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Abstract
This paper describes the factors which may affect the die off rates of the bacterium Escherichia coli (E Coli) in a waste treatment system known as high rate algal Pond (HRAP), similar to a waste stabilization pond (WSP). The disinfected water can be used in the irrigational and recreational purposes and it can reduce the risk of pathogenic contamination. The presence of the microorganisms is assessed by the E -Coli organism. There are noted evidences that the e coli die-off rate depends on the pond depth. The main reason of this effect may be the light attenuation i.e. the more the depth of the pond the lesser will be the light exposure per unit volume. The die- off rate constant of E Coli , kb which has the minimum and the maximum values of (0.36-2.34)d-1 was measured in pond saturated with nutrients broth ranging from the depth of 12 to 34cm. From the series of experiments done it was found that the E Coli die- off rate depends on the ph inattenuation of light and pond depth. The light penetration through the pond was calculated and it was shown that it plays an important role in measuring kb. Many equations related to die –off rate constant were calculated which depended on the environmental and physical factors.
Keywords
Depth, die –off rate constant, e coli, high rate algal ponds. Inattenuation, ph, waste water treatmemt
Introduction
High rate algal ponds (HRAPS ) are adaptation of waste stabilization ponds which incorporates a simple mixing system and baffles with the pond to hydraulic flow, thus increasing treatment efficiency and reduction pond area, this is achieved by optimizing condition for algal photosynthetic oxygen production (Fallowfield & Garret,1985 a). The disinfected water can be used in the irrigational and recreational purposes and it can reduce the risk of pathogenic contamination. There are few reports on the environmental and the operational factors, which affect the E- Coli, die off rate in the HRAPS.Oswald (1980) reported a 99% reduction in facial coli form numbers in ponds operating in California. Sebastian and Nair (1989) reduction from an influent of 1010 e coli 100 ml-1 to 105 and 102 e coli 100 ml-1 in the treated effluence of the HRAPs mass culture of seconesmus obloquies operated at 2 and 4 day residence times.
Various factors which affect coliform die off rates in an aquatic systems have been investigated including the irradiance (Curtis et al,1992 ,Davi Colly and Evisson 1991) temperature (Mara and Silva 1979,flint et al,1987) ph (El Hamouri et al ,1994,Liran et al,1994) dissolved oxygen (Curtis et al,1992) and predation (Meziroui et al ,1992)
In this paper we have done a series of experiments conducted in HRAPS.the main aim of the research is to determine the e coli die off rate constant in ponds operated in various depths. To make the design programme for HRAP treatment system more rational ,all the environmental and operational factors affecting kb were calculated.
Materials and Methods
HRAP design and operation
Two high rate algal ponds were prepared having each an area of 13.1sqm.the ponds were constructed in the form of meandering raceways. The HRAP was operated at the depth of 12, 17, 24 and 34cm.the nutrients are supplied as secondary treated sewage from Jopper sewage works.in all the experiments e-coli was grown in 10 l nutrient broth, aereated culture (500ml/min;25degree centigrade /24 hours. The ponds were inoculated at 9a.m.with e-coli (~10p/100ml pond) the numerated every 6 hours using MPN methods as per bacteriological examinations of water supplies.
Experiment 1
Experimental procedure: This experiment was done to check the methodology that the death of e coli obeys chick’s law.that is the there is a constant ratio between the number of cells dying and exposure time.
Experiment2
The ponds were op HRAP was operated at the depth of 12, 17, 24 and 34cm.the nutrients are supplied as secondary treated sewage from Jopper sewage works. The mean daily surface light intensity was measured and the light reaching the bottom of the pond was calculated from the attenuation equation.
Attn= is x 0.79xexp ((-dx (0.63 x od560)) + o.o301) / 2.303)
Where:
- is = irradiance at surface
- D =depth (cm)
- Od560=optical density at 560nm
The e coli die off rate constant was calculated from the slope of semi log plot against time of count at zero time (N)(N0) minus count at time t (Nt) which forms a straight line(chick’s law).
