Pergamon

Wat. Res. Vol. 29, No. I 1, pp. 2598-2600, 1995 Elsevier Science Ltd. Printed in Great Britain
0043-1354(95)00036-4

RESEARCH NOTE FATE OF F A E C A L C O L I F O R M B A C T E R I A IN A WASTE W A T E R R E T E N T I O N RESERVOIR C O N T A I N I N G L E M N A GIBBA L.

A H M E D D E W E D A R 1 a n d M A G D Y B A H G A T .2 ~Suez Canal University, Faculty of Science, Botany Department, Ismailia, and 2Suez Canal University, Faculty of Agricultural and Environmental Sciences, A1 Arish, Egypt

(First received March 1994; accepted in revised form January 1995)

Abstract--The comparative survival of total and faecal coliform bacteria was studied in a stable waste-water retention reservoir. The surface of the water reservoir was covered with a macrophyte, Lemna gibba L. Sets of dialysis sacs were suspended in an exposed site to the sun light, while other sets of sacs were suspended beneath the thick green film of L. gibba; where sunlight is almost absent. Total coliform bacteria regrow in sun and in sacs unexposed to sun, while faecal coliform cells exposed to sunlight disappeared with a decay rate 0.1768 h -~. Interestingly, faecal coliform in sacs suspended under the L. gibba green film did not decline during the period of the experiment.

Key words--faecal coliform, total coliform, Lemna gibba L., waste-water retention reservoirs

INTRODUCTION

A n u n d e r s t a n d i n g o f the survival of faecal indicators is basic to the meaningful i n t e r p r e t a t i o n of sanitary water quality data. This because the isolation of coliform bacteria is c o m m o n l y used to indicate the potential presence of intestinal p a t h o g e n s (McFeters et al., 1978). Because pathogenic m i c r o o r g a n i s m s usually a p p e a r intermittently in low concentrations, a n d then only briefly the routine e x a m i n a t i o n of water needs a more dependable a n d b r o a d e r base t h a n just p a t h o g e n detection. Ideally, it should detect other micro-organisms which co-exist with the pathogens in the faecal e n v i r o n m e n t , a n d whose presence in a body o f water will serve as an indicator o f faecal pollution, principally of w a r m - b l o o d e d animal origin. Faecal coliform bacteria (FC) are k n o w n as one of the most i m p o r t a n t indicators of potential public health h a z a r d due to faecal pollution. Cultivation o f floating aquatic plants in waste water p o n d s is increasingly being practiced in the tropics a n d subtropics with the dual objective o f purification a n d resource recovery. The presence o f aquatic m a c r o p h y t e s in a p o n d alters the physiochemical e n v i r o n m e n t of the water. Different studies have s h o w n that aquatic plants, such as water hyacinth (Echornia crassipes [Mart] Solms), water lettuce (Pistia stratiotes L.), cattail (Typha latifolia L.), a n d Lemna gibba L., are capable

of treating waste water (Reddy and DeBusk, 1985). Also, studies have discussed the potential o f aquatic plants, for reducing N a n d P levels in waste water. M o s t of studies were limited to the physiochemical characteristics of the water, a t t e n t i o n has not been given to study of the microbiological changes acc o m p a n i e d with the i n t r o d u c t i o n of m a c r o p h y t e to the water body. Previous studies (unpublished data) in a waste water retention reservoir, A b u - A t t w a , Egypt, showed t h a t the faecal coliform n u m b e r s declined within t h a t reservoir. Lemna gibba L., a floating plant, has been introduced to that reservoir causing a change in the rate of decline with evidence of regrowth. The purpose of this study is to determine the effect of Lemna gibba on the removal o f FC from a waste water retention reservoir.
MATERIALS AND METHODS

*Author to whom all correspondence should be addressed.

The investigation was carried out in a concrete waste water reservoir (15 m3). The water surface was covered with a dense thick film of Lemna gibba L. and receiving secondary treated waste water discharged out of a 200 m Gravel Bed Hydroponic (GBH) bed for sewage treatment at Abu-Attwa, Ismailia, Egypt. Description of the system was explained elsewhere (Butler and Dewedar, 1991). Purified cultures of the faecal coliform bacteria isolated from the studied reservoir were purified by the methods described in the Cowan and Steel's Manual for the Identification of Medical Bacteria (Cowan and Steel, 1993). All cultures used in this study were grown for 24 h on nutrient broth at 37"C. Cells were harvested by centrifugation (6000 rpm) for 10 min and washed twice with sterile buffer

2598

Research Note Table I. Some chemical characteristicsof the reservoir water used in the study Conductivity~ Calcium b Magnesium b NH4-Nb NO3b DOt' 1000 850 44.9 42.5 26.2 22.6 11.05 12.8 1.4 0.71 7.5 I.I

