EFFECT OF LIME STABILIZATION ON INTESTINAL PATHOGENS IN

EFFECT OF LIME STABILIZATION ON INTESTINAL PATHOGENS IN DOMESTIC SEPTAGE: - A PRE-DISPOSAL TREATMENT WACHUKWU, C. K Department of Medical Laboratory Sciences Rivers State University of Science and Technology, Nkpolu-Oroworukwo, P. M. B. 5080, Port Harcourt. Nigeria. Email: conkiwa@yahoo. com ABSTRACT #: 379

EFFECT OF LIME STABILIZATION ON INTESTINAL PATHOGENS IN DOMESTIC SEPTAGE: - A PRE-DISPOSAL TREATMENT WACHUKWU, C. K Department of Medical Laboratory Sciences Rivers State University of Science and Technology, Nkpolu-Oroworukwo, P. M. B. 5080, Port Harcourt. Nigeria. Email: conkiwa@yahoo. com BEING A PAPER PRESENTED AT THE INTERNATIONAL ASSOCIATION FOR IMPACT ASSESSMENT (IAIA) IN SEOUL-KOREA, BETWEEN 3 – 9 TH JUNE 2007.

ABSTRACT The effect of lime stabilization on intestinal pathogens from septage samples was investigated. Two septage samples, treated and untreated were analyzed microbiologically for total heterotrophic count (THC) or viable counts and Most Probable Number (MPN) for coliform estimations. Lime [Ca (OH) 2] was added to the treated septage until a p. H of 12. 0 was attained and left at that p. H for 30 minutes, while nothing was added to the untreated septage, which served as a control. This experiment is p. H and time dependent. Results of the THC obtained from treated septage showed no growth of intestinal pathogen, except few growth of Bacillus subtilis 4 4 (10 cfu/g). The untreated septage showed heavy growth of Salmonella typhi (10 cfug), Proteus vulgaris (104 cfu/g), Escherichia coli (105 cfu/g) and Bacillus subtilis (104 cfu/g). The MPN result for coliform estimation also showed a drastic reduction in faecal coliform from 1600/100 ml for untreated to 14/100 ml for treated septage. The results of the this study showed that with the application of lime or any other alkali in domestic septage, the level of intestinal pathogens will be reduced, if not completely eliminated. Keywords: Lime Stabilization, septage, pathogens, alkaline, disposal, sewage-sludge *Author for correspondence.

INTRODUCTION Human waste generation is as old as the first man created and its concern to man is dated back to the same period. As human race gradually developed from the wandering prehistoric stone age, applying himself to settlement, the problem of human waste disposal assumed a proportional status with the settlement sizes (EPA, 1991). The task of community waste disposal has become increasingly more difficult with the rapid increase in population. This population increase within some major cities like Port Harcourt in Rivers State of Nigeria, contributes to an increase in the number of septic tanks, resulting to greater volume of septage being disposed daily, without treatment. Domestic septage (material removed from septic tanks) has beneficial plant nutrients and soil conditioning properties. However, it may also contain bacteria, viruses, protozoa, parasites and other microorganisms that can cause diseases like typhoid fever, dysentery, cholera, diarrhoea, and gastroenteritis (William and Barbara, 1999, EPA, 1994). When sewage sludge is applied to land or placed on surface disposal site, humans and animals can be exposed to these pathogens directly by coming into contact with sewage sludge or indirectly by consuming drinking water or food contaminated by sewage sludge pathogens. Insects, birds, rodents and even farmers can contribute to these exposure routes by transporting sewage sludge pathogens away from the site (EPA, 1995). To protect public health from these organisms and from the pollutants that some sludge contain, it is imperative therefore, to devise an efficient and a cost effective means of treating domestic septage. The most obvious way of doing this is the lime (Calcium Hydroxide) stabilization method. The present study is aimed at reducing bacterial pathogens in faeces using lime [Ca (OH 2)] stabilization as a safe pre-disposal treatment model.

