gal/day of sewagereceives only primary treatment in a continuous-flowsettling basin, and the purified effluent is mixed with

'This forms a part of the "Studies on Sewage Puri-fication" financed by a research grant FG-In-245 fromthe Am PL 480 funds in India to C. V. Ramakrishnan,Department of Biochemistry, Maharaja SayajiraoUniversity, Baroda, India.

the remaining untreated sewage and then used for irri-gation. The raw sludge obtained from the continuousflow settling basin is digested in primary and secondarydigesters, and the gas produced thereby is used fordomestic heating. The digested sludge is dried and soldas manure.

Samples of raw sewage, of the floating scum, of theraw sludge, and of the effluent from the continuousflow settling basin, the final secondary digested sludge,and the supernatant liquid from the secondary di-gester were kept without disturbance in 1-liter'Pyrexglass beakers which were loosely covered with petridishes for preventing dust from falling on the surface.This experiment was repeated many times, and the re-sults of a typical experiment are given below.Scum was formed within 48 hr. Successive samples

of the top scum and liquid were taken from eachbeaker as soon as the top scum was noticeable. Thiswas done at least once a day and several times in thecase of some samples.A platinum loopful (0.5-cm diameter) of the surface

scum and a drop of the liquid were first examinedqualitatively for identification of the organisms andthen counts were made. To maintain uniformity, aloopful of the scum was spread over an area of 25mM2 with the use of diluted glycerol water. Five such

loopfuls or one drop of the liquid were spread care-fully over the area marked on a clean glass slide andexamined microscopically with magnification of 100.Frequencies of the organisms present are expressed inthe symbols of Fritsch and Rich (8).

For chemical analysis, standard methods published17

on Decem

ber 10, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

AMIN AND GANAPATI APPL. MICROBIOL.

EA

uF

l QI II

"0o004

0

0.*5

1).*C0O:Z az

0_0P4

UUtIjUU

U u

v I u

I

;t t:

.1 u1 1

tt u IHI I I

to11ul!

4.)

V3 uI .I 1 II4

4)0.Ac Iu Iu

I U

;Uu -U

U

cn

"o CZ q t^ X OO oL "Te

N00

_0

>_

co0 CA v

o~~~Q ,

18

14.)

boQQ

N

02C.)

*_

0

7

En

0

0

0

64)

U)

11QUu"0

II

UU

U

II

40-

V

40

II1-

_4-

"0

-0

"0C.

4-

04-

._)*-

4-0U,

>4e

4))0

ecS.

on Decem

ber 10, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

4

LI.'''S'

i AW

4.

.1..

:| .11"

*.¾ . .

I- ..I* :4.4..4.: .i.'

Zoogloea ramigera from laboratory model activated sludge process. X 64.Fingered Zoogloea colonies from untreated raw sewage. X 64.Fingered Zoogloea colonies with protozoans. X 32.Fingered branching Zoogloea colonies with Podophrya fixa. X 64.Fingered Zoogloea colonies with Vorticella microstoma and other protozoans. X 26.Fingered branch-bearing Zoogloea colonies with the filamentous Sphaerotilus nlatans. X 64.Spirilla with flagellated protozoans. X 64.Long rods with Paramecium species. X 64.

19

2

FIG. 1.FIG. 2.FIG. 3.FIG. 4.FIG. 5.FIG. 6.FIG. 7.FIG. 8.

-AOPI

on Decem

ber 10, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

TABLE 2. Physicochemical analysis of sewage samples

Physical analysis Chemical analysis (mg/liter)

Acid KMnO4 Alkalinity NitrogenSample 5-dayaSampeTemp pH Relative at D . b_ a

yats20C32mi 4 hr321. Phenol Total 0

Topscm27.76.7 O 30 14. 38.6 7.5 Ni 1,000O90.50ilENiC days

From the continuousflow settling basinRaw sewage 27.5 7.0 0 325 3.6 7.9 27.0 Nil 650 0 50 Nil NilTop scum 27.7 6.7 0 630 14.9 38.6 67.5 Nil 1,000 0 90.5 Nil NilRaw bottom 27.4 6.9 0 - 134.0 Nil 1,470 0 140 Nil Nil