2599

Determination

BOD5 b 27.4 18.6

Values of reservoir water before applying L. gibba Values of reservoir water after applying L. gibba aMicron/cm at 25C. bMilligram per liter.

solution. After the final wash, cells were suspended in the sterile buffer and diluted to the desired population density with the same diluent. Dialysis sacs were immersed in the reservoir which receives almost steady flow of water out of a 200 m GBH system. Four sets of dialysis sacs were used for both total and faecal coliform placed in exposed area to sunlight under Lemna gibba L. forming a green film floating on the water surface. One hour after the dialysis sacs were suspended in the water one sac was removed from each set and coliform and faecal coliform counts performed. These counts were used to determine Co, the number of organisms at time O. Standard plate counts were used to enumerate these bacteria on m-Endo Agar (APHA, 1985). The decay rate of faecal coliform was determined from a plot of the log of the fractional decrease in bacteria concentration over time, In (Ct/Co) versus time. The slope of the line (fit by a linear regression) was equal to the decay rate, K. The larger the number, the greater the decay rate. The decay rate of such cells was determined using the model: In (Ct/Co) = - kt, where K is the decay constant (h -j) equal to the slope of the line (fit by a linear regression); CO is the initial faecal coliform concentration; and Ct is the faecal coliform concentration at time t.

in days. N o decay was recorded in case o f the total coliform cells.

Survival o f Faecal Coliform (FC)

The survival o f pure cultures o f F C was studied in the same m e t h o d used for the total coliform (Fig. 2). N o decline in counts were recorded in case o f F C present in the set o f dialysis sacs suspended under the L. gibba, whereas, F C in dialysis sacs exposed to sun showed an initial rise in growth in the first 24 h followed by a decline to the end o f the experiment. The rate constant for decay o f faecal coliform cells exposed to the sun was 0.1768 h -~. DISCUSSION The result o f the experiment describing the survival o f indicator bacteria in the waste water retention reservoir d e m o n s t r a t e d that the total and faecal coliform bacteria remained viable except F C bacteria in dialysis sacs which were exposed to the sun. It is well d o c u m e n t e d that total coliform bacteria can regenerate in a natural environment, and m a n y workers criticized the use o f the total coliform count as an indicator o f feces-oriented water pollution (Dutka, 1973). Previous studies o f bacteria survival that were 8.0 7.8 7.6 7.4 7.2 7.0 .1
0

Chemical characteristics
Conductivity, calcium, magnesium, ammoniacal-N (NHa-N) and, nitrate-N (NO3-N), dissolved oxygen (DO) and Biological oxygen demand (BODs) concentrations were measured according to APHA (1985). RESULTS W a t e r quality parameters (Table 1) were stable and only m i n o r variations in temperatures (34-35C) were noticed during the experiment in the studied waste water retention reservoir.

./\

Survival o f Total Coliform (TC)

The survival o f natural populations o f total coliform (TC) bacteria isolated from domestic sewage was studied to obtain i n f o r m a t i o n on the population dynamics on water either exposed to sunlight or under a thick film o f L. gibba L. where sunlight is almost nil. Figure 1 is showing survival o f mixed natural populations o f total coliform bacteria exposed and unexposed to sunlight. N o linear relation was f o u n d between the natural logarithm o f the n u m b e r o f the surviving bacteria and time by the simple regression analysis using the model: lny=ln

6.8 6.6L TC covered ra TC sun

6.4 t 6.2 6.0 0

I 1

I 2

I 3

I 4

I 5

a +bx

Time (days) Fig. I. Survival of indicator Bacteria (TC) in reservoir water. Total coliform cells in dialysis sacs suspended under the L. gibba thick film exposed to sunlight.

where y = estimated bacterial counts (cfu/ml): a = intercept; b = slope; x = time o f the experiment

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8 --

Research Note /
7 -

lands (0.029h ~) (Gersberg et al., 1987). This is p r o b a b l y due to sunlight effect which have a lethal effect on coliforms. CONCLUSIONS The conclusions d r a w n from this study obtained u n d e r controlled experimental conditions, c o m p a r e d with thc actual conditions in the field should be a p p r o a c h e d with great caution. This is because there are m a n y variables in the natural aquatic environm e n t such as phage, a n d competing or predatory organisms, only one of which m a y influence bacterial survival. Even so, some general conclusion can be extracted t h a t could be of value in the interpretation of data in the environment. A m o n g these is the finding that the application of Lemna gibba L., which is a floating minute plant, to water reservoirs could be a source o f nutrients as well as shelter where bacteria can proliferate for extended periods. Also, the experiment d e m o n s t r a t e s thc need to use more than one indicator for pollution by waste water in the e n v i r o n m e n t studies.
Acknowledgement--The authors wish to thank Dr A. EI-Sayed, Suez Canal University for helpful discussions.
REFERENCES