MATERIAL AND METHODS Source of Lime: Quick Lime (Ca. O) (10 g) was purchased from commercial dealers in Port Harcourt Nigeria and slaked by sprinkling the desired quantity with water. Collection of septage sample: - The septage samples were collected aseptically from the dump-site at Owulejuan Community, Abuloma in Trans-Amadi Industrial Area in Port Harcourt, Rivers State, Nigeria. Two sterile conical flasks (250 ml capacity) were used to collect the septage samples (200 ml) directly from the rear discharge valve at the bottom of the truck’s tank, as it was being discharged in land. Hand gloves and nose masks were worn and with a gentle flow of the sewage discharge, the samples were collected carefully to avoid splash and immediately taken to the laboratory for analysis. Treatment of septage samples with slaked lime. Two separate septage samples were used for this experiment, (treated and untreated septage). In the treated sample, about 2 g of slaked lime [Ca (OH) 2] was added into 100 ml of septage sample in sterile conical flask, and mixed thoroughly. In the untreated septage sample, lime was not added. This also served as the control sample. Thereafter, the p. H of the liquid septage for the treated sample was monitored several times during the lime addition process, until p. H of 12. 0 was attained. When a p. H of 12. 0 was reached, the contents were maintained at that p. H for a period of 30 minutes. At this p. H ammonia (NH 3) is released, there by making the medium alkaline. (If the p. H was not up to 12. 0 additional alkali can be added and mixed with the domestic septage). Microbiological Analysis of Septage Samples For the microbiological analysis of septage samples, two categories of tests were carried out. They include the Total Heterotrophic Counts (THC) for viable bacteria and Most Probable Number (MPN) for monitoring faecal coliform bacteria. Total Heterotrophic Count (THC) A ten-fold serial dilution of both the lime treated septage (p. H 12. 0) and the untreated septage samples were carried out. About 1. 0 ml each of the samples was transferred to 9. 0 ml of sterile 0. 1% peptone water diluents in a test tube, giving a 10 -1 dilution. Another ten-fold dilution was made by transferring 1 ml from the 10 -1 dilution into another 9 ml of fresh peptone dilutent (10 -2 dilution).

-3 A total of 10 dilutions were serially made. Aliquots (0. lml) of various dilutions were spread – plated on surface dried Deoxycholate citrate agar, (DCA) (Oxoid CM 163), Mac. Conkey (Oxoid CM 7) and Nutrient agar (Antce product) and incubated at 37 o. C for 24 -48 hours. Growth occurred on the untreated samples, with distinct characteristics, while the treated sample did not show growth of any intestinal pathogens. This experiment was repeated three times to authenticate the results obtained. Colonies that grew were subcultured onto fresh DCA and Mac. Conkey plates for purity checks, and expressed as colony forming unit per gram (cfu/g). Standard microbiological methods were employed for the identification of the various isolates. (Buchanan and Gibbons 1995; Cheesbrough, 1984) Most Probable Number (MPN) This test was basically for monitoring of faecal coliform bacterial (FCB) in the samples. A total of 30 test tubes containing sterile Mac. Conkey broth and Durham tubes were sued for both the treated and untreated samples, each having fifteen test tubes. The first set of five test tubes contained 1 ml each of the undiluted treated sample. The second and third sets of five test tubes contained 1 ml each of 10 -1 and 102 dilutions of the samples respectively. Similar analysis was done with the untreated samples. This experiment was replicated thrice. Inoculated tubes were incubated at 37 o. C for 24 – 48 hours. Tubes showing positive, with the production of acid and gas were read using a statistical probability table (MPN index based on 95% confidence limit) (Sloat and Ziel, 1991). H p Determination The p. H of the samples, both treated and untreated were monitored as soon as the slaked lime [Ca (OH) 2] was added until a p. H of 12. 0 was obtained. The p. H was monitored with a p. H meter (Pye Unicam model ASU-118: HM 30 s).

