the of activated sludge from raw development1 ' sewage, peritrichs, holotrichs and keterotrichs steadily increased in number; that the peritrichs were eventually the most abundant forms and they continued to be the predominating type in the experimental sludge; and that 'occasional colonies of Carchesium were observed

Divergent views on protozoal activity in sewage and sludges During the last three decades, a number of investigators have pointed out generally the importance of protozoa in sewage purification, e.g. Richards and Sawyer (1922) attributed the high nitrogen content of activated sludge to the presence of large numbers of protozoa; Johnson Special Article (1922) observed that Parametria accompanied effluents of unusual purity, thereby suggesting that protozoa help in the removal of fine particles THE SPECIAL SIGNIFICANCE OF of suspended matter and the surplus bacteria; Cramer (1931) found that clarification of CERTAIN PERITRICHOUS sewage was dependent on aerobic bacterial life, PROTOZOA IN THE AEROBIC live protozoa and oxygen in solution; Viehl PURIFICATION OF SEWAGE ] (1937) inferred that while bacteria brought about the oxidation of dissolved matter in sewage, S. C. M.R.san.i. By ^PILLAI,j p1i.d., aj.i.sc., aju.c., protozoa, especially , were active in the Department of Biochemistry, Indian Institute of Science, flocculation and absorption of colloidal matter; Bangalore and Jager (1938), who also observed that the of accelerated the Schloesing and Muntz in 1877 and 1878 presence protozoa coagulation of sewage recorded that the a of the of colloids, efficiency observed during study purification _ of the pure cultures of protozoa studied was in sewage water land filters that nitrification by the order : 1Paramecium Was due to microbial and since then an following caudatum, activity, and 'small amount of attention has been devoted charan, alba, increasing \ to the investigation of micro-organisms in rela- flagellates tion to sewage purification and to the closely At the same time, others have considered the allied processes of soil metabolism. A consider- presence of protozoa useless or even harmful to on the able volume of literature has accumulated the purification process, e.g. Fairbrother and microbiology of these processes, but there is need Renshaw (1922) suggested that the protozoa in for further information on certain aspects, e.g. activated sludge tanks might be killed off selec- the aerobic process of sewage purification which tively by the use of certain dyes in order to has been of considerable interest since the early promote the activity of bacteria responsible for days of scientific study of sanitation and public the purification; Wooldridge and Standfast health. (1936), working with pure cultures of Polytoma uvella and Euglena gracilis, observed that the on protozoa exhibited less than the bacteria Earlier observations protozoa in oxidizing ' activity and that even if the under the condi- filters and activated sludge protozoa, tions of the activated sludge process, were found in filters Boyce (1902) oxidizing more active than appears from these experi- Carchesium lachmanni, a peritrichous ciliate their with that ' ments, activity in comparison in masses under similar condi- protozoan great of the very much more numerous bacteria tions to those of natans' to Spharotilus (allied usually present, must in practice be of second- forms of which is more leptothrix bacteria) ary importance'; Butterfield, Ruchhoft and gelatinous than Leptomitus lacteus, but, accord- McNamee have also the ' '. (1937) emphasized ing to him, indicates much greater pollution primary of A importance bacteria, especially similar observation was made by Fowler Zooglea ramigera (Butterfield, 1935), in the who also reported considerable (1909), develop- activated sludge process; and Jenkins (1942) ment of Carchesium lachmanni in the effluents reported that' flocculation and oxidation proceed at from the contact beds and percolating filters satisfactorily in the absence of protozoa Davyhulme, Withington and Gorton Sewage Works Ardern and Lockett Thus conflicting views have been held by (Manchester). ' (1922-23) recorded that bulking ' of activated different investigators in regard to protozoal in it is desirable to have sludge was associated in some way with activity sewage, and organisms such as Carchesium and some definite information, in the first instance, filamentous growths of the Cladothrix type. on the more important forms of protozoa that Buswell and Long (1923) reported that, during develop during the purification process. 412 THE INDIAN MEDICAL GAZETTE [Sept., 1952