61 _~. 5 4 FC o,,er~d F C sun \ q

,-.1

[]

0 0

I 1

I 2

I 3

I 4

~h 5

Time (days) Fig. 2. Survival of indicator bacteria (FC) in reservoir water. Faecal coliform cells in dialysis sacs suspended under the L. gibba thick film, on the water surface, exposed to sunlight.

carried out using either m e m b r a n e c h a m b e r s (McFeters et al., 1974) or dialysis sacs (Hendricks a n d Morrison, 1967) filled with suspensions o f the indicator bacteria a n d immersed in streams revealed t h a t fluctuations in the water composition influenced the p o p u l a t i o n dynamics observed. M i n o r changes were recorded in the water quality of the studied reservoirs. A n initial rise in coliform bacteria was noticed in m a n y studies b o t h in soil (Van Donsel et al., 1967) a n d in water (Dutka, 1973). Faecal coliform bacteria are a t h e r m o t o l e r a n t group, that declines in natural e n v i r o n m e n t s (McFeters et al., 1974). However, C o o k a n d Ruple, (1989), reported increase in the levels o f faecal coliform bacteria a c c o m p a n i e d by increases in Vibrionaceae in post-harvest shellstock oysters during commercial t r a n s p o r t a n d during storage at t e m p e r a t u r e of 10, 22 and 30C. Also, Hendricks and M o r r i s o n , (1967), showed t h a t the aquatic e n v i r o n m e n t associated with a clear m o u n t a i n stream can not only m a i n t a i n p o p u l a t i o n s of enteric bacteria but also can supply sufficient nutrients to initial multiplication and de novo protein synthesis. The Lemna gibba L. is expected to create a nutrient-rich e n v i r o n m e n t t h r o u g h p o p u l a t i o n increase and excretion o f photoassimilated organic compounds. The ability to remain within this environm e n t enables large n u m b e r s o f heterotrophic bacteria, including coliforms, to proliferate even in e n v i r o n m e n t s unsuitable for survival (Haack and McFeters, 1982). Faecal coliform cells in dialysis sacs exposed to sunlight showed typical die-off p a t t e r n experienced in different environments. The decay rate calculated in the present study (0.1768 h 1) was high c o m p a r e d with decay rate calculated in a natural cyprus wet-

APHA (1985) Standard Methods .['or the Examination of Water and Waste Water, 16th edn. American Public Health Association, Washington, D.C. Butler J. E. and Dewedar A. (1991) Gravel Bed Hydroponic Systems (GBH) for sewage treatment. In Techniques ]br Environmentally Sound Water Resources Development (Edited by Wooldridge R.), pp. 96 105. Pentach Press, London. Cook D. W. and Ruple A. D. (1989) Indicator bacteria and vibrionaceae multiplication in post-harvest shellstock oysters. J. Food Protect. U.S.A. 52, 343- 349. Cowan and Steel's Manual For The Identification of Medical Bacteria (1993) (Edited by Barrow G. I. and Feltham R. K. A.), Cambridge Univ Press. Dukta B. J. (1973) Coliforms are an inadequate index of water quality. J. Envir. Hlth. 36, 39~46. Gersberg R. M., Brenner R., Lyon S. R. and Elkins B. V. (1987) Survival of bacteria and viruses in municipal waste water applied to artificial wetlands. In Aquatic Plants for Water Treatment and Resource Recover), (Edited by Reddy K. R. and Smith W. H.). Magnolia Publishing. Haack T. K. and McFeters G. A. (1982) Nutritional relationships among micro-organisms in an epilithic biofilm community. Microbial. Ecol. 8, 115 126. Hendricks C. W. and Morrison S. M. (1967) Multiplication and growth of selected bacteria in clear mountain stream water. Wat. Res. 1,567 7-576. McFeters G. A., Bissonnette G. K., Jezeski, J. J., Thomson C. A. and Stuart D. G. (1974) Comparative survival of indicator bacteria and enteric pathogens in well water. Appl. Microbiol. 27, 823 829. Reddy K. R. and Debusk W. F. (1985) Growth characteristics of aquatic macrophytes cultured in nutrientenriched water. II. Azolla, duckweed, and salvinia. Econ. Botany 39, 200-208. Van Donsel D. J., Geldreich E. E. and Clarke N. A. (1967) Seasonal variations in survival of indicator bacteria in soil and their contribution to storm-water pollution. Appl. Microbiol. 15, 1362-1370.