Research Journal of Chemistry and Environment May Res. J ... · plants contain flavonoid chemical...

Research Journal of Chemistry and Environment_______________________________________Vol. 24 (6) June (2020) Res. J. Chem. Environ.

34

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

*[email protected]

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

mailto:[email protected]


35

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


36

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


37

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


38

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),


(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.


39

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.


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


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


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


(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.


41

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)


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



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




42

Table 4

Effect of different inoculum size on individual factors related to biosurfactant production by P. aeruginosa PAO1

Inoculum size

(ml)


Biomass

(g/l)

E24% index Oil displacement

activity (mm)


(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



Table 5

Effect of different carbon sources on individual factors related to biosurfactant production by P. aeruginosa PAO1

Carbon sources

(2%)


Biomass

(g/l)


activity (mm)


(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)


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



43

Table 7

Effect of different hydrocarbon source on individual factors related to biosurfactant production

by P. aeruginosa PAO1

Hydrocarbon

sources (2%)


Biomass

(g/l)


activity (mm)


(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


(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


44



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

(%)


Biomass

(mg/ml)


activity (mm)


(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



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 (%)


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 (%)



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)



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 (%)



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


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


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).

References 1. Ahmed E.F. and Hassan S.S., Antimicrobial activity of a

bioemulsifier produced by Serratia marcescens S10, J. AL-

Nahrain University, 16, 147-155 (2013)

2. Al-Araji Y. and Issa L., Biosurfactant production by

Pseudomonas aeruginosa 181, Ph.D. Thesis, University Putra

Malaysia (2004)

3. Amézcua-Vega C., Ferrera-Cerrato R., Esparza-García F., Ríos-

Leal E. and Rodríguez-Vázquez R., Effect of combined nutrients

on biosurfactants produced by Pseudomonas putida, J. Environ.

Sci. Health A., 39, 2983–2991 (2004)

4. Benincasa M., Contiero J., Manresa M.A. and Moraes I.O.,

Rhamnolipid Production by Pseudomonas aeruginosa LBI

Growing on Soap Stock as the Sole Carbon Source, J. Food Eng.,

54(4), 283-288 (2002)

5. Desai J.D. and Banat I.M., Microbial production of surfactants

and their commercial potential, Microbiol. Mol. Biol. Rev., 61, 47-

64 (1997)

6. Dubey K. and Juwarkar A., Distillery and curd whey wastes as

viable alternative sources for biosurfactant production, World J.

Microbiol. Biotechnol., 17, 61-69 (2001)

7. Elazzazy A.M., Abdelmoneima T.S. and Almaghrabia O.A.,

Isolation and characterization of biosurfactant production under

extreme environmental conditions by alkali-halothermophilic

bacteria from Saudi Arabia, Saudi J. Biol. Sci., 22(4), 466-475

(2015)

8. Fernandes P.L., Rodrigues E.M., Paiva F.R., Ayupe B.A.L.,

McInerney M.J. and Ttola M.R., Biosurfactant, solvents and

polymer production by Bacillus subtilis RI4914 and their

application for enhanced oil recovery, Fuel, 180, 551-557 (2016)

9. Fouda A., El-Gamal M.S., Abdel-Shakour E.H. and Radwan

A.A., Optimization and improvement of biosurfactant production

for Pseudomonas aeruginosa 4.2 and Bacillus cereus 2.3 strains

isolated from oily polluted soil sample, Int. J. Adv. Res. Biol. Sci.,

3(1), 76-87 (2016)

10. Ghribi D., Mnif I., Boukedi H., Radhouan K. and Chaabouni-

Ellouze S., Statistical optimization of medium components for

economical production of Bacillus subtilis surfactin, a biocontrol

agent for the olive moth Prays oleae, Afr. J. Microbiol. Res., 5,

4927-4936 (2011)

11. Govindammal M., Effect of carbon and nitrogen sources on the

production of biosurfactant by Pseudomonas fluorescens isolated

from Mangrove Ecosystem, Int. J. Pharm. Biol. Arch., 5(2), 108-

115 (2014)

12. Guerra-Santos L.H., Hirt T.R., Kappeli H. and Flechter A.,

Pilot plant production of rhamnolipid biosurfactant by

Pseudomonas aeruginosa, Appl. Environ. Microbiol., 51(5), 985-

989 (1986a)

13. Guerra-Santos L.H., Kaeppeli O. and Fietch A., Dependence of

Pseudomonas aeruginosa continuous culture biosurfactant

production on nutritional and environmental factors, Appl.