sludgeEffluent 27.6 7.4 0 316 4.9 20.0 28.0 Nil 800 0 56 Nil Nil

From the secondarydigesterDigested sludge 28.0 6.9 0 33 8.3 38.6 61.0 Nil 2,230 0 310 Nil NilSupernatant 27.9 7.0 0 215 23.3 9.1 72.0 Nil 1,580 0 230 Nil Nil

liquid

From the laboratorymodel activatedsludge plant

Effluent 28.0 8.6 >6 7.0 1.4 2.6 3.6 30.1 450P19.20 7.8 31.8 2.3

a BOD= biochemical oxygen demand.b Dissolved oxygen.

by the American Public Health Association (1) weregenerally employed for all the tests except the acidpermanganate values, which were done according tomethods of chemical analysis as applied to sewage andsewage effluents (13).

RESULTS AND DISCUSSION

The results of microscopic examination areshown in Table 1 and Fig. 1 to 8, and those of thephysicochemical analysis are in Table 2.

Fingered Zoogloea colonies, resembling thoseshown for the pure culture of Z. ramigera byButterfield (4) and by Crabtree et al. (5), formedin the case of samples drawn from the raw sewage,the top scum, the raw sludge, and the final effluentfrom the continuous-flow settling basin. Theydisappeared in 2 to 8 days when the top scumformation on the surface also ceased.However, they did not form in the final second-

ary digested sludge liquor and in the supernatantliquid from the secondary digester. The last twosamples were characterized by the presence of alarge amount of ammoniacal nitrogen (Table 2).It is, perhaps, possible that the high content ofammoniacal nitrogen prevented the growth ofZoogloea as suggested by Rich (23).From a study of Table 2, it will be seen that the

5-day biochemical oxygen demands at 20 C ofall the samples, except the final secondary di-

gested sludge, are very high, and their relativestability is very low. The chemical conditions ofthese samples in which colonies of Zoogloea arefound, in short, show varying degrees of purifica-tion and do not represent those of a well-con-ditioned activated sludge and effluent. Thespherical or fingered lobed zoogloeal colonieshave been discovered in raw sewage and in othersamples receiving no treatment at all. Thus, itwould seem that the presence of Zoogloea orga-nisms is not associated with the chemical con-ditions involved in purified sewage. Of course,one may ask whether this organism is the sameas the one reported to occur in the sludge of anactivated sludge plant. To test this, a laboratorymodel activated sludge plant was set up, andsimilar organisms in the well-purified sludge weredetected. Thus, Zoogloea organisms seem to bepresent in all the sewage samples where ammonia-cal nitrogen content is not very high.

Regarding the protozoans (Table 1), V. micro-stoma was found in all the samples but was pre-sent in varying numbers. Podophyrya fixa was alsoseen in a few cases. Paramecium caudatum wasfound in fairly large numbers in raw sewage alongwith fingered Zoogloea colonies. In other casesits number was comparatively less. Epistylisplicatilis was the other stalked ciliate which wasfound only in the sludge from the laboratory

20 AMIN AND GANAPATI APPL. MICROBIOL.

on Decem

ber 10, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

ZOOGLOEA IN SEWAGE

model activated sludge plant. All protozoans,except E. plicatilis, and Zoogloea were found insamples of raw sewage as well as in samplesdrawn from different places in the continuous-flow primary settling basin and in the activatedsludge plant.These observations raise an important question

regarding the role of these organisms in sewagepurification. Studies are now being carried out onthe exact biochemical composition of raw sewageas well as of samples drawn at different stages ofpurification with the aim of finding a clue abouttheir appearance and their metabolism.

LITERATURE CITrD

1. AMERICAN PUBLIC HEALTH ASSOCIATION. 1955.Standard Methods for the examination of waterand waste water, 10th ed. American PublicHealth Association, Inc., New York.

2. ARDERN, E., AND W. T. LocKETT 1936. Labora-tory tests for ascertaining the condition of ac-tivated sludge. J. Inst. Sewage Purif., Pt. 1,p. 212-215.

