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Herald Journal of Microbiology and Biotechnology Vol. 1 (1): 001 - 009 August, 2014
Available online http://www.heraldjournals.org/hjmb/archive.htm
Copyright © 2014 Herald International Research Journals
Full Length Research Paper
Microbiological assessment and physiochemical
parameters of palm oil mill effluent collected in a local
mill in Ovia North East area of Edo State, Nigeria
Okogbenin O.B*1, Anisiobi G.E2, Okogbenin E.A3, Okunwaye T3 and Ojieabu. A1
1
Plant Pathology Division, Nigerian Institute for Oil Palm Research (NIFOR), P.M.B. 1030. Edo State. Nigeria.
2
Chemistry Division, Nigerian Institute for Oil Palm Research (NIFOR), P.M.B. 1030. Edo State. Nigeria.
3
Biochemistry Division, Nigerian Institute for Oil Palm Research (NIFOR), P.M.B. 1030. Edo State. Nigeria.
Received May 21, 2014
Abstract
Accepted June 24, 2014
Changes in microbial community as well as physio-chemical properties of Palm oil mill effluent before
and after two weeks were determined using standard procedures. The results showed that the sample
contained a variety of microorganisms which include Bacillus sp. Pseudomonas sp. Micrococcus sp,
Corynebacterium sp, Apergillus niger, Penicillium sp, Candida sp. and Geothricium candidum. The
viable count of bacteria at the initial time of sample collection was 1.3 x 101 CFU/g while that of fungi
was 1.9 × 102 CFU/g. The microbial load for the final analysis of bacteria and fungi were 4.5 x 10 3 CFU/g
and 5.5 x102 CFU/g respectively. From the results, it was observed that after the duration of two weeks,
there was a reduction in all the elements from the initial reading with a percentage of 74.88% for
Calcium, 50.66% for Magnesium, 53.73% for sodium, 79.15% for phosphorus, 31.17% for Nitrogen and
10.40% for carbon. Free fatty acid content and organic matter content increased from 11.06 to 17.00%
and 0.55 to 10.42 respectively while the oil content in the samples reduced from 15.17% to a value of
10.04%. The reduction in oil content observed is an indication of the lipolytic effect by the microbial
isolates identified in this study and possible sources for bioremediation purposes both in small and
large scale.
Keywords: Palm oil mill effluent, Bacteria, Fungi, Physio-chemical, Bioremediation.
INTRODUCTION
Oil palm (Elaeis guineensis), the most productive oil
producing plant in the world is of great economic
importance in Nigeria, Okechalu, (2011); SMA Tagoe,
(2012). Palm trees may grow up to 60 feet or more in
height. The trunks of young and adult palms are wrapped
in fronds which give them a rough appearance. Each tree
produces compact bunches of fruitlets weighing about 10
kg to 25 kg with 1000 to 3000 fruits per bunch. Each fruit
which is spherical or elongated in shape turns from a
dark purple color to orange red when ripe Panapanaan et
al., (2009) and ready for milling. In general, the palm oil
milling process can be categorized into a dry and wet
(standard) process. The wet process of milling palm oil is
the most common. It is estimated that 0.5-0.75 tonnes of
*Corresponding
Author:
[email protected]
Email:
Palm oil mill effluent can be discharged from every tonne
of oil palm fresh fruit Wakil et al., (2013). The process of
extracting palm oil requires significant large quantities of
water to steam and sterilize the palm fruit bunches, clarify
the extracted oil and the cleaning processes in the mill
Nwoko and Ogunyemi, (2010). It is estimated that for
each tonne of crude palm oil (CPO) that is produced, 5.0
tonnes-7.5 tonnes of water are required and more than
50% of this water ends up as waste water sludge
commonly referred to as palm oil milling effluents POME
Ahmad et al. (2003). Phaik, Wei-Jin and Mei, (2010).
