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African Journal of Biotechnology Vol. 8 (6), pp. 1090-1109, 20 March, 2009
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2009 Academic Journals
Full Length Research Paper
Biodegradation of synthetic detergents in wastewater
Olusola A. Ojo1* and Benjamin A. Oso2
1Department of Microbiology, Lagos State University, Badagry Expressway, P.O. Box 12142, Ikeja, Lagos-Nigeria.
2Department of Botany/Microbiology, University of Ibadan, Nigeria.
Accepted 13 January, 2009
A total of 76 wastewater samples were randomly collected from pharmaceutical, textile, and detergent-
manufacturing industries as well as the Agbara Sewage Treatment Plant (STP). Thirty-eight samples
each in 2-L plastic containers were collected for morning and evening effluent used for this study.
Composite samples were later developed and the physico-chemical properties of these samples
determined. The physico-chemical properties of the composite wastewater influenced the selected
microbial population adapted to utilization of detergent components. The optimum temperature range
of the composite wastewater was 33.9 – 34.3oC while the mean optimum pH ranged from 6.9 – 8.8 for the
laboratory simulated biodegradation of test detergents. Although, the fungal consortium was eliminated
as the medium approached the alkaline pH, this is as a result of the metabolites produced. The macro-
elements, the BOD and the hydrocarbon concentration of the composite effluent were above the EU and
FEPA limits for discharged effluent. The composite effluent was thereafter spiked with test detergents
(Elephant, Omo, Klin, Ariel Persil, Teepol, and SDS) at 0.01% (w/v) and its progressive degradation
monitored for 30 days. The microbial detergent-degraders population changed between Day 0 and 15,
thereafter it stabilized. The heterotrophic bacterial count from the seventy-six randomly collected
effluent samples was 42.9 x 106 cfu/ml, while the mean bacterial detergent-degrader population was
20.94 x 106 cfu/ml. The mean fungal population from the randomly collected effluent sample was 4.5 x
106 cfu/ml. The bacterial detergent-degraders characterized and identified include Pseudomonas
aeruginosa, Escherichia coli, Enterococcus majodoratus, Klebsiella liquefasciens, Enterobacter
liquefasciens, Klebsiella aerogenes, Enterobacter agglomerans, Staphylococcus albus, Proteus sp.,
Klebsiella oxytoca and Brevibacterium sp., while the fungal detergent-degrader included;
Myceliophthora thermophila, Geomyces sp., Alternaria alternata, Fusarium sp., Aspergillus flavus and
Asperigillus oryzae. The primary biodegradability of synthetic detergent was confirmed by the
Methylene Blue–Active Substance (MBAS) method. Gas chromatography (GC) provided the convincing
evidence of synthetic detergent mineralization within the 30 day period in a sewage treatment plant. The
detection of unusual peaks in the GC profiles provided the scientific evidence of inclusion of certain
hydrocarbons in detergent formulation outside that of industry specifications. The unusual peaks are
attributable to inclusion of certain chemical optical brighteners (C17–C24). Linear alkyl benzene
sulphonates (LAS) which is the principal synthetic detergent component are thus biodegradable and its
use in detergent formulation is environment - friendly.
Key words: Biodegradation, detergents, linear alkylbenzene sulphonate, sustainable development.
INTRODUCTION
The increasing releases of organic pollutants by indus-
tries cause many health–related problems. However,
increased awareness of the harmful effects of environ-
*Corresponding author. E-mail:solayom@yahoo.com. Tel: +234
– 8055055478.
mental pollution has led to a dramatic increase in
research on various strategies that may be employed to
clean up the environment. It is now realized that microbial
metabolism provides a safer, more efficient, and less
expensive alternative to physico-chemical methods for
pollution abatement (Hebes and Schwall, 1987).
Linear alkyl benzene sulphonates (LAS) is a commonly
used anionic surfactant in detergents and it is easily bio-
degraded than non-linear alkylbenzene sulphonate (ABS)
even though, total biodegradation still requires several
days (Gledhill, 1975; Nomura et al., 1998). After soaps
linear alkylbenzene sulphonates (LAS) are the most
widely used surfactants in domestic and industrial
detergents. In 1995, the global production of LAS was ca
2.8 x 106 ton (Ainsworth, 1996).
Surfactants constitute a major ingredient of detergent
components. Usually surfactants are disposed after use
to sewage treatment plants (STPs). Here, biodegradation
processes and adsorption on sludge particles remove
these chemicals from wastewaters to a greater or lesser
extent, depending on the chemical structure of the
surfactant molecule and on the operating conditions of
the STP. After treatment, residual surfactants, refractory
co-products, and biodegradation products dissolved in
STPs effluents or adsorbed on sludges are discharged
into the environment. These chemicals through several
transport mechanisms enter the hydro-geological cycle.