Recent researches at Bangalore sludge, and they collectively produced 0.8 per cent In the course of studies at Bangalore on the sludge; the corresponding effluents were were different methods of sewage treatment over a invariably turbid), the protozoal forms much more efficient not in formation period of years, some interesting observations only sludge amounts of were : have been made on a group of closely allied (the sludge produced by them protozoa (certain peritrichous ciliates) which , 1.2 per cent; Stylonychia, 1.8 per cent; 2 seem to be particularly important in the aerobic , per cent; and Vorticella, 2.5 per but also m much better purification of sewage. cent) yielding effluents, and that the Vorticella the from among protozoa, gave Apart laboratory experiments designed cleanest effluent (Pillai, 1941; Pillai and to study the micro-organisms that develop in under different Subrahmanyan, 1945). Reynoldson (1942) sewage conditions, periodical found that under the conditions at the Hudders- observations on the activated sludges produced field Sewage Works in England, the logarithm of in the different in India and on other plants the Vorticella count in the activated aerobic in the have been carried sludge systems country followed its as out. A brief account of these observations is closely biological activity, measured the of the effluents below : by purity produced, given and reported that Vorticella might be regarded Observations on protozoa in a new activated as an indicator organism for activated sludge. sludge -plant revealing the special Natural significance of Vorticella development of peritrichous ciliates, especially the colonial forms, in activated While a new activated sludge plant was put and other aerobic into operation. in 1938 at Cossipore (near sludge systems Calcutta) under somewhat exceptional condi- causing flocculation of the tions, e.g. aerating practically sullage containing colloids little amount of faecal matter very during Further observations (Pillai, 1942) at the stages of the of the early functioning Bangalore revealed the occurrence in large plant, it was observed that the effluent was numbers of Epistylis sp. in activated sludge (in not clear it was nitrified. although considerably fluffy masses attached to the sides of the aeration Under these conditions little was very sludge tanks and in smaller colonies in the sludge flocks) formed and the material had no resemblance to and this protozoan was found more efficient, at normally activated The micro-organic sludge. least twice as efficient as Vorticella, in the clari- of the consisted of population sludge mostly fication of and in the of aerobic bacteria and few sewage production protozoa, e.g. species sludge. of Amoeba, Euglena, Gonostomum, Colpoda, Podophyra, Acineta, Stylonychia, Paramecium In the land-filtered sewage effluent at Madura and relatively few numbers of Vorticella. As masses of Carchesium sp. and considerable numbers of have been found to increasing quantities of faecal matter were intro- Epistylis sp. and Seshachar. duced into the aeration tanks, it was observed develop (Pillai, Rajagopalan In the activated at that the numbers of ciliate protozoa, particularly 1947). sludge plant where sea water is used for those of Vorticella sp., increased gradually, and Tuticorin, flushing has been simultaneously the of the effluent and the purposes, the protozoan population quality of the marine of character of the sludge improved steadily. The found to consist mainly species increased development and predominance of Vorticella and Zoothamnium (Pillai and In the activated sludge at Vorticella sp. consequent on the additions of Rajagopalan, 1948). of faecal matter were correlated with the satisfactory Ambasamudram species especially Epistylis, Carchesium and have been found to clarification of sewage and formation of sludge Opercularia (Pillai, 1938). flourish (Pillai, 1951). Further studies have shown that, depending Experimental evidence on the flocculating upon the composition of water used for flushing activity of protozoa purposes, the nature of soil washings entering the sewerage system, the extent of aeration and Following the above observation on the other factors, sp. and other peritrichous flocculating activity of protozoa and on the Epistylis e.g. of Vorticella, special significance of Vorticella in the activated ciliates, species Zoothamnium, Carchesium and develop and process, were carried out Opercularia, sludge experiments by in activated and in other isolating thirteen different strains of aerobic dominate sludges aerobic of treatment in different bacteria and four forms of protozoa (species of systems sewage of India. The occurrence of Paramecium and parts Opercularia Colpoda, Stylonychia, 1 and in activated from the activated at (plate XXXIX, figures 2)* Vorticella) sludge plant and in aerated is of further Bangalore and by introducing them in different sludge sewages interest as there seems to be no record about combinations into heat-sterilized sewage and by bubbling air through the suspensions. It was observed that the bacterial strains * although Thanks are due to Dr. B. R. Seshachar, of Central showed some sludge-forming property (the College, Bangalore, for his assistance in taking the individual strains produced 0.4 to 0.5 per cent photomicrographs. Plate XXXIX

