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Optimization and commercial production of biosurfactant from Pseudomonas aeruginosa
PAO1 using renewable resources Deivakumari M., Sanjivkumar M. and Immanuel G.*
MNP laboratory, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam – 629502,
Kanykumari District, Tamilnadu, INDIA
Abstract Microbial based rhamnolipid biosurfactants are
potentially used in many commercial industries viz.
petroleum, pharmaceuticals, biomedical and food
processing. In this study, the biosurfactant producing
bacterial strain Pseudomonas aeruginosa PAO1 was
isolated from oil contaminated areas in the fishing
harbor of Chinnamuttom, Southeast coast of
Tamilnadu, S. India. Biosurfactant production from the
isolated strain was carried out using Bushnell Hass
broth with 2% glucose as carbon source. The produced
biosurfactant was confirmed as rhamnolipid by blue
agar plate assay and it was quantified by means of
Orcinol assay. The rhamnolipid production from the
candidate strain was enhanced by using various
parameters like pH, temperature, incubation time,
inoculum size, carbon, nitrogen and hydrocarbon
sources and NaCl concentrations.
The result revealed that the strain displayed maximum
biosurfactant production at the optimized medium
condition of pH 7, temperature 30°C, incubation time
of 168 h with the inoculum size of 4%. The production
medium substituted with 4% mannitol as carbon
source, 1.5% beef extract as nitrogen source, 4% olive
oil as hydrocarbon source and 1.5 % NaCl
concentration recorded higher rhamnolipid
production. Further the rhamnolipid production was
also enhanced by using various inexpensive renewable
substrates and the result revealed that the strain
exhibited (6.04g/l) maximum biosurfactant production
in the medium supplemented with peanut oil cake as the
substrate. For maximum biosurfactant recovery, seven
different extraction methods were carried out and the
result revealed that the maximum (6.84 g/l) amount of
biosurfactant was recovered by acid precipitation and
solvent extraction method.
Keywords: Pseudomonas aeruginosa PAO 1, Rhamnolipid,
Optimization, Renewable resources.
Introduction Biosurfactants are amphiphilic and surface-active molecules
produced by a wide variety of bacteria, yeast and
filamentous fungi which either adhere to cell surface or
excreted extra cellularly in the growth medium5. Research in
the area of biosurfactants has increased widely due to its
applications in different industrial level like enhanced oil
recovery, hydrocarbon bioremediation, agriculture,
cosmetics, pharmaceutical, detergents, personal care
products, food processing, metal treatment and processing,
pulp and paper producing and paint industries,
environmental protection, crude oil recovery, food
processing industries etc.30
Almost all surfactants have been usually derived from
petroleum sources, however, these synthetic surfactants are
potential source of pollution and toxic to the environment.
Therefore, in the recent years, much interest and attention
have been directed towards biosurfactants over chemically
synthesized surfactants due to their ecological acceptance
owing to their low toxicity, biodegradable nature and
effectiveness at extreme temperature, pH, salinity and ease
of synthesis29.
Rhamnolipid is one of the glycolipid type biosurfactants
which could be produced by different bacteria.
Pseudomonas sp. is well known for its ability to produce
rhamnolipid biosurfactants with potential surface-active
properties when grown on different carbon substrates and
rhamnolipid biosurfactants produced by these species have
greater potential for industrial application and
bioremediation51.
Even though interest in biosurfactants production is
increasing due to their application, synthesis of these
compounds does not compete economically with synthetic
surfactants because of their higher production cost. To
reduce the production cost, different routes could be
investigated with respect to the increase of yield and product
accumulation27. Two basic strategies are generally adopted
worldwide to overcome the expensive cost constraints
associated with biosurfactant production:
(i) the use of inexpensive and waste substrates for the
formulation of fermentation media which would lower the
initial raw material costs involved in the process;
(ii) development of efficient and successfully optimized
bioprocesses including optimization of the culture
conditions of microbes and cost-effective recovery
processes for maximum biosurfactant production42.
Considering the importance of the above, the present study
was undertaken to enhance rhamnolipid production from
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Pseudomonas aeruginosa PAO1 through media
optimization technique and also by using various renewable
resources. Effect of different extraction methods on
maximum biosurfactant recovery was also evaluated further
in this study.
Material and Methods Microorganism and biosurfactant production: The
biosurfactant producing bacterial strain Pseudomonas aeruginosa PAO1 (Accession no. KM978038) was isolated
from the oil contaminated sediment soil samples collected
from a fishing harbor of Chinnamuttom, Southeast coast of
Tamilnadu, India. For biosurfactant production, 2% of seed
culture was inoculated in 250ml Erlenmeyer flask containing
100ml BH broth supplemented with 2% glucose and 1%
NaCl (pH 7.2). The inoculated medium was incubated at
35°C in a shaking incubator for a week. Afterwards, the
culture broth was centrifuged at 17226xg for 20min at 4°C,
subsequently the cell free supernatant was subjected to
screen the biosurfactant production by using oil
displacement method and emulsification index (E24%) assay.
The produced biosurfactant was further confirmed as
rhamnolipid by means of blue agar plate technique46.
Quantification of biosurfactant (Orcinol assay): The
orcinol assay was employed to determine the amount of
glycolipid accumulation in the sample. In this assay, 100μl
of cell free supernatant was added with 900μl of a solution
containing 0.19% orcinol (in 53% H2SO4). After heating for
30 min at 80ºC, the sample was kept for 15 min at room
temperature and the absorbance was measured at 421nm
using a U.V. spectrophotometer (Techcomb, 8500). The
biosurfactant concentration was calculated from a standard
curve prepared with L-rhamnose and expressed as rhamnose
equivalents45. Concentration of rhamnolipid was calculated
based on the assumption that 1μg of rhamnose corresponded
approximately to 2.5μg of rhamnolipid53.
Enhancement of rhamnolipid production: To optimize
rhamnolipid production by the candidate bacterial strain P.
aeruginosa PAO1, environmental parameters such as pH (4,
5, 6, 7, 8, 9, 10, 11 and 12 ), temperature (30, 35, 40, 45 and
50°C), inoculum size (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5
and 5.0%), incubation time (1 to 10 days), carbon sources
(glucose, fructose, sucrose, starch, lactose, cellulose,
mannitol, xylose, galactose and dextrose), nitrogen sources
(yeast extract, beef extract, urea, ammonium nitrate,
ammonium sulphate, peptone and glutamic acid),
hydrocarbon sources (palm oil, olive oil, coconut oil,
glycerol, kerosene, diesel, petrol, sunflower oil and crude
oil) and different NaCl concentrations (0.5, 1.0, 1.5, 2.0, 2.5
and 3%) were studied using 50ml sterile BH broth
supplemented with 2% glucose, taken in 250ml conical
flasks; in each flask 2% bacterial inoculum was added.
