discussion - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/48014/5/5.pdf · 2018-07-08 ·...
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DISCUSSION
Biosurfactants are surface active molecules produced by microorganisms as
secondary metabolites. They are classified based on their chemical composition as
glycolipids, lipoaminoacids, lipopeptides, polymers etc. They havenumerous
advantages compared to chemically synthesized surfactants, such as low toxicity,
biodegradability, possess high specificity, ease of production, ability to be synthesized
from renewable substrates, high foaming, high selectivity, specific activity at extreme
temperature, pH, salinity and can be reused through regeneration too as compared to
synthetic surfactants.
On the other hand, they have high production costs due to low yields and
fastidious purification. In the present study, an attempt was made to develop the
economically attractive biosurfactant production process by using cheapest renewable
substrates from agro-industrial wastes, and optimized the bio-processes for obtaining
maximum productivity. An attempt was also made to synthesize silver nanoparticles in
water-in-oil microemulsion, stabilized by low cost biosurfactant synthesized using
cheapest renewable substrates. Further, application of silver nanoparticles in the
production of antimicrobial textiles was studied.
Mangrove ecosystem is a bridge between terrestrial and marine ecosystem
and harbours unique microbial diversity. Mangroves are the coastal wetland forests
generally found near the intertidal regions of estuaries between creeks, lagoons,
marshes etc. Mangroves provide a unique ecological site to different microbes. Because of
richness in carbon and other nutrients, mangrove ecosystem harbours diverse microbial
communities which can adapt themselves in the extreme conditions there.
In the present study, the biosurfactant producing bacteria were isolated from the
enriched mangrove soil sediments and rhizosphere soils. Totally 63 isolates were
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screened for the biosurfactant production. Among them, two promising isolates namely
PBSC1 and KBSB1 were selected for further works. The genera of the isolated 63
organisms were as follows: Bacillus (18), Escherichia coli (6), Klebsiella (5),
Lactobacillus (3), Proteus (6), Pseudomonas (21) and Staphylococcus aureus (4).
There are several reports on the biosurfactant producing microorganisms
isolated from mangrove sediments (Maneerat et al., 2006; Rodrigues et al., 2006;
Maneerat and Phetrong, 2007; Kebbouche et al., 2009; Anandaraj and Thivakaran,
2010; Gudina et al., 2010; Burgos et al., 2011; Darvishi et al., 2011).
Saimmai et al. (2012) collected 89 sediment soil samples from mangrove
environment, from the east and west coasts of southern Thailand, screened for the
biosurfactant producers collected by an enrichment culture technique. They isolated 95
isolates positive for biosurfactant production according to the qualitative drop-
collapsing test. The 95 isolates also showed promising biosurfactant activity by
exhibiting a surface tension reduction of pure water to 20mN/m.
Govindammal and Parthasarathi (2013) also isolated five strains from mangrove
ecosystem and selected the best biosurfactant producing organism Pseudomonas
fluorescens MFS03 for biosurfactant production using renewable substrates.
5.1. Screening of biosurfactant producers
Satpute et al., (2008) reported that the single screening method was not suitable
to identify all types of biosurfactants and hence recommended more than one screening
methods as to identify potential biosurfactant producers . Therefore, in the present
study, the selected isolates were performed with different screening test to check the
biosurfactant production ability and to find the efficient biosurfactant producer by
following the standard methods described by the earlier authors viz.,glass-slide test
(Persson and Molin, 1987), drop collapse test (Jain et al., 1991), CTAB plate assay
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(Siegmund and Wagner, 1991), cell surface hydrophobicity (Pruthi and Cameotra,
1997), emulsification activity (Makkar and Cameotra, 1998), hemolytic activity
(Yonebayashi et al., 2000), oil spreading technique (Morikawaet al., 2000), surface
tension measurement (Haba et al., 2000) and lipase activity (Kiran et al., 2009).
5.1.1. Hemolytic activity
The hemolytic activity was used as a primary method to screen the biosurfactant
production (Carrillo et al., 1996). Youseff et al., (2004) reported that some organisms
excluded the haemolytic activity, so other screening methods are followed for the
confirmation of biosurfactant production. In the present study, 34 (53.9 per cent) strains
were positive for hemolysis and eighteen isolates were showed partial lysis on the
blood agar plates (28.6 per cent). Remaining 11 isolates showed negative results, they
did not produce significant results.