Microbiol. Biotechnol., 24, 443-448 (1986b)

14. Gujar R. and Hamde V.S., Separation and characterization of

biosurfactant from P. aeruginosa sp. isolated from oil mill area

MIDC, Int. Multidiscip. Res. J., 2(3), 13-15 (2012)

15. Jamal A., Qureshi M.Z., Ali N., Ali M.I. and Hameed M.,

Enhanced production of rhamnolipids by Pseudomonas

aeruginosa JQ927360 using response surface methodology, Asian

J. Chem., 26(4), 1044-1048 (2014)

16. Javaheri M., Jenneman G.E., Mcinerney M.J. and Knapp R.M.,

Anaerobic Production of a biosurfactant by Bacillus licheniformis

JF-2, Appl. Environ. Microbiol., 50(3), 698-700 (1985)

17. Jing L., Jia Y., Lu J., Han R., Li J. and Wang S., MicroRNA-9

promotes differentiation of mouse bone mesenchymal stem cells

into neurons by Notch signaling, Neuro Report, 22, 206-211 (2011)

18. Joice P.A. and Parthasarathi R., Optimization of biosurfactant

production from P. aeruginosa PBSC1, Int. J. Curr. Microbiol.

Appl. Sci., 3(9), 140-151 (2014)

19. Kaskatepe B., Yildiz S., Gumustas M. and Ozkan S.A.,

Biosurfactant production by P. aeruginosa in kefir and fish meal,

Braz. J. Microbiol., 46(3), 855–859 (2015)

20. Khopade A., Ren B., Liu X.Y., Mahadik K., Zhang L. and

Kokare C., Production and characterization of biosurfactant from

marine Streptomyces species B3, J. Colloid Interface Sci., 367(1),

311–318 (2012)

21. Kokare C.R., Kadam S.S., Mahadik K.R. and Chopade B.A.,

Studies on bioemulsifier production from marine Streptomyces sp.

S1, Indian J. Biotechnol., 66(1), 78–84 (2007)

22. Kuyukina M.S., Ivshina I.B., Philp J.C., Christofi N., Dunbar

S.A. and Ritchkova M.I., Recovery of Rhodococcus biosurfactants

using methyl tertiary-butyl ether extraction, J. Microbiol.

Methods, 46(2), 149-156 (2001)

23. Lee Y.J., Choi J.K., Kim E.K., Youn S.H. and Yang E.J., Field

experiments on mitigation of harmful algal blooms using a

Sophorolipid: Yellow clay mixture and effects on marine plankton,

Harmful Algae, 7(2), 154-162 (2008)

24. Makkar R.M. and Cameotra S.S., Utilization of molasses for

biosurfactant production by two Bacillus subtilis strain at

thermophilic conditions, J. Am. Oil Chem. Soc., 74(7), 887-889

(1997)

25. Makkar R.S. and Cameotra S.S., Biosurfactant production by

microorganisms on unconventional carbon sources-a review, J.

Surfactants Deterg., 2(2), 237–241 (1999)

26. Mani P., Dineshkumar G., Jayaseelan T., Deepalakshmi K.,

Ganesh Kumar C. and Senthil Balan S., Antimicrobial activities of


49

a promising glycolipid biosurfactant from a novel marine

Staphylococcus saprophyticus SBPS 15, 3 Biotech, 6, 163 (2016)

27. Mercade M.E. and Manresa M.A., The use of agro industrial

by products for biosurfactant production, J. Am. Oil. Chem. Soc.,

71, 61-64 (1994)

28. Morita T., Konishi M., Fukuoka T., Imura T. and Kitamoto D.,

Production of glycolipid biosurfactants, mannosylerythritol lipids,

by Pseudozyma siamensis CBS 9960 and their interfacial

properties, J. Biosci. Bioeng., 105(5), 493-502 (2008)

29. Mulligan C.N., Environmental applications for biosurfactants,

Environ. Pollut., 133(2), 183-198 (2005)

30. Mulligan C.N., Yong R.N. and Gibbs B.F., Surfactant-

enhanced remediation of contaminated soil: a review, Eng. Geol.,

60, 371-380 (2001)

31. Muriana P.M. and Klaenhammer T.R., Purification and partial

characterization of Lactacin F, a bacteriocin produced by

Lactobacillus acidophilus 11088, Appl. Environ. Microbiol., 57(1),

114-121 (1991)

32. Nalini S. and Parthasarathi R., Optimization of rhamnolipid

biosurfactant production from Serratia rubidaea SNAU02 under

solid-state fermentation and its biocontrol efficacy against

Fusarium wilt of eggplant, Ann. Agrar. Sci., 16(2), 108-115 (2018)

33. Pansiripat S., Pornsunthorntawee O., Rujiravanit R., Kitiyanan

B., Somboonthanate P. and Chavadej S., Biosurfactant production

by Pseudomonas aeruginosa SP4 using sequencing batch reactors:

effect of oil-to-glucose ratio, Biochem. Eng. J., 49(2), 185-191

(2010)

34. Parthasarathi R. and Sivakumaar P.K., Effect of different

carbon Sources on the production of biosurfactant by

Pseudomonas fluorescens isolated from mangrove forests

(Pichavaram), Tamil Nadu, India, Global J. Environ. Res., 2, 99-

101 (2010)

35. Patel R.M. and Desai A.J., Biosurfactant production by

Pseudomonas aeruginosa GS3 from molasses, Lett. Appl.