Palm oil mill effluent is an acidic, thick, brownish, colloidal
suspension with 95% to 96% of water, 0.6% to 0.7% of oil
and 2% to 4% total suspended solids (TSS) Wakil et al.,
(2013). The continuous collection of POME in particular
locations can bring about undesirable changes in soil pH,
increase in salinity Kittikun et al., (2000), soil microbial
properties and biological activities
Herald J. Microbiol. and Biotech. 002
(Thirukkumaran, and Parkison, (2000); Paredes et al.,
(2005). Soil microbiological and biochemical properties
have been considered early and sensitive indicators of
soil changes and can be used to predict long term trends
in the quality of soil Ros et al., (2003). Due to the daily
release of large amounts of POME generated, annual
disposal remains a challenge to the environment and as
such bioremediation has been considered as an
alternative for this pollution control Hipolito et al., (2008).
There is therefore need to evaluate the occurrence of
microbes
as
possible
candidates
for
bioremediation/bioconversion processes in small and
large scale. This in vitro research work was aimed at
evaluating the changes in microbial and physicochemical
properties of POME exposed under room conditions over
a period of two weeks (14days) in order to undergo
biodegradation.
MATERIALS AND METHODS
Sample collection
Palm oil mill effluent samples were collected from an oil
mill processing unit located in Ovia North East area of
Edo state on latitude 6.462861 and longitude, 5.597825.
The sludge was freshly collected at the point of discharge
during oil mill processing into pre sterilized 1000ml
conical flasks and covered with cotton wool. The samples
were left to stand on a sterile bench in the laboratory
overnight to cool and for further analysis.
The following initial physicochemical parameters were
carried out; pH, Temperature, Conductivity, Nitrogen,
Carbon, Organic matter, Phosphorus, BOD, COD,
Calcium, Magnesium, Sodium. Biochemical parameters
assayed were: Moisture content, free fatty acids (FFA),
Dirt content and Oil content.
Biodegradation of POME
This was carried out by exposing the effluent
under room temperature in sterile containers. It was
stirred every four days to allow for free flow of air inside
the sample. The mixture was allowed to stand to
degrade for 14 days after which samples for analysis
were drawn.
Preparation of Media
In this study, the two different media used were
potato dextrose agar (PDA) and nutrient agar (NA). The
media
were
prepared
aseptically
following
manufacturer’s
instructions
while
autoclavable
materials such as glassware were sterilized at
121oC for 15 minutes at 15psi.
Isolation and Identification
present in sample
of
microorganisms
The initial and final (after two weeks) isolation of micro
flora on the sample was carried out as thus; stock
solutions of the sample at day one (initial) was made by
weighing one gram of the thick sludge into sterile
MacCokney bottles containing 9ml of sterile distilled
water. A tenfold serial dilution was prepared with the aid
of sterile syringes into labeled MaCokney bottles. One (1)
ml of each dilution of 10-1 –10-4 and 10-5 – 10-8 was
pipetted with sterile syringes and plated using the pour
plate method Pepper and Gerba, (2004) into sterile
disposable Petri dishes. In the isolation of fungi, prepared
potato dextrose agar medium (PDA) was allowed
to cool to a temperature of about 35oC and
250mg/250ml of chloramphenicol was added to the
medium to inhibit the growth of any possible bacteria, and
then gently dispensed into the plates containing the
samples. The bacteria isolations were also carried out by
adopting the pour plate method Pepper and Gerba,
(2004) using sterile prepared Nutrient agar medium. The
mixtures in the plates were carefully swirled so as to
allow for even distribution of possible colonies. The
experiment was carried out in four replicates and
observations were made on a daily basis. Bacteria
colonies were enumerated using a colony counter and
values expressed as CFU/g of the sample while the
isolates were identified and characterized to the species
level based on reactions observed during gram
staining and biochemical tests carried out. The
characteristics of the bacteria isolates were also
compared with those of known taxa, using the Bergey’s
manual of determinative bacteriology and the scheme of
Cheesbrough,
(2004).
The
biochemical
tests
carried out on the isolates include; coagulase, catalase,
indole, urease, citrate utilization test, utilization of
carbohydrates such as glucose and lactose. The spores
of the bacterial colonies isolated and their gram staining
characteristics were observed under microscope. Total
viable count of the colonies was carried out according to
Collins et al., (1989) using the formula: Number of
colonies X dilution factor/ Inoculum volume and the
values expressed as colony forming units per gram
(CFU/g) of the effluent.
