Journal of Research in Biology An International Scientific Research Journal

Original Research

Remediation of crude oil polluted soil in Otuogidi town in Bayelsa

State of Nigeria using poultry manure.
Journal of Research in Biology

Authors: ABSTRACT:
Ebe TE1, Mgbemena IC2,
Njoku JD1, Ihejirika CE1, Remediation by poultry manure (RPM) is a farming treatment technology,
Emeribe E1, Edward KC3. which relies on biological processes to cleanup pollution in soil. In this study,
remediation by poultry manure was employed on oil contaminated site in Otuogidi
Institution: town at Bayelsa State of Nigeria. 200g of the polluted soil samples were distributed in
1. Environmental Technology four sterile containers labeled A, B, C, and D. 100g, 200g, 300g and 0g of manure were
Department, School of added to the soil samples respectively. Then the total heterotrophic bacteria and
Environmental Technology, fungi, hydrocarbon utilizing bacteria and fungi, total petroleum hydrocarbon, moisture
Federal University of content, sulphate content, nitrate content, phosphate content, soil pH and
Technology,P. M. B. 1526, temperature were determined. The total heterotrophic bacteria counts ranged from
Owerri, Imo State, Nigeria. 5.00 X 102 to 3.91 X 105cfug-1 and fungi ranged from 2.40 X 102 to 2.00 X 103cfug-1.
2. Biotechnology The hydrocarbon utilizing bacteria counts ranged from 3.20 X 102 to 3.02 X 104cfug-1
Department, School of and fungi which ranged from 5.00 X 101 to 3.60 X 102cfug-1. In the crude oil
Sciences, Federal University contaminated samples, the values of total petroleum hydrocarbon, pH and
of Technology, P. M. B. temperature were greatly influenced by high rate of microbial activities including high
1526, Owerri, Imo State, humidity which has a great impact in any given sample. This made the microbial and
Nigeria. physicochemical properties of the soil samples to vary with the different
3. Microbiology Department, concentrations of nutrient supplement and at different periods of remediation.
College of Natural and Therefore, poultry manure is an effective method of remediating crude oil polluted
Applied Sciences, Micheal soil.
Okpara University of
Agriculture, Umudike, Keywords:
Umuahia, Abia State, Remediation, Contamination, Heterotrophic, Physicochemical, Microbial.
Nigeria.

Corresponding author: Article Citation:

Ebe TE. Ebe TE, Mgbemena IC, Njoku JD, Ihejirika CE, Emeribe E, Edward KC.
Remediation of crude oil polluted soil in Otuogidi town in Bayelsa state of Nigeria
using poultry manure.
Email: Journal of Research in Biology (2012) 2(6): 544-548
akumzirit2@yahoo.com
Dates:
Phone No: Received: 06 Jun 2012 Accepted: 28 Jun 2012 Published: 31 Jul 2012
+2348034954739.

Web Address:
http://jresearchbiology.com/ This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
documents/RA0252.pdf. licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

544-548 | JRB | 2012 | Vol 2 | No 6

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
The discharge of crude oil into the environment
constitutes a serious pollution problem. This can threaten
human health and of beneficial organisms in the
environment (Aboribo, 2001). Increasing petroleum
exploration, refining and operation petroleum companies
in the Niger Delta region of Nigeria have led to the wide
scale contamination of most of its soils, creeks, swamps,
rivers and streams.
The main causes of oil spill in Nigeria include
criminal damage, equipment failure example wellhead
blowout, valve and flanges failures etc; corrosion: either
through chemical or biological agent, human error and
technical failure.
Consequently, there is a need for remediation
which will depend on the degree of actual or potential
environmental threat. Many substances (Hydrocarbons,
halogenated organic solvents halogenated organic
compounds, non- chlorinated pesticides and herbicides,
nitrogen compounds etc.) known to have toxic properties
have been introduced into the degree of toxicity and
danger to human health. Many of these substances come
in contact with and are sequestered by soil (Nester et al.,
2001).
Crude oil spills may cause damage to the
environment in many ways. Oil spill on land may lead to
soil compaction, disruption of land surfaces, introduction
of heavy metals into the soil, infertility for a long period
of time until natural processes re-establish stability.
Bioremediation is a destructive technique directed
towards stimulating microorganisms to grow and use the
contaminants as food and energy source. Bioremediation
is also the use of living organisms to reduce or eliminate
environmental hazards resulting from accumulation of
toxic chemicals or other hazardous wastes (Nester et al.,
2001). It is the enhancement of live soil organisms such
as fungi, bacteria etc to breakdown hydrocarbon and
organic contaminants (Adenipekun, 2008).
Metabolic processes of these organisms are
545
Ebe et al.,2012
capable of chemical contaminants as an energy source,
rendering the contaminants harmless or less toxic
products in most cases (Nester et al., 2001). A mixed
culture of acclimated bacteria added to contaminated oil
to reduce the lag period and increase the rate and extent
of bioremediation. Organic manure is considered to be a
good amendment agent for bioremediation of polluted
soil. The presence of residual crude oil in the polluted
soil boost the carbon supply in the soil, hence favored the
growth of the hydrocarbon utilizing bacteria as compared
with crude oil free soil (Ijah et al., 2003).
The main purpose of this work is to determine
the effectiveness of poultry farm manure in crude oil
polluted soil.
MATERIALS AND METHODS
Collection of Samples
Soil samples used in this research were collected
from an oil spilled area in Otuogidi Town in Bayelsa
State, Nigeria in January, 2012.
The samples were collected using soil Auger at
the depth of 10cm, stored in sterile aluminum foils and
transported to the laboratory within 48hours of
collection.
The soil reclamation material was poultry
dropping collected from the poultry house in the School
of Agriculture Agricultural Technology [SAAT], Federal
University of Technology Owerri [FUTO] and was
preserved in the laboratory at room temperature.
Sample Preparation
The soil samples were weighed and distribution
into sterile containers in 200g portions. These were
mixed with different concentrations of poultry manure as
follows:
200g of polluted soil + 100g of poultry manure [Sample A]
200g of polluted soil + 200g of poultry manure [Sample B]
200g of polluted soil + 300g of poultry manure [Sample C]
Polluted soil without nutrient supplement [Sample D]
The above samples were stored at room
Journal of Research in Biology (2012) 2(6): 544-548
Ebe et al.,2012
temperature, moderate moisture and oxygen for
12 weeks. In addition, Total Heterotrophic Bacteria and
Fungi (THB and THF) counts in the soil samples were
enumerated by adding 1g of each sample to 9ml of
distilled water and serially diluted. Hydrocarbon
Utilizing Bacteria and Fungi (HUB and HUF) were also
enumerated.
The following were also determined: Total
Petroleum Hydrocarbon (TPH), Moisture, Sulphate,
Nitrate, Phosphate, Soil pH and Temperature.
RESULTS
The following results were obtained based on
laboratory analysis conducted on bioremediation of
crude oil polluted soil using poultry manure as the
nutrient supplement.
DISCUSSION
The variation in counts of Total Heterotrophic
Bacteria (THB) over the period of 12 weeks may be due
to changes in the physicochemical properties of both the
soil and the poultry manure. However, the difference in
counts of Total Heterotrophic Bacterial (THB) and Total
Heterotrophic fungi (THF) between the biodegraded
crude oil polluted soil and the control was significant,
probably due to rapid biodegradation of the crude oil in
the soil. Hydrocarbon Utilizing Bacteria (HUB) in
biodegraded crude oil polluted soil were higher than
those of the control.
The bacterial counts in polluted soil remedied with
nutrient supplement were higher than the fungal counts
in the biodegraded crude oil polluted soil. The higher
counts of bacteria compared with fungi may be as a
result of the nutrient status of the soil (Jobson et al.,
1974) and the presence of some toxic components which
do not favor fungal growth.
The decrease in pH value may be due to
increased degradation of crude oil by microorganisms in
the soil, resulting in accumulation of acidic metabolites.
Journal of Research in Biology (2012) 2(6): 544-548
However, as the week progressed the Total petroleum
hydrocarbon (TPH) content in the samples decreased
(shown in Tables I-IV). The reduction in TPH content
may be due to the utilization of petroleum hydrocarbon
as food and energy by microorganisms. Reduction of
nutrients and increase in THB, THF, HUB,HUF counts
as the week progressed as shown in Tables I-IV indicated
the absorption of nutrients by the microorganisms for
energy and proliferation.
The moisture content of the remedied crude oil
polluted soil was lower than that of the control (shown in
Table IV). It may be due to the fact that that the dried
nutrient supplement in the samples absorbed some
amount of the moisture leading to low moisture content.
Moreover, high humidity equally can contribute to low
moisture content in the samples. Both nitrogen and
phosphorus levels were higher in the supplemented crude
oil polluted soil than non-supplemented polluted soil.
This agrees with the findings of Odu (1972) who
reported increase in nitrogen and phosphorus contents of
a supplemented crude oil polluted soil. The reason could
be due to higher organic matter content of the poultry
manure (nutrient supplement) and as well the polluted
soil.
Further more, the temperature value was higher
in the remedied crude oil polluted soil than in control
(shown in Table). This may be as a result of the activities
of microorganisms which could generate much heat in
the process of hydrocarbon degradation thereby
increasing the temperature value. Additionally, high
humidity may affect the temperature value of the
samples. The rate of crude oil biodegradation in the soil
was rapid. This may be due to the fact that the
indigenous microorganisms in the soil and those
introduced through nutrient supplement have efficient
ability in utilizing the residual crude oil as a source of
carbon and energy (ljah et al., 2003). Crude oil contains
hydrocarbon and does not resist attack by
microorganisms (Atlas 1995).
546
 Ebe et al.,2012

Table I. Trend of Change in Zero Week Table II. Trend of Change in the fourth week
Parameters A B C D Parameters A B C D
3 4 5
THB (cfug ) -1
5.00X10 2
7.40X10 3
1.02X10 5
1.20X10 2
THB (cfug ) -1
1.46x10 1.67x10 2.21x10 1.30x102
HUB (cfug-1) 3.20X102 4.1X103 7.90X103 2.20X102 HUB (cfug-1) 8.30x102 1.24x104 1.46x104 2.40x102
THF (cfug-1) 2.40X102 2.80X102 4.10X102 1.10X102 THF (cfug-1) 7.70x102 9.30x102 1.25x103 1.30x102
HUF (cfug-1) 5.00X101 7.00X101 8.00X101 3.00X101 HUF (cfug-1) 1.10x102 1.50x102 2.00x102 2.00x101
TPH 387.00 384.00 382.00 390.00 TPH 370.00 366.00 361.00 388.00
PH 5.21 5.24 5.28 4.11 PH 5.13 5.17 4.22 4.10
Temp. (C) 25.00 25.00 26.00 25.00 Temp (C) 26.00 26.00 27.00 25.00
Nitrate (%) 12.40 12.80 12.80 2.40 Nitrate(%) 11.70 12.10 12.40 2.40
Phosphate(%) 0.72 0.76 0.78 0.17 Phosphate(%) 0.51 0.60 0.69 0.16
Sulphate (%) 67.00 70.00 75.00 20.00 Sulphate(%) 58.00 65.00 72.00 18.00
Moisture 22.00 17.90 12.90 28.20 Moisture 4.10 19.30 13.60 31.20
Table III. Trend of Change in the Eight week Table IV. Trend of Change in the 12th week

Parameters A B C D Parameters A B C D
-1 3 4 5 2 -1 3 4 5
THB (cfug ) 2.01x10 2.66x10 3.17x10 1.30x10 THB (cfug ) 2.89x10 3.43x10 3.91x10 1.50x102
HUB (cfug-1) 1.33x103 1.75x104 2.05x104 2.50x102 HUB (cfug-1) 1.91x103 2.48x104 3.02x104 2.30x102
THF (cfug-1) 1.22x103 1.36x103 1.66x103 1.40x102 THF (cfug-1) 1.67x103 1.88x103 2.00x103 1.40x102
HUF (cfug-1) 1.70x102 2.10x102 2.70x102 4.00x101 HUF (cfug-1) 2.20x102 2.80x102 3.60x102 5.00x101
TPH 352.00 339.00 320.00 386.00 TPH 325.00 311.00 284.00 385.00
H H
P 4.98 4.94 5.10 4.12 P 4.84 4.83 4.94 4.11
Temp (C) 27.00 28.00 29.00 25.00 Temp(C) 30.00 30.00 32.00 26.00
Nitrate(%) 9.50 10.90 11.70 2.30 Nitrate (%) 8.30 9.20 10.50 2.20
Phosphate(%) 0.41 0.49 0.57 0.16 Phosphate (%) 0.32 0.40 0.49 0.14
Sulphate(%) 52.00 60.00 65.00 17.00 Sulphate(%) 37.00 48.00 53.00 15.00
Moisture 21.70 17.60 11.60 29.20 Moisture 21.40 16.80 10.10 29.90
There is a decrease in pH, TPH in the supplemented contaminated soil while temperature, THB,THF, HUB
and HUF increased. Also, the moisture content in the supplemented soil was lower than the control.

Finally, the biodegradation that proceeded in the
unsupplemented soil also indicated that degradation can
also be carried out without supplementation of organic
nutrient, although much slowly.
CONCLUSION
This study showed that nutrient supplement is
effective in detoxifying and as well remedying the soil
polluted with crude oil or its associated components.
547
Therefore, based on the findings of this research project
it is necessary to employ the use of nutrient supplement
(poultry manure) when there is need to remedy spilled
sites.
The study also showed that there is residual crude
oil in the soil after 12 weeks of investigation and that the
physicochemical properties of the soil were significantly
affected. Conclusively, the study has show that
Journal of Research in Biology (2012) 2(6):544-548
Ebe et al.,2012

remediation by poultry manure is effective in the cleanup

of polluted sites.

REFERENCES
Aboribo RI. 2001. Oil politics and the Niger Delta
Development Commission The tussle for control and
Domination. Afri J. Environ studies, 2:168-175.

Adenipekun C. Oyinkasola. 2008. Bioremediation of

engine. Oil polluted Soil by pleurotus tuberregium
sengre, a Nigerian white-rot fungus African Journal of
Biotechnology. 7(1):55-58.

Atlas RM. 1995. Bioremediation of petroleum

pollutants lnternational Journal of Biodeterioration and
Biodegradation; 35:317-327.

Ijah UJ and Antai SP. 2003. ``Removal of Nigerian

light crude oil in soil Over a 12 months Period
International Journal of Biodeterioration And
Biodegradation. 51:93-99.

Jobson AM, Mc Laughlin FD, Westlake DN. 1974.

``Effects of Amendment on microbial utilization of oil
Applied to soil. J. Applied microbial, 27:166-170.

Nester EW, Denise GA, C. Evans R, Jr, Nancy N,

Pear S and Martha TN. 2001. Microbiology: A Human
perspective. 3rd ed. New York: Mc Graw Hill.

Odu TK. 1972. ``Microorganisms and petroleum

pollutants``. Bioscience. 28:387-369.

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Journal of Research in Biology (2012) 2(6): 544-548 548

 Journal of Research in Biology
Peanut Oil Cake: A Novel Substrate for Enhanced Cell Growth and
Prodigiosin Production from Serratia marcescens CF-53
Journal of Research in Biology
Authors:
Chandrashekhar Naik1,
Srisevita JM1,
Shushma KN1, Farah
Noorin1, Shilpa AC1,
Muttanna CD1, Darshan N,
Sannadurgappa D2.
Institution:
1. Microbiology and
Downstream Processing
Laboratory, Department
of Biotechnology,
Sir M Visvesvaraya Institute
of Technology, Yalahanka,
Bangalore- 562 157.
2. Centre for Sustainable
Technologies, Indian
Institute of Science (IISc),
Bangalore-560 012.
Corresponding author:
Chandrashekhar Naik.
Email:
cknaikg@gmail.com
Phone No:
+91-080- 2846 7248.
Fax:
+91-080- 2846 7081.
Web Address:
http://jresearchbiology.com/
documents/RA0251.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Original Research
ABSTRACT:
Different agro-wastes such as peanut oil cake (POC), coconut oil cake (COC),
sesame oil cake (SOC), safflower seed oil cake (SFOC) and cotton seed oil cake (CSOC)
were screened for prodigiosin production through fermentation employing
Serratia marcescens-CF-53. POC was highly beneficial for the pigment production.
Fermentation parameters have been standardized; maximum amount of pigment was
obtained (~40 mg ml-1) in POC extract at 30oC for 42 h using 8% inoculum density
(1X107cell/ml) compared to PG broth (14.2 mg ml-1). The pigment yield was almost
three fold higher than that of the PG broth. Use of POC extract as a raw material for
pigment production could be of great commercial significance. This report appears to
be the first one on prodigiosin production using POC as a substrate.
Keywords:
Serratia marcescens, red pigment, prodigiosin, pea nut oil cake, fermentation.
Article Citation:
Chandrashekhar Naik, Srisevita JM, Shushma KN, Farah Noorin, Shilpa AC,
Muttanna CD, Darshan N, Sannadurgappa D.
Peanut Oil Cake: A Novel Substrate for Enhanced Cell Growth and Prodigiosin
Production from Serratia marcescens CF-53.
Journal of Research in Biology (2012) 2(6): 549-557
Dates:
Received: 02 Jun 2012 Accepted: 16 Jul 2012 Published: 03 Aug 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
549-557 | JRB | 2012 | Vol 2 | No 6
www.jresearchbiology.com

INTRODUCTION:
The interest in prodigiosin and prodigiosin like
pigments has been on the increase due to the biological
activities of these compounds. The pigment prodigiosin,
an alkaloid compound, has antibacterial, antifungal,
antimalarial, antiproliferative and immunosuppressive
properties (Montaner and Tomas, 2001; Soto et al.,
2004). It also induces apoptosis in certain cancer cells
(Furstner, 2003; Perez et al., 2003). This secondary
metabolite is produced by several microorganisms like
Serratia, Pseudomonas, Streptomyces and certain marine
bacteria (Gerber, 1975; Giri et al., 2004) through quorum
sensing (Fuqua et al., 1996; Wei et al., 2006).
The naturally occurring prodigiosin and prodigiosin like
pigments exist in cyclic and acyclic forms and contain
4 methoxy-2, 2 bipyrole ring (Manderville, 2001).
Prodigiosin, itself an acyclic form, contains
2 mehtyl-3-pentyl-pyrrole ring. The cyclic forms include
metacycloprodigiosin, nonylprodigiosin, streptorubin B
and cycloprodigiosin (Manderville, 2001).
The gram negative Serratia marcescens are
prolific producers of red pigment (Rustom et al., 1990).
From an industrial point of view and because of its
biological potentialities, there is a necessity to develop a
high throughput and cost-effective bioprocess for large
scale prodigiosin production. Earlier workers have
reported many differential, selective and synthetic media
such as, nutrient broth, peptone glycerol broth (PGB),
LB broth etc, (Montaner et al., 2000; Haddix and
Werner, 2000) for prodigiosin and prodigiosin like
pigment production. The yield of the pigment is varying:
-1
3 g L (Cang et al., 2000) incorporating ethanol in the
-1
medium, 152 mg l (Wei and Chen, 2005) in LB broth
and oil supplements, and 0.08 g-4.28
employing different carbon and nitrogen sources
(Min-Jung-Song et al., 2005). Giri et al, (2004) obtained
-1
17 to 39 g L yield and opined pea nut and sesame
powder and their oils to be good substrates for
prodigiosin biosynthesis. Our earlier works on citric acid
550
Naik et al.,2012
production had revealed that the some agro wastes could
be beneficially employed to enhance citric acid
production (Lingappa et al., 2002). It was, hence,
considered worthwhile to attempt enhancing prodigiosin
production using such agro-wastes.
The pea nut cakes obtained after extraction of oil
are at best used as a cattle feed supplementation. Our
earlier studies had revealed the presence of sufficient
sugar and fat content in these cakes. However, no
attempts appear to have been made to use these
agro-wastes for prodigiosin production. Hence, the
present work was undertaken to evaluate production of
prodigiosin and prodigiosin like pigments employing
S. marcescens CF-53.
MATERIALS AND METHODS
a Microorganism and cultural conditions
A red color pigment producing bacterial strain
was isolated from soils near dairy industry waste
disposal points in and around Bangalore and identified
as Serratia marcescens CF-53 and deposited at Microbial
Type Culture Collection and Gene Bank (MTCC),
Chandigarh, India (Accession No. MTCC7643). Sub
culturing and maintenance was on peptone glycerol agar
(PGA) slants. Seed culture of S. marcescens CF-53 to be
used as inocula for the experimental work was
maintained in PG broth.
Screening of substrates and culture medium
Different agro-based substrates, the peanut oil
cake (POC), the coconut oil cake (COC), the sesame oil
cake (SOC), the safflower seed oil cake (SFOC) and the
cotton seed oil cake (CSOC) were obtained from local
market in Bangalore and powdered. To 100 ml water, 5 g
gL-1 of each cake powder was separately added and boiled for
30 min and allowed to cool. The supernatant was further
centrifuged at 7000 rpm for 15 min to obtain a
debris-free solution. To the clarified solution, agar was
added (15-20 gL-1) after due adjustment of pH to 7.0 and
autoclave sterilized at 121C for 20 min. The agar plates
Journal of Research in Biology (2012) 2(6): 549-557
Naik et al.,2012
then prepared were employed in preliminary screening
studies to evaluate as to which substrate was most
suitable for prodigiosin and prodigiosin like pigment by
S. marcescens CF-53. After the preliminary screening the
peanut cake was similarly processed and the media
without agar served as the substrate and PG broth as the
control for the pigment production. The flasks were
7
inoculated with 0.5 ml of (1 X 10 cells/ml) 24 h cell
suspension of S. marcescens CF-53 and incubated at
30C in an orbital shaker at 200 rpm for 48 h. The initial
inoculum was 2% to avoid coloration of the starter
culture in the beginning only.
Effect of biotic and abiotic parameters
To standardize the culture parameters for
biomass yield and prodigiosin production, the effect of
varying pH, temperature, incubation period, inoculum
size, substrate concentration as well as supplementation
of different carbohydrates, nitrogen sources, salts and
metal ions were studied.
T he p ro du ct io n of pro d ig io sin by
Serratia marcescens CF-53 is very sensitive; therefore,
the selected parameters were optimized to achieve higher
yield of prodigiosin. Once a fermentation parameter was
optimized, the optimum level of that parameter was
employed in the subsequent studies wherein another
parameter was to be optimized.
Quantification of Prodigiosin
Culture broth (1 ml) was mixed vigorously with
3 ml of methanol in a vortex mixer and then centrifuged
at 5000 rpm for 5 minutes. An amount of 0.8 ml
supernatant was further mixed with 0.2 ml of acidified
methanol. The optical density of the resulting solution
was measured as per Wei and Chen (2005).
Dry cell weight determination
Cell concentration was determined by measuring
the dry cell weight (DCW). Culture broth was filtered
through a washed and preweighed micropore filter
(0.2m). The cell-loaded filter was dried at 105C and
reweighed to determine cell biomass (Wei and Chen,
Journal of Research in Biology (2012) 2(6): 549-557
2005).
Purification of pigment for Mass Spectroscopic
analysis
S. marcescens-CF53 grown in POC extract was
centrifuged at 10000 rpm for 20 minutes and the cell
biomass was collected. The pigment from the
biomass was extracted with acidified methanol
(1N HCl 1 ml: methanol 24 ml) and the extract was
centrifuged at 5000 rpm for 15 min. The supernatant was
concentrated in vacuum evaporator at 60C to obtain
crude pigment. The crude concentrated pigment was
dissolved in methanol and the solution was passed
through hexane balanced silica gel (mesh 60-120)
column. The trapped pigment within the column was
eluted with 10 M ethyl acetate to liberate the adsorbed
pigment. Further, elute was harvested and dried in a
vacuum drier at 60oC to obtain the purified product.
Small fractions of the pigment were collected and silica
gel thin layer chromatography (TLC) was performed
using the chloroform/methanol/ water (80:15:05 v/v) as
solvent system. Fractions having single red spot with
Rf value of 0.72 were pooled and the solvent was
evaporated. Pooled samples were dissolved in methanol
for molecular mass determination by ESI-MS
(Min-Jung-Song, et al., 2005).
Scale up studies in Bioreactor
The fermentations were carried out in a 2 L lab
scale bioreactor (Sartorius B-Lite, India) equipped with a
pH, temperature and DO electrode. The fermentation was
started after inoculating 24 h seed culture of the
Serratia marcescens CF53 (8% v/v) into the POC extract
broth. Fermentations were conducted under the condition
of temperature 30oC, air flow rate of 2 vvm with an
agitation speed of 200 rpm and initial pH 7.0, unless
mentioned otherwise. The working volume was
1 L with foam level controlled by automatic addition of
1% antifoam (mineral oil). Culture pH was controlled by
using 2.5 N HCL and 2.5 N NaOH as and when required.
Samples were drawn periodically to estimate the yield of
551

