the Efficacy of BrideliaFerrugineaBenth. Bark Extract in Reducing the Coliform Load and BOD of Domestic Wastewater
*Kolawole O.M1�, Oguntoye
and Olayemi A.B.3
of Microbiology, University of Ilorin,
2Department� of Chemistry, Faculty of Science, University
of Ilorin, Nigeria
3Department� of Microbiology, University
of Ilorin, Nigeria
to whom all correspondence should be addressed:
E-mail: email@example.com; Tel:- 08060088495
Issued 17 August 2006
����������� The efficacy of Brideliaferruginea
bark extract in reducing the coliform load and BOD
of Wastewater was investigated. Phytochemical
screening and chromatographic techniques revealed the bark to contain five
major compounds; polyphenols, steroids, Saponins, Tannins terpenoids
and alkaloids. Comparative studies in the reduction of coliform
load using varying concentrations (0.5% w/v, 1.0% w/v, 2.5% w/v, and 5.0%
w/v) with Alum and Ferric chloride showed that the bark extract was
effective. The optimum dose achieved was 2.5% w/v with a minimum of 24 hours
contact time. The coliform loads were reduced by
63% after 24 hours when the extract was used whereas Ferric chloride achieved
64% reductions and Alum achieved 68% reduction under similar conditions.
����������� Comparative studies of Biological
Oxygen Demand (BOD) removal from the wastewater using varying concentrations
(1%w/v, 5% w/v) with Alum and Ferric chloride showed that the bark extract
achieved 100%. The feasibility of using the bark extract as an additional
coagulant is therefore discussed.
����������� Water is one of the most important
natural resources.(1) In fact, life on earth could
not go on if deprived of this amazing liquid and evolution could never have
taken place without it.(2) Also, water is among the essential requisites that
nature provides to sustain life for plants, animals and humans and the total
quantity of fresh water on earth could satisfy all the needs of the human
population if it were evenly distributed and accessible(3). Water is an
important resource being used in a variety of ways at many different levels,
which produces social, spatial and organizational problems.
����������� There is no end to which water can
be described which led to (4) referring to water as having the peculiar
quality of being an inexhaustible natural resource which,
nevertheless is in short supply.
water demand can commonly be classified according to the nature of the user.
The ordinary classification are domestic, commercial/
industrial and public use. Generally, water can be used for drinking, food
preparation and cooking, cleaning and washing and personal hygiene, vegetable
garden watering, stock watering and other uses including waste disposal and
industrial uses (5) From the earliest times including the period of
industrial revolution, streams, lakes, lagoons and seas have been a natural
place to discharge waste leadings to pollution of these water resources. It
may be in form of solid, liquid or gases; all these adversely affect the
����������� Water quality requirement vary
according to the proposed use of the water. Turbidity which is one of the
character of water considered for it portability may come from erosion of
clay banks, domestic and also from industrial wastes. Water is classified as
polluted if it is clearly dirty in appearance and has an unpleasant taste.
����������� It may contain organic matter,
which makes it unpleasant for drinking and other uses. It may also be altered
in composition or condition, directly or indirectly as a result of man�s
activities (i.e. wastes) so that it becomes less suitable for anyone or all
of the uses it could be put. (7)
����������� The coliform
group has gained widespread acceptance among water analysts as the best
measure of faecal contamination (8). The �coliformbacteria�
belong to the family Enterobacteriaceae and
are gram-negative rods measuring some 2-5 diameters by 0.4 micrometer. The coliform group includes all the aerobic and facultative
anaerobes, non-spore forming rod-shaped bacteria, which ferments lactose with
gas formation within 48 hours at 370C. Coliforms
are generally present in large numbers in human excrement and can be detected
in numbers as small as one in 100ml of water. They are commensals
and constituted part of the intestinal microplora
and are potentially pathogenic elsewhere in the body where they produce pyogenic infections in children, elderly people and those
debilitated by other illnesses (9). Thus, the presence of coliforms
in water sample indicate that intestinal pathogens may be present although
perhaps in a fewer number. Standard of microbiological purity for portable
waters are normally quoted as total coliform or Escherichia
coli concentration per 100ml of sample water. (10)
����������� Water pollution already is a serious
problems in the majority of the developing
countries. A high proportion of domestic and industrial effluents are
untreated and discharged directly to water courses, imigation
canals, and drainage ditches. Allowed to continue, this increased pollution will
reduce the amount of water available for use in the future (11). While the
physical availability of water to each country is unique and usually
constant, demand for water will continue to increase. The problem is how to
balance demand and supply.�
����������� Producing high quality reclaimed
water from wastewater treatment plants requires a paradigm shift for
operators. Communities must explore the construction of tertiary treatment
trains on existing wastewater treatment processes, additional transmission
lines and constructions and development of reclaimed water discharge
alternatives. Planners also must develop wastewater master plans that include
reuse alternatives and treatment levels appropriate to each beneficial reuse.