Experiment 3
Experimental procedure: The hypothesis of this experiment is that if light is important for e coli die off then there may be differences in die off cultures containing more algal biomass i.e. More algae is greater than more attenuation which is in turn greater than less light penetration into the culture which is greater than reduced die-off rates.
Duplicate beakers with 800ml 0.2 m tris (hydoxylmethyl) aminomethane HCL having buffer ph 9.0 was inoculated with 14 day culture of the alga chlorella vulguris to give the final dry matter concentration of 200,400 and 600 mg/lit.which was placed on magnetic stirrers (150rpm)on the roof inoculated with E.Coli.The E-Coli was determined as experiment 2 after every 6 hours for 36 hours.the light and temperature was recorded and die –off was calculated for various time intervals i.e. Log(n0-nt).
Experiment 4
Experimental procedure: All the experimental condition was same as in experiment 3 except all the beakers were kept in the darkness which were repeated to get a reliable answer. It showed that light is required for algal photosynthesis.when proceeding rapidly the hydrogen ions are consumed and the ph of the pond rises.This rise is greater if the algal cells are using nitrate as a nitrogen source. So we can say that light may affect e-coli die-off rate via ph changes. Carbonate /bicarbonate buffer (0.2m) was used to keep the ph above 9 ( between 9.5-10).
Results
The range of values for biological and physical values are given as follows:
Experiment 1
The death of e coli obeys chick’s law ,that is the there is a constant ratio between the number of cells dying and exposure time ,and the slope of the line is kb. The graph of the above experiment is as follows:
Experiment 2
Light reaching to the bottom of the ponds were calculated using attenuation equation
Attn= is x 0.79xexp ((-dx (0.63 x od560)) + o.o301) / 2.303)
Where:
- is = irradiance at surface
- D =depth (cm)
- Od560=optical density at 560nm
The E Coli die off rate constant was calculated from the slope of semi log plot against time of count at zero time (N)(N0) minus count at time t (Nt) which forms a straight line(chick’s law)
Nt=N0 e-kbt
I.e. Rate constant ,kb= (N0 – Nt)/t
The die of rate constant of E Coli ,kb which has the minimum and the maximum values of (0.36-2.34) d-1 was found at the depth of 12 and 34 cm respect.
The graph of the experiment is as follows:
Experiment 3
Duplicate beakers with 800ml 0.2 m tris (hydoxylmethyl) aminomethane HCL having buffer ph 9.0 was inoculated with 14 day culture of the alga chlorella Vulguris to give the final dry matter concentration of 200,400 and 600 mg/lit.which was placed on magnetic stirrers (150rpm) on the roof inoculated with E.Coli.The E-Coli was determined as experiment 2after every 6 hours for 36 hours. The light and temperature was recorded and die –off was calculated for various time intervals i.e. log(N0-Nt):
Light 412 cal /cm2/d ; temp 14-280c
Red line (no algae),Blue line(200 mg algae),Green line (400mg algae),Yellow line(600mg algae).
Experiment 4
Light may affect e-coli die-off rate via ph changes. Light is required for algal photosynthesis when proceeding rapidly concentration of hydrogen ions increases and ph also increases. But this happens when nitrogen is taken as nutrient. Carbonate /bicarbonate buffer (0.2m) was used to keep the ph above 9 (between 9.5-10).Experimental conditions are all the same except that all the beakers are kept in the dark and results are repeated to get desired results.
The graph for the above experiment is as follows:
Discussions
The results of the experiments show that in high rate algal ponds kb is mainly influenced by the pond Ph , depth and penetration of the light. The Ph of the pond is obtained by the buffering capacity , algal photosynthesis and nitrogen assimilation (Fallowfield and Garret 1885). The death of E coli die –off rate is directly proportional to the exposed time. Curtis et al.(1992) suggested the interaction between irradiance, ph and reactive oxygen species exert a bacterial life in WSPs.
