Malaysian Journal of Microbiology, Vol 10(1) 2014, pp.

57-62
Malaysian Journal of Microbiology
Published by Malaysian Society for Microbiology
(In since 2011)

57 ISSN (print): 1823-8262, ISSN (online): 2231-7538


Production of biosurfactant by Pseudomonas aeruginosa PB3A using agro-
industrial wastes as a carbon source

Varadharajan Saravanan and Subramaniyan Vijayakumar*

Department of Botany and Microbiology, A.V.V.M. Sri Pushpam College, Thanjavur-613 503, Tamil Nadu, India.
Email: svijaya_kumar2579@rediff.com
Received 30 July 2013; Received in revised form 1 October 2013; Accepted 7 November 2013


Aims: To evaluate Pseudomonas aeruginosa PB3A strain for the production of biosurfactant using agro-industrial waste
as a carbon source.
Methodology and results: P. aeruginosa PB3A strain was isolated from oil contaminated soil and was found to be a
potential biosurfactant producing microorganism based on the following screening methods; hemolytic activity, drop
collapse test, emulsification activity and surface tension measurement. The identification of the isolate was confirmed by
16S rRNA sequencing. The isolate exploited for the application of agro industrial wastes such as used castor oil,
coconut oil, corn oil, motor oil, olive oil, olein, rapeseed oil, sunflower oil, barley bran, cassava flour waste, peanut cake,
potato waste, rice bran and wheat bran for biosurfactant production by replacing the existing carbon source. Among the
cheap substrates, both the used corn oil and cassava waste flour showed maximum productivity of 0.62 mg/mL and 0.60
mg/mL respectively when cultivated independently in the MSM medium.
Conclusion, significance and impact study: Present study was aimed at the application of agro-industrial wastes for
biosurfactant production. The study indicates that agro-industrial wastes can be used as inexpensive substrates by
replacing synthetic media for the production of biosurfactant.

Keywords: Toxicity, agro-industrial wastes, biosurfactant, drop collapse, hemolytic assay, surface tension.

INTRODUCTION

Surfactant belongs to amphiphatic molecules, consisting
of a hydrophobic and a hydrophilic group which helps in
reducing the surface and interfacial tension in
water/hydrocarbon systems (Prantera et al., 2002). The
application of synthetic surfactants for the treatment of
hydrocarbon-contaminated soil improves the restoration;
however, they get accumulated in the ecological system
leading to severe environmental damage. The
biosurfactants or the surface active component
synthesized by the microorganisms appears to be an
excellent alternative approach for the synthetic
surfactants. Besides possessing surface and emulsifying
activities (Van Hamme et al., 2006; Singh et al., 2007)
these bio surfactants exhibit antimicrobial and anti
adhesive properties (Singh and Cameotra, 2004;
Rodrigues et al., 2006). Microbial surfactants composed
of diverse group of surface-active components are
classified by their chemical composition and microbial
origin which include lipopeptides, glycolipids,
lipopolysaccharides, polysaccharide-protein complexes,
protein-like substances, fatty acids, phospholipids, and
neutral lipids (Van Hamme et al., 2006).
Distinct properties and physiological functions have
been observed for different groups of biosurfactants
produced by different microorganisms which include


enhanced solubility of hydrophobic compounds, heavy
metal binding, cell adhesion and aggregation, biofilm
formation and quorum sensing (Ron and Rosenberg,
2001; Singh and Cameotra, 2004). Among the various
microorganisms, bacteria are the important group of
surfactant-producers although few yeasts and fungi are
also reported (Desai and Banat, 1997). The most
common biosurfactants are glycolipids and lipopeptides
which include rhamnolipids synthesized by Pseudomonas
aeruginosa (Nitschke et al., 2005), sophorolipids released
by Candida species (Daverey and Pakshirajan, 2009),
and surfactin and iturin synthesized by Bacillus subtilis
strains (Ahimou et al., 2000).
In the present scenario, the major drawback that
prevents the widespread use of microbial biosurfactants is
the economic viability of the production costs. Currently
various strategies have been evaluated in order to reduce
the fermentation process economics and make it
competitive with chemically synthesized surfactants
(Makkar and Cameotra, 2002). Future research on
biosurfactant has to be more focused on the economics of
the fermentation processes, mainly through the usage of
alternative low-cost production media (Makkar and
Cameotra, 2002; Rodrigues and Teixeira, 2008).