prodigiosin.
RESULTS AND DISCUSSION
The results of the studies regarding prodigiosin
production by S. marcescens CF-53 employing different
substrates are presented in Figure 1. The richness of the
pigment and its production was observed to be maximum
in POC medium. Variation in the color of the pigment
produced in different substrates could be due to the
compositions of the extracts of different substrates.
Therefore, POC extract medium was the preferred
choice-medium to carry out systematic investigations on
medium improvement and process optimization for the
higher yield of prodigiosin by Serratia marcescens
CF-53.
Effect of pH
Maximum yield of the pigment was obtained at
pH 7 in POC and the control PG broth growth media
(Fig. 2A), 14.2 mg/ml and 8.5 mg /ml respectively. Thus
the pigment yield in POC was almost 1.6 times that in
PG both. It has been reported that the secondary
metabolites are generally produced in the pH range of
6-8 (Sole et al., 1997). Differences in the yield would
depend on medium composition. PG broth contains
peptone and yeast extract as nitrogen source and glycerol
as carbon source and thus lacks a direct source of carbon
(Wei et al., 2005). On the other hand, POC extract
naturally inherits oil as its carbon source and basically
contains saturated and unsaturated fatty acids,
Fig 2. Effect of pH, temperature and incubation time on prodigiosin production by Serratia marcescens CF-53;
() Pigment in POC extract; () pigment in PG broth; () Cell growth in POC extract; () Cell growth in PG broth
552
Naik et al.,2012
Fig 1. Growth and pigment production profile of
Serratia marcescens CF-53 on different substrates.
(a) COC extract agar; (b) SOC extract agar;
(c) POC extract agar; (d) PGA; (e) SFOC extract
agar and (f)CSOC extract agar
carbohydrates, minerals, vitamins etc.
Effect of temperature
The results of the influence on growth and
pigment production by S. marcescens CF-53 are
presented in Fig. 2B. Production of prodigiosin and cell
dry weight was maximum at 30C in both the POC
extract broth (18.2 mg ml-1 and cell dry weight
35.2 mg ml-1, respectively) and the PG broth
-1
(10.2 mg ml and cell dry weight 23.5 mg ml-1,
respectively). The increase is almost 1.7 times in
pigment yield and 1.4 times in cell growth in POC broth
when compared to those in the PG broth. In case of PG
broth, the viscosity of glycerol decreases at this
temperature leading to increased accessibility of the
Journal of Research in Biology (2012) 2(6): 549-557
Naik et al.,2012

Fig 3. Effect of inoculum size and substrate (POC) concentration on growth and prodigiosin production by
Serratia marcescens CF-53; A, () Pigment in POC extract; () pigment in PG broth; () Cell growth in POC
extract; () Cell growth in PG broth: B, () pigment in PG broth; () Cell growth in POC extract.

carbon source to the microorganism (Giri et al., 2004).
Pigment production was totally blocked at 40C in
PG broth (on par with the report of Giri et al., 2004) and
o
at 42 C in the POC broth. The impact of physiological
role of temperature in blocking prodigiosin synthesis
seems to vary with mediums of different substrate
composition (Giri et al., 2004).
Effect of incubation period
Incubation period plays a very important role in
the production of bacterial secondary metabolites.
Incubation period varied in the range of 6 to 60 h.
Pigment production commenced from 8th h in PG broth,
th
while the same started from 10 h of growth in POC
th nd
medium (Fig. 2C) and it was linear from 18 to 42 h in
case of POC broth and from 12th to 36th h in case of PG
broth, the maximum yields being 28.6mg ml-1 and dry
cell weight 45.8 mg ml-1 in the POC broth and the same
being ~13 mg ml-1 and 25.6mg ml-1 respectively in PG
broth. This is almost a 2.2 fold increase in pigment yield
in POC when compared to its yield in the PG broth. The
early onset of pigment production in PG broth appears to
be due to its rich nitrogen sources. PG broth contains
peptone, meat and beef extracts which are digests of
plant or animal proteins and glycerol is the carbon source
(Giri et al., 2004). The POC broth on the other hand is a
complex and undefined nutrient source of saturated and
unsaturated fatty acids yielding carbon, proteins,
minerals, vitamins and hence, the delay in pigment
production onset might have been caused.
Effect of inoculum density
Lower initial inocula in both the broths led to
lower prodigiosin production when compared with
higher initial inocula (Fig. 3A). Pigment production was
maximum with 8% initial inoculum density. It was
36.2 mg ml-1 in POC broth and only 14.2 mg ml-1 in
PG broth, almost 2.6 fold pigment production being
observed in POC broth. Pigment expression mainly
depends upon quorum sensing phenomenon
(Fuqua et al., 1996). Growth in liquid culture at low cell
density allows low level expression of a membrane
Fig 4. Electro spray Ionization Mass spectrometry
(ESI-MS) shows the prodigiosin isolated from
Serratia marcescens CF-53 has molecular weight of
325.20 Da.

Journal of Research in Biology (2012) 2(6): 549-557 553

 Naik et al.,2012

permeable positive regulator of prodigiosin expression. biomass production alone was benefited, not pigment
The intracellular concentration of the regulator remains production. Probably higher substrate concentrations
low at low cell density due to its diffusion across the cell suppress prodigiosin production. The prodigiosin
-1
membrane after synthesis. However, as the cell density production was 39.8 mg ml in POC broth compared to
increases, the intracellular concentration of regulator PG broth (14.2 mg ml-1) in 42 h, almost 2.8 times in POC
increases to a threshold needed for quicker activation of medium than in PG broth.
prodigiosin expression (Haddix and Werner, 2000). Effect of carbohydrates and Nitrogenous sources
Effect of substrate concentration Varying productions of pigment and biomass
Substrate concentration too influences the could be obtained in PG broth amended with different
production of pigment and biomass (Fig. 3B). Maximum carbon sources. Yet, maximum pigment and biomass
pigment production was observed at 8% (w/v) of POC production were obtained in POC medium alone, without
extract. But with higher substrate concentrations, any supplementary additions of carbon sources (Table 1).
Table 1. Effect of carbon source on cell growth and Supplementing different carbon sources to PG broth
pigment production by S. marcescens CF-53 yielded interesting results; in many instances, there is no
Carbon source Cell dry weight (mg ml-1) Pigment (mg ml-1) correlation between biomass yield and pigment
No carbon 20.00 1.00 production. Glucose appeared to inhibit pigment
Glucose 21.00 1.58 production, probably by catalytic repression or lowering
Galactose 22.98 3.10 the media pH as also observed by Sole et al., (1997);
Fructose 23.00 6.15 hence the use of glycerol as glucose alternative in
Sucrose 25.00 3.16 PG broth to support prodigiosin like pigments
Lactose 21.30 6.14
Maltose 23.10 5.15
Manitol 23.33 3.60
Malibiose 19.80 0.09
Rafinose 15.60 0.04
Xylose 16.60 0.04
Cellobiose 14.20 0.10
Arbinose 10.98 0.10
Rhamnnose 19.86 1.13
Soluble starch 29.08 11.50
Corn starch 35.50 10.00
Potato starch 37.60 13.08
Rice starch 37.08 12.00
POC extract 62.80 39.80
Medium PG broth: 10 g carbon source, 10 g
Bactopeptone, 10 g Beef extract, 2 g K2HPO4l-1,
2 g NaCl, 10 ml glycerol in 1 l distilled water
adjusted to pH 7 and incubated in incubator shaker
at 200 rpm at 28oC for 48 h. Two batches of growth Fig 5. Scale up studies in bioreactor with
were analyzed in replicates. POC medium by Serratia marcescens CF-53

554 Journal of Research in Biology (2012) 2(6): 549-557

Naik et al.,2012

(Montaner et al., 2000, Bae et al., 2001).
Though tryptone, yeast extract and soybean meal among
the various nitrogen sources induced better pigment and
biomass production than the PG broth, they were not
even half of those observed with POC medium alone
(Table 2). These results support similar observations of
Wei and Chen (2005). Various salts (NaCl, KCl, NaNO3,
Na2SO4 , CH3COONa) at 1% (w/v) as well as metal ions
(CuSO4, CoCl2, FeSO4, MnSO4, ZnSO4, MgSO4 and
CaCl2) at 0.1% (w/v) inhibited pigment production
(data not shown), thus supporting the observations of
Melvin and Elaine (1973).
Determination of molecular mass by ESI-MS
The purified pigment from the culture broth was
analyzed by ESI-MS (Figure 4). One major peak was
observed; its signal at 325.20 matches the molecular
weight of prodigiosin (Montaner and Tomas, 2001).
Therefore, the pigment obtained in this present study is
ascertained to be prodigiosin, the same red pigment
produced by Serratia spp. and Streptomyces spp.
(Gerber, 1975).
Scale up studies in bioreactor
Conditions for cell growth and prodigiosin
production in Ehrlenmeyer flasks become limited due to
substrate consumption as well as metabolite
accumulation. Therefore, studies were carried out in the
bioreactor employing the biotic and abiotic parameters
already standardized hitherto (Fig. 5) The maximum
amount of prodigiosin recovered was ~ 40 mg ml l-1 and
dry cell weight was 65.2 mg ml-1 (data not shown). This
yield appears to be ~3 fold higher than that reported by
Bae et al., (2001) who employed novel integrated
bioreactor with internal absorbent resins. However,
Min-jung-Song et al., (2005) reported that resins exhibit
inhibitory effects on prodigiosin production. In the
present study, neither internal nor external adsorbents
were employed; yet high levels of prodigiosin
productions could be achieved using conventional batch

Table 2. Effect of nitrogen source on cell growth and fermentation process.

pigment production by S. marcescens CF-53
Nitrogen source Cell dry weight (mg ml-1) Pigment (mg ml-1) CONCLUSION
No nitrogen 0.00 0.00 The present study has revealed the feasibility of

Yeast extract a cost-effective fermentation strategy that can lead to

24.56 8.12
(YE) enhanced production of prodigiosin pigment employing
Soybean meal 35.41 14.12 Serratia marcescens CF-53. Further, the significance of

Cas-aminoacids the present study is that an economically cheap and

26.22 10.88
(CAS) easily available byproduct can be used, without any
Tryptone 24.52 9.87 external addition of nutrients (sugars, proteins, minerals

Urea 13.23 4.01 etc.) for prodigiosin production using peanut oil cake as
substrate for fermentation. Studies are in progress
Dried yeast 23.29 6.05
for large scale pigment production employing
NH4NO3 14.70 2.03
Serratia marcescens CF-53 using pea nut oil cake as the
NH4Cl 13.15 0.01
substrate.
NH4SO4 14.20 0.02
POC extract 62.82 39.80 ACKNOWLEDGEMENT
Medium PG broth: (see Table-1) was used which The authors wish to thank the Sri
contained an indicated nitrogen source (20 g l-1) in
place of Bactopeptone and Beef extract. Two batches Krishnadevaraya Education Trust (KET) for financial
of growth were analyzed in replicates. support in executing this project. We would also like to

Journal of Research in Biology (2012) 2(6): 549-557 555

 Naik et al.,2012

express our sincere thanks to Dr. H.G. Nagendra and Dr. Haddix PL and Werner TF. 2000. Spectrophotometric
P.M. Nimbargi for their advice during manuscript assay of gene expression; Serratia marcescens
preparation. pigmentation. Bioscene, 26(4):3-12

Lingappa K, Chandrashekhar Naik, Vivek Babu CS,

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Giri AV, Anandkumar N, Muthukumaran G,
Perez-Tomas R, Montaner B, Lalgostera E, Soto-
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 Journal of Research in Biology An International Scientific Research Journal

Original Research

Effects of dietary energy levels on growth performance,

feed utilization and body composition of Rainbow trout
Journal of Research in Biology

Authors: ABSTRACT:
ABA Mustapha1,
Belghyti Driss1, In order to compare the growth performance of trout with two extruded
Elkharrim Khadija1, foods and their impact on body composition, an experimental test was conducted
Benabid Mohammed2, from June 1 to October 5, 2010 at National Center of Hydrobiology and Fish Culture.
Said Aboulfaraj3. The comparison of the two foods with different formulation and different energy is
perfomed in isoenergetic conditions. Following this study, two diets were formulated :
Institution:
the extruded diet A with 41% crude protein, 27% fat and 20% carbohydrates and the
1. Biology and Health
Laboratory, Environmental extruded food B with 39.7% CP, 24% fat and 15,7 carbohydrates, with digestible
and Parasitology Team/UFR energy respectively of 21.32 Mj and 19.32 Mj. The initial average weight of the trouts
Doctoral "Parasitology was 100 g from the same batch of eggs. They were divided randomly into six fiberglass
compared: Medical and conical tanks at open circuit . The diet was assigned to six tanks for 50 fish each with
Veterinary Applications," three replicates for each diet and the experiment was conducted for 127 days.
Sciences Faculty, The ratio DP/DE of diet influenced feed conversion ratio and specific growth
Ibn Tofail University, rate (p<0.05) . The best FCR was obtained with the extruded food A with 1.18 v.s 1.26.
Knitra B.P. 133, 14000, The higher IV was obtained with the low DP/DE (p<0.05). Final whole-body lipid
Morocco. content was positively related to dietary lipid levels and to digestible energy. Better
2. National Center of retention of protein was obtained by the diet A.
Hydrobiology and This study is consistent with current trends in the nutrition of fish and
Pisciculture (NCHP) Azrou salmonids especially designed to reduce the ratio of DP/DE in order to have better
Morocco. perfomances of growth and a better quality of the fish.
3. Les Domaines Agricoles,
Keywords:
Domaine Ain Aghbal Azrou
Morocco. growth, feed efficiency, energy ,body composition, rainbow trout.

Corresponding author: Article Citation:

ABA Mustapha. ABA Mustapha, Belghyti Driss, Elkharrim Khadija, Benabid Mohammed,
Said Aboulfaraj.
Effects of dietary energy levels on growth performance, feed utilization and body
Email: composition of Rainbow trout.
aba_mustapha@yahoo.fr Journal of Research in Biology (2012) 2(6): 558-565

Dates:
Web Address:
http://jresearchbiology.com/ Received: 10 Apr 2012 Accepted: 18 Jul 2012 Published: 07 Aug 2012
documents/RA0237.pdf.

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution, and
reproduction in all medium, provided the original work is properly cited.

558-565 | JRB | 2012 | Vol 2 | No 6

Journal of Research in Biology
An International Scientific
Research Journal www.jresearchbiology.com
 Aba et al., 2012

INTRODUCTION 1994)
The success of the aquaculture is based on the So, one of the factors affecting the optimization
provision of food containing the highest level of energy of the efficiency of the food is the balance between
and nutrients for growth. Food is the major cost for digestible protein (availability of amino acids) and
aquaculture (Kaushik, 2000). The food depends mainly energy in non-protein food. This balance is represented
on fish meal which is usually the major component of by the ratio of digestible protein (DP) and digestible
food in aquaculture, and its nutritional quality since it is energy (DE) of the food (DP / DE). To get a better
rich in essential amino acids (Medale, 2009) and the optimization of the ratio DP / DE, the rate of this ratio
effective use of these proteins are closely related to their can be reduced if an additional power source (fat or
concentration in the diet and the availability of food digestible carbohydrates) is provided to save protein.
with other non-protein sources, such as lipids (Boujard, 2004). Many studies have shown that
and carbohydrates (Kaushik and Medale, 1994;. increasing dietary DE by increased non-protein energy
Watanabe, 2002; Chaiyapechara et al., 2003., food resulted in better retention of protein with a
Morrow et al., 2004; Azevedo et al., 2004 ; Eliason decrease in the excretion of ammonia nitrogen, (Dias
2007). et al., 1999, Watanabe, 2002; Bureau, 2006, Aba et al.,
To optimize the performance of growth and 2011b).
control releases fish in fish farming, there is involvement In aquaculture, using appropriate feeding
of two key factors, feed formulation and manufacturing management is necessary, to gain an economic
method (Hardy and Barrows, 2002). During the advantage and to maximize growth and feed conversion
15 recent years, the aquaculture feed manufacturing has efficiency And the objective of this study was to evaluate
seen a great evolution which allowed food to be pressed the effects of different levels of dietary protein,fat,
with a high rate of protein, low fat and high carbohydrate carbohydrate and energy on growth, feed utilization and
to extrusion processing (Medale and Kaushik, 2008) body composition of juvenile rainbow trout and to
with which we obtain extruded fish feed, wich are determine the optimal diet for fish.
characterized by high energy and are a significant
Table 1. Proximate composition of experimental diets
substitution of fish meal with fats and carbohydrates
Extruded Extruded
(starch mainly). diet A diet B
Energy and nutrient requirements are affected by Dry matter 94.4 % 96.1 %
several factors and they may vary in different stages of Protins 41.1% 39.7 %
the life cycle of the animal. Several authors have Lipids 27.4% 24.4 %
described optimal dietary P/E ratios in some rearing carbohydrates 20.4% 15.7 %
species such as rainbow trout, Oncorhynchus mykiss Moisture 5.6 % 3.9 %
(Kim and Kaushik, 1992; Lanari et al., 1995). Gross Energy 23.73 21.70
(GE, Mj Kg-1)
But the success of the Fish culture is based on
the provision of rations containing optimal levels of Digestible energy 21.32 19.32
(DE, MJ Kg-1)
energy and nutrients for growth (Hardy and Barrows,
2002). The optimization of the ratio protein / energy DP / DE (g MJ-1) 17.35 18.48
(DP:Digestible Prtoein)
(P: E) has been therefore having an important role in
Ratio P /L 41/27 40/24
protein and energy utilization (Kaushik and Medale,
559 Journal of Research in Biology (2012) 2(6): 558-565
Aba et al., 2012
MATERIALS AND METHODS
Experimental design:
The experiment was conducted between June 1,
2010 and October 5, 2010 at National Center of
Hydrobiology and Fish Culture (NCHP) in Azrou
(Morocco).
This test was conducted in fiberglass
3
conical tanks of 0.8 m of volume at open circuit with
an initial load of 5 kg fed by spring water at a
constant temperature of around 14C 0.2 and a flow
3
rate of 1.6 m /h, with a time of renewal of water
2 times per hour with oxygen levels above 80%
saturation. The average content of dissolved oxygen in
the outlet of the ponds was 7.1 ppm, and pH around 7.
Biological materials:
300 juvenile trout females triploid of average
weight of 100g 3g from the same batch of eggs were
divided randomly into six fiberglass conical tanks.
The test was conducted in triplicate culture, fish
were fed manually and the daily ration was split into two
meals distributed at 09 am and 03 pm, seven days a week
for 127 days, according to the feeding table provided
by the supplier of food (LeGouessant). Every two weeks
8 fish of each batch have been anaesthetized after 24 h of
fasting in order to measure the size and the weight of
each fish for measurements of size and weight. The
quantities of food distributed were weighed to estimate
the consumption by the fish between two weighings.
Experimental foods
Proximate composition of experimental diets are
shown in Table 1.
The rate of feeding:
The experimental test was aimed at comparing
two non-isoenergetic foods of different formulations on
their growth performance of fish and their flesh quality
in isoenergetic condition. The amount of food distributed
is consistent with the feeding tables of two extruded
foods (ExA, ExB) that have different digestible energy
21.32 Mj, 19.32 Mj, respectively. These rates of
Journal of Research in Biology (2012) 2(6): 558-565
rationing depends on the temperature of the water close
to the site. We have set the rates according to the
temperature of the site which is about 14C so that the
quantitative ratio for the same food energy intake is:
amount of food extruded (ExA) 1.10 = amount of
extruded (ExB) food .
Gross energy was calculated using the
following values: crude protein = 23.7 kJ/g, crude
lipids = 39.5 kJ/g and carbohydrate = 17.2 kJ/g as
proposed by Brett and Groves (1979). The calculation of
digestible energy is obtained by the coefficient of
digestibility of protein, fat and carbohydrates gelatinized
or raw (Guillaume and Medale, 2001).
Body measurements:
Body mass, length and organ mass were recorded
to evaluate the Condition Factor (CF) = ([total body
weight (g)] / [total body length (cm)]3, viscerosomatic
index (VSI) = ([viscera weight (g)] / [total body weight
(g)] x 100) (Ricker, 1979).
Zootechnical parameters:
Calculations: The following variables were
calculated: Daily Weight gain (g/fish /day = (final body
weight initial body weight) / initial body weight.
Survival (%) = 100 x (final amount of fish /
initial amount of fish) Average daily growth (g) = (final
wt - initial wt) / no. Of days
Specific growth rate (SGR) = [In final mean
body wt. (g)] -[In initial mean body wt. (g)]/days x100
Feed conversion ratio (FCR) = g feed
consumption / g(final biomass - initial biomass).
BioChemical analysis:
Crude protein, crude fat and ash, at the ventral
muscle was determined, according to AOAC, (1990).
Eight fillets of final fish were sampled and
stored at -25C for proximate analyses, which were
performed according to AOAC. Dry matter was
determined after drying at 105C until a constant weight
was obtained. Ash content was measured by incineration
in a muffle furnace at 525C for 12 h. Crude protein was
560
 Aba et al., 2012