Assessment of the environmental contamination levels of
surfactants and related compounds is achieved through a
wide range of laboratory biodegradation tests and eco-
toxicological studies. There are many evidences showing
that the primary biodegradation begins with oxidation of
the external methyl group (ω-oxidation) followed by
stepwise shortening of the alkyl chain via oxidative
cleavage of C2 units (β-oxidation). This process leads to
the formation of sulpho-phenyl carboxylic acids (SPACs)
(Cook, 1998).
The second cycle (ultimate biodegradation or minerali-
zation) involves opening of the aromatic ring and/or
desulphonation of SPACs leading ultimately to CO2, H2O,
inorganic salts and biomass. It is generally accepted that
dialkyltetralin sulphonates (DATS) and iso-LAS which are
co-products of commercial LAS, form carboxylated
intermediates upon biodegradation; this detection has
been facilitated through mass spectrometric study of
sewage contaminated groundwater (Field et al., 1992).
Many researchers have studied dialkyltetralin sulpho-
nates (DATS) and iso-LAS mineralization in the
laboratory and high levels of these chemicals in ultimate
biodegradation has been detected and that many
refractory organic carbons associated with impurities
characterized LAS mineralization (Cavalli et al., 1976;
Kolbener et al., 1995a,b). Recently, laboratory simula-
tions have confirmed that the microbial populations of
domestic and industrial activated sludge were effective in
primary biodegradation of DATS and iso-LAS but were
not capable of mineralizing most of the related meta-
bolites (Nielsen et al., 1997). However, these metabolites
cannot be considered as refractory species, under
appropriate conditions, they can be utilized as a sulphur
source for bacterial growth (Cook, 1998). Liquid
chromatography/Mass Spectrometry (MS) with an
electrospray (ES) ion source and a single quadrupole is a
powerful technique (Di Corcia et al., 1999a) for charac-
terizing the structures of break-down product originated
Ojo and Oso 1091
from biotransformation of alkyl branched alcohol etho-
xylate (Di Corcia et al., 1998) and nonylphenol ethoxylate
(Di Corcia et al., 1993) surfactants.
Principally, co-products of commercial mixtures of LAS
surfactants are DATS and iso-LAS, they make-up to 15%
of LAS. A previous method based on solid – phase
extraction (SPE) and liquid Chromatography / MS has
been modified for monitoring the above analyses in
aqueous samples of STPs. The metabolites as well as
iso-LAS metabolites discharged from a STP into river
water continued to degrade in the aquatic environment
(Di Corcia et al., 1999a).
MATERIALS AND METHODS
Sources of wastewater samples
Wastewater samples were obtained from sewage treatment plant
(STP), detergent-manufacturers and industries that utilize deter-
gents as cleaning agent after production in Lagos and Ogun states,
Nigeria.
Sample collection
Sampling was done with sterile plastic container (2 L) and collection
of effluent was randomly done at all points of discharge of effluent
along the production line and stored in the refrigerator at 4°C. All
the effluent generated was untreated according to the personnel of
the companies. The experimental design was a randomized com-
plete block design.
Detergents used
Domestic detergents used included powdered ‘Omo’ which was
purchased from Unilever Nigeria Plc., ‘Elephant Extra’ from PZ,
Ariel from PT. Sayap Mas Utama, Jakarta Timur 13910, Indonesia.
‘Persil’ from Lever Brothers Ltd., Ireland. Teepol’ was obtained from
National Oil and Chemical Marketing Plc., (NOLCHEM) Lagos.
Sodium Dodecyl Sulfate (SDS) was obtained from Fischer Scientific
coy, New Jersey, USA.
Determination of anionic matter in test detergent products
The Research and Development Department (R&D) of PZ factory,
Nigeria developed a modified Methylene Blue-Active Substance
(MBAS) analysis method for detergent powders and the protocols
of that methodology was used to determine % anionic matter in
each of the test detergents (PZ R&D, 1991).
Determination of the physico-chemical properties of
wastewater samples
The physico-chemical properties of the composite (morning and
evening) wastewater samples were determined using the standard
methods for the examination of water and wastewater (APHA,
1985; 1992).
Aerobic heterotrophic microbial counts
The effluent samples collected from each sampling point at 0 – 30
cm depths were serially diluted and inoculated onto Nutrient agar
1092 Afr. J. Biotechnol.
plates in duplicates. The plates were then incubated at room tem-
perature, (28 ± 2oC) for 24 – 48 h after which colony counts were
taken (Okpokwasili and Nwabuzor, 1988; Larson and Payne, 1981).
Viable counts of detergent – utilizing microorganisms
The number of bacterial detergent-utilizers in each of the effluent
sample collected was determined by inoculating minimal salt agar
medium supplemented with test detergent at 0.01% (w/v) with 0.1
ml of the serially diluted effluent sample using spread plate techni-
que. The inoculations were done in duplicates. The control plates
were not inoculated. Incubation was at 28 ± 2oC for 48 - 72 h
(Thysse and Wanders, 1972; Okpokwasili and Nwabuzor, 1988).