The yeast isolates were characterized using the
procedures described by Barnett et al., (2000) while
Fungal isolates were examined macroscopically and
microscopically using the needle mounts technique. Their
identification was performed according to the scheme of
Barnett and Hunter (1972). The most predominant
fungi isolate was further sent to CABI, Surrey,
United Kingdom where the isolates were confirmed
using molecular techniques and given ascension
number. The pictomicrograph of the spores were
taken using a 9MP Amscope motic camera
attached to one of the eye piece of the microscope.
Okogbenin et al. 003
Physiochemical analysis and biochemical tests
All biochemical parameters were determined according to
the Malaysian Palm Oil Board (MPOB) test methods
(2004) and the analysis was carried out in triplicates.
Free Fatty Acid = 25.6 × Molarity of KOH × Volume of
KOH /mass of sample.
Where 25.6 in the formular for FFA determination is
equivalence factors (e) for palmitic acid; the predominant
fatty acid in palm oil.
Determination of Moisture and Volatile Content
Determination of temperature
A mass of 10grams of thoroughly mixed POME sample
was weighed into a known mass of clean Petri dish which
had been previously dried and cooled in a desicator. It
was placed in the oven for four (4) hours, allowed to cool
to room temperature in a dessicator for 45mins and
further weighed. This was repeated till a constant weight
was obtained. The moisture and volatile matter content
was expressed in percentage by mass using the formula:
% Moisture and Volatile
content= (Mb-Md / Mb-M) ×100
Where M=Mass (g) of Petri dish
Mb = Mass (g) of Petri dish +sample
Md = Mass (g) of Petri dish + test sample after drying.
The initial reading was determined at the point of sample
collection, while the final reading was carried out after
fourteen days. This was done by dipping the bulb of
mercury-in-glass thermometer into the POME samples
using 76MM immersion MEDALCARD thermometer and
the readings recorded.
Determination of pH.
The pH was determined with the aid of a pH meter.
Statistical Analysis
Determination of Oil Content:
Ten grams (10g) of dry POME sample was weighed into
a thimble and extraction was done with the aid of a
Soxhlet extractor for 6 hours using 150 ml of Hexane
(analytical grade) as extracting solvent. The oil extract
was dried at atmospheric pressure at 103oC for 2 hours,
cooled for 30mins and weighed. The oil content was
expressed as percentage by mass using this formula:
Mass (g) of oil in the flask after drying / mass (g) of
sample×100%.
Determination of Dirt content
The dirt content of the sample was analyzed as thus;
8.50grams of the sludge was weighed into a conical flask,
100ml of hexane was added, swirled and carefully filtered
through a glass fiber filter paper in a crucible and dried in
the oven at 103oC for 1 hour. Percentage dirt content
was expressed as:
(Mass of crucible +filter paper + dirt) – (Mass of crucible +
filter paper) / Mass of sample ×100%.
Determination of Free Fatty Acid (FFA)
A mass of 2.5grams of the sample was weighed into a
conical flask, 50ml of neutralizing solvent was carefully
added to the weighed sample in the conical flask then the
flask was placed on a hot plate at 40oC, swirled gently
and titrated with standard potassium hydroxide (0.5M).
Values were calculated using the formula:
SPSS software version 19 was used to analyze the
results statistically. A one-way analysis of variance was
carried out at α = 0.05
RESULTS
Figure 1; Shows the POME collected during milling. It
was perceived to have a smell characteristic of freshly
milled palm oil. However, during the days when it was left
to stand, it started deteriorating thereby giving an
offensive odor. A total of eight (8) microorganisms were
isolated and identified as Pseudomonas sp., Bacillus sp,
Micrococcus sp, Corynebacterium sp., Aspergillus niger,
Penicillium sp, Candida sp. and Geothricium candidum.
(Table 1). The Geothricium candidum (IMI Number;
503814) was identified by ITS DNA sequence analysis
using the FASTA algorithm with the Fungus database
from EBI. The result was confirmed using the online CBS
yeast
database.
The initial
physio-chemical
characteristics of the effluent collected and analyzed
were found to have a pH of 5.10, organic matter content
of 0.55, BOD value of 502.93mg/l and COD value of
821.45 mg/l. The percentage oil content was 10.04%,
percentage dirt content; 6.73% , nitrogen content;
3.24mg/l, carbon content; 32.70mg/l, calcium content;
605.50mg/l,
magnesium
content;
193.50mg/l,
phosphorus; 17.80mg/l, conductivity (dsm-1); 137.34,
Free fatty acids (%);11.06±0.01 , temperature of 32.80oC
(Table 3).