analyzed by the Kjeldahl method after acid digestion The Viscerosomatic index was 09,11 for the diet
using the Gerhardt system. Lipid was determined by A and 10.28 for the extruded food B; The condition
folch method (1957). factor was 1.18 and 1.26 respectively for the the extruded
Statistical studies: food A and B. The survival rate was 100% for the two
Results are expressed as mean ( SD). Our diets, the difference between the two groups was not
results are compared statistically (R Development Core significant. Dietary energy variation affects significantly
Team, 2011). All parameters of growth and yield were the chemical flesh composition of rainbow trout. The
subjected to Analysis of Variance test (ANOVA). rainbow trout fed with extruded feeds B had high levels
Tukeys multiple procedure was used to compare the of lipid and low levels of moisture compared with
differences among mean values. Differences were rainbow trout fed with extruted feeds B (p<0.05),
regarded as significant when P<0.05. probably due to high fat level in the extruded feed B and
with high energy. The crude protein levels of the fish
RESULTS fillet differ (p<0.05) among fish fed with extruded and
During the experiment no mortality and no pelleted feeds.The highest valuesof moisture was
disease were recorded. The two experimental diets were obtained with the extruded diet A with a significant
well accepted by fish throughout the trial, the water difference beween the two diets (p<0.05).
temperature varied during the test between 13.8C and
14.2C. DISCUSSION
During the experimental period, temperatures Growth and feeding performance are shown in
ranges were 13.8C and 14.2C, pH was 6.9 7.2, and table 2.
dissolved oxygen was not less than 6,9.0 mg/l. To Fish meal and fish oil are still considered key
compare growth performance, all results have been ingredients in the formulation of feeds for aquaculture
reported in the average weight of individual fish and the species. Fish meal and fish oil, combined, together
parameters are studied from the average of the three currently account for 30 to 80 percent of the weight of
tanks assigned to each diet.
In this experimental test, it is seen that the Table 2 : Results of Rainbow trout performances
obtained during this experimental test.
performance of zootechnical parameters vary
Extruded Extruded
significantly (p<0.05) between the two dietary treatments Parameters
diet 1 diet B
(Table 3). The highest values in terms of weight gain Initial mean weight (g) 100 3 100 3
Final mean weight (g) 526.66a 47 586b 34
were obtained with the extruded diet A and Duncan's test
Mean weight gain (g) 426.66a 47 486b 34
shows a significant difference between the final weights Daily Weight gain (%) 3.35a 0.09 3.82b 0.11
(p<0.05). Specific growth rate
1.30a 0.01 1.40b 0.03
(%/day)
The best feed conversion ratio (FCR) was FCR 1.26b 0.014 1.18a 0.017
obtained with extruded diet A (1.18). The extruted diet Condition factor K 1.14a 0.02 1.22b 0.03
B has the highest FCR, there is a significant difference Viscerosomatic index 09.11 0.14 10.28 0.12
between the two values of the two dies (p<0.05). The Survival rate (%) 100 100
Values are means of three replications. Data are
SGR was calculated by 3,35% for fish fed with the diet
expressed as mean SD. Values in a row with
A and 3,82 for the extruded diet B,and there was a different superscripts are significantly different from
each other (P<0.05).
significant difference (p<0.05).
561 Journal of Research in Biology (2012) 2(6): 558-565
Aba et al., 2012
most of the salmon, trout, marine fish, and shrimp feeds
sold worldwide (Bureau, 2006).
The protein requirement is dependent upon the
levels of other non protein energy sources lipids and
carbohydrates (Ruohonen and Kettunen, 2004). A recent
approach to assessing fish nutrition recognize the
importance of a mixture design (Ruohonen and
Kettunen, 2004).
The present study has shown that, the growth
performance of rainbow trout can be significantly
influenced by feeding regimes that strongly affect the
feed ingestion and assimilation. Our results revealed that
the ration levels had signicant effects on growth and
conversion efciencies in ngerling of rainbow trout
Numerous studies have demonstrated the
beneficial effect of high energy diets on growth and on
feed efficiency and fish protein (Kaushik and
Oliva-Teles, 1985; Dias et al., 1999; Medale and
Kaushik 2008; Aba et al., 2011a).
However, a supplementation of lipid rather than
carbohydrate as a non-protein energy source, is generally
more effective for increasing energy level because lipid
is readily metabolized by fish especially by the
carnivorous one (NRC, 1993). The addition of lipids to a
diet also contributes to effective utilization of dietary
protein through the sparing effect in fish (Watanabe,
1982; De Silva et al., 1991; Skalli et al., 2004).
Our results for the FCR are higher than those
obtained by Gelineau et al., (2001) and can be explained
by the richness of our food carbohydrates and trout have
a preference for protein and fat instead of carbohydrates.
The results of the present study clearly indicate that the
growth rate of trout was affected by the dietary levels of
Table 3: Fillet composition (g/100 g DM)
Parameters Diet A Diet B
Protein 19.56b 0.27 18.11a 0.17
Fat 8.47b 0.32 7.03a 0.26
Moisture 68.21a 0.24 70.01a 0.15
Means (SE) with different superscript letters were
significantly different (P<0.05).
Journal of Research in Biology (2012) 2(6): 558-565
non-protein energy (P<0.05).
Several studies (Kaushik and Medale, 1994;
Lupatsch et al., 1998) Lupatsch et al., (2001) have
shown that the optimal DP/DE ratio for rainbow trout
may be lowered from the value of 22 to 25 g MJ-1 kg
DM to 17 to 20 g MJ-1 kg DM.
Traditionally, the effect of diet quality on fish
growth is assessed using either DP:DE or the dietary
protein-to-lipid ratio. However, the sparing effect of lipid
and carbohydrate on dietary protein, using the
manufacturing technology and high energy have a
significant improvement in growth performance of
rainbow trout
This study confirms the existence of a positive
effect of high dietary energy contents on feed efficiency
and protein efficiency ratio as already described in the
rainbow trout. The FCR obtained in our test is slightly
higher than that obtained in the tests of De Francesco
et al., (2004 ) because the test foods are higher in fat and
carbohydrates (Gelineau et al., 2001).
The specific growth rate (SGR, 1.30-1.40) values
obtained in this study are indicative of good growth in
rainbow trout and our results are similar to those
reported by Chaiyapechara et al., (2003) who found that
rainbow trout fed diets containing a low ratio DP/DE
had a significantly greater SGR compared to fish fed
diets containing high ratio. The same results are obtained
by Morrow et al., (2004), yildiz (2004), Eliason (2007).
Our results for the condition factor K indicates
that the trout have a good growth in weight rather than
size especially when fed with diet A and these results are
similar with those of Yildiz (2004).
The extruded diet can be to have a greater
incorporation of lipids,(Kaushik, 2000; Aba et al., 2012)
which is probably increased by the IVS and increased
visceral fat is seen in the viscera that there are more
deposition of fat in the rainbow trout (Richard et al.,
2006; Medale, 2009) and IV obtained in this study is
almost similar with the results of Dias et al.,(1999) and
562

Gelineau et al., (2001).
For body composition generally,
accumulation in fish increases with higher levels of
dietary lipid;(Jobling et al., 1998; Rasmussen et al.,
2000; Chaiyapechara et al., 2003). As reported for
several fish species such as rainbow
(Chaiyapechara et al., 2003), sea bass;
et al.,2000) and Atlantic salmon (Hamre et al., 1998), .
Lipid concentration in fish body (fillets),
reflected dietary lipid concentration (Chaiyapechara
et al., 2003; Medale, 2009; Aba et al., 2012). Increasing
the lipid concentration in the feed from 24-27%
increased the fillet lipid concentration from 7,03-8.47%.
The trout composition at the end of the test shows an
increased content of lipids independently of protein
levels (Dias et al., 1999).
Increasing dietary energy intake leads, in almost
all species, to an increase in body. In addition, deposition
of lipids is associated with a lower deposition of water
(Jobling, et al., 1998; Medale, 2010). Moisture content,is
inverse ly c o r r e la t e d to lip id
(Quillet et al., 2007). According to studies by De
Francesco et al., (2004), Quillet et al., (2007),Eliason
(2007), Aba et al., (2012) ,the moisture content increases
as the fat content of fillet is low, Similar results were
obtained for Sea bass by Yildiz (2004) and these results
are consistent with results obtained in this trial.
CONCLUSION
The present study demonstrated that diets
containing high energy and low ratio DP/DE affect the
growth performance and body composition. The high
energy in diet may be an indicator of the good flesh
quality. These results suggest that feeding the fish with
diets containing more of fat and carbohydrate are the
most effective.
The protein sparing effect of lipid and to a lesser
extent carbohydrates have been studied by numerous
researchers. In salmonids, up to 30% lipid diets have
563
Aba et al., 2012
been shown to improve feed and protein utilization
lipid efficiencies and to reduce N excretion.
This study is consistent with current trends in the
nutrition of fish and salmonids and especially designed
to reduce the ratio DP / ED in order to have better
trout perfomances of growth and a better quality of flesh fish.
Pirini
ACKNOWLEDGEMENT
This research was funded by Les Domains
Agricoles, Domaine Ain Aghbal, Truites de l'Atlas
Azrou Morocco. The authors wish to thank the managers
and staff Domaines Agricoles Morocco for funding
and assistance in this study.
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 Journal of Research in Biology An International Scientific Research Journal

Original Research

Honey bee Passiflora edulis Sims. (Passifloraceae) pollination

found in the Pachamalai Hills, Eastern Ghats of Tamil Nadu.
Journal of Research in Biology

Authors: ABSTRACT:
Nandagopalan V2,
Anburaja V2,
Johnson Gritto M2 and Pollination is the process of fertilization in higher plants. In flowers, pollen is
Uma Maheswari R1.
delivered to the stigma through a wide range of mechanisms that insure an
Institution: appropriate balance in the genetic makeup of the species. For many plants, flowers
1. Department of Plant are vital organs of reproduction containing both male and female gametes. For bees
Science, Bharathidasan and other nectar-feeding animals, flowers are a source of food. In Passiflora edulis,
University, Trichy, 24, India. pollen is distributed by bees. The flower is the device by which the plant recruits the
bee. The bees and P. edulis have evolved an interdependent relationship.
2. PG and Research
Department of Botany,
National College,
Tiruchirappalli 620 001.

Corresponding author: Keywords:

Anburaja V. Passiflora edulis, honey bee pollination, Pachamalai Hills, Eastern Ghats.

Email: Article Citation:

vanburaja@gmail.com Nandagopalan V, Anburaja V, Johnson Gritto M and Uma Maheswari R.
Honey bee Passiflora edulis Sims. (Passifloraceae) pollination
found in the Pachamalai Hills, Eastern Ghats of Tamil Nadu.
Phone No: Journal of Research in Biology (2012) 2(6): 566-571
+91 9976516006.
Dates:
Web Address: Received: 24 May 2012 Accepted: 01 Jun 2012 Published: 14 Aug 2012
http://jresearchbiology.com/
documents/RA0246.pdf.

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

566-571 | JRB | 2012 | Vol 2 | No 6

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com
 Nandagopalan et al, 2012

INTRODUCTION peak of flowering season, comparative studies were also

The mechanism of pollination among the higher carried out on another population growing about 4 km
plant groups has been under investigation from very away from this population. To study floral phenology,
early times and it is highly significant in biological flower buds that would open the next day were tagged
studies. It is particularly important among other aspects (N=502) and were kept under observation
of biological studies in ecology, co-evolution, variation, (every hour on the first day and every morning, noon and
speciation, classical and applied genetics and plant evening on subsequent days until senescence) to record
breeding. The knowledge of pollination biology is a the time of anthesis, anther dehiscence and structural
prerequisite in plant breeding and for obtaining better changes associated with ageing of flowers.
yields of crops. Variability is controlled by the breeding A few pollinators were caught using sweeping
system, of which pollination mechanism forms an net, immobilized in ethyl acetate vapours and used to
integral component (Roy and Choudhury, 1981). study pollen load on their body. The time and day of
Pollination biology has shown a new phase of their opening were recorded and the samples were kept
synthesis and correlation. During its entire flowering under observation. Initial observations revealed that
period, a plant species needs several flower visitors for honey bee and fly visit the flowers throughout the day
its pollination. In the same way, the flower visitors from 0600 to 1800 h. The frequency of visits and
require a number of plant species to provide them foraging time were continuously recorded from
continuous nourishment. It is considered that the 0600 to 1800 h for three days (36 hrs of total
pollination relationships take place on a community basis observation). Pollinators were distinguished from floral
(Baker, 1963). Plant pollinator interactions are crucial visitors based on their landing site, method of foraging,
in determining community structure and its functioning
(Heithaus, 1974; Frankie, 1976).
Pollen productivity of a plant species indicates
the biological efficiency of a particular plant. Pollination
biology of some angiospermic plants has been studied
(Ashoke and Sudhendu, 2000). But an adequate
knowledge about the pollination biology of
Passiflora edulis Sims of the family Passifloraceae is
lacking since the plant is economically important on
account of its edible fruits, medicinal uses, production of
beverages and etc. The present investigation was done
with the principal objectives to acquire detailed
knowledge about the floral biology, pollen dispersal,
pollen-pollinator interaction, pollination mechanism,
morphology and receptivity of P. edulis.

MATERIALS AND METHODS

The pollination studies were carried out during Figure: Shows various stages of Passiflora edulis
two seasons (January- May 2007 and 2008). During the flowering and Pollination.

567 Journal of Research in Biology (2012) 2(6): 566-571

Nandagopalan et al., 2012
their contact with the anthers and stigma, presence of
pollen load on their body and their efficiency in
transferring pollen to the stigma. For studying the
transfer of pollen to the stigma, flowers visited by the
insects were excised and observed under the microscope
for pollen load on their stigma. An extensive field
exploration has been done with a view of finding out the
flowering period and floral nature of the selected plant.
Flower colour, odour and nectar were observed visually.
Anthesis and anther dehiscence were observed using a
fluorescent lamp at night and a hand lens, following the
standard methods (Reddi and Janaki, 1981;
Mathur and Mohan, 1986). Reproductive success was
assessed on the basis of fruit and seed set. As the bracts,
bracteoles and sepals are persistent and continue to be
green until all the flowers in the flowers have opened and
the fruits have reached maturity, it was possible to count,
from the older flowers, the number of flowers produced
and the number of fruits developed from each branch.
Plant morphology and floral characters were studied by
following standard floras (Matthew, 1983).
RESULTS AND DISCUSSION
The P. edulis is a native of southern Brazil
through Paraguay to northern Argentina. The yellow
form of passion fruit is probably native of the Amazon
region of Brazil (Morton 1987). Passion fruit is
cultivated today throughout the tropics and subtropics
and has naturalized and escaped in many areas
TABLE: Vegetative and Flowering characteristics of P. edulis.
S. NO PARAMETER
1. Height of the Plant
2. Number of Branches in an Individual
3. Number of Branch initiating flowers
4. Number of flower in a branch
5. Number of flowers opens
6. Time of opening
7. Time of closing
8. Foraging time
9. Foraging duration
10. Number of fruits Produced Per branch
11. Number of seeds per fruit
Journal of Research in Biology (2012) 2(6): 566-571
(Acevedo-Rodrguez, 1985). In our study P. edulis is
found as a perennial climber in the riverine ecosystems
of mid elevation forests of Pachamalai Hills.
The flower buds of P. edulis start to open
7.461.17 hrs in the morning after receiving sufficient
intensity of the sunlight (Figure 1). At the time of
opening the anther facing downwards and the stigma is
almost straight (Figure 2). Till the dehiscence of anther,
the stigma stands away from them. Later on it bends
towards the anther. Even though the bending takes place,
the self pollination is not possible because the dehiscence
anthers are facing downwards and contains the pollen
grains which could not be moved by wind.
As the plant P. edulis produces sticky and heavy
pollens without any aids to fly in the wind, this plant is in
the need of a vector for the pollen transfer from the
anther to stigma. In this case, honey bees are playing an
important role as the pollen transferring devices. Honey
bees are one of the members of the insect family Apidae.
They are having very specific body structures with
feather-like hairs called setae. Flowers are the main
support for the survival of honey bees. The nectar
secreted in the flowers give carbohydrates, which gives
energy to fly and their activities like hive making and
reproduction. The Pollen grains are the main source of
proteins, fats, vitamins and minerals to build their body
and for growth. A worker bees foraging for pollen grains
over the flower and store it in their legs specialized for
pollen collection (Figure 3). The bees have three pairs of
OBSERVATIONS
According to the supporting plants
8 2.77
Almost all
7 3.34
Almost all
7.46 1.17 hrs
15.2 1.48 hrs
Between 8.06 1.11 hrs and 14 1.22 hrs
9.8 4.14 seconds
6 1.81
37 6.38
568

legs, which are evolved to comb pollen from the bee
hairs and pack it into the pollen basket for transport to
the hive.
The forelegs are equipped to clean their antenna
and brush pollen from the antennae. With the mid legs,
the bee clears the pollen from its head, thorax and
forelegs. And then the pollen grains are brushes and
packed into the baskets like structures in the hind legs.
The petals of the flower attracting them to the
flower with their colourful markings, act as landing pad
for the bees and guiding them to the nectarines and
anthers. The bee drives their head very deep into the
flower to get the sweet nectar. At this time, pollen grains
are entrapped in its body hairs and then brushes against
the anthers and stigma.
As bees move one plant to another, the pollens
are also carried from anther to stigma and perform the
pollination. Some pollen grains are deposited on the
sticky surface of each stigma and germinate in to a
pollen tube through the style of the ovule to complete
th
fertilization. After the 50 14.28 hours of fertilization,
petals fall down and the pistil stars to develop into a fruit
and the seeds develop inside. The vegetative,
reproductive and pollination details are given in the
table.
It is also noted that some flies come to the
flowers to collect nectar only. As they are comparatively
small in size, they could not get contact with the anther
during the collection of nectar from the inner part of the
flower. And usually they are not having the habit to
collect the pollen grains. Thus the flies are not involved
in the pollination of P. edulis and they get the nectar only
from the flower, in this case it may be considered as
nectar robbers (Figure 4).
The fruit of the P. edulis is used to make
delicious juice. It is also collected by local people in the
forests. The fruit pulp contains protein, fat,
carbohydrates. The fruit stands as food for numerous
wild birds and mammals.
569
Nandagopalan et al., 2012
Plants were once the main source of all
medicines in the world and they continue to provide
mankind with new remedies. Natural compounds found
in plants and their derivatives make up more than
50% of all drugs in clinical use in the world
(Marius Hedimbi et al., 2012 and Igoli et al., 2005).
The P. edulis is also used as a diuretic to treat urinary
infections. The earlier reports focused on the
antibacterial properties of Passiflora species by different
methods. The crude materials of Passiflora were
separated into several fractions; passicol was obtained,
which had antimicrobial activity (Birner and Nicolls,
1973). On the other hand, the antibacterial activity of
Pseudomonas tetrandra, which has got activity against
E. coli, B. subtilis and P. aeruginosa, the potential plant
derived antibiotic (Perry et al., 1991).
The type of fragrant produced by the flower
determines the type of pollinators. P. edulis produces
pleasant fragrance and it attracts honeybee. In the case of
Caralluma umbellata a fleshy odour is produced during
the peak flowering season. This odouring attracts the
house fly towards the flowers and the pollination also
performed by the house fly only (Anburaja et al., 2011).
In the other hand, like P. edulis, most flowering
plants depend on animals for effective pollination and
sexual reproduction (Buchmann and Nabham, 1996).
Although animal vectors improve pollen transfer to
stigmas such evolutionary dependence on mutualists for
reproduction has increased plant susceptibility
to fragmentation and other forms of habitat disturbance
(Aizen et al., 2002). The majority of studies conducted
so far indicate that insect pollinator guilds are
particularly sensitive to habitat fragmentation
(Aizen and Feinsinger, 2002).
CONCLUSION
The Passiflora edulis is found as a perennial
climber in the riverine ecosystems of mid elevation
forests of Pachamalai Hills. The opening of the flower
Journal of Research in Biology (2012) 2(6): 566-571
Nandagopalan et al., 2012
starts after receiving sufficient intensity of the sunlight.
At the time of opening the anther facing downwards and
the stigma is almost straight. Later on stigmas bend
towards the anther. Even though, the bending of stigma
does not facilitate the self pollination. In this case, honey
bees are playing an important role as the pollen
transferring devices. The petals of the flower attract them
towards the flower with their colourful markings.
It is also noted that some flying insects come to the
flowers to collect nectar only. As they are comparatively
small in size, they could not successfully transfer the
pollen from anther to stigma. And usually they are not
having the habit to collect the pollen grains. Thus the
flies are not involved in the pollination of P. edulis and
they get the nectar only from the flower, in this case it
may be considered as nectar robbers.
ACKNOWLEDGEMENT
The authors are thankful to National College,
Tiruchirappalli, Tamil Nadu, India and the Rapinat
Herbarium, St. Josephs College, Tiruchirappalli, Tamil
Nadu, India for providing necessary facilities to
complete the research work.
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 Journal of Research in Biology An International Scientific Research Journal