Bacterial isolates were characterized using standard and conven-
tional methods. These tests were according to the methods of
Gerhardt et al. (1981) and Bergey’s manual of systematic bacterio-
logy (1984). The fungal isolates were characterized using standard
and conventional methods (Smith, 1981).
Microbial growth in wastewater spiked with detergents
Composite effluent sample (1 L) was dispensed into 2 L Erlenmeyer
flask. A total of 16 flasks were filled with the composite effluent. The
flasks were in duplicates. Then, 5 mg/L of test detergent was spiked
into each wastewater flask with perforated plug for aeration. These
were kept at ambient temperature (28 ± 2oC) for 30 days.
Samples were taken at Day 0, 5, 10, 20 and 30 from the Erlen-
meyer flasks containing composite effluent samples spiked with 5
mg/L of detergent; this was to determine the pH, LAS concentration
and total aerobic viable counts (Okpokwasili and Olisa, 1991).
Determination of LAS concentration using the Methylene Blue-
Active Substance (MBAS) method
The method for determining the concentration of MBAS in the
detergents was that adapted from Standard Methods for the
Examination of water and wastewater (APHA, 1985; 1992). This
involved the preparation of a series of ten separatory funnels for
each of the test detergents. Each series of funnels contained
different volumes, 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5 7.5 and 10 ml of
solutions of the test detergents each made up to 50 ml with de-
ionized water such that with the exception of sodium dodecyl
sulphate (SDS), the concentration of the detergents in the above
solution were 0.51, 1.60, 2.60, 3.76, 4.95, 6.18, 7.50, 8.8 12.5 µg/ml
respectively. In the case of SDS, dilutions were prepared such that
the concentration of detergents in the resultant solution after
making up to 50 ml with de-ionized water corresponded to 0.1,
0.31, 0.53, 0.75, 0.99, 1.24, 1.49, 1.77, 2.50 µg/ml respectively. The
lower SDS concentration was because it contained more surfactant
than the other detergents under test. The tenth funnel in each
series contained no detergent and served as control since a total of
eight detergents were under test and needed determination, each
test detergent sample was diluted taking a series of ten different
volumes, a total of eighty samples were analyzed for MBAS
determination to generate standard curve prior to the ultimate
biodegradation studies.
The solutions of detergents in each series of separatory funnels
were made alkaline by adding 1 N NaOH using 1 drop of 1%
phenolphthalein solution as indicator to obtain a change in colour
from colorless to pink. Then, 1 N H2S04 was added in droplets to
make the solution acidic thereby reverting the colour from pink to
colorless. Thereafter, 5 ml of chloroform and 13 ml of methylene-
blue reagent were added respectively to the funnel after which each
funnel was shaken vigorously for 30 s for the contents to mix. The
flasks were then kept still for 30 min for the phases to separate.
The chloroform layer was drawn off into a 100 ml Erlenmeyer
flask. Extraction was performed three times employing 5 ml of
chloroform each time. All extracts were pooled in the 100 ml
Erlenmeyer flask. Extracts collected were later transferred back to
the separatory funnel and 25 ml wash solution (6.7 mM Phosphate
buffer, pH 7.1) was added to each funnel. The funnels were
vigorously shaken for 30 s after which they were allowed 30 min to
settle before the chloroform layer was drawn off through glass wool
into 50 ml volumetric flasks. The chloroform extracts were finally
shaken to ensure uniform mixing; Absorbance measurements of the
extracts were done using Ultra Violet - visible Spectrophotometer
(PHOTOMECH – 301 D+ Model 100 – 20 U–V Spectrophotometer,
OPTMA Co., Japan) set at 652 nm wavelength against blank
chloroform. The concentration of the residual surfactant present in
each test detergent in terms of methylene blue - active substance
(MBAS) were then plotted against the time (Days) for the 30 day
biodegradation period. The result obtained with the SDS served as
the standard.
To determine the primary and ultimate biodegradation of test
detergent samples using the river die–away method, 16 (2 L)
Erlenmeyer flasks each holding 1000 ml of freshly collected 24 h
composite effluent samples from both domestic and industrial
sources, southwest Nigeria were obtained. The composite effluent
samples in each flask were spiked with 5 mg/L test detergents
coded as: AK17 (Klin), AK 27 (Omo), AK37 (Elephant), AK47
(Persil), AK57 (Ariel), AK77 (Teepol) respectively, while AK 67
(SDS) served as the standard containing 1.0 µg/ml of (SDS)
detergent. The control flask was spiked with no detergent. The
flasks were then left still under room temperature for 30 days.
One milliliter (1 ml) samples were drawn from each of the sixteen
flasks and diluted with de-ionized water twenty times (x20) at day 0,
5, 10, 20 and 30 in order to determine the residual surfactant
concentration in terms of MBAS for each test detergent (Larson and
Payne, 1981; Okpokwasili and Olisa, 1991).