*Corresponding author
Mal. J. Microbiol. Vol 10(1) 2014, pp.57-62
58 ISSN (print): 1823-8262, ISSN (online): 2231-7538

MATERIALS AND METHODS

Materials

All the chemicals and reagents used in the present study
were of analytical grade and purchased from SRL
Chemicals and Merck Chemicals, India. Used residual oil
wastes were collected from various shops and household;
agro-wastes were collected from local market and
industries from Chennai and nearby area.

Isolation and screening of biosurfactant producers

Bacterial strains used in this study were isolated from oil
contaminated soils at Chennai as described by
Saravanan and Vijayakumar (2012). The samples were
serially diluted and preserved in nutrient agar slant for
further screening of biosurfactant production. Hemolytic
activity was performed as described by Carrillo et al.,
(1996) with slight modifications. Isolated bacterial strains
were screened for their hemolytic activity on blood agar
plates with 5 vol. % of blood and incubated at 37 C for 24
h. The hemolytic activity was distinguished based on the
presence of a clear zone around the colonies. Using a 96
micro-well plate the drop collapse test was carried out as
described by Bodour and Maier (1998). Two microliter of
crude oil was added to the well and left to equilibrate for
24 h, followed by 5 L of 48 h cell free supernatant of
bacterial strain and the drop size was observed after 1
min with the aid of a magnifying glass. Positive result
shows a flat drop and rounded drops were scored as
negative which indicate a negative result for biosurfactant
production. The emulsifying activity of biosurfactant was
carried out according to Cooper and Goldenberg (1987).
After 48 h, 2 mL of cell free supernatant and hydrocarbon
(oil) was taken in a test tube and vortexed at high speed
for 1 min and left to stand for 24 h. Emulsifying (E24)
activity was expressed as the percentage of the total
height occupied by the emulsion (Bodour et al., 2004).
Surface tension (ST) was determined using a Kruss
processor tensiometer by the plate method (Paza et al.,
2011). The bacterial cultures were centrifuged at 10,000
rpm for 20 min and the cell free supernatant was
analyzed for the determination of surface activity. All the
assays were performed in triplicate with appropriate
positive and negative controls.

Identification of bacterial strain

The selected isolate PB3A was identified by
morphological and biochemical analysis based Bergeys
Manual of Systemic Bacteriology (Holt et al., 1994). The
identity of bacterial strain PB3A was further confirmed by
16S rRNA gene sequence analysis. The total genomic
DNA was extracted from an overnight culture in Luria
Bertani broth using CTAB (Cetyltrimethylammonium
Bromide) method (Ausubel et al., 1994). The 16S rRNA
gene amplification was done by universal 16S rDNA
primers 27f (5- GAGTTTGATCCTGGCTCAG-3) and
1492r (5-TACGGYTACCTTGTTACGACTT-3) followed
by purification using QIAquick Gel Extraction kits from
Qiagen. DNA sequencing was performed using DTS cycle
sequencing kit and Applied Biosystems automated DNA
sequencer (Marchesi et al., 1998). Nucleotide sequences
were determined for both strands. Sequence obtained
was aligned and used for similarity comparisons using the
BLAST program (Altschul et al., 1990).