Original Research

The combined effects of temperature and salinity on survival of larvae and

juveniles of tropical abalone Haliotis asinina under laboratory conditions
Journal of Research in Biology

Authors: ABSTRACT:
Nilnaj Chaitanawisuti1,
Sirinun Nunim2 and This paper reports on a 3 x 3 factorial design experiment conducted to
Wannanee Santhaweesuk1. examine the combined effects of temperature and salinity on survival of larvae and
juveniles of tropical abalone Haliotis asinina under laboratory conditions for 96 h. The
temperatures used were 25, 30 and 35C and the salinities were 27, 30 and 33 ppt.
Response surface contour diagrams were generated from the survival data to estimate
optimal conditions. The highest survival of newly-hatched larvae, newly-settled
Institution:
1.Aquatic Resources juvenile and fully juveniles of H. asinina was obtained at the lowest temperature
Research Institute, tested (25C) with the highest salinity tested (33 ppt), while the lowest survival was
Chulalongkorn University, obtained at the highest temperature tested (35C) with the lowest salinity tested
Bangkok, Thailand 10330. (27 ppt). Two - way ANOVA showed that survival of larvae, newly - settled juveniles
and fully juveniles were significantly affected by temperature and salinity. A significant
2. Department of interaction between both factors occurred in newly - settled juveniles and fully
Environmental Science, juveniles but not for larvae. Multiple regression analysis indicated a higher correlation
Graduate School, between salinity and survival of larvae H. asinina but a higher correlation between
Chulalongkorn University, temperature and survival for newly - settled juveniles and fully juveniles. This study
Bangkok, Thailand 10330. indicated that the optimal conditions for maximum survival of larval, newly - settled
juvenile and fully juveniles were 27-30C and 31-33 ppt, 26-29C and 27-33 ppt, and
26-27C and 31-33 ppt, respectively.

Corresponding author: Keywords:

Nilnaj Chaitanawisuti. Tropical abalone, H. asinina, temperature, salinity, survival, early life stages.

Web Address: Article Citation:

http://jresearchbiology.com/ Nilnaj Chaitanawisuti, Sirinun Nunim and Wannanee Santhaweesuk.
documents/RA0218.pdf. The combined effects of temperature and salinity on survival of larvae and juveniles of
tropical abalone Haliotis asinina under laboratory conditions.
Journal of Research in Biology (2012) 2(6): 572-579

Dates:
Received: 14 Mar 2012 Accepted: 03 Apr 2012 Published: 17 Aug 2012

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

572-579 | JRB | 2012 | Vol 2 | No 6

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
The tropical abalone (Haliotis asinina) is
distributed along the East coast of the upper Gulf of
Thailand, and in the Andaman Sea. Tropical abalone
culture in Thailand is presently the very early stages of
basic and applied research conducted in small-scale
operations. The economic viability of commercial
tropical abalone farming depends on the system design
and techniques used for grow-out culture. H. asinina has
fast growth rates and a relatively high salinity tolerance
in comparison to other species (Jarayabhand and
Paphavasit 1996). In general, major factors affecting
growth and survival rates of the early life stage and
mature of various aquatic animals are temperature and
salinity. Temperature and salinity are considered to be
the most important physical factors influencing marine
organisms, and the biological effects of these factors are
complex and wide ranging. Temperature is one of the
most critical external factors of development in the early
life stages of shellfish. There are two ways in which
temperature affects ontogeny. Firstly, temperature, if
within a viable range, strongly affects the rate of
ontogeny. A temperature beyond this range is lethal for
the species. Secondly, temperature affects the hatch rate,
incubation period, the size of the newly hatched larvae,
larval yolk absorption and utilization, larval feeding
behavior, larval survival and larval growth (Shi et al.,
2010). In addition, salinity could modify the effects of
temperature and alter the temperature range of many
biological processes. In turn, temperature can also
modify the effects of salinity (Albuquerque et al., 2009).
Temperature and salinity affect larval and juvenile
growth and survival of many marine invertebrates. The
combined effects of temperature and salinity on the
survival of marine animals have been demonstrated in
many marine mollusks such as bivalves (Taylor et al.,
2004, Robert et al., 1988, Tettelbach and Rhode, 1981)
and gastropods (Lu et al., 2004, Davis, 2000, Chen and
Chen 2000, Zheng et al., 2000). However, there is no
573
Chaitanawisuti et al., 2012
information on the effects of environmental parameters,
such as temperature, salinity on survival of early life
stages for tropical abalone H. asinina particularly the
combined effects of temperature and salinity.
Determination of the optimal salinity and temperature for
larvae and juvenile culture of Haliotis asinina is an
important step in developing more efficient large-scale
hatchery culture techniques for this species. In addition,
result of this study provides greater precision for
assessing the interactions between different factors for
survival of various life stages for this species under
natural conditions particularly the effects of climate
change. The objective of this study is to examine the
combined effects of temperature and salinity on survival
of larvae and juveniles of tropical abalone
Haliotis asinina under laboratory conditions.
MATERIALS AND METHODS
This laboratory experiment was designed to test
the combined effects of three temperatures (25, 30 and
35C) and three salinities (27, 30 and 33 ppt) on survival
for newly-hatched larvae, newly-settled juveniles and
fully juveniles of H. asinina. The experiment was a
3x3 factorial design, with all nine temperatures and
salinity combinations were tested. The study was
conducted during summer season from February to April
2011 in which temperatures ranged 28-29C and it was
the spawning season for H. asinina. All temperature and
salinity combination treatments for newly-hatched
larvae, newly-settled juveniles and fully juveniles were
not run simultaneously (newly-hatched larvae in
February, newly-settled juveniles in March and fully
juveniles in April).
Seawater brought from the shore, and filtered
through a series of filter net down to 0.5 m was used in
all combination treatments. Ambient seawater was
lowered to salinity of 27 ppt by dilution with de-ionised
water, and it was increased salinity of 33 ppt by
concentration using air evaporating. Treatment water was
Journal of Research in Biology (2012) 2(6): 572-579
Chaitanawisuti et al., 2012

made in the containers for 24 hrs in advance to allow practice for this species so that stocking density was not
thoroughly mixing of seawater and the temperatures to a limiting factor for survival (Jarayabhand and
adjust to treatment conditions. Salinity and temperature Paphavasit 1996). In this study, the animals were
were monitored every 6 hrs. Salinity was maintained suddenly exposed to each temperature and salinity
within +0.5 ppt and water temperature was maintained combination. There were three replicate containers for
within +0.2C using thermostatically controlled water each combination treatment. No food was served to the
baths. Gently aeration was provided in the container larvae during the experiment but the newly-settled
during the experiment and a normal photoperiod of juveniles and fully juveniles were fed at once daily with
12L:12D was adopted throughout the experiment. There cultivated benthic diatom mainly (Navicula sp.) and
were no water exchange during experiment for all commercial shrimp (Penaeus monodon) pellet,
treatments. Salinity and temperature in all experimental respectively so that food availability was not a limiting
units were measured every six hours using a portable Table 1. Percentage survival rate of larvae, newly- settled
refracto-salinometer and a mercury thermometer, juveniles and fully juveniles H. asinina through 96 h under
different temperature and salinity combinations
respectively. All experimental containers were covered
with aluminum foil to prevent evaporation and Temperature (C) Salinity () Larvae

consequent salinity increase. Larvae

This study was conducted at the hatchery of 25 27 29.94+3.33
30 50.09+8.59
Research Unit for Abalone Cultivation, Aquatic
33 62.20+2.90
Resources Research Institute, Chulalongkorn University. 30 27 30.41+9.09
The newly-hatched larvae, newly-settled juveniles and 30 56.16+0.81
33 59.30+1.97
fully juveniles H. asinina used for all combination
35 27 18.81+1.40
treatments were obtained from the same batch of 30 23.89+2.89
spawning. One male and female mature breeder was 33 41.94+3.12
Newly-settled juveniles
selected for induced spawning using dry method 25 27 94.67+1.15
(Jarayabhand and Paphavasit 1996). The larvae were 30 94.67+1.15
33 97.33+1.15
then incubated to reach the desired life stage for
30 27 94.67+1.15
the experiment. The mean size (+SD) and age of 30 93.33+1.15
newly-hatched larvae, newly-settled juveniles and fully 33 93.33+3.06
35 27 10.00+5.29
juveniles H. asinina used in this experiment were
30 24.00+12.17
1200+0.04 m in shell length (1 days after fertilization), 33 48.67+4.16
2.20+0.06 mm in shell length (45 days after fertilization) Fully juveniles
25 27 83.34+3.34e
and 5.10+0.12 mm in shell length (50 days after 30 91.12+6.94c
settlement), respectively. The most active and healthy 33 98.89+1.92a
30 27 58.89+7.70f
animals of each life stage were chosen for the
30 85.56+3.05d
combination treatments. A sample of 100 newly-hatched 33 93.56+3.67b
larvae, 50 newly-settled juveniles and 50 fully juveniles 35 27 1.11+1.93i
30 12.22+6.94h
were initially stocked in each experimental units of
33 34.45+5.09g
1000-mL transparent, glass container. This stocking Mean in the same column with different superscript
density is lower than that used in standard aquaculture letters are significantly different (P<0.05)

Journal of Research in Biology (2012) 2(6): 572-579 574


factor for survival (Jarayabhand and Paphavasit 1996).
Each experimental unit was initially examined for the
dead larvae and juveniles after 6 h, and every 12 h
thereafter. The experiments lasted for 96 h. The number
of larvae which sank down to bottom of the
aquaria / empty shell or no movement of velum were
observed microscopically and considered as dead as well
as the juveniles did not react to the touch of a needle
were considered as dead. The mean percentage of
survival was calculated by combining the data from three
replicates at the end of the experiment.
To investigate the combined effect
temperature and salinity on survival of
newly-settled juveniles and fully juveniles, a two-way
analysis of variance (ANOVA) (fixed
temperature and salinity) with a 95% confidence interval
was used. All data were tested for normality and
homoscedasticity. If significant difference
indicated, then tukey test was used to verify the
difference among the treatments. The correlation
between the survival, temperature and salinity was
estimated by multiple regression analysis. Response
surface contour diagrams were generated
experimental data on survival rate using the SigmaPlot
Version 7.0.
RESULTS
The highest survival of newly-hatched larvae
(62.20+2.90%), newly-settled juvenile (97.33+1.15%)
and fully juveniles (98.89+1.92%) of H. asinina was
obtained at the lowest temperature tested (25C) with the
highest salinity tested (33 ppt), while the lowest survival
(18.81+1.40, 10.00+5.29 and 1.11+1.93%, respectively),
was obtained at the highest temperature tested (35C)
with the lowest salinity tested (27 ppt) (Table 1).
Two-way ANOVA showed that survival of newly-
hatched larvae, newly-settled juvenile and fully juvenile
larvae of H. asinina were significantly affected by
temperature (P = 0.000) and salinity (P = 0.000).
575
Chaitanawisuti et al., 2012
Significant interaction between temperature and salinity
was found in newly-settled juvenile and fully juveniles
(P = 0.000) but not for the newly-hatched larvae.
(P = 0.087) (Table 2). Results of tukey test showed that
the survival of fully juveniles of H. asinina was
significantly different among temperature treatments but
survival at 25C treatments and 30C treatments of
newly-hatched larvae and newly-settled juveniles were
not significantly different. In addition, significant
difference in survival among salinity treatments was
found in newly-hatched larvae and fully juveniles of
of H. asinina but survival at 27 ppt treatments and 30 ppt
larvae, treatments of newly-settled juveniles was not
significantly different (Table 3).
factors: Multiple regression analysis showed that both
temperature and salinity were statistically significant,
showing a negative correlation with the percentage
were survival of newly-hatched larvae, newly-settled juveniles
and fully juveniles of H. asinina. Standard coefficient
(Beta) of temperature was higher than that of salinity,
indicating a higher correlation between temperature and
survival of newly-settled juveniles and fully juveniles of
from H. asinina but not for those of newly-hatched larvae. The
positive values of beta also indicated that the higher
salinity provided the higher survival for newly-hatched
larvae, newly-settled juveniles and fully juveniles
H. asinina (Table 3). The multiple regression equation
on survival of newly-hatched larvae, newly-settled
juveniles and fully juveniles H. asinina over the
combined effects of temperatures and salinity were
estimated as following:
Survival (newly-hatched larvae) = -41.545 1.919
Temperature + 4.682 Salinity
Survival (newly-settled juveniles) = 209.630 6.800
Temperature + 2.222 Salinity
Survival (fully juveniles) = 156.658 7.704
Temperature + 4.568 Salinity
The response surface plots summarizing
percentage survival of H. asinina under different
Journal of Research in Biology (2012) 2(6): 572-579
Chaitanawisuti et al., 2012

Table 2. Results of two-way ANOVA for survival of larvae, newly-settled juveniles and fully juveniles
H. asinina through 96 h under different temperature and salinity combinations (95% confidence interval)
Parameters Sum of square df Mean square F-value P-value
Larvae
Intercept 30875.297 1 30875.297 1.384E 0.000
Temperature 1572.865 2 786.432 35.256 0.000
Salinity 2401.797 2 1200.899 53.837 0.000
Temperature x salinity 255.891 4 63.973 2.868 0.087
Error 200.757 9 22.306
Newly settled juveniles
Intercept 141122.370 1 141122.370 6067.363 0.000
Temperature 27037.630 2 13518.815 581.223 0.000
Salinity 835.852 2 417.926 17.968 0.000
Temperature x salinity 1481.481 4 370.370 15.924 0.000
Error 418.667 18 23.259
Fully juveniles
Intercept 106024.480 1 106024.480 5312.744 0.000
Temperature 30103.963 2 15051.982 754.235 0.000
Salinity 3388.020 2 1694.010 84.885 0.000
Temperature x salinity 573.429 4 143.357 7.183 0.001
Error 359.219 18 19.957
temperature and salinity combinations over 96 h showed salinity at 30C or a higher temperature survived
that high survival of newly-hatched larvae (60%), salinities lower than 14 when salinity is
newly-settled juveniles (100%) and fully juveniles decreased. Cheng et al., (2002) also found that
(100%) were obtained at 27-30C and 31-33 ppt, hemo lymph osmo lalit y of Taiwan abalo ne
26-29C and 27-33 ppt, and 26-27C and 31-33 ppt, Haliotis diversicolor supertexta stabilized within two
respectively (Fig 1). days after they were transferred to different salinities
from 33 psu and hemolymph osmolality (Cl-, Na+ and K+
DISCUSSION concentrations) increased directly with medium salinity.
It is clear that for the tested ranges of In addition, Romo et al., (2010) found that oxygen
temperature and salinity; it is latter which mostly affect consumption rate of pink abalone Haliotis corrugate was
survival of the newly-hatched larvae, newly - settled not affected by temperature and salinity. Ammonium
juveniles and fully juveniles of H. asinina, especially at excretion was inversely related to salinity. The O:N ratio
the lower ranges. The lowest survival was found at the indicated that abalone maintained in lower salinities had
lowest salinity tested of 27 ppt and the highest an interval of 4.9-7.7, which is indicative of protein-
temperature tested of 35C for larvae, newly - settled dominated metabolism, whereas the O:N ratio in 35
juveniles and fully juveniles. The results of this study was 28.8-35.5 for temperatures of 20 and 24C,
suggested that salinity has a strong effect on larvae and suggesting that carbohydrates were used as energy
juveniles of abalone H. asinine, which agreed with substrate. He also concluded that optimized culture of
the study in Haliotis diversicolor supertexta. pink abalone should be cultivated at 24C in a salinity of
Chen and Chen (2000) found that juvenile abalone 35. The results of this study were in agreement with
Haliotis diversicolor supertexta maintained 35 or various studies that salinity has a strong effect on larvae
higher at 20C or lower survival salinity higher than and juveniles of various mollusks such as spotted
45 when salinity is increased. They have also babylon Babylonia areolata (Xue, 2010), scallop
suggested that juveniles maintained in 25 or a lower Argopecten purpuratus (Soria et al., 2007), green mussel

Journal of Research in Biology (2012) 2(6): 572-579 576

 Chaitanawisuti et al., 2012
33
Table 3. Turkey test applied to different temperature 50 40
and salinity treatments for survival of larvae, 45
55
newly-settled juveniles and fully juveniles H. asinina 32
60

at 95% confidence interval 60

40 35
31
Temperature (C) Post-test Salinity () Post-test 50
45
Larvae 60 55

Salinity (ppt)
55 30

25 35 27 30; 33 30
50
30 35 30 27; 33 55
50 40 35 25

35 25; 30 33 27; 30 29
45
50 45

Newly-settled juveniles 40 45
30

25 35 27 33 28 40
40
35

30 35 30 33 35
35
25

30 20
35 25; 30 33 27; 33 27

Fully juveniles 26 28 30 32 34

Temperature(C)
Temperature (0C)
25 30; 35 27 30; 33
30 25; 35 30 27; 33 33
100
100 80 60
35 25; 30 33 27; 30 100
32

Perna viridis (Nair and Appukuttan, 2003), sydney rock 31

100
100
40

80 60

oysters Saccostrea glomerata (Dove and OOconner Salinity (ppt)

2007), European flat oyster Ostrea edulis (Robert et al., 30

40
100 20
100
1988), conch Strombus gigas (Davis, 2000), black-lip 29
80 60

pearl oyster Pinctada margaritifera (Doroudi et al.,

28 100
1999), silver-lip pearl oyster Pintada maxima
40
100 20

80 60

(Taylor et al., (2004), northern Bay scallop 27

26 28 30 32 34

Argopecten irradians (Tettelbach and Rhode, 1981). Temperature (0C)

Temperature(C)
Xue, (2010) found that survival of juvenile spotted 33
100
100
40
babylon Babylonia areolata was significantly different 60

80
32 100
among temperature and salinity combination treatments 100

due to temperatures but not due to salinities. The optimal 31

60 40
condition for culturing juvenile B. areolata was obtained 20
Salinity (ppt)

80

at temperatures from 26 to 30C and salinity from 30

26 to 30 g/l. Soria et al., (2007) explained that higher 40

29 60
20
survival rates of juvenile scallop Argopecten purpuratus 80

at higher salinity was due to an increase in salinity that 28

produced a reduction in NH3 - N proportion and under 80

60 40
20
hypersaline conditions juvenile scallop tend to decrease 27
26 28 30 32 34

excretion as a way of osmoconformation. Nair and Temperature (0C)

Appukuttan, (2003) found that total mortality of the
green mussel larvae Perna viridis occurred after 24 hr
at the temperature of 33C and 35C and no significant
Temperature(C)
Fig 1. Response surface contour diagram showing maximized
survival rate of larvae (upper), newly-settled juvenile
(middle) and fully juvenile (lower) H. asinina through 96 h
under different combinations of temperature and salinity.

difference in settlement of larvae at 29C and 31C. rock oysters Saccostrea glomerata whereas temperature
Dove and OOconner (2007) showed that salinity had a significantly affected survival of both D-veliger and
significant effect on D-veliger larval survival of Sydney pediveliger larvae. There was an interaction between
577 Journal of Research in Biology (2012) 2(6): 572-579
Chaitanawisuti et al., 2012