Microbiol, 25(2), 91-94 (1997)

36. Pornsunthorntawee O.P., Arttaweeporn N., Paisanjit S.,

Somboonthanate P., Abe Rujiravanit R. and Chavadej S., Isolation

and comparison of biosurfactants produced by Bacillus subtilis

PT2 and Pseudomonas aeruginosa SP4 for microbial surfactant-

enhanced oil recovery, Biochem. Eng. J., 42(2), 172–179 (2008)

37. Raza Z.A., Khan M.S. and Khalid Z.M., Physicochemical and

surface-active properties of biosurfactant produced using molasses

by a Pseudomonas aeruginosa mutant, J. Environ. Sci. Health A.

Tox. Hazard, 42, 73–80 (2007)

38. Rismani E., Fooladi J. and Ebrahimi Por G.H., Biosurfactant

production in batch culture by a Bacillus licheniformis isolated

from the Persian Gulf Pakistan, J. Biol. Sci., 9, 2498-2502 (2006)

39. Robert M., Mercade M.E., Bosch M.P., Parra J.I., Espiny

M.J., Manaresa A. and Guinea J. Effect of the carbon source

on biosurfactant production by Pseudomonas

aeruginosa 44TI, Biotechnol. Lett., 11(12), 871-874 (1989)

40. Rodrigues L., Banat I.M., Teixeira J. and Oliveira R.,

Biosurfactants: potential applications in medicine, J. Antimicrob.

Chemoth., 57(4), 609–618 (2006)

41. Rosenberg E., Zuckerberg A., Rubinovitz C. and Gutnick D.L.,

Emulsifier Arthrobacter RAG-1: Isolation and Emulsifying

Properties, Appl. Environ. Microbiol., 37(3), 402- 408 (1979)

42. Saharan B.S., Sahu R.K. and Sharma D., A review on

biosurfactants: fermentation, current developments and

perspectives, Genet. Eng. Biotechnol. J., 1, 1-14 (2011)

43. Sahoo S., Datta S. and Biswas D., Optimization of culture

conditions for biosurfactant production from Pseudomonas

aeruginosa OCD1, J. Adv. Sci. Res., 2(3), 32-36 (2011)

44. Sarachat T., Pornsunthorntawee O., Chavadej S. and

Rujiravanit R., Purification and concentration of a rhamnolipid

biosurfactant produced by Pseudomonas aeruginosa SP4 using

foam fractionation, Bioresour. Technol., 101, 24–30 (2010)

45. Saravanan V. and Vijayakumar S., Isolation and screening of

biosurfactant producing microorganisms from oil contaminated

soil, J. Acad. Ind., 1(5), 264-268 (2012)

46. Satpute S.K., Bhawasar B.D., Dhakephalkar P.K. and Chopade

B.A., Assessment of different screening method for selecting

biosurfactant producing marine bacteria, Indian J. Mar. Sci., 37(3),

243-250 (2008)

47. Sen R. and Swaminathan T., Response surface modeling and

optimization to elucidate the effects of inoculum age & size on

surfactin production, Biochem. Eng. J., 21(2), 141-148 (2004)

48. Shah V., Badia D. and Ratsep P., Sophorolipids having

enhanced antibacterial activity, Antimicrob Agents Chemother,

51(1), 397-400 (2007)

49. Shah M.U.H., Sivapragasam M., Moniruzzaman M. and Yusup

S.B., A comparison of recovery methods of rhamnolipids produced

by P. aeruginosa, Procedia Eng., 148, 494-500 (2016)

50. Sim L., Ward O.P. and Li Z.Y., Production and characterization

of a biosurfactant isolated from Pseudomonas aeruginosa UW-1,

J. Ind. Microbiol. Biotechnol., 19(4), 232-238 (1997)

51. Tahzibi A., Kamal F. and Assadi M.M., Improved production

of rhamnolipid by a Pseudomonas aeruginosa mutant, Iran

Biomed. J., 8(1), 25-31 (2004)

52. Thavasi R., Nambaru S.M., Jayalakshmi S., Balasubramanian

T. and Ibrahim M.B., Biosurfactant production by Azotobacter

chroococcum isolated from the marine environment,

Mar. Biotechnol., 11(5), 551-556 (2009)

53. Wang Q.H., Fang X.D., Bai B.J., Liang X.L., Shuler P.J.,

Goddard W.A. and Tang Y.C., Engineering bacteria for production

of rhamnolipid as an agent for enhanced oil recovery, Biotechnol.

Bioeng., 98(4), 842–853 (2007).

(Received 08th January 2020, accepted 12th March 2020)

Research Journal of Chemistry and Environment May Res. J ... · plants contain flavonoid chemical...

Documents

Transcript of Research Journal of Chemistry and Environment May Res. J ... · plants contain flavonoid chemical...