Consumers also will need assurance that the reclaimed water is safe and they
are adequately protected from deleterious substances. The initial success of
reuse projects will be sustained only if the public perceives that the reuse
of wastewater is healthy and necessary (11).
����������� There are very many methods of
domestic wastewater treatment, which follows the same trend. Chemical
precipitation, an early wastewater treatment method, involved the addition of
early wastewater treatment method, involved the addition of lime, iron sulphate and other coagulants to cause organic and
inorganic solids to settle out of wastewater (12).
����������� Further treatment (tertiary) of a
biological treated effluent is carried out to remove BOD5,
bacteria, suspended solids, specific toxic compounds or nutrients to enable
the final effluent to comply with a standard more stringent than before discharge. This
work intend to study the use of Brideliaferugineabenth bark
extract as a coagulating agent in wastewater treatment.
����������� Brideliaferrugineabenth bark
is the most common savannah Bridelia. It is
usually a gnarled shrub which sometimes reaches the sizes of a tree in
suitable condition. Its common names are Kizni, Kirni (Hausa); Marehi (Fulani),
Iralodan (Yoruba), Ola
(Igbo), Kensangeabia (Boki) (13). However, the flocculating value of the plant
has recently attracted the attention of non-governmental organization (NGO)
and research centres in developing countries.
Recently, the bark extract of the plant has been used for the coagulations of
milk and also lime juice for the formulation of a traditional gargle �Ogunefu� (14). A decoction of
the leaves is used to treat diabetes. It is also used as a purgative and a vermifuge (Cimangaet al.,
1999). (15). The effect of the stem bark and leaf extracts of Brideliaferrugineaon
skeletal muscles has been studied. (16)
����������� The bark extract of the plant has
demonstrated antimicrobial activity against microorganisms commonly known to
cause enteric and secondary upper respiratory tract infections.(17) Also, the
ability of the bark extract in the reduction of total bacterial count,
significant sedimentation of total solids and clarification of river water
has been reported. (18) The reduction of total bacterial count, significant
sedimentation of total solids and clarification of domestic wastewater using
the bark extract has also been reported (19).
����������� Further research into it�s
efficacy in the reduction of coliform load and BOD
removal in wastewater treatment is desirable. With rapid population growth
and the accompanying urbanization, there is bound to be greater demand for
water for public utilities, which in turn, may require more direct water
re-use or intensification of indirect re-use (20). In these circumstances,
removal of harmful coliform from wastewater
discharged into public water course becomes extremely important.
����������� Cost benefits ratios will be
increasingly rewarding for water reuse and recycle research throughout this
millennium. In this study, the effect of Bridelia
on coliform load in wastewater was determined by
the standard plate count. The Biological Oxygen Demand was also determined by
the standard dilution technique.
MATERIALS AND METHODS
����������� Brideliaferruginea bark was collected from the tree of Brideliaferrugineabenth family Euphorbiaceae
from the residential quarters of the University of Ilorin,
Nigeria. A voucher sample was deposited at the Biological Sciences herbarium
of the University.