Production of biosurfactant using agro-industrial
wastes

In the present study, various solid and liquid wastes were
used as cheap substrates for biosurfactant production.
Different waste substrates such as used castor oil,
coconut oil, corn oil, motor oil, olive oil, olein, rapeseed
oil, sunflower oil, barley bran, cassava flour waste, peanut
cake, potato waste, rice bran and wheat bran were
amended in MSM medium by replacing glucose at a
concentration of 1 % in the final composition according to
Tahzibi et al. (2004) (g/L of distilled water: NaNO3, 15;
NaCl, 1.1; KCl, 1.1; KH2PO4, 3.4; K2HPO4, 4.4;
MgSO47H2O, 0.5; FeSO47H2O, 0.00028; yeast extract,
0.5; glucose 1%). The selected strain PB3A was then
inoculated into the medium amended with different
wastes substrates and kept in shaker incubator (100 rpm)
at 37 C. After 48 h of incubation, cell-free supernatant
was obtained by centrifuging the culture broth at 10,000
rpm for 20 min. The biosurfactant concentration in the
cell-free culture broth was estimated using orcinol assay
method (Tuleva et al., 2002). One hundred microliter of
each sample was mixed with 900 L of a 0.19% orcinol
solution (in 53% H2SO4) and heated at 80 C for 30 min.
The samples were then cooled to room temperature and
absorbance was measured at 421 nm and compared with
the standard curve prepared with L-rhamnose and
expressed as rhamnose equivalents (RE) (mg/mL).

Statistical analysis

The data represent the arithmetical averages of at least
three replicates. The results were represented as mean
value standard deviation (SD). The statistical analysis
was performed using MS office Excel 2007 for calculating
mean, standard deviation and standard error.

RESULTS AND DISCUSSIONS

Various bacterial isolates obtained from oil contaminated
soil were screened for the presence of biosurfactant
activities using blood hemolytic test, drop collapse test,
emulsification index (E24) and Surface tension according
to Satpute et al. (2008) who described the significance of
more than one screening method for the primary
screening of potential biosurfactant producers. Among the
different bacterial strain, PB3A showed hemolytic colonies
(Figure 1) on blood agar which was similar to the report
done by Mulligan et al. (1984). The strain also showed a
flat drop appearance (Figure 2) in the drop collapse, a
sensitive method to detect biosurfactant production as
suggested by Jain et al. (1991).
Mal. J. Microbiol. Vol 10(1) 2014, pp.57-62
59 ISSN (print): 1823-8262, ISSN (online): 2231-7538



Figure 1: PB3A showing hemolytic colonies on Blood
agar.



Figure 2: PB3A showing flat drop in (C) in drop collapse
test. A, Negative control; B, positive control.

Although the use hemolytic activity and drop collapse test
constitute an easy method to screen biosurfactant
production, determination of surface tension and
emulsification activity (E24) was performed to confirm the
biosurfactant producers (Plaza et al., 2011). Among the
isolates, the strain PB3A reduced the surface tension of
the culture medium up to 43 mN/m and also showed the
highest emulsification index of about 65.5% against crude
oil.
The efficient isolate PB3A was selected based on the
results of screening tests and identified using partial
sequence obtained from their 16S rRNA gene. The 16S
rRNA sequences were aligned and showed that the
isolate was Pseudomonas aeruginosa. The sequence
PB3A showed high similarity of more than 97% with P.
aeruginosa when compared with the other sequences
existing in the GenBank using BLAST search. The 16S
rRNA sequence was then deposited in the GenBank
database under the accession number of KF029593.1.
The results of morphological and biochemical tests of the
selected isolate PB3A is summarised in the Table 1. The
isolate PB3A was motile, Gram negative bacilli and
showed positive result for oxidase, citrate, urease and
gelatine liquefaction and negative for other tests. The
isolate was able to ferment mannitol, xylose and
galactose. The results of the morphological and
biochemical tests confirmed that the isolate PB3A was P.
aeruginosa (Holt et al., 1994).

Table 1: Morphological and biochemical characteristics of
the strain PB3A.