Ultimate biodegradation of the linear alkyl benzene sulphonates
(LAS) the active matter in detergents was monitored using a Gas
Chromatogram (GC). Samples from MBAS analyses on days 0, 5,
10, 20 and 30 were used. Calibration of the GC was done with
Acenaphthelene, an aromatic compound. The Gas Chromatogram
(SRI 8610 instrument, Model USA) was fed with samples by syringe
injection. The residual surfactant–chloroform extracts were desulfo-
nated by boiling in 5 ml concentrated phosphoric acid. The evolved
volatile materials were trapped in 3 ml n-Hexane using soxhlet
apparatus and a condenser heated electrically. The evolved volatile
materials were brought to 25 with n-Hexane; they were cooled for
30 min before they were decanted in glass bottles and then taken to
the GC laboratory for analysis.
The content of the glass bottles were allowed to evaporate for 24
h, leaving behind concentrates (that is, LAS biodegradation
residues). The volume of sample injected into GC was 1- to –2 ,
and this was in a split ratio 10 – to– 1 for the GC; SRI 8610
instrument, 200ft x 0.01 in. (60m x 0.25 mm) FID channel 1 packed
capillary column, 3% OV – 17 carrier gas of Nitrogen at 30ml/min.
Components: STD – Mix CPT, Temp. 800C (Sullivan and Swisher,
1969).
Gas chromatographic analysis
The gas chromatographic analyses for days 0, 10 and 20 were
determined as reported by Sullivan and Swisher (1969). The GC
(Perkins Elmer Auto–System Gas Chromatography, USA) analysis
of the total hydrocarbon was carried out using a GC equipped with
flame ionization detector (FID). A 30 m fused capillary column with
internal diameter 0.25 mm and 0.25 m film thickness was used and
the peak areas were analyzed with a SRI Model 203 Peak Simple
Chromatography Data System. The column temperature was 60°C
for 2 min to 300°C programmed at 12°C/min. Nitrogen was used as
Ojo and Oso 1093
0
20
40
60
80
100
120
TEST DETERGENTS
% ANIONIC MATTER
KLIN
OMO
ELEPHANT
PERSIL
ARIEL
SDS
TEEPOL
Figure 1. Determination of anion.
carrier gas at 37 psi. Hydrogen and air flow rates were 9 and 13 psi
respectively. The injector port and detector temperatures were 250
and 320°C respectively as well as 1 – 2 µl of sample was injected.
Effect of detergent on the growth of four fungal isolates
Four fungal detergent-degraders were transferred independently
and aseptically from Sabouraud dextrose-detergent agar slant and
inoculated onto sterile yeast-extract peptone dextrose (YEPD)
medium supplemented with detergent at 0.01% (w/v). The quadru-
plicate test tubes containing the 100 ml of medium were incubated
at room temperature for 5 days. These isolates were B1 (Aspergillus
oryzae) B2 (Myceliophthora thermophila), B3 (Geomyces sp.), and
B4 (Alternaria alternata). After 5 days of incubation, day 6, 7, 8 and
9 harvested mycelia’s weight were determined intermittently after
24 h, by filtering the content of each set of test tubes using sterile
filter paper while the mycelia were Oven-dried (LTE G150 Oven,
UK) at 85oC for 24 h. Thereafter, the mycelia were cooled and
weighed. This process was repeated for days 6, 7, 8 and 9. The
control was YEPD-detergent medium without inoculants (Gerhardt
et al., 1981).
Detergent degradation by microbial isolates (microcosm
experiment)
The microbial detergent- utilizers obtained from river die-away
methods were tested in a laboratory simulated biodegradation
study. Nutrient broth (1 L) supplemented with test detergent product
(Elephant) at 0.1 mg/ml was dispensed into eight conical flasks
(250 ml) prior to sterilization at 121°C for 15 min using the
autoclave, such that each flask had 250 ml of nutrient-detergent
broth. This was inoculated with five bacterial isolates and four
fungal isolates in this pattern; X1 (fungal consortium), X2, X3, X4,
X5, X6 (bacterial isolates) and X50 (microbial consortium) all
duplicated. The control flasks were uninoculated. This experiment
was monitored over a period of 30 days with samples taken on
days 0, 10, 20, and 30 for microbial population count, pH measure-
ment (Metrohm 780 pH Meter, UK) and Absorbance readings at
652 nm on U-V spectrophotometer (Helos Gamma & Delta
Spectrophotometer Model 9423 UVG, Spectronic Unicam Ltd.,
Mercers Row, UK.) (Okpokwasili and Olisa, 1991).
RESULTS AND DISCUSSION
The results of this study were predicated on the fact that
microorganisms are ubiquitous. Hence, the detection of
the microbial consortium involved in detergent degra-
dation. The anionic matter (LAS) content of SDS (sodium
dodecyl sulphate) was the highest of the seven detergent
products used, while the least was found with Persil. The
LAS concentration in both Persil and Teepol which are
foreign products were relatively low compared with those
of other detergent products analyzed (Figure 1). The
microbial detergent-utilizers were characterized using
standard and conventional methods (Table 1).