The hemolytic activity of biosurfactants was first discovered when
Bernheimer and Avigad (1990) reported that the biosurfactant produced by
B. subtilis, surfactin, lysed red blood cells. Reason for using hemolytic assay in
this study as a criterion for biosurfactant production was because it is a widely
used method to screen biosurfactant production and in some reports it is the sole
method used to screen biosurfactant production (Banat, 1993; Yonebayashi et al.,
2000). Carrillo et al. (1996) found an association between hemolytic activity and
surfactant production and hence, it was recommended the use of blood agar lysis
as a primary method to screen biosurfactant production. None of the studies
reported in the literature mention the possibility of biosurfactant production
without a hemolytic activity (Carrillo et al., 1996; Moran et al., 2002; Youssef et al.,
2004; Afshar et al., 2008; Satpute et al., 2008; Walter et al., 2010). However, in some
studies hemolytic assay excluded many good biosurfactant producers and in some
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reports strains with positive hemolytic activity were found negative for
biosurfactant production (Youssef et al., 2004). In addition, not all biosurfactants have
hemolytic activity and compounds other than biosurfactants may cause hemolysis.
Dhail and Jasuja (2012) studied that hemolytic activity assay, oil displacement
activity and emulsification activity measurement was used to screen the biosurfactant
producer.
5.1.2. Drop collapse test
Among the 63 strains screened, 41 (65.1 per cent) strains were positive for drop
collapse activity. 12 isolates showed positive to hemolytic and negative to the drop
collapse test. The reason behind the negative drop collapse and positive hemolytic
results obtained with above 12 strains might be that some bacterial cells act as
biosurfactant themselves (Hommel, 1994) and have high cell hydrophobicity, but do
not produce extracellular biosurfactants.
In this experiment cell free culture broth was used as the biosurfactant source.
For strains which produce extracellular biosurfactant there was a drop collapse activity
and for strains which do not produce biosurfactant the results were negative, which also
inferred that to check the extracellular biosurfactant production of any microbial strain,
cell free culture broth should be used instead of using culture broth with cells. This
criterion will exclude microbial strains having high cell hydrophobicity and hemolytic
activity but no biosurfactant production. Accuracy and reliability of results obtained in
drop collapse assay, in this study, was similar to the results reported by Thavasi et al.
(2011c). Another merit associated with drop collapse assay is that the very low sample
volume is required for checking the drop collapse. To further confirm the biosurfactant
production of above strains with positive and negative results, cell free culture broth
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from all 63 bacterial strains was subjected to oil spreading and surface tension
measurement experiments.
5.1.3. Oil spreading assay
Oil spreading assay results were in corroboration with drop collapse assay
results. Strains found with positive drop collapse results were positive for oil spreading
assay also. These results confirmed the presence (for strains with positive results) and
absence (for strains with negative results) of surface active compound (biosurfactant) in
the cell free culture broth. Morikawa et al. (2000) reported that the area of oil
displacement in oil spreading assay is directly proportional to the concentration of the
biosurfactant in the solution. However, in this study there was no quantitative study
conducted on biosurfactant concentration versus oil spreading activity, but a qualitative
study to check the presence of biosurfactant in the cell free culture broth was in
concurrence with the above mentioned earlier report. As found in drop collapse assay,
13 strains showed no oil spreading activity and in total out of 63 strains, 40 (63.5 per
cent) strains were positive for the oil spreading assay. Similar results with drop collapse
and oil spreading assay was reported by Youssef et al. (2004) while screening bacteria
for biosurfactant production and also recommended that both drop collapse and oil
spreading assay methods as reliable techniques for testing biosurfactant production.
5.1.4. Emulsification Assay
Emulsification assay is an indirect method used to screen biosurfactant
production. It was assumed that if the cell free culture broth used in this assay contains
biosurfactant will emulsify the hydrocarbons present in the test solution. In this study,
crude oil was used as the hydrophobic substrate. Results observed in this study revealed
that from 63 strains screened, 45 (71.4 per cent) strains showed positive emulsification
activity. No emulsification activity was found with the following 18 (28.5 per cent)
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strains. Out of 18 strains fewer strains were positive for hemolytic and drop collapse
test but negative for emulsification assay. Thus, hemolytic and drop collapse assays are
not very reliable methods to test the biosurfactant production and which also inferred
that extracellular products other than biosurfactants are responsible for the positive
hemolytic and drop collapse activity observed with the strains showing negative
emulsification activity.
