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 – 9TH 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 pH of 12.0 was attained
and left at that pH for 30 minutes, while nothing was added to the untreated septage, which served
as a control. This experiment is pH 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 from1600/100ml
for untreated to 14/100ml 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
(OH2)] stabilization as a safe pre-disposal treatment model.
MATERIAL AND METHODS
Source of Lime: Quick Lime (CaO) (10g) 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 2g of slaked lime [Ca (OH) 2] was added into 100ml 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 pH of the liquid septage for the treated sample was monitored several times during the lime
addition process, until pH of 12.0 was attained. When a pH of 12.0 was reached, the contents were maintained at
that pH for a period of 30 minutes. At this pH ammonia (NH3) is released, there by making the medium alkaline.
(If the pH 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 (pH 12.0) and the untreated septage samples were carried
out. About 1.0ml 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 transferring1ml from the 10-1 dilution into
another 9ml of fresh peptone dilutent (10-2 dilution).
A total of
dilutions were serially made. Aliquots (0.lml) of various dilutions were spread –
plated on surface dried Deoxycholate citrate agar, (DCA) (Oxoid CM 163), MacConkey (Oxoid
CM7) and Nutrient agar (Antce product) and incubated at37oC 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 MacConkey plates for
purity checks, and expressed as colony forming unit per gram (cfu/g).
-3
10
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 MacConkey 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 1ml each of the undiluted treated sample. The second and third sets of five test tubes
contained 1ml 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 37oC 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 pH of the samples, both treated and untreated were monitored as soon as the slaked lime [Ca
(OH) 2] was added until a pH of 12.0 was obtained. The pH was monitored with a pH meter (Pye
Unicam model ASU-118: HM 30s).
RESULTS
Four different genera of microorganisms were isolated from the untreated sample, while only one was
isolated from the treated (Table 1). Bacillus Subtilis
while Salmonella typhi
5
(10
cfu/g), E. coli
5
(10
4
(10
cfu/g) was isolated from the treated sample,
cfu/g), Proteus vulgaris
3
(10
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 1600/100ml for untreated to 14/100ml for treated septage. The application of lime [Ca
(OH) 2] stabilization reduced the intestinal pathogens to innocuous level.
TABLE 1. EFFECT OF LIME STABILIZATION ON BACTERIAL PATHOGENS.
Type of organism
Bacillus subtilis
Proteus vulgaris
Salmonella typhi
Escherichia coli
()
Untreated Domestic
Septage (cfu/g)
10
1.5 x 10 (4.18)
5
2.0 x 10 (3.30)
5
2.0 x 10 (5.30)
5
3.0 x 10 (5.48)
Treated Domestic
Septage (cfu/g)
10
1.4 x 10 (4.15)
-
Figures in parenthesis represent Log10 of bacterial numbers -= No growth.
TABLE 2. CHARATERIZATION OF MICROORGANISMS ISOLATED FROM
UNTREATED DOMESTIC SEPTAGE.
Physical and
Biochemical
Characteristics
ISOLATES
1
2
3
4
Colony on DCA,
MacConkey media
Pale-raised, cleared
zone on Medium
with black center
and entire edge
Flat, dried colony
Smooth
Muciod
and
raised
colony
Fishy smell
swarming
appearance
Cell characteristics
GM-Ve rods
GM +Ve spore
forming rods
GM-Ve
GM –ve rods
rods
Motility
+
+
+
+
Catalase
-
+
+
+
Indole
-
+
+
+
Urease
-
+
+
+
Suophide (H2S)
+
-
-
+
Citrate
+
-
-
+
Acid from Lactose
-
-
AG
-
Glucose
A
AG
Ag
-
Mannitol
AG
AG
Ag
-
Sucrose
A
Ag
Ag
-
ND
Positive
ND
A
Salmonella typhi
Bacillus subtilis
E.coli
Proteus vulgaris
Hydrogen
Nitrate Reduction
Probable identity
ND = Not Determined, AG = Acid and Gas, += Positive, -= Negative = weak positive reaction
TABLE 3. MOST PROBABLE NUMBER (MPN) INDEX OF DOMESTIC SEPTAGE
BASED ON 95% CONFIDENCE LIMITS.
Number of tubes giving Positive reactions
H
Type of sample
Untreated (Control)
Treated septage
p
Range
5 undiluted
(Neat)
5 Dilutions
-1
(10 )
5 Dilutions
-2
(10 )
ND
5
5
5
1600
H
5
5
2
540
H
3
2
0
14
p 10.0
p 12.0
ND = Not Determined
MNP
Index per
100ml
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 (NH3) 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 (NH3) 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 pH of 12.0 agrees with the finding of Henry et al., (1983) who reported that pH
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.
REFERENCES
1. Buchanan, R. E and Gibbons, N. E., 1995. Bergey’s Manual of Determinative
Bacteriology(18thEdition) William and Wilkins, Baltimore, U.A Waverly Press Inc.
2. Cheesbrought, M., 1984. Medical Laboratory Manual for Tropical Countries, (Vol.
II) Elbs, Butterworthe U.K. 40-411.
3. EPA. 1991. Environmental Protection Agency. Supplemental Manual of the
Development and Implementation of Local Discharge Limitations Under The Pretreatment Programme Washington D.C. Office of Water Publication. No En-336.
4. EPA. 1994 Environmental Protection Agency. Handbook. Septage Treatment and
Disposal. Cincinnati, OH. EPA/625/6-84-009.
5.
6.
5. EPA. 1995. Environmental Regulations and Technology. Control of Pathogen and
Vector Attraction in Sewage Sludge. Process Design Manual, 1-10.
6. Henry, D. P., Frost, A. J., Samuel, J. L., Boyle, D. A. and Thomson, R. H., 1983.
factors Affecting The Survival of Salmonella and Escherichia coli in Anaerobically
Fermented Pig Waste. J of Appl. Bact. 55:89-95.
7. Sloat, S. and Ziel, C., 1991. The Use of Indicator Organisms to Assess Public Water
Safety. American Public Health Association Washington D. C. Pp 882-884.
8. Tortora, G. J., Funke, B. R. and Case C. L., 1992. Aquatic Microbiology and Seqage Treatment. In
Microbiology – An Introduction (4th Edition). Benjamin/Cummings Publishing
Company, U.K. pp 684-685.
9. Wachukwu, C. K., 1998. Production by Process and Substrate Modification of Ugba
and Daddawa-two Alkaline Fermented Nigerian Food Condiments Ph.D Thesis, Rivers
State University of Science and Technology, Port Harcourt, Nigeria.
1
10. William, P. C. and Barbara, W. S., 1999. Human Waste Disposal. In:Environmental
Science (5th Edition) McGraw-Hill Publishers. USA 452-454.