Cultural characteristics of the bacteria isolates were
observed and recorded based on their elevation, margin,
colour and shape (Table1). The morphological
Herald J. Microbiol. and Biotech. 004
Figure 1. Freshly collected POME from milling site
Table 1. Cultural, morphological and biochemical characteristics of bacteria isolated from Palm Oil Mill Effluent.
Characteristics
Cultural
Elevation
Margin
Color
Shape
Morphological
Gram staining
Cell type
Cell arrangement
Spore staining
Biochemical Test
Catalase
Oxidase
Coagulase
Urease
Indole
Citrate
Glucose
Lactose
Isolates
Isolate 1
Isolate 2
Isolate 3
Isolate 4
Low convex
Entire
Green
Circular
Convex
Entire
Orange
Circular
Flat
Entire
Cream
Circular
Convex
Entire
Cream
Circular
Rod
Single
-
+
Cocci
Single
-
+
Rod
Chains
+
+
Rod
Single
-
+
+
+
+
+
Pseudomonas sp.
+
+
+
+
+
Micrococcus sp.
+
+
+
+
+
Bacillus sp.
+
+
+
+
+
Corynebacterium sp.
Key= + Positive, - negative
characteristics were recorded using gram staining
techniques as described by Christopher K and Bruno E
(2003).
The
gram
staining
was
aimed
at
differentiating gram reactions, sizes, shapes and
arrangement of cells of each bacteria. The results are
shown in table 1. Confirmation of the isolates were
carried using the biochemical tests Christopher K and
Bruno E, (2003) as shown in table 1.
Okogbenin et al. 005
Table 2: Microscopic and cultural characteristics of fungi isolated from unsterilized freshly milled palm oil
Fungi Isolates
Aspergillus sp.
Candida sp.
Penicillium sp.
Geotrichium candidum
(IMI Number; 503814)
Microscopic Morphology
Presence of septate hyphae; long and smooth
conidiophores, long unbranded sporangiospores with
large, round head.
Cultural characteristics
Brownish-black
mycelium with dark
spores on the surface
Microscopic morphology shows spherical to subspherical Colonies are white to cream colored,
budding yeast-like cells or blastoconidia, 2.0-7.0 x 3.0-8.5 smooth, glabrous and yeast-like in
um in size. Long branched pseudohyphas were also seen.
appearance.
Presence of septate and fruity mycelium and branched
conidiophores.
A rapid dark green colored growth
with the edge surrounded by whitish
margin was observed on the surface
of the organism and pale yellow on
the reverse side.
They are either rectangular in shape or rounded at the
produce rapidly growing, white, dry,
ends resembling the barrel shape. They posses
powdery to cottony colonies
Arthroconidia which are unicellular, in chains, hyaline, and
result from the fragmentation of undifferentiated hyphae by
fission through double septa.
Figure 2: Penicillium sp. grown on PDA medium
At the initial stage of day one, the microbial load was very
low with a few bacteria species isolated with a load of
1.25 x 103 CFU/g. The isolation after the fourteen days
period, revealed the predominance of Geotrichum
candidum (IMI Number; 503814) which dominated and
covered the entire plates. The initial result could be due
to the nature of the sludge as it was freshly collected in
sterile beakers and hadn’t started undergoing
biodeterioration as the initial time of analysis while the
later could be attributed to the fact that yeast are more
Figure 3. G.candidum grown on PDA
fastidious than fungi and hence the multiplication and
total colonization of the plates. (table 2)
The plates below show some of the fungi isolated from
POME and grown on PDA medium (Figure 2-5)
Physio-chemical
parameters
including
BOD
measurements were prepared according to the modified
method explained in American Public Health Association
APHA (1992). COD determination was based on closed
reflux dichromate oxidation colorimetric method and was
read out in DR 2000 Spectrophotometer as explained in
Herald J. Microbiol. and Biotech. 006
Figure 4: Aspergillus niger isolated from POME Figure 5: Pictomicrograph of A.niger (x40) captured using a motic camera
Table 3. Physio-chemical parameters of the sample at the initial and final stage
(after fourteen days) under the same conditions of room temperature.