PREPARATION OF THE BARK EXTRACT
����������� The bark pieces were cut into
small pieces and dried in an oven at 400C for 48 hours (Gallenkamp Oven Bs Size two). The dried pieces were then
pulverized using the laboratory mill (Christy and Norris limited, machine
type 8) and the powder obtained were stored as stock from which appropriate
amounts were taken for experiment.
Two hundred grams of the powdered bark were extracted with solvent
combination of water and ethanol in the volume ratio of 1:2 at room
temperature for 48 hours. (2 days).
����������� The suspension was then decanted
and filtered using sterile Whatsmann Paper No. 1.
The filterate was concentrated to dryness at 450C
in a rotary evaporator. The residue obtained served as the bark extract (21).
����������� The screening procedures were
adapted from those of (22). The extract were
screened for the presence of Alkaloids, Tannins, Terpenoids,
Glycosides, Flavonoids, Saponins,
Anthraquinones and steroids.
1.ALKALOIDS: 1.5ml of 10% HCl
was added to about 5ml of the extracts in a test tube. The mixture was heated
for 20 minutes. It was cooled and filtered 1ml of the filterate
was tested with few drops (5 drops) of Mayers and Draggendorff�s reagents. A whitish yellow and reddish
precipitate observed in the extract tested as indication of the presence of
alkaloids in the extracts.
2.TANNINS: 3 drops of 5% ferric chloride was
added to 1ml of the extract. A greenish black precipitate observed in the
extract was taken as indication of the presence of tannins in the extract.
3.GLYCOSIDES: 10ml of 50% HCl
was added to 2ml of the extracts in a test tube. The mixture was heated in
boiling water for 30minutes. 5ml of fehling�s
solution was added and the mixture was boiled for 5 minutes. A brick-red
precipitate observed in the extract tested as indication of the presence of
glycosides in the extract.
4.SAPONINS: Frothing test: 2ml of the extract in
a test tube was vigorously shaken for 2 minutes. The frothing which persisted
for 5 minutes and when warmed on water bath was taken as indication of the
presence of saponig in the extract.
5.STEROIDS: Liebermann�s Burchard
test: 1ml of the extract was dissolved in 0.5ml of acetic anhydride and
cooled well in ice. This was mixed with 0.5ml of chloroform and 1ml of
concentrated H2S04 was then carefully added by means of
a pipette. At the separating level of the two liquids, a reddish-brown ring
was formed, as indication of the presence of steroids.
6.TERPENOIDS: Ketonicterpenoids were located by dissolving 0.5g of 2,4- dinitrophenylhydrazine in
100ml of 2M HCl. 1ml of the mixture was added to
2ml of the extract. A yellow-orange colouration was
observed as indication of the presence of a terpenoid.
7.FLAVONOIDS: Shibata�s reaction: 3ml of extract
was warmed with three pieces of magnesium turning�s and mixed with 3 drops of
concentrated HCl; An orange pink colouration was taken as indication of the presence of flavonoids.
8.ANTHRA QUINONES: Borntrager�s
test: 5ml of the extract was dried and shaken with 3ml petroleum ether. The filterate was added to 2ml of a 25% ammonia solution. The
mixture was shaken and a red colouration observed
was taken as indication of the presence of anthraquinone.
THIN LATER CHROMATOGRAPHY
����������� The water and ethanol crude
extract (1:2) was spotted and examined using TLC precoated
plates (silica gel Gf 254, 0.25mm Merck W. (Germany).
These were developed using a mixture of petroleum ether and diethylether (3:1). After the development, the
chromatogram was dried and viewed under UV lamp at 366nm and 254nm
respectively. Five components were observed and their corresponding Rf valves were noted.
PREPARATIVE THIN LAYER CHROMATOGRAPHY (PTLC)
����������� This was used to isolate and
purify the phytocompounds in the extract. Glass
plates (20 x 20cm) were coated (0.5mm) with silica gel, (Gf
254, 60mesh) used according to the method described by (23). The solvent
system was petroleum ether �diethylether (3:1).