The biosurfactant produced by Aeromonas sp., LAMI005 showed high
emulsification index (E24> 50 per cent) on kerosene and soybean oil, but not
against gasoline. Most microbial surfactants are substrate specific, solubilizing or
emulsifying different hydrocarbons at different rates (Ilori et al., 2005). Best results
for emulsification index (E24 ) were obtained by using kerosene (67 per cent),
followed by soybean oil (64 per cent).
Das et al. (2008) reported that the biosurfactant production by marine
Bacillus circulans in glycerol mineral salt medium and antracene supplemented
glycerol mineral salt medium, which emulsified various hydrocarbons such as
diesel, hexadecane, kerosene, benzene and petrol in the range of 30–80 per cent .
Generally, low molecular weight biosurfactants cannot make stable
emulsions and used as flocculants while high molecular weight bio-surfactants
act as emulsion stabilizers (Sobrinho et al., 2008). Similar results were obtained by
other authors (Rocha et al., 2009), after 72 h of cultivation, 65 percent of
kerosene emulsification was obtained, indicating that this biosurfactant has an
emulsifying activity.
Formation of stable emulsion was observed with xylene, toluene, carbon
tetrachloride, dichloromethane and cotton seed oil. The product of starch
containing medium yielded maximum biosurfactant, high viscosity, enhanced
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reduction in the surface tension and high emulsification indices proving it the
best carbon source. It was reported that high viscosity enhanced the
emulsification abilities of hydrocarbons (Freitas et al., 2009).
Khopade et al. (2012) reported almost 80percent E24 against hydrocarbon,
within 8–9 days by biosurfactant produced by a marine Nocardiopsis sp. B4. In
our study higher emulsification was observed ( ≥50 per cent) with long chain
hydrocarbons such as crude oil,which could most probably play an essential role
in enhancing oil recovery.
The emulsifying activity was determined by its strength in retaining the
emulsion of hydrocarbons or oils in water. Cell free supernatant of starch
containing medium exhibited high emulsification indices in the range from 80 per
cent to 100per cent with promising organic solvents and oils screened (Jain et al.,
2012).
It was also reported that uronic acid and proteinaceous components of
biosurfactant play an important role in the emulsification, apart from functional
groups (acetyl) present in the biopolymer, which provide hydrophobicity which
imparts enhanced emulsifying activity (Bramhachari et al., 2007; Jain et al., 2012).
5.1.5. Surface tension measurements
The measurement of surface tension has traditionally been used to detect
biosurfactant production and most of the other methods that measure the surface
properties of biosurfactant use surface tension reduction as the standard (Willumsen
and Karlson, 1997; Makker and Cameotra, 1998). Surface tension measurement of cell
free culture broth revealed that out of 63 strains screened, 40 (63.5 per cent) strains
showed reduction in surface tension and maximum surface tension reduction were
observed with six strains. There was a direct correlation found between drop collapse,
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oil spreading and surface tension assays. Strains highly active in any one of these
methods were active in other two methods. Similar direct correlation between drop
collapse method and surface tension was reported by Bodour and Miller-Maier (1998)
and direct correlation between drop collapse and oil spreading method by Youssef et al.
(2004). Above earlier reports Morikawa et al. (2000); Batista et al. (2006); Thavasi et
al. (2011c) and results from this study indicated that drop collapse and oil spreading
assays are easy, reliable and sensitive methods to check the biosurfactant production.
As a confirmation, two strains from six potential strains showing positive activity in
drop collapse, oil spreading and surface tension assays were further identified for the
species level conformation.
5.1.6. Bacterial adherence to Hydrocarbond (BATH)
Rosenberg et al. (1980) developed a procedure to estimate the cell
hydrophobicity. Cell adherence to hydrophobic compounds like crude oil is
considered as an indirect method to screen bacteria for biosurfactant production,
because cells attach themselves with oil droplets by producing surface active
compounds called biosurfactants. Strains of Pseudomonas genus showed highest cell
adherence with crude oil than other bacterial strains screened, which is
complemented by other earlier reports on cell hydrophobicity and biosurfactant
production by Pseudomonas strains (Zhang and Miller, 1992; Deziel et al., 1999;
Tuleva et al., 2002). Visualization of bacterial cells adhered to crude oil confirmed the
affinity of cells towards crude oil droplets. In the present study, the maximum bacterial
adherence to the hydrocarbons i.e. hydrophobicity index was observed as 0.91 per cent
by PBSC1 followed by KBSB1 as 0.94 per cent. The lower in percentage of
hydrophobicity index indicated the higher affinity of cells towards hydrocarbons. The
present finding was supported by the earlier work of Liu et al. (2004).