PARAMETERS
BOD (mg/L)
COD (mg/L)
Electrical Conductivity (ds m-1)
pH
Organic Matter
Moisture content (%)
Oil content (%)
Dirt content (%)
Free fatty acids (%)
Temperature (o C)
INITIAL READING
502.93±0.04
821.45±0.65
137.34±0.00
5.10±0.05
0.55±0.02
77.98±0.00
15.17±0.02
6.73±0.00
11.06±0.01
32.80±0.00
FINAL READING
741.98±0.00
1186.22±0.5
93.70±0.00
5.70±0.04
10.42±0.00
72.00±0.02
10.04±0.2
6.08±0.10
17.00±0.08
30.50±0.00
*Values are means of triplicates ± SEM
APHA (1998). The electrical conductivity (1:5w/v) was
measured according to Jackson, (1962) while pH of the
sample was determined using Hach-one pH meter in a
ratio of 1:2. Total organic carbon was determined by
oxidation with K2Cr2O7 in a concentrated H2SO4 medium
followed by measurements of dichromate excess using
(NH4)2Fe(SO4)2 Yeomans and Bremmer,(1989).
From the results in table 3, it was observed
that there was an increase in some physicochemical
parameters
of
the
sludge
from
the
initial
experiment and the final experiment. The BOD and
COD content of the POME increased tremendously,
this could be an indication of serious pollution
when the POME is actually left on d soil. This finding is
similar to work done by Dhouib et al., (2006), who
suggested that the use of anaerobic digestion with white
rot fungus could decrease COD and BOD levels in
POME. The organic content of the sludge increased from
0.55 to 10.42 this confirms the findings of Logan et al.,
(1997). A slight increase in pH from the initial experiment
and the final experiment showed that the medium was
still acidic.
The results in Table 4 show the initial and final
elements present in the sample. From the results,
it was observed that after the duration of two weeks,
there was a reduction in all the elements from
the initial reading and the final reading with following
percentages
74.88% for Calcium, 50.66% for
Magnesium, 53.73% for sodium, 79.15% for phosphorus,
31.17% for Nitrogen and 10.40% for carbon.
Okogbenin et al. 007
Table 4. Elements at the initial and final experimental period
ELEMENTS
Calcium
Magnesium
Sodium
Phosphorus
Nitrogen
Carbon
INITIAL
READING(mg/l)
605.50±0.05
193.50±0.02
255.50±0.01
17.80±0.03
3.24±0.05
32.70±0.65
FINAL
(mg/l)
151.80±0.08
95.76±0.04
118.49±0.07
3.71±0.03
2.23±1.00
29.30±0.8
READING
DISCUSSION
Fresh POME collected from the palm oil mill was thick
yellow in color, colloidal suspension, oily and viscous with
a sweet smell. The isolation of lipase producing bacteria;
Bacillus sp. from the initial microbial analysis of this
study, revealed that the oily nature of POME serves as a
good medium for lipolytic microorganisms to thrive
Mukesh et al., (2012), this is in agreement with the work
of Gowland et al., (1987) who also isolated lipase
producing Bacillus sp. from POME. The presence of this
organism in POME is most likely an indication that the
microorganisms can be isolated, multiplied and applied to
POME contaminated sites, as it will aid in bioremediation
of the contaminated site. Studies by Kanokrat et al.,
(2013), revealed that some bacteria such as Bacillus sp,
Pseudomonas sp, Corynebacterium sp. and some others
isolated from palm oil contaminated soils had the ability
to produce biosurfactants. Biosurfactants are amphiphilic
(containing both hydrophilic and hydrophobic moieties)
surface active agents produced by microorganisms
Kanokrat et al., (2013) that have the ability to reduce
surface and interfacial tensions by accumulating at the
interface between two immiscible fluids such as oil and
water Nitschke and Coast, (2007). The presence of
biosurfactants can increase the solubility of oil and hence
potentially increase their bioavailability for use as carbon
and energy sources Mulligan, (2009) in soils. The ability
of the biosurfactant producers to reduce interfacial
surface tension in oil contaminated sites that are most
important in tertiary oil recovery and bioremediation
processes Lin, (1996); Rouse et al., (1994), Volkering et
al., (1998) and many of the known biosurfactant
producers are also hydrocarbon-degrading organisms
Rouse et al., (1994); Willumsen and Karlson, (1997);
Volkering et al., (1998). The bacteria isolated from this
work may have the potential of producing biosurfactants,
hence, consideration should be given to this area of
research as it will help reduce the continuous use of
chemical surfactants as well as aid in bioremediation
processes especially in Nigeria.