After development and viewing under UV lamp: The observed bands were Scraped
having correlated their respective Rf values with
the TLC Rf values before elution was done.
EFFECT OF BRIDELIA ON COLIFORM LOAD
load of the wastewater was estimated using the plate count method (24). One
milliliter of a 10-4 dilution was plated on Eosin methylene Blue agar (oxoid) and
incubated at 370C for 48 hours. At 0,24, 48 and 96 hours, 1ml was
carefully measured from the treated portion of the wastewater (Varying
concentrations of the coagulants added to wastewater sample) serially diluted
and enumerated for total coliform on the same
media. The same procedure was followed for the Alum and Ferric chloride
treated water. Experiments were conducted in duplicates.
EFFECT OF BRIDELIA ON BOD
����������� Preparation of Dilution water:
Before used, the distilled water in cotton-plugged bottles was stirred long
enough to permit it to become saturated with D.O (Dissolved Oxygen). The desired
volume of distilled water was then placed in suitable bottle and 1ml each of
phosphate buffer, Magnesium sulfate, Calcium chloride and Ferric chloride
solutions were added for each litre of water (24).
Dilution technique: Several dilutions of the prepared samples were made so as
to obtain the required depletions. The following dilutions were made; 2% and
3.0% in the course of this research work, which falls within the acceptable,
range of 1-5% dilutions for raw and settled sewage as recommended by (24).
����������� Standard dilution water was
carefully siphon into a graduated cylinder of 1,000 to2,000ml
capacity, filling the cylinder half full without entrainment� of air. Carefully mixed samples and
coagulants were added to make the desired dilution and diluted to the
appropriate level with dilution water. The mixture was mixed well with a
plunger �types mixing rod avoiding entrainment of air. The mixed dilutions
were then siphon into two BOD bottles (300mls); one for incubation and the
other for determination of the initial DO in the mixture; stopper tightly and
incubated for 5 days at 200C. The BOD bottles should be
water-sealed by inversion in a tray of water in the incubator or by the use
of a special water-seal bottle. Succeeding dilutions of lower concentrations
were prepared in the same manner.
����������� Titration: 20ml of the liquid
supernatant were dispensed into a conical flask and starch solutions were
added to it, giving a blue-black colouration.
0.025N Sodium sulphate solution was titrated with
the blue-black solution to the starch �iodide end point, which is colourless.
����������� The results of
the chromatographic techniques and phytochemical
screening is represented in Table 1.�
The result revealed that alkaloids, terpenoids,
saponins, steroids, and tannins were present in the
extract. Anthraquinone, flavonoids
and glycosides were not found in the extract.
����������� This layer and preparative thin
layer chromatographic techniques were used to isolate different phyto-components from the crude extract. The crude
extract developed with petroleum ether-diethyl ether (1:3) revealed four
components which were thoroughly purified using PTLC to give the phytocompounds as shown in Table 1.
����������� The results of
the effect of the coagulants on the coliform load
of the wastewater sample is represented in Table 2. Result showed that
there was significant reduction in coliform load at
any particular dosage of the coagulant for the processed samples compared to
the control (raw wastewater sample). However, after a holding time of 96
hours, a higher percentage reduction was achieved with 2.5% w/v dose of bark
extract compared to Alum and Ferric chloride. The greater the amount of the
coagulants, the more the acidity and the more the reduction in coliform load until an optimum dose is achieved.
����������� The results of
the effectiveness of the coagulants in the reduction of Biological Oxygen
Demand (BOD) is shown in Table 3. It was revealed that there was
appreciable reduction in BOD5 of the test wastewater sample at any
concentration of coagulants used. Brideliabark extract exhibited the best performance regarding BOD5
removal. It was also found to be more effective than others at any dosage. Brideliabark extract achieved total
depletion (100% reduction) of BOD5 in the wastewater sample.