All the flasks were incubated in a shaking incubator at
180rpm for 7 days at room temperature. After incubation, the
growth of the organism in the individual flasks was
measured by means of biomass production and for
determination of biosurfactant production, the individual
cell free supernatant was subjected to carry out the standard
assays such as E24% index, oil displacement assay and
Orcinol assay.
Determination of biomass production: To determine the
dry cell biomass, the culture broth of P. aeruginosa PAO1
was centrifuged at 7656xg for 20min. The cell pellet was
washed thoroughly with n-hexane to remove any slimy
materials attached on the cell surface that might cause error
in the assessment. The washed cells were resuspended in
sterilized distilled water and centrifuged again. Then the
pellet was oven-dried at 105°C for 4 h and weighed37.
Oil displacement test: The oil displacement test is an
indicative for the surface and wetting activities of the
biosurfactants. This technique was done according to the
modified method of Rodrigues et al40. In this method, 1ml of
crude oil was added on the surface of 50 ml distilled water
in a Petri dish (15cm diameter) followed by 20µl of culture
supernatant was gently placed on the center of the oil layer,
oil displacement was formed out within 30 seconds. The
diameter of the zone of displacement in the oil was measured
and it evidenced the presence of biosurfactant.
Determination of emulsification index (E24 %):
Emulsification index was determined by the method of Patel
and Desai35. 2ml of diesel was added to a screw cap test tube
containing 2ml of culture supernatant and vortex mixed for
2min. The reaction mixture was allowed to stand for 24h at
room temperature. The E24 index was calculated by using the
following formula:
Height of emulsion
E24 (%) index = ×100
Total height
where height of emulsion = height of emulsified layer and
total height = total height of the liquid column.
Production of rhamnolipid using renewable resources:
Effect of various solid renewable agro industrial wastes on
rhamnolipid production by P. aeruginosa PAO1was studied.
Different renewable resources such as coconut oil cake,
gingili oil cake, rubber seed cake, castor seed cake, neem
seed cake, peanut oil cake, orange peel, rice bran, rice straw
and sugar cane baggase were tested individually for
biosurfactant production. These substrates were individually
ground well in dry form to increase the exposed surface area
for the microbial activity amended in mineral salt medium
(BH broth) as sole carbon and nitrogen sources.
Simultaneously a control setup was kept without carbon and
nitrogen sources, however inoculated with the test organism
according to the modified method of Tahzibi et al51.
Further, the individual flasks inoculated with the test
organism were incubated in a rotary shaker at 180rpm for 9
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days. Once in 2 days interval of incubation, the cell-free
supernatant were collected from the individual flask by
centrifuging the culture broth at 11963xg for 20min. Then
the cell free supernatants were subjected to determine the
biosurfactant production through emulsification index and
Orcinol assays.
Standardization of extraction methods to maximum
recovery of biosurfactant: The optimized parameters were
subjected for mass production of biosurfactant by the
selected organism P. aeruginosa PAO1. After incubation,
the culture broth was centrifuged at 17226xg in 4°C for
10min; subsequently the cell free supernatant was subjected
for the extraction of biosurfactant. For standardization of
extraction method to maximum biosurfactant recovery,
different extraction methods were carried out. The
biosurfactant extracted by following individual methods was
quantified by means of dry weight. The dry weight of the
biosurfactant was calculated by the following formula:
Dry weight of the biosurfactant (g/l) = weight of plate
with biosurfactant after drying - weight of empty plate
Chloroform/ Methanol/ Butanol extraction method: In
this method, the cell free supernatant was extracted using a
combination of solvents mixture with methanol/chloroform/
1-Butanol at 1:1:1 ratio. The mixture was continuously
shaken at 200rpm in 30ºC for 5h23. After 5 h, 2 layers of
precipitation were obtained.
The upper layer was discarded and the lower layer was
poured on to a clean glass Petri dish. The Petri dish with
biosurfactant was put inside the fume hood until fully dried
to get a brown- colored powder. Further the extracted crude
biosurfactant was quantified by means of dry weight.
Ethyl acetate extraction method: In this method, the cell
free supernatant was extracted using equal volume of ethyl
acetate28. The extracted solvent was kept overnight in a
rotary evaporator. After evaporation of the solvent, the dry
weight of the extracted crude biosurfactant was determined.
Methanol/Chloroform extraction method: In this method,
the cell free supernatant containing biosurfactant was
extracted with chloroform/methanol at the ratio of 2:122. The
extracted solvent was kept overnight in a rotary evaporator.
After evaporation of the solvent, the dry weight of the
extracted crude biosurfactant was determined.
Diethyl ether extraction method: In this method, the cell
free supernatant containing biosurfactant was extracted with
an equal volume of diethyl ether. The resulting solution was
then poured into a separating funnel. After vortex mixing,
the solution was kept stable for a while. Then, the top water
layer was removed and the emulsion layer was collected in
a sterile glass Petri dish. Afterwards, it was dried in an
incubator at the temperature of 40-45◦C. Finally, the dry
weight of the obtained biosurfactant was determined1.
Chilled acetone precipitation method: In cold acetone
precipitation method, one volume of cell free supernatant
was mixed with 3 volume of ice-cold acetone (1:3 ratio) to
precipitate biosurfactant which was further suspended in
phosphate buffer. Then the mixture was incubated at 4°C for
15–20h to get the precipitate of biosurfactant. The
precipitate was collected by centrifugation and evaporated to
dryness to remove residual acetone41. The biosurfactant
recovery rate was determined using dry weight of the
extracted product.
Ammonium sulphate precipitation method: In this
method, the cell free supernatant containing biosurfactant
was precipitated with 40% (w/v) ammonium sulphate and
incubated overnight at 4°C. The precipitate was then
collected by centrifugation at 10,000 xg for 10 min at 4°C31.
Further, the precipitate was dried and the recovery rate was
determined using dry weight of the extracted product.
Acid precipitation method: In this method, the supernatant
containing biosurfactant was acidified with 6N HCl until it
reached to pH 2.0 and then the mixture was incubated
overnight at 4°C. Then it was centrifuged at 26916xg for
20min and the precipitate was collected and re dissolved
using Milli-Q water (pH 7.0). Further it was lyophilized and
extracted with chloroform and methanol (2:1). The extracted
honey colored biosurfactant was considered as partially
purified biosurfactant and it was dried to the weight
consistent (w/v). The biosurfactant recovery rate was
determined using dry weight of the extracted product16.
The extracted biosurfactants through the above individual
methods were evaluated with fluorescence microscope (100
xs) and photographed.
Statistical analysis: The data obtained in the present study
were expressed as Mean ± SD and were analyzed using One-
way ANOVA test and subsequently conducted post hoc
multiple comparison with SNK test at 5 % level of
significance using computer software STATISTICA 6.0
(Statosoft, Bedford, UK).
Results Rhamnolipid production (Orcinol assay): The
biosurfactant produced by the candidate strain P. aeruginosa
PAO1 was quantified through Orcinol assay, it revealed that
the strain utilized the substrate glucose and produced 2.58 ±
0.02 g/l of biosurfactant on 7th day of incubation period.