Table 4. Multiple regression analysis on survival of larvae, newly-settled juveniles and fully juveniles
H. asinina through 96 h under different temperature and salinity combinations on 95% confidence interval
Parameters B Standard error Beta t-value p-value
Larvae
Intercept -41454 26.984 -1.536 0.145
Temperature -1.919 0.462 -0.499 -4.158 0.001
Salinity 4.682 0.769 0.731 6.085 0.000
R2 = 0.784; F = 27.155; p < 0.000
Newly-settled juveniles
Intercept 209.630 50.826 4.124 0.000
Temperature -6.800 0.870 -0.836 -7.820 0.000
Salinity 2.222 1.449 0.164 1.533 0.138
R2 = 0.726; F = 31.755; p < 0.000
Fully juveniles
Intercept 156.658 38.096 4.112 0.000
Temperature -7.704 0.652 -0.878 -11.820 0.000
Salinity 4.568 1.086 0.312 4.205 0.000
R2 = 0.868; F = 78.695; p < 0.000
salinity and temperature for D-veliger larval survival. ACKNOWLEDGMENTS
While spot survival was significantly affected by salinity This research was a part of the Research
only and no interaction was detected between salinity University Program funded by Chulalongkorn University
and temperature for spat survival. Davis, (2000) showed (CC103A). The authors thank Sichang Marine Science
that at the end of 0 to 7 day interval, percent mortality of Research and Training Station, Aquatic Resources
tropical gastropod veligers Strombus gigas was highest Research Institute, Chulalongkorn University, in
for veligers grown at 20 and 24C and at salinity of particular Mr. Soontorn Thepmoon for his help and
45, while percent mortality was low and not different suggestion during the experiments. The authors also
for veligers grown at 24C and at salinity of 30, 35 and thank to Associated Professor Gullaya Wattayakorn for
40. Doroudi et al., (1999) reported that optimal valuable comments and co-ordinations during the
conditions for maximum larval survival of the black-lip preparation of this research project.
pearl oyster Pinctada margaritifera were 26-29C and
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Romo ZM, Re AD, Diaz F and Mena A. 2010. Affordable Charges
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579 Journal of Research in Biology (2012) 2(6): 572-579

 Journal of Research in Biology An International Scientific Research Journal

Original Research

A Study On Uropathogens In Diabetic Patients from a tertiary care

Hospital in Kanchipuram
Journal of Research in Biology

Authors: ABSTRACT:
Sivasankari S, Diabetes has long been one of the predisposing factors of UTI. There were
Senthamarai S, Anitha C, several studies about the role of DM in etiology and resistance pattern of
Amsavathani Sk, uropathogens with UTI. Hence this study was aimed to know the prevalence of
Venugopal V. Asymptomatic Bacteriuria (ASB) among diabetic patients
Materials and Methods:
This Study was conducted from May 2008 - Oct 2009. A total of 220 urine
samples were collected from patients above 40yrs who attended the OPD with the
Institution:
Dept of Microbiology, history of diabetes and 100 urine samples were collected from non diabetics patients
Meenakshi Medical screened for and asymptomatic bacteriuria (>105 CFU /ml Urine). All urine samples
College and Research were processed according to the standard protocol. Antibiotic sensitivity was done by
Institute, Enathur, kirby Bauer disc diffusion method.
Kanchipuram - 631 552. Results:
Out of 220 diabetic patients, 52 had significant bacterial growth, and out of
100 non diabetic patients 22 had significant growth. UTI was more common in female
patients in diabetes but more common in males in non diabetes. E.coli was the
commonest isolate in both diabetic and non diabetic patients. The antibiotic
Corresponding author: resistance pattern in diabetics and non diabetics were found to be similar.
Anitha C. Conclusion:
Our study with asymptomatic UTI in diabetes mellitus, shows the
antimicrobial resistance patternfor formulating antibiotic policies.

Keywords:
Email:
ani.phd@gmail.com Diabetes, UTI, Asymptomatic bacteriuria.

Web Address: Article Citation:

http://jresearchbiology.com/ Sivasankari S, Senthamarai S, Anitha C, Amsavathani Sk, Venugopal V.
documents/RA0265.pdf. A Study On Uropathogens In Diabetic Patients from a tertiary care Hospital in
Kanchipuram.
Journal of Research in Biology (2012) 2(6): 580-584

Dates:
Received: 14 Jul 2012 Accepted: 28 Jul 2012 Published: 20 Aug 2012

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

580-584 | JRB | 2012 | Vol 2 | No 6

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com
 Sivasankari et al.,2012

INTRODUCTION: patients. Inclusion criteria is all patients with Type-2

Urinary tract infection is a major problem DM or proven cases of diabetes. Exclusion criteria
in d e ve lo p ing c o u nt r ie s of all ages include the pregnancy, known urinary tract anomalies,
(Eshwarappa et al., 2011). Diabetes mellitus is use of antimicrobial drugs. The fasting blood sugar level
considered to be a predisposing factor for urinary tract of the patients was estimated. Midstream clean voiding
infection (Jenifer and Geethalakshmi, 2009). Most UTIs Urine samples were collected and streaked on to
are asymptomatic especially in women. Asymptomatic bloodagar, MacConkey agar plates and were incubated
infections go unnoticed by the patient himself due to lack aerobically at 37C for 24 hours. Bacteriuria was
of signs and symptoms. Bacteruria is the second most confirmed as the presence of atleast 105CFU/ml of urine
common infect ion in developing countries and they were subjected to a repeat culture for
(Jhan and Singh, 2009). The role of DM in etiology and confirmation. Presence of 3 or more than 3 types of
in the antimicrobial resistance of uropathogens with UTI colonies were considered as contamination of urine and
has not been studied by many authors in India were asked for repeat culture. All the organisms were
(Eshwarappa et al., 2011). Diabetes causes several identified as per standard protocols.
abnormalities in the host defence system that might Antibiogram was done by kirby Bauer disc
result in higher risk of UTI (Bokyko et al., 2005). diffusion method as per CLSI protocol 2009.
Several studies showed a higher prevalence of
asymptomatic bacteriuria among women with diabetes RESULTS:
(Bonadio and costarella, 2006). Hence asymptomatic Out of 220 diabetic patients, only 52(24.8%) had
infections are important in regard to health hazard. significant bacterial growth, 124(56.3%) had
Hence this study was aimed to know the insignificant growth and 44(20%) had no growth.
prevalence of Asymptomatic bacteriuria (ASB) among Among 100 non diabetic patients only 18(18%) had
elderly diabetic patients for the bacteriological profile significant growth.
and the antimicrobial resistance pattern of uropathogens.
DISCUSSION:
MATERIALS AND METHODS: In our study we tried to find whether there is any
Our st udy was co nduct ed fr o m difference in the bacteriological profile of UTI and the
May 2008 - Oct 2009. A total of 220 diabetic patients antibiogram pattern of diabetic and non diabetic patients.
above 40 yrs of age attending our OPD were included in The risk of UTI is higher in diabetic patients due to
the study. A brief clinical history was taken from all the abnormalities in the metabolic control which leads to

Table I: Age and sex distribution of Diabetics and Non diabetics with ASB
Diabetes N = 52 Non Diabetes N = 18
Age% Male% Female% Total% Male% Female% Total%
40 49 yrs 4 7 11 1 1 2
50 59 yrs 7 10 17 4 2 6
>60yrs 11 13 24 6 4 10
Total 22(42.3%) 30(57.7%) 52(100%) 11(61.1%) 7(38.8%) 18 (100%)
From the above table, it is clear that ASB is present more in females in diabetic patients where as in
non diabetics the males have more predominance. The incidence is more after the 6th decade of life in both
diabetics and non diabetics.
581 Journal of Research in Biology (2012) 2(6): 580-584
Sivasankari et al.,2012

Table II: Isolation of Uropathogens in male and This is concordant with other studies. In non diabetics
female patients of diabetes.
also ASB is seen more after the 6th decade of life
Diabetic Male Diabetic Female (Ramzan et al., 2004). This could be due to neutropathic
N = 22 N = 30
Uropathogens complications (incomplete bladder emptying) and
Percentage Percentage
No No
% %
increased glucose concentration in urine, which may
E.coli 12 35.2% 18 46.1%
Klebsiella spp. 9 26.4% 12 30.7% provide a good culture medium which favours the
Proteus spp 9 26.4% 5 12.8% seeding of bacteria and genesis of infection in urinary
Enterococci spp 2 5.8% 3 7.6%
S.aureus 2 5.8% 1 2.5% tract in diabetics (Thomas and Jeyaraman, 2010).
Total 34 100% 39 100% Evidence from various epidemological studies
The gram negative organisms were more common showed that UTI is more common in women with
than gram positive organisms in both Male and
Female Diabetic patients. With E.coli to be the diabetes (Patterson and Andriole, 1997). Our study is
predominant isolate in both the sexes, there were also concordant with the other studies which report to be
more than one isolate in some patients.
that UTI is more common in women with diabetes. But
weakness in the host defense and glycosuria provides a in non diabetes UTI is more common in males here our
favourable environment to micro organsims in the study is concordant with (Moorthy et al., 2011). Our
urinary tract (Joshi and Gregory, 1999). study reveals that GNB is more common than GPC in
Out of 220 diabetic patients only 52(23.62%) had UTI in diabetic and non diabetic patients. E.coli is the
significant bacterial growth, 124(56.4%) had common isolate in both the diabetics and non diabetics.
insignificant growth and 44(20%) had no growth. This is concordant with other studies done
Among the 100 control patients only 18(18%) had (Bonadio and costarella, 2006).
significant bacterial growth, 27(27%) had insignificant A study done by Jeniffer et al showed that
growth and 12(12%) had no growth. E.coli (71%) and Klebsiella spp (13%) and
In our study ASB are seen in 23.6% of people. Enterobacter spp (4%) were isolated from female
This is slightly lower than (Jenifer and Geethalakshmi, diabetic patients. Several other studies showed that E.coli
2009) who had reported ASB 30.3%. Many reports is the common isolate (Goswami and Tejasmi, 2001).
world wide reported ASB ranging from 5% to 30% The higher prevalence of E.coli may be due to poor
(Sarah Wild et al., 2004). hygenic condition of the patients and especially higher
In our study ASB is seen in diabetic patients who among females due to the contamination of perineum
are suffering from diabetes for a longer duration and through fecal flora (Park K, 2008).
th
bacteriuria more after the 6 decade of life.

Table-III Isolation of Uropathogens in Male and Female patients of non diabetics

Non diabetic(18)
Uropathogens Male n = 11 Female n =7
No Percentage % No Percentage %
E.coli 5 38.4% 4 50.0%
Pseudomonas spp. 3 23.1% 1 12.5%
Klebsiella spp 3 23.1% 2 25.0%
S.aureus 2 15.3% 1 12.5%
Total 13 100% 8 100%
Among the non diabetic population E.coli was found to be the predominant
isolate followed by Pseudomonas spp.

Journal of Research in Biology (2012) 2(6): 580-584 582

 Sivasankari et al.,2012

Table IV Resistance pattern of various isolates from diabetic and non diabetic patients

Diabetics n = 52 Non Diabetics n = 18

Klebsiella

Proteus n = 14

S.aureus n=3

Klebsiella n=5

S.aureus n=3
E.coli n = 30

Enterococci n=5

E.coli n=9

Pseudomonas n=4
Pseudomonas n=4
n=21

Enterococci n=5
Proteus n = 14

Klebsiella n=5
S.aureus n=3

S.aureus n=3
E.coli n = 30
Antibiotic used

E.coli n=9
Klebsiella
Ciprofloxacin 22% n=21
11.1% 8.5% 2.1% 1% 24% 10.1% 8.5% 1%
Amikacin 9.8% 7.1% 9.1% 1% 1% 10.4% 6.4% 9.1% 1%
Gentamycin 20% 6.2% 5.2% - - 18.4% 5.1% 5.2% -
Norfloxacin 12.4% 9.1% 3% 14% 8% 1% -
Ofloxacin 10.4% 6.2% 3% 1% 11.5% 5.4% 1% -
Ceftrioxone 11.9% 8.9% 5.2% - - 9.8% 7% 5% -
Ceftazidime 15.4% 3.1% 6.1% - - 13.2% 4.1% 4.1% -

The antimicrobial resistance pattern in diabetics risk of UTI. Hence diabetic patients must be regularly
was found to be higher. screened for urine examination for detection of ASB
(Akram et al., 2007) reported the ciprofloxacin along with blood sugar.
resistance of 47 to 69% among gram negative organisms
in India. Our study showed 22% resistance to REFERENCES:
ciprofloxacin. Akram M, Shahid M, Khan AU. 2007. Etiology and
In our study all the gram negative isolates were antibiotic resistance patterns of community-acquired
resistant to amikacin and gentamicin. This is concordant urinary tract infections in JNMC Hospital Aligarh.
with (Eshwarappa et al., 2011) who has also reported India. Ann Clin Microbiol Antimicrob. 6:4.
nearly half of uropathogens showing resistance to
Bokyko, Edward, Stephan D fink. 2005. Risk of UTI
amikacin and gentamicin. All our GNB isolates were
and asymptomatic bacteriuria among diabetic and non
sensitive to Imipenem.
diabetic post menopausal women. American Journal of
In the non diabetics gram negative isolates
Epidermology. 161:557-564 .
showed maximum resistance to aminoglycosides and
quinolones. Eshwarappa M, Dosegowda R, Vrithmani Aprameya
In our study with asymptomatic UTI, diabetes I, Khan MW, Shiva Kumar P, Kempegowda P. 2011.
mellitus could not be considered as the risk factor for Clinico-microbiological profile of urinary tract infection
UTI. in South India. India journal of Nephrology. 21:30-36

Goswami Tejasmi. 2001. Prevalence of UTI and renal

CONCLUSION:
Scar in patients with DM. Dia Res clin pract 53:181-6.
From our study ASB is more common after the
6th decade of life in diabetic females. Jenifer J, Geethalakshmi S. 2009. Prevalence of lower
E.coli was the most frequent isolate in diabetics urinary tract infection in South Indian type 2 diabetic
and non diabetics. Diabetics has large post void residual subjects. Indian Journal of Nephrology 19:107.
urine than non diabetic-this difference may be the higher
583 Journal of Research in Biology (2012) 2(6): 580-584
Sivasankari et al.,2012

Jhan BK, Singh Y. 2009. Prevalence of asymptomatic

bacteria among elderly patients residing in Chitwan.
Kattmandu Medical Journal. 7:157-161 .

Mario Bonadio, Sibia costarella. 2006. The influence

of diabetes mellitus on the specturm of uropathogens and
the antimicrobial resistance in elderly adult patients with
UTI. Journal of Infections Disease. 6:54

Moorthy K, Pargavi B, Thamarai A. 2011. Prevalence

of urinary tract infection among diabetic patients in
Vandavasi. International Journal of Biological
Technology. 2(2):42-45.

Nihal Thomas, Kanakamani Jeyaraman. 2010. A

practical guide to diabetes mellitus 5th edition. 255-256.

Nirmal Joshi MD, Gregory. 1999. Infection in patients

with Diabetes mellitus. The New England Journal of
Medicine. 341:1906-1912 .

Park K. 2008. Infections Disease. In: K. Park, editor

Parks test book of Preventive and social medicine
311-15.

Patterson JE, Andriole VT. 1997. Bacterial Urinary

tract infection in diabetes Infect Dis Clin North Am.
11(3):735-50.

Ramzan M, Bakhsh S, Salam A, Khan GM. 2004.

Ghulam Mustafa. Risk factors in Urinary tract infections.
Gomal Journal of medical sciences. 2:1-4.

Sarah Wild, Bchir MB, Richard Sicree. 2004. Global

prevalence of Diabetes, Estimates for the year 2000 and Submit your articles online at jresearchbiology.com

projections for 2030. Diabetic care. 27. Advantages

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Journal of Research in Biology (2012) 2(6): 580-584 584

 Journal of Research in Biology An International Scientific Research Journal

Original Research

New record of common Banded Peacock Papilio crino in Seshachalam

Biosphere Reserve, Eastern Ghats, Andhra Pradesh, India
Journal of Research in Biology

Authors: ABSTRACT:
Bubesh Guptha M,
Chalapathi Rao PV, The Common Banded Peacock is a member of the family Papilionidae. They
Srinivas Reddy D, Sekhar accomplish pollination, a key stone ecological process in natural sustainability
Maddala SRSC and throughout the world. Preliminary survey was conducted to identify areas with large
Madhu Babu P. population of butterflies. Six locations were selected which were visited every week
from August 2011 to January 2012. Mostly photographic documentation was done, we
sighted Common Banded Peacock Papilio crino species near the (CBET) complex
Mamandur, Talakona, Balapalli and Tirumala of Chittoor District, Andhra Pradesh. This
Institution: species is very rare in Eastern Ghats. During the time of sighting, it was sunny, and few
Wildlife Management Circle, of the recorded places are mud puddle. We recommend further studies in this species
Tirupati, Andhra Pradesh in Eastern Ghats at the earliest opportunity. Also we insist on the protection of
517 507, India. habitat is an important aspect in the conservation of such species.

Keywords:
Corresponding author: Seshachalam Biosphere Reserve, Common Banded Peacock, New Record,
Bubesh Guptha M. Andhra Pradesh.

Article Citation:
Email:
Bubesh Guptha M, Chalapathi Rao PV, Srinivas Reddy D,
bubesh.guptha@gmail.com.
Sekhar Maddala SRSC and Madhu Babu P.
New record of common Banded Peacock Papilio crino in Seshachalam
Biosphere Reserve, Eastern Ghats, Andhra Pradesh, India.
Phone No: Journal of Research in Biology (2012) 2(6): 585-588
+91 9908594183.
Dates:
Received: 02 Apr 2012 Accepted: 15 Apr 2012 Published: 08 Sep 2012

Web Address:
http://jresearchbiology.com/
documents/RA0224.pdf.
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

585-588 | JRB | 2012 | Vol 2 | No 6

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com

INTRODUCTION
The Common Banded Peacock is a member of
the family Papilionidae. Butterflies (Lepidoptera:
Rhopalocera) are lovely and graceful insects that provide
economic and ecological benefits to the human society.
Having multihued colors on their wings, they enhance
the earths beauty incontestably, and add immense
aesthetic value to the ambient environment. They
accomplish pollination, a key stone ecological process in
natural sustainabilit y throughout the world
(Venkata Ramana, 2010). An estimated 20,000 - 25,000
species of butterflies occur in the world (Wynter Blyth,
1957). The Indian sub-region hosts about 1,504 species
of butterflies (Gaonkar, 1996; Smetacek, 1992;
Kunte, 2009; Roy et al., 2010) of which peninsular India
hosts 351, in central India 177, in Western Ghats 334,
(Ashish et al., 2009) and in Eastern Ghats nearly 150
(Venkata Ramana, 2010).
METHOD
Preliminary survey was conducted to identify
areas with large population of butterflies. Six locations
were selected which were visited every week from
August 2011 to January 2012. Data on butterfly fauna, its
abundance and seasonality is based on observation from
0700-1130 hr and 1400-1700 hr, mostly photographic
documentation was done. Species identification was
made using various field guides and other available
literature (Antram, 2002; Evans, 1932; Gunathilagaraj,
1998; Haribal, 1992; Kunte, 2000).
RESULTS AND DISCUSSION:
With this background, A detailed survey of
butterflies was conducted from August 2011 to January
2012. The objective of the survey is to determine the
presence of butterflies. The reserve area has different
habitats like scrub jungle, open forest and trial path. Each
zone was explored on the basis of possibility and
availability of butterflies. Six locations namely Talakona,
586
Guptha et al., 2012
Mamandur, Balapalli, Tirumala, Jungle Book and
Divyaram, were selected which were visited every week.
Out of six location, Common Banded Peacock
Papilio crino were sighted at four places frequently,
especially in the month of October 2011 (Fig-1and 2).
During the time of sighting, it was sunny, and few of the
recorded places are mud puddle.
The reserve located in southern Eastern Ghats,
are spread over the Seshachalam hills of Kadapa district
and Tirumala hills of Chittoor district. Tirumala hills
which are popularly known as the seven hills Lord Sri
Venkateswara. The elevation ranges from 150 to 1,130
m, the terrain is undulating, with deep forest-covered
valleys. Most of the rainfall is received from the
northeast monsoon and a little from the southwest
monsoon. The vegetation is a unique mix of the Dry
Deciduous and Moist Deciduous types. This Park is a
home to six endemic plant species: Cycas beddomei,
Pterocarpus santalinus, Terminalia pallida,
Syzygium alternifolium, Shorea tambaggia and
Boswellia ovalifoliolata. It is the richest floristic hot spot
harboring many endemic and rare plants. The entire
sanctuary is an uninhabited large chunk of dry deciduous
Red Sanders bearing forest, forming catchments to
Swarnamukhi and Penna rivers, both in Chittoor and
Kadapa districts.
Fig 1. Common Banded Peacock Papilio crino
Journal of Research in Biology (2012) 2(6): 585-588
Guptha et al., 2012
Fig 2. Area where Papilio crino was sighted in
Seshachalam Biosphere Reserve
DESCRIPTION
The body is black and dark brown, the fore
wings are black. The whole wing is powdered by green
scales. In the middle of the wing there is a green band
and underside is brown. The bigger part of wing is
powdered by white scales and to the margin there is a
white stripe. The hind wings are black and have tails.
The margin is ridged. In back there is an orange and
black eye. The top of tails are green. Next to the margin
there is a chain of small yellow and blue spots. The
wingspan is about 7.0 - 9.0 cm.
We recommend further studies in the Eastern
Ghats at the earliest opportunity. Also everyone should
realise that the protection of habitat is an important
aspect in the conservation of such species.
ACKNOWLEDGEMENT
The authors are very much thankful to Sri S.V.
Kumar, IFS, Principal Chief Conservator of Forest and
Chief Wildlife Warden, Andhra Pradesh. Special thanks
to Sri T. Chakrapani, SFS, Divisional Forest Officer,
Journal of Research in Biology (2012) 2(6): 585-586
WLM Tirupati and Sri N.V. Sivaram Prasad, Asst.
Conservator of Forests, Biodiversity Research Centre,
Tirupati. We thank Dr.Rajasekar and Dr.Kishore
Department of Zoology, S.V.University Tirupati for
valuable suggestion for this paper. Finally, we would like
to thank all forest staffs from S.V.National Park, Tirupati
helping field trips.
REFERENCES
Antram CB. 2002. Butter flies of India. A Mittal
Publications, New Delhi. 226.
Ashish D, Tiple, Arun M, Khurad A. 2009. Butterfly
Species Diversity, Habitats and Seasonal Distribution in
and Around Nagpur City, Central India, World Journal
of Zoology 4(3):153-162.
Evans WH. 1932. The Identification of Indian
butterflies. 2nd ed. Bombay Natural History Society,
Bombay, x+454pp+32pls.
Gaonkar H. 1996. Butterflies of Western Ghats with
notes on those of Sri Lanka. A report to the Center of
Ecological Sciences, Indian Institute of Science,
Bangalore, Zoologi-cal Museum, Copenhagen and
Natural History Museum, London, 89.
Gunathilagaraj K, Perumal TNA, Jayaram K,
Ganesh Kumar M. 1998. Some south Indian Butterflies.
Field Guide. Published under Project Lifecape, Indian
Academy of Science, Bangalore, 270 .
Haribal M. 1992. The Butterflies of Skkim Himalaya
and their Natural History. Nataraj Publishers, Dehradun,
217.
Kunte K. 2009. Occurrence of Elymnias obnubila
Marshall and de Nicville, 1883 (Lepidoptera:
Nymphalidae: Satyri-nae) in southern Mizoram: Range
extension of the species and an addition to the Indian
butterfly fauna. Journal of Threatened Taxa 1(11):567-
568.
587
 Guptha et al., 2012

Kunte K. 2000. India- A Lifescape Butterflies of

Penisular India. Indian Academy of Science, Bangalore,
University Press, 270.