The physico-chemical properties of the composite
wastewater used for this study showed that it was heavily
polluted with organic matter, hence, the relatively high
BOD value. Comparatively, the COD falls short of
Federal Environmental Protection Agency (FEPA),
European Union (EU) and World Health Organization
(WHO) standards (Table 2). This might be the reason for
the slow rate of mineralization of xenobiotic compounds
in this ecosystem. The NO3 – N, SO42-, PO43-, NH4 – N
and total hydrocarbon (THC) content of the composite
wastewater used exceeds the WHO and EU limits which
is suggestive of high organic chemical pollution and this
is the reason for the longer time required for minera-
lization to be effected, since high concentration of N and
P may be toxic to microorganisms. Although, the dissolv-
1094 Afr. J. Biotechnol.
Table 1. Micromorphology and biochemical characterization of bacterial detergent–degraders.
Isolate code
Gram reaction
Cellular – morphology
Catalase
Oxidase
Indole test
Motility test
MR
VP
Citrate utilization
Urease activity
Starch hydrolusis
Gelatin hydrolysis
Growth on MacConkey
NO
3
reduction
Coagulase test
Spore test
Glucose
Xylose
Lactose
Suscrose
Aratinose
Galactose
Maltose
Mannitol
Sulicin
Raffimose
Probable identity
X1 + O + - - - + - - - - - - - - - + + + + - - - + - - E. majodoratus
X2 - R + - - - + - + + + + + - - - + + + + + - - + + + K. liquefasciens
X3 - R + - - + - + + + + + + - - - + + + + + - - + + - E. liquefasciens
X4 - R + - - - + - - + + + + - - - + + + + + - - + - + K. aerogenes
X5 - R + - + + - - - - - - + + - - + + + + + - + + - - E. coli
X6 - R + - - + - + - - - - + + - - + + - + + - + + - - E. agglomerans
A + C + + - - - + - - - - - - - - + - + + + - + - - - S. albus
B - R + - - + - + - - - - + + - - + + - + + - + + - - E. agglomerans
C - R + - - + - - - - - + + + - - + + - + - - + + - - Proteus sp.
X55 - R + - + - - + + + - - + + - - + + + + + + + + + + K. oxytoca
U + R + - - - - - - - - + - - - - + - - - - - - - - - Brevibacterium sp.
X8 - R + + - + + - + - - + - - - - + + - - - + - - - - P. aeruginosa
R = Rods, O = oval, C = cocci, + = positive, and – = negative.
ed O2 was relatively adequate, it was due to
presence of aerators in the Agbara STP. This is
suggestive of the fact that optimal presence of a
single physico-chemical factor does not determine
the rate of mineralization of xenobiotics in the
environment. In temperate climate, mineralization
of synthetic detergent products in wastewater has
been achieved under 25 days (WWI, 2005, 2004),
whereas under tropical climatic conditions this
study showed that for some of the commercial
detergents it would take more than 30 days for
some of them to be mineralized by microorga-
nisms which might be due to absence of optimal
physico–chemical conditions in wastewater and
the archaic technology being used in sub-Sahara
African countries STP.
Compliance with EU regulations on discharged
effluent (WWl, 2005) is yet to be met by any
country in sub-Saharan Africa due to problem of
system design as regards STPs and heavy
discharge of synthetic organic materials in both
domestic and industrial sewers.
Centralized wastewater treatment plants can
achieve total nitrogen concentrations of 3 mg/L for
discharged effluent from STP which is the curren-
tly set limit of technology in the United State of
America as at 2004 (US EPA, 2000), under
natural conditions or during treatment processes,
the degradation of pollutants is controlled often by
a variety of physical and chemical parameters
such as temperature, pH and availability of the
substrate, and not by the presence or absence of
the appropriate population of microorganisms.
The presence of optimal physical and chemical
conditions will allow eventual evolution and growth
of the best-adapted microbial population (WWI,
2005, 2004). This fact was corroborated when
similar strains of detergent-degraders from Cen-
tral Medical Laboratory, Nigeria were subjected to
detergent degradation under similar physico-
chemical conditions and they were able to utilize
the detergent but for longer acclimatization time.