Optimization of biosurfactant production by P.
aeruginosa PAO1
Effect of different pH on biosurfactant production: The
effects of various pH (4 to12) on the growth of candidate
strain P. aeruginosa PAO1 and subsequent biosurfactant
production are presented in table 1. The result indicated that
in acidic pH, the growth and biosurfactant production of the
candidate strain were very low i.e. at pH 4, the biomass
production, E24% index, oil displacement activity and
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biosurfactant weight were observed as 0.78 ± 0.06g/l, 54.2 ±
2.05%, 0.00 ± 0.00mm and 1.34 ± 0.13g/l respectively.
When the pH increased, the growth and biosurfactant
production were simultaneously increased and reached the
maximum at pH 7 and 8.
Here the maximum biosurfactant production (3.32 ± 0.22
g/l) and E24% index (85.60 ± 2.81%) were achieved at pH
7.0 and maximum oil displacement activity (81.30 ±
2.86mm) and the biomass production (2.01 ± 0.20g/l) were
achieved at pH 8. Further increase in medium pH, growth
and biosurfactant production gradually decreased i.e. at pH
10, the biomass production, E24% index, oil displacement
activity and biosurfactant production were recorded as 0.85
± 0.09 g/l, 72.0 ± 2.16%, 72.0 ± 2.81mm and 1.42 ± 0.01
respectively.
Effect of temperature on biosurfactant production: The
results on the effect of different temperature (25 to 50°C) on
growth of the test strain P. aeruginosa PAO1 and
biosurfactant production are represented in table 2. The
result revealed that at lower temperature, the growth and
biosurfactant production of the candidate strain were very
low i.e. at 25°C, the biomass production, E24% index, oil
displacement activity and biosurfactant weight were
observed as 0.17 ± 0.001g/l, 23.0 ± 1.70%, 22.0 ± 1.01mm
and 1.24 ± 0.001 g/l respectively.
When the incubation temperature increased, the growth of
the organism and the biosurfactant production were
simultaneously increased and attained maximum at 30°C i.e.
at this temperature, the biomass production, E24% index, oil
displacement activity and biosurfactant weight were found
to be 0.25 ± 0.006 g/l, 79.0 ± 2.58%, 70.0 ± 2.01mm and
3.47 ± 0.04 g/l respectively. Further increase in incubation
temperature, growth and biosurfactant production were
gradually decreased.
Effect of incubation period on biosurfactant production:
The results on the effect of incubation period (24 to 240 h)
on growth of P. aeruginosa PAO1 and biosurfactant
production are given in table 3. Here at the beginning of
incubation period, the growth and biosurfactant production
of the candidate strain were very low i.e. at 24h, the biomass
production, E24% index, oil displacement activity and
biosurfactant weight were observed as 0.72 ± 0.01 g/l, 52.33
± 2.40%, 0.00 ± 0.00mm and 0.24 ± 0.008 g/l respectively.
Whereas when the incubation period increased, the growth
and biosurfactant production were simultaneously increased
and reached the maximum at 168 and 192 h. Here the
maximum E24% index (88.5 ± 2.67%) and the biosurfactant
production (2.80 ± 0.20g/l) were observed at 168h and
maximum biomass production (2.58 ± 0.36g/l) and oil
displacement activity (84.34 ± 2.82mm) were achieved at
192h.
Further increase in incubation period resulted in decreasing
level of growth and biosurfactant production i.e. at 240 h, the
biomass production, E24% index, oil displacement activity
and biosurfactant weight were observed as 1.60 ± 0.38g/l,
13.60 ± 0.76%, 61.60 ± 2.24 mm and 0.96 ± 0.05g/l
respectively.
Effect of inoculum size on biosurfactant production: The
effects of different inoculum size (0.5 to 5%) on the growth
of the test organism and biosurfactant production were
determined (Table 4). The result revealed that at the lowest
inoculum size of 0.5%, the growth of the test organism and
simultaneously the biosurfactant production were very low
i.e. at 0.5% inoculum size, the biomass production was
determined as 0.17 ± 0.02g/l, E24% index was nil whereas
the oil displacement activity and biosurfactant weight were
observed as 22.30 ± 1.05mm and 0.46 ± 0.02g/l respectively.
However, when the inoculum size increased, the growth and
biosurfactant production also correspondingly increased and
reached the maximum at 4%. Here, the biomass
concentration, E24% index, oil displacement activity and
biosurfactant weight were found to be 1.24 ± 0.09g/l, 70.66
± 2.58%, 85.00 ± 3.26mm and 3.77 ± 0.62 g/l respectively.
Further increase in inoculum size, the growth and
biosurfactant production were gradually decreased.
Effect of carbon sources on biosurfactant production:
The results on the influence of various carbon sources on
growth of P. aeruginosa PAO1 and biosurfactant production
are given in table 5. Among the tested carbon sources, the
candidate strain displayed maximum biomass production
(2.74 ± 0.06g/l), E24% index (77.33 ± 2.18%), oil
displacement activity (78.66 ± 2.47mm) and biosurfactant
production (3.26 ± 0.08g/l) in mannitol substituted medium.
The strain displayed minimum biomass production (0.26 ±
0.003g/l), E24% index (17.2 ± 1.14%), oil displacement
activity (3.66 ± 0.04mm) and biosurfactant production (0.11
± 0.02g/l) in xylose substituted medium. Moreover, the
biosurfactant production was absolutely nil in lactose,
cellulose and galactose substituted media.
The result on the effect of different concentrations (0.5 to
5%) of mannitol on growth of P. aeruginosa PAO1 as well
as biosurfactant production is given in fig. 1a and b. Among
the tested mannitol concentrations, the candidate strain
produced maximum biomass production (3.74 ± 0.06g/l),
E24% index (86.12 ± 2.18%), oil displacement activity
(84.66 ± 2.47mm) and biosurfactant weight (4.25 ± 0.33 g/l)
in medium supplemented with 4% mannitol. Further on
increase in the concentration of mannitol, the growth and
biosurfactant production were decreased simultaneously, for
instance at 5% mannitol concentration, biomass production,
E24% index, oil displacement activity and biosurfactant
weight were found to be 2.70 ± 0.05g/l, 62.93 ± 2.09%, 83.0
± 2.63mm and 2.63 ± 0.05g/l respectively.
Effect of different nitrogen sources on biosurfactant
production: The effects of different nitrogen sources on
growth of the test organism P. aeruginosa (PAO1) and
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biosurfactant production are varied much (Table 6). Among
the tested nitrogen sources, the candidate strain exhibited
maximum biomass production (2.13 ± 0.06g/l), oil
displacement activity (68.00 ± 2.16 mm), E24% index (70.53
± 2.00%) and biosurfactant weight (2.92± 0.08g/l) in beef
extract substituted medium. The strain displayed minimum
biomass production of 0.54 ± 0.009g/l, E24% index of 20.20
± 0.92%, oil displacement activity of 18.00 ± 0.86mm and
biosurfactant weight of 0.11 ± 0.003g/l in NH4NO3
substituted medium. However, there was no biosurfactant
production observed in glutamic acid substituted medium.