THE SPECIAL SIGNIFICANCE OF CERTAIN PERITRICHOUS CILIATE PROTOZOA IN THE AEROBIC PURIFICATION OF SEWAGE : S. C. PILLAI. (S. A.) PAGE 411

?

of a of sp. jrjo- i1.?Photomicrograph Photomicrograph of a colony of Opercularia sp. Fig. at X 70. isolated from serrated sewage at Bangalore. X 70.

Fig-Fi

this organism in the literature bearing on the media. The exact mechanism of the clarifica- is Protozoa in Indian soils, waters and sewages. tion of the media by the protozoal activity still under study. Further observations on the development of Many years ago, Greenwood (1894), in the course of a academic into the colonial peritrichs in sewage under purely enquiry made some aerobic conditions processes of digestion in protozoa, interesting observations on Carchesium With a view of collecting further information when the was introduced treated polypinwm organism ?n the development of protozoa in sewage into suspensions of finely granular precipitate in different ways to some extent the (simulating1 from diluted white of egg, the smaller fatty conditions during dilutionland filtration, globules of milk, carmine and Indian ink broad irrigation, and the activated sludge suspended in water. She observed that both Process), experiments were carried out with the nutritious matter (e.g. the coagulation of (a) domestic sewage varying strengths by precipitate separated from the diluted white of diluting the sewages with fresh water in suitable fresh and the innutritious matter (e.g. of the eggs) Proportions depending upon the strength Indian ink suspended in water) were swallowed and the in sewage successively replacing samples by Carchesium with indiscriminating eagerness, 24 shallow basins at intervals of 8 to hours; {b) and that both the substances exhibited alike a the material columns (6 ft. 3 ' filtering through or ', while the of sand centripetal clustering aggregation inches deep and 4 ft. 2 inches diameter) intracellular sojourn of innutritious material was and soil and successively replacing and exposing however curtailed, the vacuoles in which they the filtrates as in up a fairly ' (a); (c) pumping lay at first tended to disappear quickly, and weak' into sewage (about 10,000 gallons daily) there was but rarely that re-formation of fluid an overhead gently sloping channel (160 ft. long concerned in the solution of true foodstuffs. and 1 cemented ft. wide, made of granite slabs Quite recently, Sugden and Lloyd (1950), while and mounted on granite pillars, 8 ft. to 2 ft. high) the of turbid waters by and investigating clearing and allowing it flow down continuously Carchesium polypinum, demonstrated that 30 ml. numerous rapidly through ordinary irrigation of 0.3 per cent Indian ink suspension was almost channels (to about 100 plots at different levels) completely clarified in two hours by the organism over an area of acres in farm about 2\ the sewage colonized on a hay stalk 5 cm. long. at the Institute (during night time a very dilute sewage was allowed to stand in these channels Summary and conclusion the and blowing during experimental period); {d) A study of the literature that has accumulated air continuously through successive portions of ' ' during the last fifty years on the microbiology sewage by the fill-and-draw method. In the of the aerobic purification of sewage would show course of three weeks, in the x diluted sewages, that although some interesting observations have soil sewage and in the artificially filtrates, flowing been made since the turn of the on the aerated of century samples, vigorous development occurrence and of in sewage such as the of and activity protozoa protozoa, species Epistylis there has been no definite was found to take causing purification systems, Opercularia, place information on the of and on the clarification of the media and'oxidation type protozoa rapid in the Lack in them. considerable part they play purification process. changes Thus, very of such information has led to the view evidence is accumulating at Bangalore that probably that protozoa may not have any role in, or may certain peritrichous ciliates, e.g. species of even have an adverse effect on, the purification Vorticella, Carcliesium, Zoothamnium, Epistylis of and Opercularia, invariably develop in sewage sewage. under aerobic conditions and play a vital role Recent work at Bangalore has shown that as of in the purification process. peritrichous ciliates, such the species Vorticella, Carchesium, Zoothamnium, Epistylis and are more Observations on the protozoal activity, digestion Opercularia, the important forms, and that about clarification of and flocculation they bring rapid sewage and related changes during aerobic Microscopic observations on the protozoa purification of the material. when introduced into raw (e.g. Epistylis sp.) from their in and other soil Apart importance sewage puri- sewage suspensions, e.g. suspen- fication and kindred the on a slide showed that the move- operations, protozoa sion, ciliary referred to above seem to a valuable ment of the in such media was very provide organisms material for some of the fundamental considerably stimulated, the cilia having been studying on unicellular life processes and found to work most and In problems bearing rapidly vigorously. their possible a few minutes the turbid liquid around the applications. was found actively functioning protozoan cells REFERENCES to become clear. When the movement ciliary Ardern, E., and Lockett, Manchester Rivers Dept. of the was reduced protozoa very considerably W. T. (1922-23). Report, Appendix II, p. 38. or the inactive stopped by rendering organisms Boycb (1902) .. Second Report of the Royal or it heat or by killing (by application of Commission on Sewage, chemicals), there was little, or no clarification of p. 104. 414 THE INDIAN MEDICAL GAZETTE [Sept., 1952