Ojo and Oso 1095
Table 2. Mean physico-chemical properties of composite wastewater
Parameter Morning Evening FEPA/WHO standards EU standards
General appearance Cloudy foaming Foaming NS NS
Colour Blue Light green NS NS
Odour Soapy smell Soapy smell NS NS
pH (H20) 10.54 11.08 6 – 9 7.5 – 8.5
Conductivity @ 250C 204 Usm-1 185 Usm-1 NS 340
Temperature 34.30C 33.90C 400C 20 – 250C
PO43- 99.9 mg/L 90.3 mg/L 5 mg/L 10 – 25 mg/L
SO42- 92.7 mg/L 88.6 mg/L 500 mg/L NS
NO3-1 26.29 mg/L 22.86 mg/L 20 mg/L 20 mg/L
Total suspended solid (TSS) 170 mg/L 200 mg/L 30 mg/L 35 mg/L
COD 57.51 mg/L 52.01 mg/L 200 mg/L <125 mg/L
Specific gravity 1.009 1.022 NS NS
NH4 – N 193.5 mg/L 178.7 mg/L NS 15 mg/L
Cl-1 36.18 mg/L 37.95 mg/L 600 mg/L 600 mg/L
Dissolved oxygen (DO) 9.05 mg/L 9.45 mg/L >2 mg/L 2 mg/L
BOD 38.08 mg/L 34.41 mg/L 30 mg/L <25 mg/L
Total hydrocarbon (THC) 15.0 mg/L 13.6 mg/L 10 mg/L <10 mg/L
DO5 36.04 mg/L 32.67 mg/L >2 mg/L NS
Total dissolved solid (TDS) NS NS NS NS
NS = Not Specified
(Source: FEPA, 1991; Degremont, 1991; WWI, 2005).
The mean aerobic heterotrophic bacterial count from
effluent was 42.9 x 106 cfu/ml, while the mean aerobic
heterotrophic fungal population count was 4.5x106 cfu/ml.
The total viable count (TVC) for Detergent–utilizing
bacterial population was 209.4 x 105 cfu/ml. These were
determined with composite wastewater samples from all
the sampling points including the Agbara STP.
Acclimatization of this microbial population to detergent
components enhances the biodegradation efficiency of
the microorganisms. Although, bacterial population was
more than fungal detergent-degrader population in
tropical wastewater, this agrees with the previous findings
of researchers like Okpokwasili and Olisa, (1991); Amund
et al. (1997). The adaptability of native microbial
population in wastewater to detergent component would
be the reason for their success at mineralizing LAS
component in effluent where the physico-chemical
properties of the wastewater ecosystem were supportive
of the survival of these microorganisms (Spain and van
Veld, 1983).
Alkaline pH range as well as mesophilic temperature
range was observed to favor the acclimatization process
for the native detergent–utilizing microbial population as
soon as the optimum conditions became prevalent within
the wastewater ecosystem (Figure 2). These physico-
chemical factors were particularly important for the
survival of detergent–utilizing microbial consortium in the
wastewater. These findings in connection with the pH and
temperature range corroborated the findings of
Okpokwasili and Olisa (1991). Responding to changes in
the environment is a fundamental property of a living cell
and chemo taxis is the best studied bacterial behavioral
response that navigates the bacteria to niches that are
optimum for their growth and survival (Bren and
Eisenbach, 2000). Bacterial chemo taxis (Bacterial
heterotrophic population) was in this order KLIN >
PERSIL > OMO > ELEPHANT > ARIEL > SDS and least
with TEEPOL, while PERSIL attracted the highest fungal
heterotrophic population (Figures 3 and 4). TEEPOL
attracted the least fungal heterotrophic population from
the field experiment while SDS has the highest anionic
matter (LAS) content of all the test detergent products
and it’s the most easily mineralized because of its
chemical structure (Figure 3). This corroborated the
submission of Willets (1973a). SDS is being used as the
standard in this study. In the course of the field study, the
composite wastewater was spiked with each of the
different test detergents, the chemical changes were
monitored via the pH changes. At Day 0, pH changes
was in this order KLIN>ARIEL >OMO >ELEPHANT
>TEEPOL >PERSIL > SDS while at Day 30, OMO had
the highest value with ELEPHANT having the least value
(Figure 2) whereas during the microcosm study the pH
range was adjusted by microbial metabolism to the range
6.9 – 8.8 (Figure 6). Thus, alkaline pH range supported
the microbial consortium that mineralizes synthetic
detergents. This explains the absence and reducing
population of some detergent–utilizing fungal species
after day 10 during the laboratory simulated biodegra-
dation of test detergents (Figures 5 and 7), the pH shifted
1096 Afr. J. Biotechnol.
9.2
9.4
9.6
9.8
10
10.2
10.4
10.6
10.8
11
11.2
0 5 10 20 30
TIME(DAYS)
pH VALUES
CONTROL
KLIN
OMO
ELEPHANT
PERSIL
ARIEL
SDS
TEEPOL
Figure 2. pH readings of primary biodegradation from field experiment (shake-flask).
(µg/ml)
Figure 3. Biodegradation residues from (shake flask) field experiment.
to the alkaline range as a result of generation of alkaline
intermediates which accounted for the initial pH
increases. Although, the pH falls as the number of days
increased further probably as a result of production of
some acidic metabolites (SO42-), this has been reported
by other researchers (Hales et al., 1986; Okpokwasili and
Olisa, 1991). Macro nutrients such as P and S are
fundamentally essential in microbial cell physiology and
biochemistry, being a part of such important bio-
molecules as phospholipids, nucleic acids, proteins as
well as nucleotides, cofactors involved in energy
transport and catalysis of many cell processes (Hales et
Ojo and Oso 1097
0
20
40
60
80
100
120
140
160
180
200
0 5 10 20 30
TIME (DAYS)
COUNTS x10
2
KLIN
OMO
ELEPHANT
PERSIL
ARIEL
SDS
TEEPOL
CONTROL
Figure 4. Mean aerobic bacterial detergent-degrader count (shake-flask experiment).