Bacterial degradation of organic mater to ammonia also affects the equilibrium Ph and ultimately influences kb (Pearson et al, 1987 ). If light is important for E -Coli die off then there may be differences in die off cultures containing more algal biomass i.e. More algae is greater than more attenuation which is in turn greater than less light penetration into the culture which is greater than reduced die-off rates. Light may affect E-Coli die-off rate via ph changes. Light is required for algal photosynthesis when proceeding rapidly, concentration of hydrogen ions decreases and ph also increases. But this happens when nitrogen is taken as nutrient. Algal photosynthesis causes an increase in the ph due to the simultaneous removal of the CO2 and H+ ions (Fallowfield et al. 1996). Domestic buffering capacity, which ultimately affects the ph, depends on the wastewater being treated. Domestic sewage is poorly buffered by the carbonate –bicarbonate equilibrium. Whereas the piggery waste is buffered by its phosphate content (Hill& Lincoln 1981). Croma et al. (1996) reported the occurrence of the nitrification within HRAP treating a synthetic sewage, consequently nitrate may be from the predominant form of the nitrogen available for the algal growth. The HRAPs used in the study showed variations in the ph and may attain ph value of Ph above 9 (i.e.9.5-10). From the experiments done it is clear that there is a relationship between ph and kb. As ph drop below 9 there is a increase in E-Coli concentration but as the ph becomes greater than 9 there is a rise in E- Coli die off rate.
Thus we can say that the E-Coli die off rate depends on many factors mainly ph , depth and light attenuation. But we are not sure which one is more effective on the die –off rate constant. There are many opinions regarding the importance of various factors affecting rate of death of algae but all the factors contribute towards it.This is supported by ( Davis –Colly et al.1999 ). So the factors responsible for the rate of death of algae are, values of ph greater than 9 (Sebastian & Nair, 1984 ; pearson et al. 2004).
Reference
Arthur J.P.(1983) notes on the designs a d operation of the waste stabilization ponds in warm climate of developing countries, world bank technical page no.7,the world bank Washington D.C U.S.A.
Bailey , C.A, Nerhof, R.A and Tabor, p.s.(1983) effect of solar radiation on amino acid uptake in Chesapeake bay bacteria. Applied and environmental microbiology, 46, 44-49.
Cromar,N.J.(1994) composition of biomass and computer modeling of high rate algal ponds. PhD thesis, Napier University, Edinburgh.
Cromar, N.J., Fallowfield,H.J. And Martin, N.J.(1996) influence of environmental parameters on biomass production and nutrient removal in a high rate alga pond operated by continuous culture. Wat. Sci. Tech. 34(11).
Curtis, T.P, Mara, D.D, And Silva, S.A. (1992) influence of ph oxygen and humic substances on ability of sunlight to damage faecal coliforms in waste stabilization pond water. Applied and environmental microbiology, 58 ,1335-1343.
Davies-Colley, C.M. And Evison, l.m. (1991) sunlight and the survival of enteric bacteria in natural waters. Journal of applied bacteriology, 70, 265-274.
El Hamouri, B., Khallayoune, K, Bouzoubaa, K, Rahallabi, N And Chalabi, m.(1994) high rate algal pond performances in faecal coliform and helminth egg removal. Water research, 28, 171-174.
Fallowfield H.J. & Garrett, M.K. (1985a) the treatment of wastes by algal culture. Journal of applied bacteriology symposium supplement, 59, s187-s205.
Fallowfield H.J. and Garrett, M.K. (1985b) the photosynthetic treatment of pig slurry in temperate climatic conditions: a pilot plant study. Agricultural wastes, 12 , 111-136.
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Falowfield H.J.,N.J.Cromar & L.H.Evision 1996 published by Elsivier science ltd.
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