Roy AB, Ghosh U, Kunte K. 2010. Sighting of

Elymnias panthera (Lepidoptera: Nymphalidae:
Satyrinae) in West Bengal, eastern India. Journal of
Threatened Taxa 2(1):670-671.

Smetacek P. 1992. Record of Plebejus eversmanni

(Stgr.) from India. Journal of the Bombay Natural
History Society 89:385-386.

Venkata Ramana SP. 2010. Biodiversity and

Conservation of Butterflies in the Eastern Ghats. The
Ecoscan, 4 (1):59- 67.

Wynter Blyth MA. 1957. Butterflies of the Indian

region. Bombay Natural History Society, Bombay. 523.

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588 Journal of Research in Biology (2012) 2(6): 585-588

 Journal of Research in Biology An International Scientific Research Journal

Original Research

Medicinal values of Elaeis guineensis and Raphia hookeri wines

Journal of Research in Biology

Authors: ABSTRACT:
Ibegbulem CO1, Alisi CS1,
Nwankpa P2, Amadi BA2, The medicinal values of fresh Raphia hookeri and Elaeis guineensis wines
Agiang MA3and were evaluated. Face-to-face interview questionnaire-based ethno-medical survey on
Ekam VS.3 1000 randomly selected families in some southeastern and southsouthern states in
Nigeria on the use of the palm wines as antimicrobial agents, vehicles for antimicrobial
agents, galactogogues in postpartum mothers and prophylactic agents against malaria
Institution:
in ethno-medicine were carried out. The presence of bioactive phytochemical and
1. Department of
Biochemistry, Federal biochemical constituents with reported pharmacological activities were detected and
University of Technology, their biochemical modes of action were proposed. In conclusion, the antimicrobial
Owerri, Nigeria. values of the wines are phytochemical and ethanol mediated, their lactogenic effects
are saponin-mediated increases in serum prolactin content and their prophylactic
2. Department of effect is by the inhibition of the intra-erythrocytic plasmodial growth.
Biochemistry, Imo State
University, Owerri, Nigeria.

3. Department of Keywords:
Biochemistry, University of Concoction, ethno-medicine, medicinal value, palm wines, proposed
Calabar, Calabar, Nigeria. mechanisms.

Corresponding author: Article Citation:

Ibegbulem CO. Ibegbulem CO, Alisi CS, Nwankpa P, Amadi BA, Agiang MA and Ekam VS.
Medicinal values of Elaeis guineensis and Raphia hookeri wines.
Journal of Research in Biology (2012) 2(6): 589-595
Email:
ibemog@yahoo.com
Dates:
Received: 27 Jul 2012 Accepted: 09 Aug 2012 Published: 10 Sep 2012
Phone No:
+2348037239349.

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

Web Address: licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
http://jresearchbiology.com/ reproduction in all medium, provided the original work is properly cited.
documents/RA0266.pdf.

589-595 | JRB | 2012 | Vol 2 | No 6

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com
 Ibegbulem et al.,2012

INTRODUCTION bioactive phytochemical and biochemical constituents

Palm wine is a sweet, effervescent drink and antimicrobial tests were carried out with these wines
obtained from the fermented sap of the tropical and their concoctions. Tests on microbial dehydrogenase
palm tree (Elaeis guineensis) and raphia palm trees activity (DHA) were run with the wines and
(Raphia species) (Ukhun et al., 2005). Palm wine is used 4-methyl hydroxyl benzoic acid (4-MHBA).
in ethno-medicine as prophylactic against malaria and The empirical and experimental evidences were then
also used (mainly that of E. guineensis) to increase milk used to propose their mechanisms of action.
flow in postpartum mothers (C. Ibegbulem, personal
communications). Medicinal herbs can be infused in MATERIALS AND METHODS
palm wine to remedy a wide variety of physical Procurement of samples
complaints (http://en.wikipedia.org/wiki/Palm_wine; The fresh palm wines used were tapped from
Obute, 2005) and are used to drink some medicines, like Raphia hookeri G. Mann. and H. Wendl. trees and
the exceptionally bitter ones, such as the mixture with Elaeis guineensis trees by palm wine tappers at Orodo,
sequestered extracts of Vernonia amygdalina Del. Mbaitoli Local Government Area of Imo State, Nigeria.
(bitter leaf); the mixture is rubbed on the body together The V. amygdalina leaves were collected from a private
with drinking a glass daily in the course of curing garden and authenticated by Dr. F.N. Mbagwu,
measles, small pox and chicken pox (Obute, 2005). Wine a taxonomist at the Department of Plant Science and
prepared from E. guineensis is called Nkwu in Igbo while Biotechnology, Imo State University, Owerri, Nigeria.
that from the Raphia species is called Ngwo. A voucher specimen was deposited at his laboratory with
The measurement of microbial enzyme activity is voucher number: IMSUH 028.
used in the assessment of ecotoxicological impacts of All the chemicals used were of analytical-reagent
environmental pollutants. The most often studied groups grade and were purchased locally.
of enzyme are oxidoreductases e.g.dehydrogenases. Ethno-medical survey
Total microbial dehydrogenase assays involving the The survey was carried out on 1000 randomly
reduction of 2,3,5-triphenyltetrazolium chloride (TTC) selected rural families many of who rely on them as
and 2-(p-iodophenyl)-5-phenyltetrazoliumchloride (INT) home-made remedies (including those of traditional
to their formazans have been used to measure microbial medicine practitioners) in the southeastern and
activity (Gong, 1997; Mathew and Obbard, 2001; southsouthern states of Nigeria using face-to-face
Nweke et al., 2006). interview-based questionnaires. This method was used
Most plant materials used in ethno-medicine are because most of the rural families interviewed are
of unknown biochemical modes of action other than that illiterates. There was also the need to create the
they are efficacious at treating the debilitating malady. atmosphere conducive for them to freely air their
Their phytochemical and biochemical constituents may views without restrictions. The survey entailed
be responsible for their acclaimed effects. This study gathering information on the (i) use of the samples in
carried out a randomized, face-to-face interview ethno-medicine (ii) major types of ailment treated with
questionnaire-based ethno-medical survey on 1000 each sample (iii) most popular combinations (if any).
randomly selected rural families in some southeastern Qualitative analysis for phytochemical and
and southsouthern states in Nigeria on the use of Nkwu biochemical constituents
and Ngwo in ethno-medicine. The presence of some Tests for the presence of tannins, flavonoids and
590 Journal of Research in Biology (2012) 2(6): 589-595
Ibegbulem et al.,2012

Dehydrogenase Activity (mg formazan/mg cell dry weight/ hr)

Dehydrogenase Activity (mg formazan/mg cell dry weight/ hr)

Concentration (g/ml) Concentration (g/ml)

Fig 1. Effect of Ngwo on Dehydrogenase Activity of Fig 2: Effect of Nkwu on Dehydrogenase

Microorganism Activity of Microorganism

catechins were determined according to the methods of
Evans (2002). The presence of tannins was confirmed
using 17% Na2CO3 and Folin-Denis Reagent as
described by Ibegbulem et al., (2011a). Saponins were
detected using the frothing and red blood cell haemolysis
tests described by Harborne (1973). Ethanol suppresses
frothing (C. Ibegbulem, personal communications), so
ethanol-containing samples were initially heated to 80C
to distill it off. The presence of ethanol in each sample
was detected by adding 0.5 ml of Jones Reagent to a
mixture of 1 ml of acetone and 0.5 ml of sample which
led
precipitate or emulsion. Test for the presence of
4-methylhydroxybenzoic acid (4-MHBA) was carried
out using the method of ASEAN (2005).
Treatment of wine and preparation of concoction
mortar and pestle. 10.0 g of the ground leaves was mixed
with 30.0 ml of distilled water and sieved with muslin
cloth. A 50.0 ml aliquot of the respective wine was
treated with 20.0 ml sequestered extract of the
V. amygdalina leaves.
Microbial culture and sensitivity tests
The pathogenic Pseudomonas aeruginosa and
Staphylococcus aureus bacteria used were obtained from
degenerated wound. Isolates were purified on nutrient
agar (Fluka) plates and characterizations were done using
standard microbiological methods. Identification to the
generic level was carried out using the methods of
Holt et al., (1994). The microbial culture and sensitivity
to formation of a green or blue-green tests were carried out as described by Ekwenye and
Ijeomah (2005) using the disc diffusion method.
Inhibition of total dehydrogenase activity of
microorganism
The effects of the wines and oils on the total
The wet V. amygdalina leaves were ground using DHA of the test microorganisms were evaluated using
2,3,5-triphenytetrazolium chloride (TTC) as an artificial
electron acceptor as described by the methods of

Table 1: Sample in ethno-medicine, ailment treated or prevented, response on ethno-medical

usage and scientific data
Sample in Ailment treated or Response on Ethno-medical Scientific data on
ethno-medicine prevented usage (%)* usage
Nkwu or Ngwo Malaria (prophylaxis) 47 -
Nkwu or Ngwo Postpartum milk flow 100 -
Nkwu or Ngwo and Chicken pox, 68 Obute (2005)
V. amygdalina concoction small pox, measles
*Responses are of 1000 families (including those of traditional medicine practitioners).

Journal of Research in Biology (2012) 2(6): 589-595 591

 Ibegbulem et al.,2012

Dehydrogenase Activity (mg formazan/mg cell dry weight/ hr)

Table 2: Phytochemical and biochemical constituents
of the samples*
Parameter Sample
Nkwu Ngwo V. amygdalina

Tannins + + +

Flavonoids + + +

Catechins + + +

Saponins + + +
Ethanol + + -

Concentration (g/ml)
4-MHBA + + +

*Values are mean of triplicate determinations.

Fig 3. Effect of 4-MHBA on Dehydrogenase
Key: + = detected, - = not detected.
Activity of Microorganism

Alisi et al., (2011). A standard antimicrobial agent, phytochemicals. This may be due to differences in assay
4-MHBA (Zimmer and Huyck, 1961), was also used. methods. The phytochemical and biochemical contents
Statistical analysis of Ngwo corroborated that reported by Ibegbulem et al.,
Data generated were analysed using the students (2011b).
t-test of significance. Values were declared significantly Table 3 shows that the palm wines were potent
different at p<0.05. antimicrobial agents. This may be due to their ethanol
and phytochemicals contents. Ethanol is antiseptic.
RESULTS AND DISCUSSION Tannins, saponins and flavonoids are antimicrobials
The medicinal values of the samples were (Evans, 2002). In ethno-medicine, concoctions made
confirmed in the survey (Table 1). Researchers have also from these wines are more potent.
confirmed the medicinal values of some of them. The effects of the wines and 4-MHBA on DHA
Responses were also influenced by use of alternative of the microorganisms are shown in Figures 1-3.
traditional and orthodox medicines and occurrence of the The Ngwo was a better antimicrobial agent (Fig 1) than
ailment so treated. Nkwu (Fig 2). The antimicrobial activity of 4-MHBA
The phytochemicals and biochemicals detected was confirmed in Fig 3. In all, the P. aeruginosa was a
in the wines are presented in Table 2. The Nkwu however more resistant organism than S. aureus when the slopes
contained tannins and saponins contrary to the report of of Figures 1-3 are considered. The variations in
Dioha et al., (2005) whose sample did not present these resistance may be due to differences in cell wall

Table 3: Sensitivity of microorganism to wine and concoction

Microorganism
Sample Pseudomonas aeruginosa Staphylococcus aureus
Nkwu + -
Ngwo + +
Nkwu and V. amygdalina concoction + +
Ngwo and V. amygdalina concoction + +
4-MHBA + +
Key: + = sensitive; - = insensitive.

592 Journal of Research in Biology (2012) 2(6): 589-595

Ibegbulem et al.,2012

compositions or different dehydrogenase systems.
Staphylococcus is a Gram-positive microorganism while
the Pseudomonas is a Gram-negative microorganism.
Different microorganisms have been reported to have
different dehydrogenase systems (Praveen-Kumar,
2003). The responses of the bacterial DHAs to the wines
and 4-MHBA were both concentration dependent and
organism dependent.
The biochemical modes of action of these wines
as galactogogues and prophylactic agents are largely
unknown. Even though we did not confirm the lactogenic
and prophylactic effects in our laboratory, hypothetical
modes of action are suggested and were however based
on the suggested bioactive principles in the wines
(Table 4) since most of their acclaimed effects had been
confirmed in Table 1.
The biochemical basis for the lactogenic
(or galactogogue) effect of palm wine in postpartum
women may be mediated by their saponin contents.
The presence of steroidal saponins and sapogenins in
Asparagus racemosus had been reported to be
responsible for its lactogenic effect, which increased
serum prolactin content (Oketch-Rabah, 1998;
Goyal et al., 2003; Okasha et al., 2008). Flavonoids
could have been mentioned as another bioactive
galactogogue in the palm wines. Di Pierro et al., (2008)
reported that silymarin (a flavonolignan) increased
production of breast milk in healthy women after
delivery. Silymarin is generated by the oxidative
combination of a lignan and a flavonoid (Di Pierro et al.,
2008). However, the lignan component may be needed
for bioactivity. The bioactive galactogogues contained in
palm wines may increase prolactin-releasing factor
(PRF) which eventually increase serum prolactin (PRL)
thereby resulting in increased milk production.
On the possible use of palm wines as
prophylactic agents against malaria when large quantities
are ingested, the hypothesized mechanism is that their
ethanol contents, which diffuse freely across biological
membranes, may increase membrane fluidity, altering
their receptors and ion channels of the infected
erythrocytes thereby impairing motor performance of
plasmodia. The ethanol may even lyse the infected red
blood cells thereby threatening the survival of
intra-erythrocytic parasites. Chi and Wu (1991) reported
that ethanol increased membrane fluidity, caused the
leakage of erythrocytic potassium ions before lysing the
red blood cells. Devlin (2006) posited that ethanol
altered membrane receptor and ion channel activities and
impaired motor performance. The mechanisms may also
include other intra-erythrocytic conditions which limit
the metabolism and growth of the parasites. von Brand
(1966) reported that erythrocytic forms of
Plasmodium berghei, separated from host cell, showed a
much more vigorous metabolism when the K+/ Na+ ratio
corresponded to that of the erythrocytes rather than the
blood stream. Lell et al., (2000) on the other hand
reported that ethanol inhibited the growth of
P. falciparum, in vitro, adding that the growth of
malarial parasites was strongly inhibited by ethanol
concentrations which were attainable by extensive
alcohol consumption. Neuberger (1997) had reported that
the distribution of ethanol between blood and expired air
was 2100:1. This meant that there is increased blood

Table 4: Suspected bioactive principle in ethno-medicine

Ethno-medicine Ailment treated or prevented Suspected bioactive principle*
Nkwu or Ngwo Malaria (prophylactic agent) Ethanol
Nkwu or Ngwo Postpartum milk flow Saponins
Nkwu or Ngwoand V. amygdalina Chicken pox, small pox, Ethanol, tannins, flavonoids, saponins,
concoction. measles 4-MHBA
*Based on Table 2.

Journal of Research in Biology (2012) 2(6): 589-595 593

 Ibegbulem et al.,2012

ethanol residency time which may inhibit Devlin TM. 2006. Biological membranes: structure and
intra-erythrocytic plasmodial growth. membrane transport, in: Devlin, T.M. (Ed.), Textbook of
In instances when the wines are mixed with Biochemistry with Clinical Correlations (6th edn).
herbs to treat infections, the wines act as vehicles, their Wiley-Liss, New Jersey. 443-487.
ethanol is considered antiseptic and the herbs
Di Pierro F, Callegari A, Carotenuto D and Tapia
antimicrobials because of their phytochemical contents.
MM. 2008. Clinical efficacy, safety and tolerability of
Many of their phytochemicals like tannins, flavonoids
BIO-C (micronized Silymarin) as a galactagogue. Acta
and saponins are both antioxidants and antimicrobials
Biomedica 79:205-210.
(Evans, 2002) and confer such medicinal property on the
plant and its product(s). Dioha IJ, Agho MO and Sambo AS. 2005. Evaluative
In conclusion, the medicinal values of the wines comparison of palm wine analogue and oil palm wine.
are based on the antimicrobial effects of their ethanol and Nigerian Journal of Chemical Research 10:1-7.
phytochemical contents, saponin-mediated lactogenic
Ekwenye UN and Ijeomah CA. 2005. Antimicrobial
effects and their ethanol-mediated prophylactic effects.
effects of palm kernel oil and palm oil. KMITL Science
Journal 5(2):502-505.
ACKNOWLEDGEMENT:
We thank all the traditional medicine Evans WC. 2002. Trease and Evans Pharmacognosy
practitioners who, at the risk of exposing the secrets of (15th edn). W.B. Saunders, Edinburgh.
their trades, gave insights into the applications of these
Gong P. 1997. Dehydrogenase activity in soil: a
samples.
comparison between the TTC and INT assay under their
optimum conditions. Soil Biology and Biochemistry
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Journal of Research in Biology (2012) 2(6): 589-595 595

 Journal of Research in Biology An International Scientific Research Journal

Original Research

Hypothesized biochemical modes of action of palm oils used in

ethno-medicine
Journal of Research in Biology

Authors: ABSTRACT:
Ibegbulem CO1,
Egbung GE2, Okoro AA2,
Kalu NN3, Nwaogu LA1 The biochemical modes of action of palm oil (PO) and palm kernel oil (PKO)
and Igwe KO1. that are used in ethno-medicine were hypothesized. One thousand randomly selected
families in the southeastern and southsouthern parts of Nigeria were used in a
Institution:
face-to-face interview questionnaire-based ethno-medical survey on the use of the
1. Department of
palm oils and the ointments made from them to treat infections and febrile seizures in
Biochemistry, Federal
University of Technology, ethno-medicine. The presence of bioactive phytochemical and biochemical
Owerri, Nigeria. constituents with the desired pharmacological activities was detected and their
biochemical modes of action hypothesized. When PKO is used to treat febrile seizures,
2. Department of transdermally transported antipyretic agents inhibit the expression or activities of
Biochemistry, University of cyclooxygenase (COX) isoforms. In conclusion, the hypothesized modes of action are
Calabar, Calabar, Nigeria. that the oils are antimicrobials and increase trans-dermal transport of bioactive
agents.
3. Department of
Biochemistry, Ambrose Alli
University, Ekpoma,
Nigeria.
Keywords:
Corresponding author: Biochemical, ethno-medicine, hypothesis, modes of action, palm oil, palm
Ibegbulem CO. kernel oil.

Email: Article Citation:

ibemog@yahoo.com Ibegbulem CO, Egbung GE, Okoro AA, Kalu NN, Nwaogu LA and Igwe KO.
Hypothesized biochemical modes of action of palm oils used in ethno-medicine.
Phone No: Journal of Research in Biology (2012) 2(6): 596-601
+2348037239349.
Dates:
Web Address: Received: 06 July 2012 Accepted: 18 Jul 2012 Published: 10 Sep 2012
http://jresearchbiology.com/
documents/RA0263.pdf.

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.