3. BARKER, A. N. 1949. Some microbial aspects ofsewage purification. J. Inst. Sewage Purif., Pt.1, p. 7-22.

4. BurTRFIELD, C. T. 1935. Studies on sewage puri-fication. II. A zoogloea forming bacterium iso-lated from activated sludge. Public HealthRept. (U.S.) 50:671-684

5. CRABTREE, K., E. McCoYL, W. C. BOYLE, ANDG. A. ROHLICH. 1965. Isolation, identification,and metabolic role of the sudanophilic granulesof Zoogloea ramigera. Appl. Microbiol. 13:218-226.

6. DiAS, F. F., AND J. V. BHAT. 1964. Microbialecology of activated sludge. I. Dominant bac-teria. Appl. Microbiol. 12:412-417.

7. DUGAN, P. R., AND D. G. LUNDGREN. 1960. Isola-tion of the floc-forming organism Zoogloearamigera and its culture in complex and syn-thetic media. Appl. Microbiol. 8:357-361.

8. FRITSCH, F. E., AND A. RICH. 1913. Studies onthe occurrence and reproduction of Britishfresh water algae in nature. 3. A four years' ob-servation of a fresh water pond. Ann. Biol.Lacus. 6:33-115.

9. HARTMAN, L. 1963. Ciliates as indicators in theactivated sludge process. Environmental Health5:31-40.

10. HAwKES, H. A. 1960. Ecology of activated sludgeand bacteria beds, p. 52-97. In P. C. G. Isaac[ed.], Waste treatment. Proc. Symp. Treatment

of Waste Waters, 2nd, Newcastle Tyne. Perma-gon Press, London.

11. HEUKELEKIAN, H., AND M. L. LITTMAN. 1939.Carbon and nitrogen transformation in thepurification of sewage by the activated sludgeprocess II. Morphological and biochemicalstudies of zoogloeal organisms. Sewage WorksJ. 11 :752-763.

12. JENKINS, S. H. 1942. Role of protozoa in acti-vated sludge process. Nature 150:607.

13. MINISTRY OF HOUSING AND LOCAL GOVERNMENT.1956. Methods of chemical analysis as appliedto sewage and sewage effluents. H. M. Sta-tionery Office, London.

14. PILLAI, S. C. 1941. The function of protozoa inactivated sludge process. Current Sci. (India)10:84-85.

15. PILLAI, S. C., AND V. SUBRAHMANIAN. 1942. Roleof protozoa in activated sludge. Nature 150:525.

16. PILLAI, S. C., AND V. SUBRAHMANIAN. 1943. Pro-tozoa in relation to bulking of activated sludge.Sci. Cult. (Calcutta) 8:376-378.

17. PILLAI, S. C., AND V. SUBRAHMANIAN. 1944. Roleof protozoa in the aerobic purification ofsewage. Nature 154:179-180.

18. PILLAI, S. C., AND V. SUBRAHMANIAN. 1945. Pro-tozoa in relation to sewage purification. Sci.Cult. (Calcutta) 11:75-77.

19. PILLAI, S. C., T. K. WADHAWANI, M. I. GURU-BAXANI, AND V. SABRAHAMANIAN. 1947. Rela-tive efficiency of bacteria and protozoa inflocculation and oxidation of organic mattersuspended in water. Current Sci. (India) 16:340-341.

20. PILLAI, S. C., R. RAJAGOPALAN, AND B. R.SESHACHAR. 1947. On the occurrence of pro-tozoa in land-filtered sewage effluent. CurrentSci. (India) 16:254-256.

21. PILLAI, S. C., G. J. MOHAN RAO, A. V. S.PRABHAKARA RAO, C. A. SAsrRI, AND P. V. R.SUBRAMANIAN. 1960. Natural purification offlowing sewage. Current Sci. (India) 29:461-465.

22. REYNOLDSON, T. B. 1942. Vorticella as an indica-tor organism for activated sludge. Nature 149:608-609.

23. RICH, L. G. 1955. Respiration studies on the organicnitrogen preferences of Zoogloea ramigeraAppl. Microbiol. 3:20-25.

24. WATrIE, E. 1943. Cultural characteristics ofZoogloea forming bacteria isolated from acti-vated sludge and trickling filters. Sewage WorksJ. 15:476-479.

VOL. 15, 1967 21

on Decem

ber 10, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from