0
2
4
6
8
10
12
14
0 5 10 15 20 25 30 35
Time (Days)
Count x10
2
KLIN
OMO
ELEPHANT
PERSIL
ARIEL
SDS
TEEPOL
CONTROL
Figure 5. Mean fungal detergent-degrader count (shake-flask experiment).
1098 Afr. J. Biotechnol.
0
1
2
3
4
5
6
7
8
9
10
0 10 20 30
TIME (DAYS)
pH VALUES
Fungal consortium
Ps. aeruginosa
Pseudomonas sp.
K. aerogenes
E. majodoratus
E. coli
Microbial consortium
Control
Figure 6. Mean pH readings of biodegradation of test detergents (Microcosm experiment).
Figure 7. Mean fungal detergent-degrader count (microcosm experiment).
al., 1999). Thus, the overall increase in microbial num-
bers in the 30-day biodegradation period may be
attributed to the availability of carbon source and
sulphate in the detergent product for energy and growth
(Figures 7 and 8) (Kertesz et al., 1994; Zurrer et al.,
1987). The microbial culture media lacks C and sufficient
SO42- sources. Hence, commercial detergent products
with relatively high SO42- concentrations exhibit rapid
degradation because this enhances both biomass accu-
mulation and increase in cell number of the detergent-
degraders (Konopka et al., 1996). This supports the
observations of Higgins and Burns (1975) who stated that
the relationship between surfactants and microbes is
complex and involves factors other than biodegradation
Ojo and Oso 1099
0
20
40
60
80
100
120
140
160
180
200
0 10 20 30
TIME (DAYS)
Count x10
2
Ps. aeruginosa
E. majodoratus
K. liquefasciens
E. liquefasciens
K. aerogenes
E.coli
E.agglomerans
S. albus
Proteus sp.
K. oxytoca
Brevibacterium sp.
Control
Figure 8. Bacterial colony count (microcosm experiment).
(µg/ml)
Figure 9. Biodegradation residues (microcosm experiment).
1100 Afr. J. Biotechnol.
Figure 10. GC profile of detergent residues (shake flask experiment). Control: no detergent.
and that under appropriate conditions, surfactants can act
as bactericides and bacteriostats. However, the ability of
a surfactant to be bactericidal depends largely on the
microbial species, size of the hydrophobic portion of the
surfactant molecule, purity of the water sample in terms
of organic matter such as sewage and the presence of
divalent metal ions (Higgins and Burns, 1975).
The microbial isolates from the shake-flask experiment
capable of utilizing the test detergents as C and energy
sources were Enterococcus majodoratus, Klebsiella
liquefasciens, Enterobacter liquefasciens, Klebsiella
aerogenes, Escherichia coli, Enterobacter agglomerans,
Staphylococcus albus, Pseudomonas aeruginosa,
Proteus sp, Klebsiella oxytoca, Brevibacterium sp.,
Myceliophthora thermophila, Geomyces sp, Alternaria
alternata, Verticillium alboatrum, Aspergillus flavus,
Trichoderma sp, and Aspergillus oryzae.
Some of these isolates have been reported as capable
of utilizing pure anionic surfactant molecule (Gledhill,
1974; Sigoillot and Nguyen, 1992; Schleheck et al., 2004)
and surfactant components of detergents (Okpokwasili
and Nwabuzor, 1988; Amund et al., 1997; Kertesz et al.,
1994).
When the test synthetic detergents were subjected to
Ojo and Oso 1101
Figure 11. GC Profile of detergent residues (shake flask experiment). AK 67 = Sodium dodecyl sulphate (SDS).
ultimate biodegradation in both the shake – flask and
laboratory simulated experiments, the native micro-
organisms metabolized the detergent components for
growth and biomass accumulation (Figure 9), as a result
the gas chromatography was used at intervals to analyze
the samples within a 30 day period to monitor the tran-
sitory intermediates formed as well as to provide the con-
vincing evidence for the mineralization of the detergent
spiked into wastewater and nutrient broth (Larson and
Payne,1981; Swisher, 1987; Di Corcia et al., 1999a,b;
Konopka et al., 1996). Although, unusual peaks in GC
profiles were detected by other researchers but it was
1102 Afr. J. Biotechnol.
Figure 12. GC Profile of detergent residues (shake flask experiment). AK 67 = Sodium dodecyl sulphate (SDS).
sparsely reported, these unusual peaks were not strange
because previous researchers had also observed it
(Kertesz et al., 1994).