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Bacterial strains with high cell hydrophobicity are reported as potential biosurfactant
producers (Tuleva et al., 2002; Volchenko et al., 2007) and in some reports, BATH
assay was used as a principle method to screen biosurfactant production (Volchenko
et al., 2007). Positive cell hydrophobicity was reported as an indication of biosurfactant
production (Franzetti et al., 2009a).
5.1.7. Cetyl Trimethyl Ammonium Bromide (CTAB)
The Cetyl Trimethyl Ammonium Bromide (CTAB) method is highly specific
for anionic surfactants; it cannot be used as a general method of screening for
biosurfactant producers (Siegmund and Wagner, 1991). On the other hand, the CTAB
method is used to differentiate the rhamnolipid producing and non-producing strains
while studying the P. aeruginosa fermentation samples (Pinzon and KwangJu, 2009).
Likewise in the present study also, 18 isolates showed positive and the rest of the
isolates showed negative results for the CTAB.
Methylene blue detection is one of the efficient methods to detect anionic
surfactants and the biosurfactants produced from microbes react with methylene blue
and form anionic surfactant ion pair (Siegmund and Wagner, 1991). This was migrated
into the chloro-form layer and confirmed the production of biosurfactants in the
production medium. Aparna et al. (2012) detected the di-rhamnolipid type of
biosurfactants from Pseudomonas sp. 2B using the CTAB-Methylene blue agar
medium based method.
5.2. 16S r RNA Sequencing for the identification of bacterial isolates
The most efficient bacterial strain was identified by studying the
morphological and physiological characteristics (Cappuccino and Sherman 1999)
and sequencing 16S r DNA. The characterization of morphological and
biochemical characters of the isolates PBSC1 and KBSB1 was studied according
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to the Bergey’s Manual of Determinative Bacteriology and the 16S r RNA sequencing
was examined to determine the precise taxonomic position of the strain and identified
as Pseudomonas aeruginosa PBSC1 and Bacillus cereus KBSB1. Pseudomonas sp. is
the most common producers of biosurfactants, isolated from the petroleum-
contaminated soil samples (Pornsunthorntawee et al., 2008; Price et al., 2009; Oliveria
et al., 2009; Singh et al., 2011; Aparna et al., 2012).
Biosurfactant producing bacterium was isolated from Tunisian soil and it was
identified as B. subtilis SPB1 (HQ392822) by morphological, biochemical and 16S
Ribosomal deoxyribonucleic acid (rDNA) sequence analysis (Ghribi et al., 2011).
Saikia et al. (2012) also studied the morphological and physiological patterns of
the strain showed similarity to P. aeruginosa (99 per cent). When partial 16S rDNA
gene sequence was aligned with the NCBI GenBank and RDP databases, 10 of the top
10 matches were to Pseudomonas aeruginosa strains.
5.3. Extraction of Biosurfactant
Recovery and/or purification of biotechnological products in downstream
processing costs usually account for approximately 60 per cent of the total production
costs which make commercial production of biosurfactantquite expensive. Methods to
reduce costs through the use of inexpensive and renewable substrates are, therefore,
necessary (Desai and Banat, 1997; Makkar and Cameotra, 1997; Banat et al., 2000).
However, a great deal of monetary input is required in the purification processes
(Rodrigues et al., 2006).
The most common biosurfactant recovery methods are either extracted with
solvents (chloroform-methanol, dichloromethane- methanol, butanol, ethyl acetate,
pentane, hexane, acetic acid, ether) or acid precipitation at low temperature. There are
several extraction methods for the recovery of biosurfactant including acid
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precipitation, solvent extraction, centrifugation (Banat et al., 2010). In the present
study, the acetone precipitation method was the best method for extracting
biosurfactant from KBSB1 and methanol: chloroform (2:1 ratio) was the best
combination to extract the biosurfactant from PBSC1.
5.4. Estimation of Macromolecules
Aparna et al. (2012) reported that Pseudomonas sp. 2B produced a glycolipid
which consisted of a mixture of lipid and carbohydrate combination of 65 per cent: 32
per cent (w/w) respectively.