596-601 | JRB | 2012 | Vol 2 | No 6

Journal of Research in Biology
An International
Scientific Research Journal www.jresearchbiology.com
 Ibegbulem et al., 2012

INTRODUCTION purchased from Camela Vegetable Oil Limited,

Fru it s of tro pica l palm tree Irete-Owerri, Nigeria. Fresh PO, Akanwu and
(Elaeis guineensis Jacq.) yield two types of oils. The O. gratissimum were purchased from Nkwo-Ukwu
palm fruits are cooked, mashed and palm oil (PO) Market, Ihiagwa, Nigeria. The O. gratissimum leaves
pressed out and the kernels fried or crushed and pressed were authenticated by Dr. F.N. Mbagwu, a taxonomist at
for palm kernel oil (PKO) (Ekwenye and Ijeomah, 2005; the Department of Plant Science and Biotechnology, Imo
Ugbogu et al., 2006). In ethno-medicine, PO is mixed State University, Owerri, Nigeria. Voucher specimen
with the acid salt, sodium sesquecarbonate was deposited; with number: IMSUH 029.
(Na2CO3.NaHCO3.2H2O; Akanwu in Igbo) to treat skin All the chemicals used were of analytical-reagent
diseases on domestic animals. On the other hand, PKO is grade and were purchased locally.
mixed with ground wet or dried leaves of Ethno-medical survey
Ocimum gratissimum (scent leaf) to treat febrile The survey was carried out on one thousand
seizures (C. Ibegbulem, personal communications). randomly selected rural families, many of who rely on
Ugbogu et al., (2006) had reported the anti-microbial PO, PKO and their ointments in ethno-medicine as
roles of PKO against Staphylococcus aureus and home-made remedies (including those of traditional
Streptococcus specie. medicine practitioners) in the southeastern and
Traditional medicine provides most of the southsouthern parts of Nigeria, using face-to-face
health-care needs of most rural dwellers in Nigeria. This interview-based questionnaires. This method was used
involves the use of local gin, oils and assorted local herbs because most of the rural families interviewed are
(Ekwenye and Ijeomah, 2005). The biochemical basis for illiterates and there was need to create the conducive
the modes of action of the plant materials used in environment for them to freely air their views without
ethno-medicine is not understood. It is thought that their restrictions. The survey entailed (i) confirmation of the
phytochemical and biochemical constituents may be use of the oils in ethno-medicine (ii) most popular
responsible for their acclaimed effects. This study carried combinations (if any) (iii) major types of ailment treated
out a randomized, face-to-face interview, questionnaire- with each sample and (iv) knowledge of biochemical
based ethno-medical survey on one thousand randomly mechanism other than the efficacy.
selected rural families in some southeastern and Qualitative analysis for phytochemical and
southsouthern states in Nigeria on the use of PO and biochemical constituents
PKO and their ointments in ethno-medicine. The oils Tests for the presence of tannins, flavonoids and
were screened for the presence of bioactive catechins were determined according to the methods of
phytochemical and biochemical constituents with the Evans (2002). Saponins were detected using the frothing
desired pharmacological actions and anti-microbial tests and red blood cell haemolysis tests described by
were carried out using the oils and their ointments. Such Harborne (1973). Test for the presence of lipid was
information was then used to hypothesize their carried as described by Plummer (1971). Test for the
biochemical modes of action. presence of 4-methyl hydroxyl benzoic acid (4-MHBA)
was carried out using the method of ASEAN (2005).
MATERIALS AND METHODS Treatment of sample and preparation of ointment
Procurement of samples A quantity (5.0 ml) of PO was mixed with 3.0 g
The refined PKO used in the study was of ground Akanwu while 5.0 ml of PKO was mixed with
597 Journal of Research in Biology (2012) 2(6): 596-601
Ibegbulem et al., 2012

Table 1: Ethno-medicine, ailment treated, response to ethno-medical usage and scientific data
Ethno-medicine Ailment treated Ethno-medical survey usage (%)* Scientific data on usage
PO Skin infection 93 Ekwenye and Ijeomah
(2005)
PKO Skin infection 96 Ekwenye and Ijeomah
(2005); Ugbogu et al.,
(2006)
PKO and O. gratissimum Febrile seizures 98 NA
ointment (convulsion)
PO and Akanwu Skin infection 72 NA
ointment on domestic animals
*Responses are of 1000 families (including those of traditional medicine practitioners).
Key: NA = not available.
3.0 g of sun dried and ground leaves of O. gratissimum. antimicrobial and antioxidant properties (Evans, 2002).
Microbial culture and sensitivity tests The paraben, 4-MHBA, is also an antimicrobial agent
The pathogenic Pseudomonas aeruginosa and (Zimmer and Huyck, 1961). The lipid detected in the oils
Staphylococcus aureus bacteria used were obtained from and ground O. gratissimum may have been composed of
degenerated wound. Isolates were purified on nutrient different fatty acids. Wardlaw and Kessel, (2002)
agar (Fluka) plates and characterizations were done using reported that palmitic acid is the major fatty acid in PO
standard microbiological methods. Identification to the while lauric acid is the major fatty acid in PKO.
generic level was carried out using the methods of The composition of the lipid in O. gratissimum is
Holt et al., (1994). The microbial culture and sensitivity suggested for further studies.
tests were carried out using the oils and their ointments The palm oil and PKO used did not inhibit the
as described by Ekwenye and Ijeomah (2005) using the growth of the test microorganisms (Table 3) possibly due
disc diffusion method. to their limited solubility and diffusions into the
agar edia. Ekwenye and Ijeomah (2005) reported the
RESULTS AND DISCUSSION same observations. These observations do not however
The ethno-medical importance of the oils was preclude the fact that the oils are used in ethno-medicine
confirmed in the survey (Table 1). Usage of some of for treating infections. It may be that their phytochemical
them had also been confirmed by scientific data. The and biochemical contents were not enough to inhibit the
responses were influenced by the use of alternative growth of the test microorganisms. Again, the test
traditional and orthodox medicines and occurrence of the microorganisms may have been more resistant because
ailment so treated.
Table 2: Phytochemical and biochemical
The desired phytochemicals and biochemicals constituents of pharmacological importance*
detected in our samples are presented in Table 2. Sample
Parameter
Ibegbulem and Chikezie (2012) had also reported the PO PKO O. gratissimum
Tannins + + +
presence of these phytochemicals and biochemicals in
Flavonoids + + +
PO and PKO. Table 2 also shows that the oils had Catechins + + +
Saponins + - +
property of antimicrobial agents because they contained
Lipid + + +
phytochemicals and biochemicals with reported 4-MHBA + + +
antimicrobial and antioxidant property.
Tannins, *Values are mean of triplicate determinations. Water
saponins and flavonoids have been reported to have soluble fraction. Key: + = detected, - = not detected.

Journal of Research in Biology (2012) 2(6): 596-601 598

 Ibegbulem et al., 2012

Table 3: Sensitivity tests using oils and ointments

Microorganism
Sample Pseudomonas aeruginosa Staphylococcus aureus
PO - -
PKO - -
PO and Akanwu + +
PKO and O. gratissimum ointment + +
4-MHBA + +
Key: + = sensitive; - = insensitive
they were hospital isolates; being more resistant than antipyretic nature of the unctuous PKO-based ointment,
environmental isolates. However, the ointments made which is rubbed on the body, soles, palms and into the
from the oils had antimicrobial effects (Table 3). The rectum of patients, stems largely from the ability of the
ground O. gratissimum leaves contained all the fatty acid contents of the oil to increase the percutaneous
phytochemicals and biochemicals detected in the oils absorption of the antipyretic agents contained in the
(Table 2), so, its effect may have been additive. The oils febrifuge (like O. gratissimum). Fatty acids like myristic,
may also have been emulsified by the Akanwu and capric, oleic and lauric acids increase transdermal
ground O. gratissimum thereby increasing their delivery of highly lipophilic drugs (Pathan and Setty,
solubilities. Traditionally, ointments made from these 2009). Antipyretic agents inhibit the activities of
oils are more potent than just the oils and may justify cyclooxygenase (COX) isoforms thereby reducing the
their usage amongst the populace in some parts of concentration of prostaglandin E 2 within the
Nigeria. Ugbogu et al., (2006) reported that lauric acid is hypothalamus which cause the elevation of body
the antimicrobial agent in PKO and that the antimicrobial temperature (Aronoff and Neilson, 2001). Flavonoids
effect of fatty acids are additive. like wogonin, anthocyanidins, cyanidin, delphinidin have
Though we did not prove the antipyretic property been reported to inhibit the expression of COX-2 genes
of the PKO-based ointment here, the biochemical (Hou et al., 2005; Chen et al., 2008) while baicalin and
mechanisms were however suggested. The hypothesis catechin inhibited the expression of COX-1, COX-2 and
was based on the presence of bioactive principles of 5-lipoxygenase (5-LOX) genes in dogs (Burnett et al.,
pharmacological interest (Table 4). Most of their 2009). The antipyretic properties of O. suave and
acclaimed effects had been confirmed by empirical O. lamiifolium have also been reported (Makonnen et al.,
evidence (Table 1). 2003). The very high body temperatures that are the
Febrile seizures occur between the ages cause the seizures are commonly lowered by tepid
of six months and six years in children sponging, which principle is based on latent heat of
when the body temperature exceeds 38o C vaporization.
(http://en.wikipedia.org/wiki/Febrile_seizure). The
Table 4: Suspected bioactive principle in ethno-medicine
Sample Ailment treated Suspected bioactive principle*
PO Skin infection Lipid, tannins, flavonoids, saponins, 4-MHBA
PKO Skin infection Lipid, tannins, flavonoids, saponins, 4-MHBA
PKO and O. gratissimum Febrile seizures (convulsion) Lipid, flavonoids (catechins)
ointment.
PO and Akanwu ointment. Skin infection on domestic animals Lipid, tannins, flavonoids, saponins, Akanwu,
4-MHBA
*Based on Table 2.
599 Journal of Research in Biology (2012) 2(6): 596-601
Ibegbulem et al., 2012

CONCLUSION gene expression in human lung epithelial cancer cells.

In conclusion, we hypothesize that when PO and
PKO and their ointments are used to treat infections,
their phytochemical and biochemical constituents inhibit
microbial growth. When their oinments are used to treat
febrile seizures, they increase percutaneous absorption of
antipyretic agents which inhibit the expression or
activities of COX isoforms.
ACKNOWLEDGEMENT
We acknowledge the assistance received from all
the traditional medicine practitioners who, at the risk of
exposing the secrets of their trades, gave insights into the
ethno-medical applications of these oils.
Molecular Nutrition and Food Research 52(11):1349-
1357.
Ekwenye UN and Ijeomah CA. 2005. Antimicrobial
effects of palm kernel oil and palm oil. KMITL Science
Journal 5(2):502-505.
Evans WC. 2002. Trease and Evans Pharmacognosy
(15th edn). W.B. Saunders, Edinburgh.
Febrile Seizure. http://en.wikipedia.org/wiki/
Febrile_seizure. Retrieved 28/02/2012.
Harborne JB. 1973. Guide to Modern Technique of
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601 Journal of Research in Biology (2012) 2(6): 596-601

 Journal of Research in Biology
The microflora of Eagle Island mangrove swamp, southern Nigeria
Journal of Research in Biology
Authors:
Okpokwasili GC1,
Ifenwanta CE1 and
Nweke CO1,2.
Institution:
1. Department of
Microbiology, University of
Port Harcourt, P.M.B.5323,
Port Harcourt, Nigeria.
2. Department of
Microbiology, Federal
University of Technology,
P.M.B. 1526, Owerri,
Nigeria.
Corresponding author:
Okpokwasili GC.
Email:
gidsilman@yahoo.com
Web Address:
http://jresearchbiology.com/
documents/RA0241.pdf.
Journal of Research in Biology
An International
Scientific Research Journal
An International Scientific Research Journal
Original Research
ABSTRACT:
The occurrence, abundance and distribution of bacteria (nitrifying,
nitrogen-fixing and heterotrophic), yeasts, moulds, algae and actinomycetes in
mangrove and freshwater swamps were studied. Microbial abundance and diversity
were greater in freshwater than in mangrove swamps. Actinomycetes were the
dominant organisms in sediments while algae occurred widely in water. A total of 57
microbial genera were isolated from the mangrove swamp, out of which the algae had
greatest diversity comprising 20 genera. It was followed by the moulds, which had
16 genera, then bacteria (10 genera), yeast (6 genera) and actinomycetes (5 genera).
The organisms were widely distributed in all parts of the swamp, though
actinomycetes did not occur in the water samples and yeasts occurred sparsely in
freshwater. The study implicates the genera Aeromonas, Proteus, Serratia and
Citrobacter (bacteria); Thermoactinomyces and Streptomyces (actinomycetes);
Aspergillus, Trichoderma, Penicillium, Mucor, Fusarium, Geotrichum, Verticillium and
Botrytis (moulds); Rhodospiridium, Trigonopsis and Pichia (yeasts); Gomphonema,
Fischerella, Asterionella, Borgea, Nostoc, Chlamydomonas, Laminaria, Spirulina,
Chlorobotrys and Vaucheria (algae) as autochthonous members of the mangrove
swamps of the Niger delta. Mangrove swamps therefore harbour a wide range of
microorganisms some of which are indigenous to this slightly acidic habitat and occur
in varying proportions.
Keywords:
Mangrove swamp, microbial ecology, rhizosphere.
Article Citation:
Okpokwasili GC, Ifenwanta CE and Nweke CO.
The microflora of Eagle Island mangrove swamp, southern Nigeria.
Journal of Research in Biology (2012) 2(6): 602-616
Dates:
Received: 14 May 2012 Accepted: 01 Jun 2012 Published: 22 Sep 2012
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
602-616 | JRB | 2012 | Vol 2 | No 6
www.jresearchbiology.com

INTRODUCTION
Mangroves are woody plants that grow at
the interface between land and sea in tropical and
sub-tropical latitudes. These plants, and the associated
microbes, plants, and animals, constitute the mangrove
forest community or mangal. The mangal and its
associated abiotic factors constitute the mangrove
ecosystem (Kathiresan and Bingham, 2001). The plants
refer to evergreen salt-tolerant species of shoreline trees
and shrubs belonging to numerous unrelated families that
share similar habitat preferences, physiognomy,
physiology and structural adaptations (Kinako, 1977).
Mangrove ecosystems cover roughly 60-75% of
the worlds tropical and subtropical coastlines
(Holguin et al., 2001). The coastal region of Nigeria is
dominated by swamp forests of which mangrove swamps
cover a sizeable proportion. The mangrove swamp zone
lies between latitude 4.0 and 7.6N and is made up of
three subzones i.e. the freshwater, saltwater and
brackishwater swamps. In southern Nigeria, they consist
of an area of low-lying land bordering the coastal
swamps and creek area (Onofeghara, 1990). The Eagle
Island houses one of the mangrove swamps of the Niger
Delta. It is inundated by saltwater for 2-10 months in a
year (Onofeghara, 1990). The swamp is extensively
covering a wide area of land occupied chiefly by
mangrove plants.
Mangrove communities are highly productive
ecosystems that provide large quantities of detrital
organic matter to nearby coastal waters. The detritus
serve as a nutrient source and constitutes the base of an
extensive food web involving many organisms of
commercial importance. Basically, microbes are
responsible for nutrient transformations within mangrove
ecosystem (Alongi et al., 1993). In tropical mangrove,
bacteria and fungi constitute 91% of the total microbial
biomass while algae and protozoa represent only 7 and
2% respectively (Alongi, 1988). In temperate, intertidal
surface sediments, algae are the dominant contributors to
603
Okpokwasili et al.,2012
total microbial biomass and usually account for 1-2% of
total sediment organic carbon (Rublee, 1982).
In the other parts of the world, the microbial
productivity and diversity have been widely studied and
documented. The microbial ecology of Australian
mangrove ecosystem has been widely investigated by
Alongi and co-workers (Alongi, 1988; Alongi et al.,
1993). Kathiresan (2000) has reviewed studies on the
microbial composition of Pichavaram mangrove,
southeast India. However, little is known about the
ecology of microorganisms in Nigerian mangrove
ecosystems. Literature on the microbial ecology of Eagle
Island mangrove ecosystem is even scantier. Available
publication have been on the phytoplankton flora of the
Warri-Forcadoes estuaries (Opute 1990, 1991), lower
reaches of the Nun river (Yakubu et al., 1998),
epipsammic and epipellic algae of Qua Iboe River
estuary (Ubom and Essien, 2003; Essien and Ubom,
2003; Essien et al., 2008) and the density of hydrocarbon
utilizing bacteria in Iko River mangrove ecosystem
(Udotong et al., 2008). Not much is known on the
microflora of Eagle Island mangrove ecosystem.
This study therefore presents the composition, abundance
and distribution of the microbial community of
Eagle Island mangrove swamp.
MATERIALS AND METHODS
Study area
All samples were collected from the Eagle Island
located behind the Rivers State University of Science
and Technology, Nkpolu, Port Harcourt, Nigeria. Like
the other mangrove swamps of the Niger Delta, it lies
between Latitude 4 and 7.6 North. The swamp is
extensive, covering a wide area of land with a top layer
of mud slurry overlying a relatively hard substratum.
It is dominated with mangrove vegetation and conforms
to the characteristics of mangrove swamps.
Collection of samples
The soil samples were collected from the
Journal of Research in Biology (2012) 2(6): 602-616
Okpokwasili et al.,2012

rhizosphere and non-rhizosphere areas. Soils from these Identification of organisms

areas were dug up using sterile hand trowel and collected The morphological characteristics of the moulds
in sterile bags. The sediment samples were collected were macroscopically and microscopically ascertained
using a sediment grab. The water samples were collected using needle mount method and the moulds were
in sterile screw cap bottles. All samples were taken to the identified according to Larone (1976), Hunter and
laboratory in a cooler and analyzed within six hours of Benneth (1973) and Sampson et al., (1984). The yeasts
collection. were characterized biochemically and identified
Enumeration and isolation of microorganisms following the scheme of Laskin and Lechevelier (1977).
Ten-fold serial dilutions of the water, sediment The heterotrophic bacteria were identified to generic
and soil samples were prepared in sterile 0.85% level following the scheme of Holt et al., (1994).
NaCl solution. Small aliquots (0.1 ml) of water and The nitrifying bacteria were identified based on the
suspensions of soil and sediment were aseptically method used by Colwell and Zambruski (1972).
spread-inoculated onto triplicate plates of different Nitrogen-fixing bacteria were characterized
microbiological media and incubated at room biochemically (Smibert and Krieg, 1981) and identified.
temperature (28 2C). The fungal populations were Actinomycetes were identified based on the scheme of
enumerated on Sabouraud dextrose agar (Difco) Krieg and Holt (1984) and Laskin and Lechevalier
supplemented with streptomycin at 50 g/ml to suppress (1977). Identification of algae was based on the scheme
bacterial growth. Incubation was at room temperature for of Fritsch (1975).
48 h and five days for yeasts and moulds respectively.
The hererotrophic bacterial population was enumerated RESULTS AND DISCUSSION
on nutrient agar plates after 48 h incubation. Nitrifying The Eagle Island mangrove swamp has been
and nitrogen-fixing bacteria were enumerated on subjected to various contaminating materials capable of
Winogradsky medium of Colwell and Zambruski (1972) impairing water and sediment quality. The pH of the
and the Azotobacter medium of Krieg (1981) freshwater and mangrove swamp samples showed that
respectively following four days of incubation. the areas were slightly acidic (Table 1). The pH values
Actinomycetes were enumerated on starch-casein agar varied from 5.25 0.22 in the non-rhizosphere soil to
(APHA, 1985) following seven days of incubation. 6.05 0.33 in the water samples of the mangrove
The starch-casein agar was amended with 50 g/ml each swamp. In the freshwater swamp, the pH values varied
of nystatin and cyclohexamide to suppress fungal from 6.51 0.17 in the sediment to 6.88 0.11 in the
growth. The algal population was enumerated on algae
Table 1: pH values of mangrove and freshwater
medium of Deft (1988) as modified by Odokuma and samples
Okpokwasili (1993). The medium was amended with Sample pH
50 g/ml each of streptomycin and amphotericin B and Mangrove ecosystem
Rhizosphere soil 5.67 0.16
incubation was for seven days. In each of these media, Non-rhizosphere soil 5.25 0.22
the different microbial species were counted upon Sediment 5.93 0.32
Water 6.05 0.33
growth. The different morphotypes of organisms were Freshwater ecosystem
subcultured onto freshly prepared medium, stored at Rhizosphere soil 6.88 0.11
Non-rhizosphere soil 6.67 0.21
4C and later identified.
Sediment 6.51 0.17
Water 6.59 0.19

Journal of Research in Biology (2012) 2(6): 602-616 604

605
mangrove
The

heterotrophic

swamp
5
(Nweke and Orji, 2009).
abundance

bacterial
of

sample.
count
bacteria,

Moreover,
6
ranged
fungi

environments. Also, the fungal counts in the mangrove

more acidic than the freshwater samples. The value is

ecosystem. In the freshwater ecosystem, total aerobic

and
lower than the pH value of seawater and estuaries but is

heterotrophic bacteria ranged from 2.42 ( 0.11) x 104 to

freshwater samples fell within the range reported for Imo

are shown in Table 2. The population of total aerobic

has made similar observation on Pichavaram mangrove.

recorded for the freshwater swamps, the bacterial loads

of inorganic compounds and settlement of dead organic
sediments

River, Nigeria (Ihejirika et al., 2011). The bacterial

obtained from the various mangrove samples fell within

sediment were reported, to be 6.40 and 6.62 respectively
(Environmatics, 1995). In a study of Elechi creek, close

(Obire et al., 2005). The occurrence of bacterial

CFU/ml. Generally, there were more bacteria, fungi or

counts obtained in the mangrove swamp ecosystem are

the range reported by Austin (1988) in similar

organisms. Although these counts were lower than those
4.87 ( 0.71) x 10 to 2.25 ( 0.19) x 10 CFU/g or
actinomycetes in the mangrove and freshwater samples

lower than the counts reported for Elechi creek

matter in the sediments. The bacterial counts of the
(Obire et al., 2003). The pH of brackish water bodies

3.06 ( 0.51) x 104 CFU/g or CFU/ml in the mangrove

This is attributed to nutrient accumulation, precipitation

actinomycetes in the freshwater samples than in the

harboured more bacteria than water. Ravikumar (1995)

to the Eagle Island, a pH range of 6.3 to 7.7 was reported
rhizosphere soil. The mangrove swamp samples were

The pH of brackish New Calabar River water and

fro m

populations followed a particular pattern. Among the

samples, the rhizosphere soil had the highest number of
not unusual for environments like mangrove swamps

stated by Imevbore (1983) ranged from 6.5 to 7.4.