It has long been recognized that susceptibility to
primary biodegradation is insufficient in itself to prove the
environmental acceptability of a compound. Information
on the intermediates formed in the course of bio-
degradation is needed as well. Hence, the desire for a
conclusive evidence for the ultimate biodegradation of
synthetic detergent component in open-rivers, this was
provided by the GC analysis (Swisher, 1987). Although,
few high peaks were detected in the chromatograms
suggesting inclusion of certain hydrocarbons in detergent
formulations outside that of industry prescriptions
(Figures 11, 13, 18 and 19). It has been legislated by the
international committee on synthetic detergents that
commercial synthetic detergents should be manufactured
with C10 – C14 atoms (CLER, 1999) but this study
discovered some other C atoms up to C20 from the GC
profiles of analyzed samples from the shake – flask
experiment while in the microcosm study, the GC profile
revealed presence of C21 atoms (Figure 17, 18 and 19). It
is either other chemical substances were included in
detergent formulation which are undisclosed to con-
Ojo and Oso 1103
Figure 13. GC profile of detergent residues (shake flask experiment). AK 67 = Sodium dodecyl sulphate (SDS).
1104 Afr. J. Biotechnol.
Figure 14. GC profile of detergent residues (shake flask experiment). AK 37 = ELEPHANT detergent.
sumers which is certainly the case because substances
such as toluene sulphonate has been reported in some
detergent formulations (Schoberl and Huber, 1988).
Shake–flask experiment with wastewater samples when
subjected to GC analysis after 0, 5, 10, 20 days
biodegradation process showed that C14 LAS homolo-
gues were mineralized faster than C12 homologues while
during the laboratory simulated (microcosm study)
biodegradation process the result was the same, thus
corroborating the fact that increased distance between
sulphonate group (phenyl position and chain length) and
the far end of the hydrophobic group increases the speed
of primary biodegradation (Huddleston and Allred, 1963;
Swisher, 1970; Swisher, 1975). The residual total hydro-
carbon content (THC) from extracted samples for the
laboratory simulated biodegradation was from 0.13 x10-6
– 1.82 x 10-6 mg/ml for the 30 day biodegradation
process. The more sophisticated desulphonation and gas
Ojo and Oso 1105
Figure 15. GC profile of detergent residues (shake flask experiment). AK 37 = ELEPHANT detergent.
chromatographic method enables quantization of the LAS
present as well as the relative concentration of each of
the chain length. The performance level for the microbial
consortium was assessed with fungal consortium (X1)
and microbial consortium (X50). The microbial consortium
(X50) (Figure 20) was second to the best in performance
because bacterial isolate Ps. aeruginosa was able to
metabolize detergent product with only 1.86 x10-6 mg /ml
remaining after 20-day incubation period in this study.
The best culture of detergent–utilizing bacterial strains
were Ps. aeruginosa and K. oxytoca while bacterial
isolate E. coli was the slowest in terms of rate of
detergent – utilization as shown by the GC profile
(Figures 18 and 19). SDS was found to be the most
rapidly biodegraded of all the test detergent products
utilized for this study followed by Elephant (Figures 11 –
16). This is due to the fact that straight chain LAS are
rapidly biodegraded than branched chain LAS, also SDS
is a purer detergent of analytical grade often used in the
laboratory with over 95% purity level while Elephant’s
1106 Afr. J. Biotechnol.
Figure 16. GC Profile of detergent residues (shake flask experiment). AK 37 = ELEPHANT detergent.
Figure 17. GC profile of detergent residues (microcosm experiment). X1 = Fungal consortium.
Ojo and Oso 1107
Figure 18. GC profile of detergent residues (microcosm experiment). X2 = Pseudomonas aeruginosa
Figure 19. GC Profile of detergent residues (microcosm experiment). X6 = Escherichia coli.
1108 Afr. J. Biotechnol.
Figure 20. GC profile of detergent residues (microcosm experiment). X50 = Microbial consortium.
purity level cannot be guaranteed up to 90% this was true
for other commercial test detergents too. In comparisons,
SDS relatively degraded faster than all the test
detergents in the presence of the microbial consortium
apart from the fact that it contains C9 – C12. Under 10
days, SDS was almost completely mineralized (except
C17) (Figure 12), while as at Day 10, ELEPHANT had
components of C11, C12 and C17 unmineralised (Figure
16).
The inefficiency associated with the local technology
used in STPs has made the change to membrane
bioreactor technology inevitable. WWI (2006) reported
that most countries are upgrading their effluent treatment
plant to Membrane Bioreactor Technology (MBR) which
improves the quality of domestic sewage and wastewater
discharged without increasing the plant foot-print. This
MBR has a single line designed to handle effluent flow of
1000 fold/day more than that of conventional STPs. The
up -graded process design increases the quality of
discharged effluent to satisfy consent levels and achieve
effluent of unrestricted irrigation re-use standard (WWI,
2005, 2006).
The introduction of LAS (C10-C14) into detergent formu-
lation as the principal surfactant component is thus
environment-friendly, since it is biodegradable and it
would enhance sustainable development processes.
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