RESULTS Four different genera of microorganisms were isolated from the untreated sample, while only one was isolated from the treated (Table 1). Bacillus Subtilis 4 (10 cfu/g) was isolated from the treated sample, while Salmonella typhi (105 cfu/g), E. coli (105 cfu/g), Proteus vulgaris (103 cfu/g), and Bacillus subtilis (104 cfu/g), were isolated from the untreated domestic septage. Table 2 shows the morphological and biochemical characteristics and probable identities of the microorganisms isolated from the untreated sample. The Most Probable Number (MPN) index of Escherichia Coli, based on 95% confidence limits is given in Table 3. The coliform numbers were reduced from 1600/100 ml for untreated to 14/100 ml for treated septage. The application of lime [Ca (OH) 2] stabilization reduced the intestinal pathogens to innocuous level.

DISCUSSION A major environmental concern is the pollution of the environment through indiscriminate dumping of untreated domestic septage into rivers, streams or land. Such domestic septage generally contains major human pathogenic organisms such as bacteria, viruses, protozoa and helminthes (EPA, 1995). Only bacterial pathogens were sought for and no attempt was made to look for viral or parasitic pathogens, as the method of treatment used may have little or no effect on them. The dangerous consequences of dumping untreated septage material into the river or land cannot be over emphasized, as these are known to result into outbreaks of epidemics and eutrophication of the rivers (Tortora et al. , 1992). The simplest and most economical technique for treating domestic septage is the lime stabilization method. Lime (Calcium Hydroxide) stabilization is a treatment technique designed to reduce the level of pathogenic bacteria, lower the potential for putrefaction and reduce objectionable odours. Lime [Ca(OH)2] an alkaline material, was added to the domestic septage in sufficient amount, to H H H raise the p to 12. 0, and holding it at that p for 30 minutes. At this p of 12. 0 ammonia (NH 3) is released, hence making the environment alkaline (Wachukwu, 1998). The addition of lime caused a drastic reduction in bacteria pathogens particularly the intestinal pathogens, as they could not grow in its presence. Only Bacillus subtilis was able to survive the lime stabilization treatment. This could be as a result of the heat resistant nature and endospore formation of the organisms, the addition of lime into the septage material produced an exothermic reaction where little heat was produced. The release of ammonia (NH 3) at the end of the reaction, H leading to the elevation of p inhibited the growth of most intestinal pathogens, like Salmonella typhi, that is capable of causing typhoid fever. Other organisms that could not survive the lime treatment effect are Proteus vulgaris and Escherichia coli (Table 1). The inability of pathogens to grow at an elevated p. H of 12. 0 agrees with the finding of Henry et al. , (1983) who reported that p. H of 4. 0 and 9. 0 are inimical to the growth or survival of Salmonella typhimurum, a food poisoning H organism. It therefore, means that an increased p and ammonia gas were critical in determining the survival of these organisms in domestic septage. Similarly, there was a reduction in coliform estimation of the treated septage. The growth of a typical faecal coliform (E. coli) was drastically reduced in the presence of lime stabilization. (Table 3).

The implication of finding E. coli in faeces is a normal phenomenon since the small intestine is its habitat but it also means that other intestinal pathogens like Salmonella typhi, Vibrio cholerae or Shigella dysenteriae that are capable of causing typhoid fever, cholera or dysentery, could be present. Therefore, if such untreated faeces or domestic septage was dumped into the river or land, it may create a potential for human exposure to these organism. To protect public health from these organisms and from the pollutant that some domestic septage contains, sewage sludge or faeces should be treated before disposal. Instead of looking for a more sophisticated or complex technique to use for sewage treatment, lime stabilization method, which is cheap, economical, readily available and less cumbersome to prepare, could be used as a pre-disposal treatment. All haulage vehicle operations responsible for disposal of sewage and occupants of residential houses with septic tank should be educated or trained on how to apply lime or an alkaline to their truck’s tank or septic tanks before and after haulage. With the application of lime or any other alkali in domestic septage, the level of intestinal pathogens will be reduced, if not completely eliminated. ACKNOWLEDGEMENT The author wishes to thank Prof. N. O. Isirima-Director of Institute of Pollution Studies, Rivers state University of Science and Technology, Port Harcourt, for allowing me to use their facilities for the work.

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