Buswell, A. M., and J. Amer. Water Works Long, H. L. (1923). Assoc., 10, 309. Butterfield, C. T. (1935). Pub. Health Rep., 50, 671. Butterfield, C. T., Ibid., 52, 671. Ruchhoft, C. 0., and' McNamee, P.D. (1937). Cramer, R. (1931) .. Indian and Eng. Chem., 23, 309. Fairbrother, T. H., and J. Soc. Chem. Industry, 41, Renshaw, A. (1922). 134 T. Fowler, G. J. (1909) .. J. Roy. Sanitary Inst., 30, 513. Greenwood, M. (1894). Phil. Trans., B., 185, 355. Jager, G. (1938) .. Zeitschr. Hyg. Injektkrank- heit-, "'20, 620. Jenkins, S. H. (1942) .. Nature, 150, 607. Johnson, H. (1922) .. Proc. Manchester S. D. Works ^Issoc., p. 54. Pillai, S. C. (1938) .. Report on the New Acti- vated Sludge Plant at Cossipore, Calcutta. Idem (1941) .. Current Sci., 10, 84. Idem (1942) .. Ibid., 11? 437. Idem (1951) Ibid., 20, 75. Pillai, S. C., and Raja- Ibid-, 17, 329. GOPALAN, R. (1948). Pillai, S. C., Raja- Ibid., 16, 254. gopalan, R., and Seshachar, B. R. (1947). * Pillai, S. C., and Subrah- Science and Culture, 11, 75. manyan, V.' (1945). Reynoldson, T. B. Nature, 149, 608. (1942). Richards, E. H., and J. Soc. Chem. Industry, 41, Sawyer, G. C. (1922). 62 T. Schloesing, Th., and Compt. Rend. Soc. Biol., 84, Muntz, A. (1877). 301. Idem. (1878). Ibid., 85, 1018; 86, 892. Sugden, B., and Lloyd, The Water and San. En- L. (1950). gineer, 1, 22. Viehl, K. (1937) .. Zeitschr. Hyg. Infektkrank- heit., 119, 383. Wooldridge, W. R., and Biochem. J., 30, 1542. Standfast, A. F. B. (1936).