The result on the influence of different concentrations (0.25
to 2%) of beef extract on growth of P. aeruginosa PAO1 and
biosurfactant production is given in fig. 2a and b. Here the
strain exhibited maximum biomass production of 2.78 ± 0.02
g/l, E24% index of 78.13 ± 2.86%, oil displacement activity
of 75.12 ± 2.81mm and biosurfactant weight of 3.25± 0.03
g/l in medium containing 1.5% beef extract. Further on
increase in concentration of beef extract, the growth and
biosurfactant production were decreased simultaneously i.e.
at 2% beef extract concentration, the biomass production,
E24% index, oil displacement activity and biosurfactant
weight were observed to be 1.75 ± 0.04g/l, 52.29 ± 1.62%,
59.66 ± 1.47mm and 1.05 ± 0.05g/l respectively.
Effect of different hydrocarbon sources on biosurfactant
production: There were nine different hydrocarbon sources
tested to determine the growth and production of
biosurfactant by the test strain P. aeruginosa PAO1 and the
results obtained are summarized in table 7. The candidate
strain could be able to utilize majority of the tested
hydrocarbons for its growth and production of biosurfactant.
Among the tested hydrocarbon sources, the candidate strain
exhibited maximum biomass production (2.83 ± 0.02g/l),
E24% index (80.60 ± 1.27%), oil displacement activity
(82.66 ± 2.12mm) and biosurfactant weight (4.32 ± 0.06g/l)
in olive oil substituted medium.
The strain displayed minimum biomass production of 0.16 ±
0.004g/l, E24% index of 18.30 ± 1.00%, oil displacement
activity of 1.00 ± 0.00mm and biosurfactant weight of 1.12
± 0.08g/l in kerosene substituted medium.
The results on the effect of different concentrations (0.5 to 5
%) of olive oil on growth of P. aeruginosa PAO1 and
biosurfactant production are given in fig. 3a and b. Among
the tested olive oil concentrations, the candidate strain
produced maximum biomass production of 3.74 ± 0.06g/l,
E24% index of 85.80 ± 2.89%, oil displacement activity of
86.6 ± 2.27mm and biosurfactant weight of 5.26 ± 0.21 g/l
in medium containing 4% olive oil. Further on increase in
concentration of olive oil, the growth and biosurfactant
production were decreased considerably. For instance, at 5%
olive oil concentration, biomass production, E24% index, oil
displacement activity and biosurfactant weight were found
to be 2.91 ± 0.01g/l, 69.20 ± 2.94%, 82.00 ± 2.16 mm and
2.44 ± 0.10 g/l respectively.
Effect of different NaCl concentrations on biosurfactant
production: The result on the effect of NaCl concentrations
(0.5 to 3%) on growth and biosurfactant production by P. aeruginosa (PAO1) is represented in table 8. Among the
tested NaCl concentrations, the candidate strain produced
maximum biomass (3.12 ± 0.02g/l), E24% index (71.0 ±
2.16%), oil displacement activity (72.33 ± 2.69mm) and
biosurfactant weight (3.15 ± 0.08g/l) in the medium
containing 1.5% NaCl. Further increase in NaCl
concentration, the growth and biosurfactant production were
decreased positively i.e. at 3% NaCl concentration, the
biomass production, E24% index, oil displacement activity
and biosurfactant weight were found to be 1.41 ± 0.005g/l,
13.83 ± 0.84%, 21.33 ± 1.05mm and 1.06 ± 0.001g/l
respectively.
Production of biosurfactant by P. aeruginosa PAO1
using renewable resources: The influence of different
renewable resources on individual factors related to
biosurfactant production by the candidate organism is
discussed here with (Fig. 4 to 6). The result indicated that
among the tested renewable resources, the strain exhibited
maximum oil displacement activity of 84.33 ± 2.27mm,
E24% index of 79.66 ± 1.14% and biosurfactant production
of 6.04 ± 0.02g/l in peanut oil cake substituted medium. At
the beginning of 24h of incubation, the biosurfactant
production and the oil displacement activity were observed
as low in all the tested renewable resources.
Here, the oil displacement activity was observed to be from
0.00 to 44.3 ± 2.08g/l, the E24% index was recorded from
0.00 to 26.22 ± 1.37mm and the biosurfactant production
was recorded from 0.016 ± 0.001 to 1.27± 0.003g/l in all the
tested renewable resources.
When the incubation period increased, the factors related to
biosurfactant production were increased simultaneously and
could be observed maximum during 168th h of incubation
period i.e. during this period, the oil displacement activity
was observed to be 12.00 ± 1.00 to 84.33 ± 2.57mm, the
E24% index ranged from 17.6 ± 0.80 to 79.66 ± 2.52% and
the biosurfactant production was recorded between 0.54 ±
0.002 and 5.43 ± 0.01g/l in all the tested renewable
resources. Further increase in incubation period resulted in
decreasing level of biosurfactant production and its related
factors.
Recovery of biosurfactant through different extraction
methods: The result on the recovery of biosurfactant by
following different extraction methods is presented in table
9. Among the tested extraction methods, highest amount of
biosurfactant recovery (6.84 ± 0.12 g/l) was achieved by
using acid precipitation and solvent extraction method
followed by methanol/chloroform extraction (6.25 ± 0.32
g/l) method. In the case of chloroform/methanol/butanol
extraction method, the recovery rate of biosurfactant was
5.30 ± 0.28g/l. The minimum biosurfactant recovery of 1.68
± 0.008g/l was noted at diethyl ether extraction method.
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Figure 7 shows the crystalline appearance of biosurfactant
observed under fluorescence microscope (100xs).
Discussion Use of media optimization strategy has resulted an increase
in production of biosurfactant and lowered the production
cost thereby making the process economical. In the present
study, the biosurfactant production by the potent strain was
enhanced by adopting several parameters such as pH,
temperature, inoculum size, incubation period, types and
concentrations of carbon sources, nitrogen sources,
hydrocarbon sources and different NaCl concentrations.
Desai and Banat5 proposed that the pH has a significant role
in affecting biosurfactant production through their effect on
cell growth and metabolic activity. In the present study, the
pH 9 and 10 have shown significant influence on the growth
and biosurfactant production by the selected isolate. Here the
maximum biomass production and oil displacement activity
were observed at pH 7.0 and maximum E24% index and
biosurfactant production were achieved at pH 8.