Several lipases from Bacillus thermoleoverans, Lee et
al.,
(2001)
Markossian
et
al.,
(2000),
B.
stearothermophilus
Sinchaikul
et
al.,
(2001),
thermoacidophilic bacteria, B. acidocaldarius have been
identified and characterized. Lipases (triacylglycerol
acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and
the synthesis of esters formed from glycerol and longchain fatty acids thereby leading to release of free fatty
acids (FFA) Koshimizu et al. (1997); Onilude et al.
(2010). Microbial lipases extensively applied in food and
dairy industry for the hydrolysis of milk fat, cheese
ripening, flavor enhancement and lipolysis of butter fat
due to their shorter generation time; ease of bulk
production and ease of manipulation, and are
economical. This work and the works of others show that
palm oil mill effluent provides a good source for abundant
lipase producing bacteria for the confectionary industries.
Growth and multiplication on any substrate is often
considered as the first step towards its bioconversion
Molla et al., (2002). The presence of Aspergillus sp. in
the sample may not be unconnected with its high lipase
producing ability as POME serves as a good substrate for
the fungus. However, at the final (after 14days) isolation
of the sample, Geotrichum candidum (IMI Number;
503814) was most predominant and had a very rapid
growth over the rest fungi. The results of this work gives
an indication that this rapidly growing yeast specie can be
applied to palm oil mill effluent contaminated soils as they
tend to breakdown oil components and utilize them
rapidly as substrates for their proliferation. The increase
in organic matter may be attributed to increase in
biomass of resident microorganisms in the POME during
the fermentation. The Organic matter from Palm oil mill
effluent application can be applied to soil to help increase
some beneficial soil chemical and physical characteristics
such as organic carbon and some major nutrients. Free
fatty acid content of fermented POME increased with
fermentation days, this was probably due to the activities
of lipolytic bacteria isolated from the samples and it is in
agreement with Onilude et al., (2010) who had earlier
made such observation in a similar study. Mineral
nutrients are inorganic elements that have essential and
specific functions in oil palm plant metabolism due to this
reason, some of minerals content from palm fruits might
have been retained in POME during the milling and
extraction process Mohd Rizal Abas et al., (2013). The
reduction in the levels of the nutrient elements may be
due to the activities of microbes as they utilize the
nutrient sources for proliferation. The low nitrogen
content in POME was validated by previous literature
where it was also reported that nitrogenous content in
POME was significantly in low amount Chow (1991). The
presence of the elements determined in this study
Herald J. Microbiol. and Biotech. 008
revealed that POME is rich in nutrients; however, the
amount of nutritional contents varies according to the
location of palm oil plantation and milling factories Wu et
al., (2009).
The presence of these degrading microorganisms
seemed to utilize the available nutrients during the
fermentation process. The increase in pH during
fermentation could be correlated with proteolytic activity
which yields ammonia into the medium that causes
increase in pH Parihar, (2012). This rise in pH also raises
the possibility that POME may be used in the form of
liming material. Shamshuddin et al., (1995) reported that
when raw POME is discharged, the pH is acidic but
seems to gradually increase to alkaline as biodegradation
takes place as observed in this study. The reduction in
the oil content of the sample was observed, this is likely
due to the breakdown of oil content by microbial isolates
Koshimizu et al., (1997). Reports show that, Ahmed et
al., 2011 utilized the bacterium, Pseudomonas sp. in cocomposting process of Oil palm frond with Palm Oil mill
anaerobic sludge.
CONCLUSION
The exploitation of the microorganisms isolated from
palm oil mill effluent can be considered as potentials for
biodegradation or bioremediation of palm oil spills. Once
their remediation potential has been established on a
large scale, it may serve as a cheap and efficient tool for
purifying contaminated effluent water and soils.
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