Tests Results
Gram stain
Motility +
Oxidase +
Catalase
Indole production
MR test
VP test
Citrate +
H2S
Gelatin liquefaction +
Mannitol fermentation +
Glucose fermentation
Xylose fermentation +
Maltose fermentation
Galactose fermentation +
Lactose fermentation
Sucrose fermentation
Nitrate reduction
Urea hydrolysis +

The carbon source present in the production medium
influences the bio surfactant production (Davis et al.,
1999; Adamczak and Bednarsk 2000). Patel and Desai
(1997) used whey wastes from dairy industries for the
microbial growth and biosurfactant production at
commercial scale against synthetic medium. Youssef et
al. (2004) also screened for various substrates such as
brewery effluents, molasses and fruit and vegetable
decoction for production of biosurfactants using Bacillus
strains.
The biosurfactant production depends upon the type
of carbon substrate present in the production medium
(Davis et al., 1999). In the present study, the various
agro-industrial wastes were used as carbon sources for
the biosurfactant production. Different waste substrates
such as used castor oil, coconut oil, corn oil, motor oil,
olive oil, olein, rapeseed oil, sunflower oil, barley bran,
cassava flour waste, peanut cake, potato waste, rice bran
and wheat bran were used as alternative source for
carbon in the biosurfactant production medium. Among
the cheap substrates used corn oil and cassava flour
waste shows the maximum biosurfactant production of
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60 ISSN (print): 1823-8262, ISSN (online): 2231-7538

0.62 mg/mL and 0.60 mg/mL respectively (Figures 3 and 4).



Figure 3: Effect of agro-industrial cheap substrates (used oils) on biosurfactant production.



Figure 4: Effect of agro-industrial cheap substrates (solid wastes) on biosurfactant production.
Similarly surface tension values of the used corn oil
and cassava flour waste amended medium after 48 h
showed 41 mN/m and 42 mN/m respectively which were
similar to the study conducted by Patel and Desai (1997)
and Dubey and Juwarkar (2004). Among the cheap
substrates, used Castor oil, motor oil, olive oil, olein, and
wheat bran showed lower amount of biosurfactant
production ranging from 0.28 mg/mL to 0.38 mg/mL
compared to coconut oil, rapeseed oil, sunflower oil,
barley bran, potato waste, peanut cake and rice bran
which showed significant production ranging from 0.42
mg/ml to 0.56 mg/mL. Sheppard and Mulligan (1987)
studied the utilization of peat hydrolyzate for the
production of biosurfactant production and Mercad et al.,
(1993) along with his co-workers also reported application
of olive oil mill effluent for production of rhamnolipid by
Pseudomonas sp. Manersa et al. (1991) studied the
production of rhamnolipids by P. aeruginosa using olive
oil as the carbon source in the production medium.
Similar studies were also reported by Rashedi et al.
(2006) and Raza et al. (2007) who have utilized molasses
as a cheap carbon substrate for the production of
biosurfactant. The present work on the application of
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61 ISSN (print): 1823-8262, ISSN (online): 2231-7538

cheap substrate for the biosurfactant production was in
agreement with the other studies reported in the literature.

CONCLUSION

The present study deals with application of industrial
wastes as substrates for the biosurfactant production.
Efforts have been made to use different cheap substrates
such as used oil and solid wastes for bio surfactant
production. This approach would be economic in
developing new strategies to increase the volume of
productivity. Among the waste cooking oils corn oil and in
case of solid wastes, cassava waste flour were suitable
substrates for biosurfactant production using P.
aeruginosa PB3A, a strain isolated from oil contaminated
soils. Further optimization and production strategy have to
be studied for the culture medium to increase the
productivity of biosurfactant. However, further research is
needed to develop biotechnological processes for
increase productivity using complex wastes with
nutritional potential for biosurfactant production.

ACKNOWLEDGEMENT
The authors are thankful to the Management, A.V.V.M. Sri
Pushpam College for their grateful support and
encouragement for the completion of work.

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