Similarly, Joice and Parthasarathi18 suggested that the strain
P. aeruginosa PBSC1 exhibited the highest (29.19mN/m)
surface tension reduction at pH 6.5 and maximum
biosurfactant production (5.13g/l) and emulsification index
(75.12%) at pH 7. Jing et al17 revealed that the strain B.
subtilis JA-1 isolated from an oil reservoir displayed
optimum biosurfactant production and emulsification
activity at the pH range of 7-8. Elazzazy et al7 revealed that
when the pH of the fermentation medium increased,
simultaneously the biosurfactant production was also
increased, however beyond pH 10, the biosurfactant
production started decreasing.
Sahoo et al43 proposed that temperature is one of the
important parameters that greatly affected the culture growth
and the biosurfactant production. A decrease or increase in
the incubation temperature leads to lower growth of
organism and biosurfactant production. Makkar and
Cameotra24 reported that the B. subtilis exhibited maximum
biosurfactant production in sucrose substituted fermentation
medium at 45°C. In the present study, the candidate strain
displayed maximum cell biomass (0.25 ± 0.006 g/l), E24%
index (79.00 ± 2.58%), oil displacement activity (70.00 ±
2.01mm) and biosurfactant production (3.47 ± 0.04 g/l) at
30°C.
The present study was supported by Joice and Parthasarathi18
who stated that the strain P. aeruginosa PBSC1 exhibited
maximum biosurfactant production of 5.12 g/l at the
temperature of 30°C. Similarly, Guerra-Santos et al12
documented that maximum rhamnolipid production by P.
aeruginosa cultured at 34.5ºC with a higher reduction at
temperatures above 36ºC.
Amezcua-Vega et al39 suggested that biosurfactant
production is a secondary microbial metabolic process.
They reported that the strain Candida ingens produced
maximum amount of biosurfactant (4.84 g/l) in the
stationary growth phase on 7th day of incubation. Similarly,
in the present investigation, the maximum E24% index (88.50
± 2.67%) and biosurfactant production (2.80 ± 0.20g/l) were
observed at 168h (7th day) of incubation whereas maximum
biomass production (2.58 ± 0.36g/l) and oil displacement
activity (84.34 ± 2.82mm) were achieved at 172h by the
candidate strain. Al-Araji and Issa2 portrayed that maximum
biosurfactant production by P. aeruginosa 181 was achieved
after 120 h of incubation at pH 7.0 and temperature at 37°C.
Kaskatepe et al19 documented that, P. aeruginosa ATCC
9027 produced maximum amount of rhamnolipid at pH 6.8,
temperature 35°C, agitation rate of 150rpm and incubation
time of 7 days. Fouda et al9 reported that the bacterial strains
P. aeruginosa 4.2 and B. cereus 2.3 reached their maximum
biosurfactant production during 60 - 72 h and 48 - 72 h of
incubation, respectively.
Sen and Swaminathan47 suggested that adequate density of
the inoculum was determinant for high biosurfactant
production. In the present study, it was observed that up to
4% inoculum size, the biomass production, E24% index, oil
displacement activity and biosurfactant production were
increased and thereafter it decreased with increasing level of
the inoculum size.
Pansiripat et al33 revealed that the biosurfactant produced by
B. subtilis PT2 and P. aeruginosa SP4 displayed the highest
surface tension reduction and oil displacement activity at 2%
inoculum size. In accordance with the present study, Sahoo
et al43 reported that the strain P. aeruginosa OCD1 exhibited
maximum reduction of surface tension and highest
emulsification index at l% inoculum size. Likewise, Nalini
and Parthasarathi32 revealed that optimum conditions for
reduction of surface tension by Serratia rubidaea SNAU02
were in 7.78g mahua oil cake substituted medium,
2.4 ml inoculum size (1 × 108 cells/ml), pH 7 and
30°C temperature.
Types and concentrations of carbon sources play an
important role in the production of rhamnolipids by
microorganisms including P. aeruginosa strains12. In the
present study, among the tested carbon sources, the strain
displayed maximum growth and biosurfactant production in
the medium supplemented with mannitol (4%) as the carbon
source. Similarly, Parthasarathi and Sivakumar34
documented that considerable amount of rhamnolipid
biosurfactant production by P. fluorescens by
utilizing glucose, fructose, mannitol, glycerol, olive oil and
cashew juice as carbon sources.
Khopade et al20 found the maximum biosurfactant
production by Streptomyces sp. by using sucrose as a sole
carbon source. Similarly, Govindammal11 reported that P. fluorescens exhibited maximum rhamnolipid production of
8.76 g/l when grown in glucose substituted medium.
Research Journal of Chemistry and Environment_______________________________________Vol. 24 (6) June (2020) Res. J. Chem. Environ.
40
It was reported that rhamnolipid production is more efficient
under nitrogen limiting conditions4. Robert et al39 revealed
that Pseudomonas 44Ti exhibited maximum rhamnolipid
production in the presence of sodium nitrate as the nitrogen
source. In the present study, among the tested nitrogen
sources, the candidate strain exhibited maximum biomass
production (2.78 ± 0.02g/l), E24% index (78.13 ± 2.86%), oil
displacement activity (75.12 ± 2.81mm) and biosurfactant
production (3.25 ± 0.03g/l) in 1.5% beef extract substituted
medium.
Similarly, Ghribi et al10 reported that B. subtilis SPB1
exhibited maximum (720 mg/l) biosurfactant production,
when using urea as nitrogen source. Fernandes et al8
obtained highest concentration of biosurfactant produced by
B. subtilis RI4914 when this strain was cultured in a mineral
salt medium amended with sucrose and ammonium nitrate.
Makkar and Cameotra25 recommended that the petroleum
hydrocarbons and vegetable oils have been used to enhance
the production of biosurfactants and bioemulsifiers from
microbes. In accordance with this, Kokare et al21 portrayed
that among the tested hydrocarbons, toluene (1% v/v)
enhanced maximum biosurfactant production by
Streptomyces sp.
Similarly, Sim et al50 pointed out that a concentration of
11g/l of rhamnolipid was produced when P. aeruginosa
UW-1 was grown in medium containing Canola oil as
hydrocarbon source. Likewise, P. aeruginosa LB1 produced
4.9, 5.4 and 4.8 g/l of rhamnolipid when it was cultivated in
the medium containing sunflower oil, olive oil and soybean
oil respectively as hydrocarbon sources4. In the present
study, P. aeruginosa PAO1 produced maximum biomass
(3.74 ± 0.06g/l), E24% index (85.8 ± 1.89%), oil
displacement activity (86.6 ± 2.27mm) and biosurfactant
production (5.26 ± 0.21g/l) in the medium substituted with
4% olive oil.
In agreement with the present findings, Gujar and Hamde14
reported that P. aeruginosa isolated from oil mill area
exhibited maximum E24% index (70%) and biomass
production (0.21g/l) in the medium containing olive oil as
the hydrocarbon source.