Table 2: The Microbial Population of the mangrove and freshwater ecosystems

Sample
Table 2: The microbial population of the mangrove and freshwater ecosystems
Counts (Cfu/ml or Cfu/g)2
Sample Bacteria Counts(Cfu/ml or Cfu/g)a Fungi Algae Actinomycetes
Bacteria Fungi Actinomycetes
Heterotrophic Nitrifying Moulds Yeasts Algae
Heterotrophic Nitrifying
NN2-fixing
-fixing Moulds Yeasts
2

Mangrove Ecosystem

Rhizosphere soil (RS)b 3.06 0.51 x 104 7.82 2.07 x 103 1.74 0.14 x 104 5.3 1.5 x 102 1.2 0.16 x 104 5.93 2.25 x 103 1.60 0.32 x 104
Non-rhizosphere soil (NR)b 2.42 0.11 x 104 6.06 2.95 x 103 1.12 0.13 x 104 2.0 1.0 x 102 7.5 2.13 x 103 4.8 3.21 x 103 1.09 0.30 x 104

Sediment 2.99 0.09 x 104 7.51 1.58 x 103 1.31 0.20 x 104 1.6 0.58 x 102 1.2 0.13 x 104 3.26 1.02 x 103 4.56 2.60 x 104
4 3 4 2 3 3
Water 2.98 0.60 x 10 6.10 1.31 x 10 1.59 0.18 x 10 4.0 2.6 x 10 5.0 0.33 x 10 3.06 1.40 x 10 NIL

Freshwater Ecosystem

Rhizosphere soil (RS)c 2.25 0.19 x 106 4.97 0.50 x 104 1.29 0.16 x 105 2.73 0.9 x 104 2.77 0.40 x 105 4.13 1.45 x 104 4.00 2.04 x 105

Non-rhizosphere soil (NR)c 1.13 0.14 x 106 3.73 0.15 x 104 1.17 0.16 x 105 1.97 0.32 x 104 1.67 0.66 x 105 2.37 2.28 x 104 2.13 0.55 x 105
Sediment 8.40 2.07 x 105 4.73 1.06 x 104 1.36 0.13 x 105 1.20 0.17 x 104 0.60 0.98 x 104 2.77 0.21 x 104 5.60 2.08 x 105

Water 4.87 0.71 x 105 4.00 0.60 x 104 1.45 0.07 x 105 1.37 0.3 x 104 1.37 0.30 x 104 3.23 0.21 x 104 NIL
a
Values are means of triplicates S.D
b
Rhizosphere effect (RS/NS) for: heterotrophic bacteria = 1.26; nitrifying bacteria = 1.64; N 2-fixing bacteria =1.55; moulds = 2.65; yeasts=1.60; algae =1.23; actinomycetes = 1.47
aValues are means of triplicates S.D
c
Rhizosphere effect (RS/NS) for: heterotrophic bacteria = 1.99; nitrifying bacteria = 1.33; N 2-fixing bacteria =1.10; moulds = 1.38; yeasts=1.66; algae =1.74; actinomycetes = 1.88
b Rhizosphere effect (RS/NS) for heterotrophic bacteria = 1.26; nitrifying bacteria = 1.64 N2- fixing bacteria = 1.55; moulds = 2.65; yeasts = 1.60; algae = 1.23 ; actinomycetes = 1.47
C Rhizosphere effect (RS/NS) for heterotrophic bacteria = 1.99; nitrifying bacteria = 1.33 N2- fixing bacteria = 1.10; moulds = 1.38; yeasts = 1.66; algae = 1.74 ;

Journal of Research in Biology (2012) 2(6): 602-616

actinomycetes = 1.88
Okpokwasili et al.,2012
Okpokwasili et al.,2012

soil is similar to the counts reported for mangrove soil of occurrence of both Gram negative and Gram positive
Suva, Fiji Islands (Kumar et al., 2007). The occurrence bacteria. Futhermore, the results implicated Serratia,
of higher bacterial count in the rhizosphere soils could be Proteus, Aeromonas and Citrobacter species as
attributed to edaphic changes induced by plants, which indigenous organisms of mangrove swamps since they
could influence the proliferation of certain groups of did not occur in the freshwater swamps. However, the
bacteria. In mangroves, root exudates fuel the microbial occurrence of the same organisms in both environments
community in sediments (Alongi et al., 1993; could be attributed to the fact that the Eagle Island
Nedwell et al., 1994). Also mangrove trees can supply mangrove swamp is diurnally flooded by freshwater. In a
oxygen to the anaerobic subsoil through their aerial roots bacteriological water quality assessment of Elechi creek,
and thus remedy the detrimental effects of hydrogen Citrobacter freundii, Corynebacterium jeikeium,
sulphide in the soil (Sherman et al., 1998; Thibodeau and Escherichia coli, Enterobacter aero genes,
Nickerson, 1986). In terrestrial environments, rhizoplane Flavobacterium balustinum, Proteus mirabilis,
bacteria induce root exudation, which stimulates Staphylococcus aureus and Enterococcus faecalis were
microbial activity by providing bacteria with nutrients isolated. Other bacteria isolated include Pseudomonas,
(Lynch and Whipps, 1990). Aeromonas, Bacillus, Klebsiella, Micrococcus,
In this work, soils, sediments and water samples Aeromonas and Vibrio species (Obire et al., 2005).
collected from mangrove and freshwater swamps were Benka-Coker and Olumagin (1995) have isolated drilling
analyzed to determine the types of microorganisms that fluid-ut ilising Staphylococcus, Acinetobacter,
inhabit them, their abundance and distribution. Alcaligenes, Serratia, Clostridium, Enterobacter,
The freshwater samples served as controls. The data Nocardia, Bacillus, Micrococcus and Pseudomonas
obtained indicate that a wide range of microorganisms species from mangrove swamp in Niger delta area of
proliferate in the various areas of the swamps in varying Niger ia. The hydro car bo no clast ic bact er ia
proportions. The ten bacterial genera isolated from the S t a p h y l o co c cu s a u r eu s, Bacillus c er eu s,
mangrove swamp belonged to six different families and Flavobacterium breve, Pseudomonas aeruginosa,
they all occurred in the soil. Out of the ten genera, all of Erwinia amylovora, Escherichia coli, Enterobacter sp.,
which occurred in the rhizosphere soil, four belonged to Desu l f o vi b ri o sp . , A ci n et o b a ct e r i wo f f i i ,
the Enterobacteraceae, two to Nitrobacteraceae and one Chromobacterium violaceum, Micrococcus sedentarius,
each to the Vibrionaceae, Pseudomonadaceae, Corynebacterium sp., and Pseudomonas putrefaciens
Azotobacteraceae and Bacilaceae. In the non-rhizosphere were isolated from Iko river mangrove ecosystem,
soil, the same organisms found in the rhizosphere soil Nigeria (Udotong et al., 2008). Characterization of
also occurred except for Azotobacter and Bacillus bacterial isolates from Suva mangrove soil revealed
species. The members of the Enterobacteraceae, Bacillus as the dominant genera. Other genera such as
Pseudomonadaceae and Nitrobacteraceae occurred in the Micrococcus, Listeria and Vibrio were also encountered
sediment while only genera from Enterobacteraceae and in soil samples of the Suva mangrove ecosystem
Bacillaceae families were isolated from the water (Kumar et al., 2007). In Pichavaram mangrove, southeast
samples. From the results presented in Table 3, India, common bacterial genera are Vibrio, Bacillus,
Gram negative rods are the dominant organisms in M icrococcus, Pseudomonas, A e r o m o n a s,
mangrove swamps whereas in their freshwater Flavobacterium etc. (Sathiyamurthy et al., 1990).
counterpart, there appears to be a balance in the Escherichia coli, Micrococcus and Bacillus species have

Journal of Research in Biology (2012) 2(6): 602-616 606

 Okpokwasili et al.,2012

Table 3: Actinomycetes and bacterial population of mangrove swamp and freshwater ecosystems
Occurrence
Bacteria/ Actinomycetesa Soil
Rhizosphere Non rhizosphere Sediment Water
Mangrove Ecosystem
Bacteria
Proteus sp. + + + +
Bacillus sp. + + +
Pseudomonas sp. + +
Serratia sp. + + +
Escherichia coli + + + +
Azotobacter sp. +
Nitrobacter sp. + +
Nitrosomonas sp. + + +
Aeromonas sp. + +
Citrobacter sp. + + +
Actinomycetes
Actinomyces sp. + + +
Nocardia sp. + +
Streptomyces sp. + + +
Thermoactinomyces sp. +
Freshwater Ecosystem
Bacteria
Klebsiella sp. + + +
Clostridium sp. + + +
Corynebacterium sp. +
Pseudomonas sp. + +
Listeria sp. + + +
Bacteriodes sp. + + +
Nitrosomonas sp. + +
Nitrobacter sp. + + +
Agrobacterium sp. + +
Escherichia coli + +
Nitrosolobus sp. +
Arthrobacter sp. + + +
Bacillus sp. + + +
Azotobacter sp. +
Flavobacterium sp. + + +
Nitrospira sp. +
Lactobacillus sp. +
Actinomycetes
Streptomyces sp. + + +
Nocardia sp. + +
Actinoplanes sp. + +
Actinomyces sp. + + +
a
+, isolated; , not isolated
been isolated from body surfaces of a marine edible agents of early transformation of organic matter and
fish Harpodon nehereus of Mumbai coast, India regeneration of nutrients and also serve as food source
(Shingadia, 2011). for higher trophic level. By participating in various steps
Bacteria and other microorganisms densely and of the decomposition and mineralization of litter,
ubiquitously colonize freshwater and marine ecosytems. sediment microorganisms play essential roles in
In these environments, bacteria constitute the primary mangrove ecosystem and make an important contribution
607 Journal of Research in Biology (2012) 2(6): 602-015
Okpokwasili et al.,2012
to the productivity of the mangrove ecosystem
(Holguin et al., 2001).
Mangroves provide a unique ecological
environment for diverse bacterial communities. Bacteria
fill a number of niches and are fundamental to the
functioning of these habitats. They are particularly
important in controlling the chemical environment of the
mangal, for instance, nitrogen-fixing bacteria. In this
work, nitrogen-fixing bacteria including species of
Azotobacter and Nitrobacter were isolated from soils of
mangrove and freshwater ecosystems (Table 3). Other
bacterial isolates of the mangrove and freshwater
samples capable of fixing atmospheric nitrogen include
Bacillus, Klebsiella and Clostridium
Nitrogen-fixing microorganisms can colonize both
terrestrial as well as marine environments (Sahoo and
Dhal, 2009). Nitrogen-fixing bacteria such as members
of the genera Azospirillum, Azotobacter, Rhizobium,
Clostridium and Klebsiella were isolated from the
sediments, rhizosphere and root surfaces of various
mangrove species (Sengupta and Chaudhuri, 1990;
1991). The possibility of atmospheric nitrogen fixation
by Pseudomonas stutzeri asso ciat ed
Languncularia racemosa have been reported (Krotzky
and Werner, 1987; Alongi et al., 1992; 1993).
Pseudomonas species are among the most widely
distributed bacteria. In this study, Pseudomonas species
was isolated from soils of both freshwater and mangrove
ecosystems. Another noteworthy bacterium is Bacillus
species, that is isolated from the soils, sediments and
riverwaters of freshwater and mangrove
ecosystems. Bacillus species exhibit phosphatase
activity, capable of solubilizing phosphate. In an arid
Mexican mangrove ecosystem, bacterial strains including
Bacillus amyloliquefaciens and Bacillus licheniformis
were isolated from black and white mangroves
(Vazquez et al., 2000).
Species of actinomycetes belonging to the
fa m i l ie s Act ino m yc et ace ae, No car d i ace ae,
Journal of Research in Biology (2012) 2(6): 602-616
Streptomycetaceae, Micromonosporaceae and the
coryneform group of bacteria were isolated only from
soil and sediment samples of the mangrove and
freshwater swamps. No species of actinomycetes was
obtained from the water samples. The occurrence of
actinomycetes in soils and sediments has been reported
by Takizawa et al., (1993). The freshwater soils habour
more actinomycetes than mangrove sediment. This
corroborated the report of Goodfellow and Williams
(1983), that the actinomycetes population density is less
common in marine sediments relative to terrestrial soils.
The isolation of a member of the Micromonosporaceae
and Streptomycetaceae in this work agrees with the
species. reports of Watson and Williams (1974), that
Micromonosporaceae constitute the dominant
actinomycetes in beach sand and Jensen et al., (1991)
that Streptomycetes are the predominant species at
shallow depths in near shore tropical marine
environments. Actinomycetes belonging to the family
Streptomycetaceae was also found to dominate in the
environmental samples from the sediments at coastal and
offshore area of Nagasaki, Japan (Anzai et al., 2008).
wit h Micromonosporaceae have been found to be the
dominant actinomycetes group in a range of aquatic
environments, particularly in the deeper mud layers as
well as in deep sea sediments (Mincer et al., 2002).
Thus, Thermoactinomyces and Streptomyces are by this
work proffered as autochthonous members of the
mangrove swamp of the Eagle Island. The population of
actinomycetes obtained from the sediments and soils in
swamp this work is slightly higher than those recorded by
Weyland (1969) and Okami and Okazaki (1978) in
coastal sediments but agrees with the figures of
Takizawa et al., (1993). Similarly, there are reports of
the occurrence of actinomycetes in terrestrial soils
(Nolan and Cross, 1988), thus buttressing their isolation
in mangrove soils in this work. One interesting aspect of
the result presented in Tables 2 and 3 is the absence of
actinomycetes in the water samples. This might suggest
608
 Okpokwasili et al.,2012

Table 4: Fungal population of mangrove swamp and freshwater ecosystems

Occurrence
Fungia Soil
Rhizosphere Non rhizosphere Sediment Water
Mangrove Ecosystem
Moulds
Aspergillus niger + + + +
Aspergillus flavus + +
Aspergillus ochraceus + + +
Rhizopus stolonifer + + +
Mucor hiemalis + + + +
Fusarium verticilloides + + + +
Fusarium moniliforme + + + +
Fusarium oxysporum + + +
Botyritis cinerea + +
Trichoderma viride + +
Verticillium lecanii + + +
Penicillium caseicolum + + +
Penicillium brevicompactum + + +
Penicillium digitatum + + + +
Geotrichum candidum + + + +
Sterilia mycelia +
Yeasts
Candida sp. + + + +
Pichia sp. + + +
Saccharomyces sp. + + + +
Kluveromyces sp. + + +
Trigonopsis sp. + + + +
Rhodospirillum sp. + + + +
Freshwater Ecosystem
Moulds
Aspergillus niger + + + +
Aspergillus flavus + + +
Aspergillus ochraceus +
Penicillium brevicompactum + +
Penicillium expansum + + + +
Penicillium digitatum + -
Trichoderma harzianum + + +
Monascus rubber + +
Verticillium trifidum + + +
Byssochlamys nivea + + +
Chalaropsis sp. + +
Phialophora hoffmanii +
Trichothecium roseum + +
Fusarium oxysporum + + + +
Fusarium solani +
Aureobasidium pullulans + +
Bdellospora helicoides +
Chrysonilia sitophila +
Yeasts
Candida sp. + + + +
Saccharomyces sp. +
Schizosaccharomyces sp. + +
Saccharomycopsis sp. +
Yarrowia sp. +
Kluveromyces sp. + + +
Brettanomyces sp. + +
Zygosaccharomyces sp. +
a
+, isolated; , not isolated

609 Journal of Research in Biology (2012) 2(6):602-616

Okpokwasili et al.,2012
that they are unable to survive in brackish water habitats
such as those found in the mangrove swamps of the
Niger Delta. While high counts of bacteria and
actinomycetes were expected in environments such as
this, the relatively low counts (excepts for the sediments)
obtained could be attributed to the harsh conditions
presented by regular mixing of fresh and seawater which
ultimately create an environment with a wide range of
continually fluctuating conditions which may be
unfavourable for bacterial colonization. Furthermore, the
higher actinomycete population is expected, since it is
known that actinomycetes do better than most bacteria in
such stressed environments (Kobayashi and Rittman,
1982).
The result presented in Table 4 shows that from
the mangrove swamp, 16 species of fungi belonging to
several groups especially the ascomycetes were isolated.
More species were isolated from the freshwater swamp.
This agrees with the report that fungi are poorly
represented in marine environments, since the marine
fungi account for only 5% of the total fungal flora
(Purushothaman and Jayalakshmi, http://ocw.unu.edu).
The lower counts of fungi (Table 2) in mangrove than in
freshwater ecosystem also buttressed this fact. In this
work, the most frequently encountered fungi were
Aspergillus and Penicillium species, which occurred on
virtually all the areas sampled. The predominance of
Aspergillus and Penicillium in Pichavaram mangrove,
southeast India has been reported (Mohamed- Salique et
al., 1985; Venkatesan and Natarajan, 1986). Fungi
identified as Alternaria maritima, Aspergillus flavus,
Aspergillus n i g e r, Aspergillus sulfurus,
Aureobasidium pullulans, Bispora sp., Cladosporium sp.,
Humicola sp., Mucor sp., Penicillium sp., Phoma sp.,
Pythium sp. and Rhizopius sp. were isolated from black
mangrove (Avicennia marina) growing in Korangi creek
and Clifton areas of Karachi, Pakistan (Mehdi and
Saifullah, 1992). This report shows the occurrence of
Aspergillus, Trichoderma, Mucor, Fusarium,
Journal of Research in Biology (2012) 2(6): 602-616
Verticillium, Botrytis and Penicillium species as
inhabitants of mangrove swamps. The rhizosphere soil
had more organisms than any other area. Other
prominent group isolated in this work was oomycetes.
Similar results by Fell et al., (1980) suggest that fungi
particularly the oomycetes play a substantial role in the
breakdown of mangrove litter. Total heterotrophic mould
counts (Table 2) revealed higher values for the
freshwater than the mangrove swamp. However, the
counts in mangrove swamp agreed with those reported
by Odokuma and Okpokwasili (1993) in a Niger Delta
brackish water ecosystem.
Species of yeasts belonging to the Ascomycotina,
Basidiomycotina and Deuteromycotina were isolated
from the mangrove swamp in this study. These yeasts
were found in the soil, sediment and water samples.
However, there were greater diversity of yeast in soil
samples than in the sediment and water samples from
mangrove and freshwater ecosystems. The results
(Table 2) also showed that the rhizosphere soil harboured
more yeasts than the other areas. The presence of
nutrients from root exudates may have accounted for
higher yeast load in the rhizosphere. It appears that
Rhodospiridium, Trigonopsis and Pichia species are
natural inhabitants of mangrove swamps. Candida and
Kluveromyces species were widely distributed in the
mangrove and freshwater ecosystems. In a Brazilian
mangrove ecosystem, Kluyveromyces aestuarii
predominated the ascomycetous yeast communities of
detritus feeding crabs (Araujo et al., 1995).
In the algal composition of the mangrove swamp,
20 species were isolated of which the Cyanophyceae had
higher number of taxa, followed by Bacillarophyceae,
Chlorophyceae, Chrysophyceae, Euglenophyceae,
P haeo p h yceae, Cho lo ro co ccaceae and t he
Xanthophyceae. Species belonging to the above families
were also isolated from the freshwater habitat. This result
is similar to the results of Chindah (1988) and Chindah
and Pudo (1991) on the algal composition found in the
610
 Okpokwasili et al.,2012

Table 5: Algal population of mangrove swamp and freshwater ecosystems

Occurrence
Algaea Soil
Rhizosphere Non rhizosphere Sediment Water
Mangrove Ecosystem
Chlamydomonas chrenbergi + + +
Nostoc sp. + +
Protococcus viridis + + +
Euglena gracilis +
Euglena acus +
Borgea plantonica +
Synura uvella +
Oscillatoria tenium +
Spirogyra adnata +
Spirulina maior +
Laminaria sp. +
Denticula valida + +
Gleocapsa quarternata +
Fischerella sp. + +
Chlorobotrys regularis +
Gomphonema olivaceum +
Asterionella gracillima + +
Asterionella formosa +
Vaucheria sessilis +
Chromulina nebulosa +
Freshwater Ecosystem
Oscillatoria redeki +
Chromulina zartensis + +
Synedra ulna + +
Ochromonas mutabilis +
Ankistrodesmus septatus +
Chlamydomonas angulosa + +
Chlamydomonas elegans +
Chlamydomonas grandis +
Chlamydomonas pertusa +
Spirogyra mirabilis +
Pinularia viridis + + +
Cosmasium birretum + +
Euglena acus + + +
Amphora ovalis + +
Chlorella variegata +
Urothrix oscillatoria + + +
Enteromorpha compressa + +
Stuarastrum tumidum + + +
Scenedesmus acuminatus +
Cymbella cistula + +
Ovulites margaritula + +
Coscinodiscus excentricus +
Denticula valida +
Oscillatoria redeki + +
Navicula pelliculosa +
Euastrum didelta +
Botrydiopsis arrhiza +
Chloromeson agile +
a
+, isolated; , not isolated
611 Journal of Research in Biology (2012) 2(6): 602-616
Okpokwasili et al.,2012

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