����������� During the past decade, there has
been growing concern that the world is moving towards a water crisis. Water
is increasingly scarce in dry climate regions (for example, Africa
and South Asia), and there are major political
implications for the scarcity of water in some regions (for example, the
middle East) (25). Issues of both water quantity and quality are of concern.
The reuse of wastewater is one of the main options being considered as a new
source of water in regions where water is scarce. The standards required for
the safe use of wastewater and the amount and type of wastewater treatment
needed are contentious. The cost of treating wastewater to conform to high
microbiological standards can be so prohibitive that in many developing
countries, the use of untreated wastewater is effectively unregulated (25).
����������� It has been shown in many studies
that domestic wastewater usually contain various pathogenic organisms. Some
studies revealed that species such as the agents causing typhoid fever,
bacillary dysentery, amoebic dysentery, ascaris and
other protozoan and helminthic diseases were
isolated from wastewater. Other studies have indicated the detection of major
enteric viruses in raw domestic wastewater (Ademoroti,
1980). Detection of total coliforms in any kind of
water implies the possible presence of pathogenic microorganisms in water and
indicates that faecal pollution of the water has
occurred. Removal of total coliforms from water,
therefore safeguards public health risks (26).
����������� In this work, the coagulating
properties of Brideliaferrugineabenth bark have
been established and found to compare favourably
with other coagulants such as Ferric chloride and Alum. The result of the
chromatographic techniques and phytochemical
screening revealed the bark extract to contain tannins and alkaloids as its
major bioactive constituents while other active agents includes steroids, terpenoids and saponins (Table
1). This observation agrees with the result obtained by (27).
����������� Coagulating properties of Brideliaferrugineabenth bark have been established and found to
compare favourably with other coagulants such as
Ferric chloride and Alum in the reduction of total bacteria count of river
water (18). In this study, the ability of the bark extract to reduce the coliform load of wastewater can be explained in two ways;
one, it made the wastewater acidic, thereby assisting in the removal of
sizeable percentage of total coliform load. Also,
the cations forms complexes with the enteric bacteria, which caused
further reduction in the total coliform population.
(20) The percentage removal of the enteric bacteria was appreciable even at
lower doses of the coagulants, (Table 2). This result is in consonance with
the work done by (20) who explained the removal of coliform
from sewage through chemical coagulation and flocculation. The effectiveness
of the coagulants in Biological Oxygen Demand (BOD) removal was revealed in
this study. Brideliabark extract
showed outstanding activity of 100% total depletion of Biological Oxygen
Demand (BOD) of the wastewater sample at any concentration compared to Ferric
chloride and Alum (Table 3).
����������� These encouraging findings further
support the recommendation of Brideliaferruginea bark extract as an additional coagulant
because it is cheap, readily available and serves as an easy means of reuse
alternatives of wastewater, especially in remote areas. Driven by high water
demand, research and technology for wastewater treatment and monitoring will
continue to break the barriers of affordability, ease of operation, safety
and efficiency (11).
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++ = Highly Present,
Table 2. Effect
of Coagulants on the Reduction of Coliform Load of
Contact time (hrs)
Percentage concentration (%
Percentage Reduction %
Table 3. Effects on
Biological Oxygen Demand (BOD) Using Varying Concentrations of the Coagulants
in Wastewater Sample.
Concentrations of coagulants/ sample
Percentage dilution (%)
Sample volume (ml)
Control (wastewater alone)
1% w/v Alum + Wastewater
6mls + 3mls
5% w/v Alum + wastewater
3mls + 3mls
6mls + 3mls
1% w/v FeCl3 +
3mls + 3mls
6mls + 3mls
5% w/v FeCl3 +
6mls + 3mls
1% w/v EXTRACT + wastewater
3mls + 3mls
6mls + 3mls
5% w/v Extract + wastewater
For 2% v/v Dilution � 3mls of wastewater + 3mls
of coagulant in solution.
3% v/v Dilution � 6mls of Wastewater + 3mls
of coagulant in solution.