In the present investigation, the candidate strain was able to
grow in a medium with a wide range (0.5 to 4%) of salinity
and exhibited maximum biomass production (3.12 ±
0.02g/l), E24% index (71.0 ± 2.16%), oil displacement
activity (72.33 ± 2.69mm) and highest biosurfactant
production (3.15 ± 0.08g/l) in the medium containing 1.5%
NaCl. The present finding was supported by Guerra-Santos
et al13 who stated that limiting the concentrations of salts of
magnesium, calcium, potassium, sodium and trace elements
resulted in a better yield of rhamnolipid by P. aeruginosa
DSM2659. Likewise, Rismani et al38 documented that the
growth of B. licheniformis was affected by different
concentrations of NaCl and optimal cell growth was found
at 2% NaCl. Similarly, Elazzazy et al7 stated that the
thermophilic strain Virgibacillus salarius exhibited
maximum biosurfactant production in the presence of 4%
NaCl, temperature 45-50ºC and at pH 9.
Although biosurfactants exhibit several advantages than
synthetic surfactants, they have not been employed
extensively in industrial application because of relatively
high production costs. Makkar and Cameotra24 suggested
that the choice of inexpensive raw materials is ideal to
reduce 50% of the final product cost. A variety of cheap raw
materials such as plant derived oils, oil wastes, starchy
substances and lactic whey have been reported to support
biosurfactant production34.
In accordance with this, in the present study, an attempt was
made to synthesize biosurfactant by using renewable
resources like coconut oil cake, gingili oil cake, rubber seed
cake, castor seed cake, neem seed cake, peanut oil cake,
orange peel, rice bran, rice straw and sugar cane baggase
testing individually for the production of biosurfactant by the
candidate strain P. aeruginosa PAO1. Among the tested
renewable resources, the test organism displayed maximum
E24% index (79.66 ± 2.27%), oil displacement activity
(84.33 ± 1.14%m) and biosurfactant production (6.04 ±
0.02g/l) in the BH medium supplemented with peanut oil
cake as the carbon source after 168h of incubation.
Similarly, Thavasi et al52 tested the effect of waste motor
lubricant oil and oil cake on biosurfactant production by P.
aeruginosa isolated from sea water sample of Tuticorin
harbor. Their study revealed that the strain exhibited
maximum biosurfactant production of 8.6mg/l in the
presence of peanut oil cake at 132h of incubation.
Likewise, Mani et al26 represented that the novel marine
bacterium B. simplex exhibited most economical lipopeptide
biosurfactant production with sunflower oil cake after
54thh of incubation. Shah et al48 studied the production of
sophorolipid by Candida bombicola in both batch and fed
batch fermentation, they achieved a yield of 34 g/l of
sophorolipids in the medium containing restaurant oil waste
as the carbon source
Desai and Banat5 documented several methods used for the
recovery of biosurfactant including acid precipitation,
solvent extraction and centrifugation. In the present study,
among the tested extraction methods, maximum
biosurfactant (6.84 ± 0.12g/l) recovery was achieved by
means of acid precipitation and solvent extraction method.
Similar to that of the present study, Jamal et al15 reported that
by using acid precipitation and solvent extraction method,
8.3g/l of biosurfactant was extracted from the culture
supernatant of P. alcalifaciens. Pornsunthorntawee et al36
portrayed that about 2.17g/l of the biosurfactant was
extracted from the cultured medium grown with P.
aeruginosa by acid precipitation and solvent extraction
method.
Research Journal of Chemistry and Environment_______________________________________Vol. 24 (6) June (2020) Res. J. Chem. Environ.
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Table 1
Effect of different pH on individual factors related to biosurfactant production by P. aeruginosa PAO1
pH Factors related to biosurfactant production
Biomass
(g/l)
E24 % index Oil displacement
activity (mm)
Biosurfactant
weight (g/l)
4 0.78 ± 0.06a 54.20 ± 2.05a 0.00 ± 0.00a 1.34 ± 0.13a
5 1.03 ± 0.07b 64.30 ± 2.69b 52.00 ± 1.60b 1.95 ± 0.02b
6 1.62 ± 0.17c 72.60 ± 2.13c 57.30 ± 1.86c 2.50 ± 0.14c
7 1.76 ± 0.26d 85.60 ± 2.81d 72.30 ± 2.69d 3.32 ± 0.22d
8 2.01 ± 0.20e 84.30 ± 2.49d 81.30 ± 2.86e 2.66 ± 0.24e
9 1.72 ± 0.12df 77.30 ± 2.40e 79.60 ± 2.05ef 1.84 ± 0.09f
10 0.85 ± 0.09g 72.00 ± 2.16ef 72.00 ± 2.81d 1.42 ± 0.01g
Each value is the Mean ± SD of triplicate analysis; within each row means with different superscript letters are
statistically significant (One-way ANOVA test; P< 0.05 and further post hoc multiple comparison with SNK test)
Table 2
Effect of different temperature on individual factors related to biosurfactant production by P. aeruginosa PAO1
Temperature (°C) Factors related to biosurfactant production
Biomass
(g/l)
E24 % index Oil displacement
activity (mm)
Biosurfactant
weight (g/l)
25 0.17 ± 0.001a 23.00 ± 1.70a 22.00 ± 1.01a 1.24 ± 0.01a
30 0.25 ± 0.006b 79.00 ± 2.58b 70.00 ± 2.01b 3.47 ± 0.04b
35 0.21 ± 0.002c 70.00 ± 2.86c 62.00 ± 2.05c 2.98 ± 0.02c
40 0.18 ± 0.004d 34.20 ± 1.70d 35.00 ± 1.01d 1.34 ± 0.02d
45 0.10 ± 0.002e 20.00 ± 0.93e 0.00 ± 0.00e 1.03 ± 0.03e
50 0.02 ± 0.002f 0.00 ± 0.00f 0.00 ± 0.00e 0.16 ± 0.006f
Each value is the Mean ± SD of triplicate analysis; within each row means with different superscript letters are
statistically significant (One-way ANOVA test; P< 0.05 and further post hoc multiple comparison with SNK test)
Table 3
Effect of various incubation time on individual factors related to biosurfactant production by P. aeruginosa PAO1
Incubation time
(hr)
Factors related to biosurfactant production
Biomass
(g/l)
E24 % Oil displacement
activity (cm)
Biosurfactant weight
(g/l)
24 0.72 ± 0.01a 52.33 ± 2.40a 0.00 ± 0.00a 0.24 ± 0.008a
48 0.87 ± 0.05b 61.66 ± 2.12b 61.6 ± 2.05b 0.77 ± 0.004b
72 1.19 ± 0.44c 65.46 ± 2.03c 71.23 ± 2.05c 1.17 ± 0.016c
96 1.26 ± 0.64d 70.32 ± 2.85d 76.33 ± 2.69d 1.29 ± 0.06d
120 1.34 ± 0.32e 76.72 ± 2.42e 78.16 ± 2.92de 2.26 ± 0.19e
144 1.73 ± 0.40f 82.50 ± 2.40f 80.24 ± 2.46ef 2.46 ± 0.08f
168 2.34 ± 0.24g 88.50 ± 2.67g 82.10 ± 2.28fg 2.80 ± 0.20g
192 2.58 ± 0.36h 75.00 ± 2.46eh 84.34 ± 2.82gh 2.64 ± 0.12h
216 2.17 ± 0.25i 33.60 ± 1.82i 80.36 ± 2.17efg 1.38 ± 0.04i
240 1.60 ± 0.38j 13.60 ± 0.76j 61.60 ± 2.24bi 0.96 ± 0.05j
Each value is the Mean ± SD of triplicate analysis; within each row means with different superscript letters are
statistically significant (One-way ANOVA test; P< 0.05 and further post hoc multiple comparison with SNK test)
Research Journal of Chemistry and Environment_______________________________________Vol. 24 (6) June (2020) Res. J. Chem. Environ.
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Table 4
Effect of different inoculum size on individual factors related to biosurfactant production by P. aeruginosa PAO1
Inoculum size
(ml)
Factors related to biosurfactant production
Biomass
(g/l)
E24% index Oil displacement
activity (mm)
Biosurfactant weight
(g/l)
0.5 0.17 ± 0.02a 0.00 ± 0.00a 22.30 ± 1.05a 0.46 ± 0.02a
1.0 0.34 ± 0.03b 13.00 ± 0.64b 28.66 ± 1.49b 0.95 ± 0.02b
1.5 0.37 ± 0.05c 18.32 ± 0.98c 42.66 ± 1.05c 1.23 ± 0.10c
2 0.44 ± 0.04d 28.33 ± 1.63d 51.00 ± 1.16d 1.65 ± 0.24d
2.5 0.62 ± 0.03e 32.66 ± 1.62e 55.00 ± 1.63e 2.13 ± 0.43e
3 0.69 ± 0.02f 43.66 ± 1.24f 63.00 ± 1.81f 2.43 ± 0.16f
3.5 0.73 ± 0.01g 68.22 ± 1.86g 72.66 ± 2.05g 2.59 ± 0.25g
4 1.24 ±0.09h 70.66 ± 2.58gh 85.00 ± 3.26h 3.77 ± 0.62h
4.5 0.99 ± 0.01i 52.33 ± 1.05i 79.00 ± 2.81i 1.44 ± 0.18i
5 0.26 ± 0.02j 16.80 ± 0.84j 46.66 ± 1.86j 0.72 ± 0.08j
Each value is the Mean ± SD of triplicate analysis; within each row means with different superscript letters are
statistically significant (One-way ANOVA test; P< 0.05 and further post hoc multiple comparison with SNK test)
Table 5
Effect of different carbon sources on individual factors related to biosurfactant production by P. aeruginosa PAO1
Carbon sources
(2%)
Factors related to biosurfactant production
Biomass
(g/l)
E24% index Oil displacement
activity (mm)
Biosurfactant weight
(g/l)
Fructose 1.33 ± 0.08 57.4 ±1.65 40.66 ± 1.29 1.20 ± 0.01
Sucrose 1.70 ± 0.03 16.6 ± 0.89 58.66 ± 2.18 1.44 ± 0.09
Starch 1.21 ± 0.08 6.80 ±0.18 0.00 ± 0.00 0.24 ± 0.02
Lactose 1.13 ± 0.06 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
cellulose 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Mannitol 2.74 ± 0.06 77.33 ± 2.18 78.66 ± 2.47 3.26 ± 0.08
Xylose 0.26 ± 0.003 17.20 ± 1.14 3.66 ± 0.04 0.11 ± 0.02
Galactose 1.17 ± 0.04 0.00 ± 0.00 0.00 ± 000 0.00 ± 0.00
Dextrose 1.26 ± 0.02 36.80 ±1.14 46.66 ± 1.86 2.22 ± 0.04
Each value is the Mean ± SD of triplicate analysis
Table 6
Effect of different nitrogen sources on individual factors related to biosurfactant production by P. aeruginosa PAO1
Nitrogen sources (1%) Factors related to biosurfactant production
Biomass
(g/l)
E24% index Oil displacement
activity (mm)
Biosurfactantweig
ht (g/l)
Yeast extract 1.18 ± 0.02 63.02 ±1.10 48.00 ± 1.63 2.26 ± 0.02
Beef extract 2.13 ± 0.06 70.53 ± 2.00 68.00 ± 2.16 2.92 ± 0.08
Urea 0.22 ± 0.002 65.46 ± 1.22 52.30 ± 2.05 1.29 ± 0.06
(NH4)2SO4 0.55 ± 0.005 22.60 ± 1.13 20.00 ± 1.12 0.17 ± 0.001
NH4NO3 0.54 ± 0.009 20.20 ± 0.92 18.00 ± 0.86 0.11 ± 0.003
Peptone 1.85 ± 0.04 34.60 ± 1.46 0.00 ± 0.00 0.14 ± 0.005
Glutamic acid 0.22 ± 0.003 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Each value is the Mean ± SD of triplicate analysis
Research Journal of Chemistry and Environment_______________________________________Vol. 24 (6) June (2020) Res. J. Chem. Environ.
43
Table 7
Effect of different hydrocarbon source on individual factors related to biosurfactant production
by P. aeruginosa PAO1
Hydrocarbon
sources (2%)
Factors related to biosurfactant production
Biomass
(g/l)
E24% index Oil displacement
activity (mm)
Biosurfactant weight
(g/l)
Palm oil 0.52 ± 0.003 17.20 ± 0.86 22.33 ± 1.05 1.21 ± 0.008
Olive oil 2.83 ± 0.02 80.60 ± 1.27 82.66 ± 2.12 4.32 ± 0.06
Coconut oil 0.21 ± 0.008 16.60 ± 0.64 19.66 ± 1.20 1.64 ± 0.07
Kerosene 0.16 ± 0.004 18.30 ± 1.00 1.00 ± 0.00 1.12 ± 0.08
Diesel 0.22 ± 0.002 63.00 ± 2.08 0.00 ± 0.00 1.14 ± 0.06
Petrol 0.34 ± 0.005 62.00 ± 2.71 60.00 ± 2.16 1.24 ± 0.02
Sunflower 1.52 ± 0.04 66.00 ± 2.26 73.00 ± 2.94 2.52 ± 0.08
Fried oil 1.33 ± 0.06 19.66 ± 0.84 50.00 ± 1.44 1.14 ± 0.01
Crude oil 2.55 ± 0.03 36.30 ± 1.14 60.00 ± 2.52 1.20 ± 0.02
Each value is the Mean ± SD of triplicate analysis
(a) Biomass and Biosurfactant production
\
(b) E24% index and Oil displacement activity
Fig. 1: (a and b) Effect of different concentrations of mannitol on individual factors related to biosurfactant
production by P. aeruginosa PAO1
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5B
iosu
rfa
cta
nt
pro
du
ctio
n (
g/l
)
Bio
ma
ss
pro
du
ctio
n (
g/l
)
Concentrations of mannitol (%)
Biomass Biosurfactant
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Oil
dis
pla
cem
ent
act
ivit
y(m
m)
E2
4%
in
dex
Concentrations of mannitol (%)
E24% Oil displacement activtiy
Research Journal of Chemistry and Environment_______________________________________Vol. 24 (6) June (2020) Res. J. Chem. Environ.
44
(a) Biomass and Biosurfactant production
(b) E24% index and Oil displacement activity
Fig. 2: (a and b) Effect of different concentrations of beef extract on individual factors
related to biosurfactant production by P. aeruginosa PAO1
Table 8
Effect of different concentrations of NaCl on individual factors related to biosurfactant production
by P. aeruginosa PAO1
NaCl
concentrations
(%)
Factors related to biosurfactant production
Biomass
(mg/ml)
E24 % index Oil displacement
activity (mm)
Biosurfactant weight
(g/l)
0.5 1.46 ± 0.004a 62.26 ± 2.75a 51.66 ± 1.24a 2.18 ± 0.006a
1 1.62 ± 0.002b 66.20 ± 2.39b 65.66 ± 2.26b 2.94 ± 0.004b
1.5 3.12 ± 0.02c 71.00 ± 2.16c 72.33 ± 2.69c 3.15 ± 0.08c
2 2.61 ± 0.008d 63.00 ± 2.81ad 62.33 ± 2.43d 2.48 ± 0.02d
2.5 2.06 ± 0.006e 21.00 ± 1.41e 25.33 ± 1.52e 1.32 ± 0.005e
3 1.41 ± 0.005ad 13.83 ± 0.84f 21.33 ± 1.05f 1.06 ± 0.001f
Each value is the Mean ± SD of triplicate analysis; within each row means with different superscript letters are
statistically significant (One-way ANOVA test; P< 0.05 and further post hoc multiple comparison with SNK test)
0
0.5
1
1.5
2
2.5
3
3.5
4
0
0.5
1
1.5
2
2.5
3
3.5
0.25 0.5 0.75 1 1.25 1.5 1.75 2
Bio
surf
act
an
t p
rod
uct
ion
(g
/l)
Bio
ma
ss p
rod
uct
ion
(g
/l)
Concentrations of beef extract (%)
E24% Oil displacement activtiy
0
10
20
30
40
50
60
70
80
90
0
10
20
30
40
50
60
70
80
90
0.25 0.5 0.75 1 1.25 1.5 1.75 2
Oil
dis
pla
cem
ent
act
ivit
y(m
m)
E24%
in
dex
Concentrations of beef extract (%)
E24% Oil displacement activtiy
Research Journal of Chemistry and Environment_______________________________________Vol. 24 (6) June (2020) Res. J. Chem. Environ.
45
Table 9
Effect of different extraction methods on recovery of biosurfactant from production medium
Extraction methods Yield of biosurfactants (g/l)
Diethyl ether extraction 1.68 ± 0.004a
Chilled acetone precipitation method 2.08 ± 0.06b
Ammonium sulfate precipitation 3.20 ± 0.08c
Ethyl acetate extraction 4.60 ± 0.14d
Chloroform/Methanol/Butanol extraction 5.30 ± 0.28e
Methanol/Chloroform extraction 6.25 ± 0.32f
Acid precipitation and solvent extraction 6.84 ± 0.12g
Each value is the Mean ± SD of triplicate analysis; within each row means with different superscript
letters are statistically significant (One-way ANOVA test; P< 0.05 and further post hoc multiple
comparison with SNK test)
(a) Biomass and Biosurfactant production
(b) E24% index and Oil displacement activity
Fig. 3: (a and b) Effect of different concentrations of olive oil on individual factors related
to biosurfactant production by P. aeruginosa PAO1
0
1
2
3
4
5
6
0
0.5
1
1.5
2
2.5
3
3.5
4
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Bio
surf
act
an
t p
rod
uct
ion
(g
/l)
Bio
ma
ss
pro
du
ctio
n (
g/l
)
Concentrations of olive oil (%)
Biomass Biosurfactant
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Oil
dis
pla
cem
ent
act
ivit
y (
mm
)
E2
4%
in
dex
Concentrations of olive oil (%)
E24% Oil displacement activtiy
Research Journal of Chemistry and Environment_______________________________________Vol. 24 (6) June (2020) Res. J. Chem. Environ.
46
Fig. 4: Effect of various renewable resources on oil displacement activity by P. aeruginosa PAO1 during
different days of incubation period (24 to 216h)
Fig. 5: Effect of various renewable resources on E24% index by P. aeruginosa PAO1
during different incubation period
0
10
20
30
40
50
60
70
80
90O
il d
isp
lacem
en
t a
cti
vit
y (
mm
)
Renewable resources
24h 72h 120h 168h 216h
0
10
20
30
40
50
60
70
80
90
E24
% i
nd
ex
Renewable resources
24h 72h 120h 168h 216h
Research Journal of Chemistry and Environment_______________________________________Vol. 24 (6) June (2020) Res. J. Chem. Environ.
47
Fig. 6: Effect of various renewable resources on biosurfactant production by P. aeruginosa PAO1
during different incubation period
Fig. 7: Appearance of biosurfactant under fluorescence microscope (100 xs)
Similarly, Dubey and Juwarkar6 also reported that 0.92 g/l of
rhamnolipid was obtained from the culture supernatant of P.
aeruginosa BS2 by acid precipitation and solvent extraction
method. Likewise, Shah et al49 studied the effect of various
extraction methods on recovery of rhamnolipid from P. aeruginosa. In their study, they revealed that organic solvent
extraction method was found to be the best recovery
technique and giving the highest yield (7.5 g/l) of
biosurfactant, while acid precipitation yielded the least
(3.5g/l) amount of biosurfactant.
Conclusion The present study depicted the enhancement of rhamnolipid
production from P. aeruginosa PAO1 through media
optimization and also by using renewable resources. The
candidate strain showed maximum biosurfactant production
at pH 7, temperature 30°C, incubation time of 168 h, 4%
inoculum size, 4% mannitol as carbon source, 1.5% beef
extract as nitrogen source, 4% olive oil as hydrocarbon
source, 1.5 % NaCl concentration and peanut oil cake as the
waste substrate.
0
1
2
3
4
5
6
Bio
surf
act
an
t p
rod
uct
ion
(g
/l)
Renewable resources
24h 72h 120h 168h 216h
Rhomboid crystal
appearance
100
px
Research Journal of Chemistry and Environment_______________________________________Vol. 24 (6) June (2020) Res. J. Chem. Environ.
48
Further the produced biosurfactant was recovered using
different extraction methods and the result revealed that 6.84
g/l of rhamnolipid was recovered by means of acid
precipitation and solvent extraction method.
Acknowledgement The authors gratefully acknowledge the DST-SERB, New
Delhi, Govt. of India, for financial support in the form of
research grant (Grant No: EMR/2017/001453).
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(Received 08th January 2020, accepted 12th March 2020)