Jain et al. (2013) reported that the sugars (total and reducing), uronic acid
and proteins were major constituents of the purified biosurfactants produced in
different carbon substrates. Earlier, it was reported that bacterial biosurfactants
were comprised of carbohydrates, uronic acids, proteins and sulphates (Parikh and
Madamwar, 2006; Bramhachari et al., 2007; Jain et al., 2012). Monosaccharide
composition analysis revealed heteropolysaccharide nature of the biosurfactants
including both hexose and pentose sugars in varying proportions.
The chemical composition analyses of the biosurfactant produced by
P. cepacia revealed the presence of 75 per cent lipids and 25 per cent
carbohydrates, suggesting once again the glycolipid nature of the compound, as
demonstrated by TLC. A minor fraction of protein was found in the samples,
likely resulting from remaining culturemedia co-precipitated with the biosurfactant
during the extraction process. According to the literature, most surfactants
produced by species of Pseudomonas are glycolipids in nature (Haba et al., 2000;
Monteiro et al., 2007; Silva et al., 2010).
Rufino et al. (2014) reported that the preliminary chemical characterization of
biosurfactant revealed that the examined agent was a lipoprotein material which
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consisted of protein (50 per cent), lipid (20 per cent) and carbohydrate (8 per cent).
Differently the emulsifier liposan produced by C.lipolytica grown in hexadecane as
substrate is composed of 83 per cent carbohydrate and 17 per cent of protein (Cirigliano
and Carman, 1985) and glycolipid produced by C.sphaerica consisted of 70 per cent
lipid and 15 per cent carbohydrate (Luna et al., 2013). Similar results were obtained in
our present study the isolate Bacillus cereus KBSB1 recorded 50.5 per cent protein,
10.3 per cent carbohydrate and 39.2 per cent lipid respectively. The isolate PBSC1
extract contain 68.26 µg/ml protein, 258.67µg/ml carbohydrate and 286.2 µg/ml lipid.
The study clearly revealed that the biosurfactant produced from PBSC1 was glycolipid
in nature and KBSB1 was lipopeptide.
5.5. Critical Micelle Concentration (CMC)
One of the most important properties of a surfactant is their spontaneous
aggregation in water and the formation of well-known structures such as spherical
micelles, cylinders, etc. the surface tension decreases gradually with increasing
surfactant concentrations. At a certain concentration called critical micelle
concentration (CMC), this decrease stops. Above the CMC, the surface tension remains
almost constant (Butt et al., 2004).
El-Sheshtawy and Doheim (2013) reported that the surface tension decreased
from 60 to 32 mN/m with small increases in the rhamnolipids concentrations up to 50
mg/l. Further the addition of rhamnolipids concentration had no effect until 70 mg/l.
This result was found to be in agreement with other workers like (Nitschke and Pastore,
2006; Pornsunthorntawee et al., 2008). Abbasi et al. (2013) demonstrated the surface
tension of distilled water decreased gradually with increasing biosurfactant
concentrations to 32.5 mN/m, with CMC values of 10.1 mg/l.
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Zhang and Miller (1992) reported that the concentration of biosurfactant
required to reach the CMC is typically between 1 and 200 mg/l, while the interfacial
tension (oil/water) is around 1 and 30 mN/m. surface active compounds can
reduce the surface tension of water to values around 27 - 37 mN/m and their
CMCs range from 15 to 180 mg/l. Purified surfactin (standard) is even more
efficient since its CMC could reach 7.8 mg/l.
Biosurfactants produced by P. aeruginosa strains were found to reduce the
surface tension of distilled water from 72 to 30 mN/m with CMCs in the range of
5 - 200 mg/l (Finnerty, 1994; Healy et al., 1996).
Variations in the values of CMC (13, 22 and 17 mg/l) for surfactin have
been described by other authors (Kikuchi and Hasumi, 2002; Carrillo et al., 2003;
Sen and Swaminathan, 2005).
Pornsunthorntawee et al. (2008) reported that Pseudomonas aeruginosa sp., the
extracted biosurfactant in the culture supernatant could decrease the surface tension of
distilled water from 72 to 28.3 mN/m and the CMC was estimated to be 120 mg/l. In
the present study the CMC for the isolated biosurfactant calculated from the breakpoint
of surface tension verses the log of its concentration curve was 80 mg/l for the
biosurfactantproduced by P. aeruginosa PBSC1 and 81.5 mg/l for biosurfactant
produced by B. cereus KBSB1 and the corresponding surface tension was 30.4 mN/m,
29.8 mN/m respectively.
5.6. Rhamnose test
The rhamnose test was positive for the isolate PBSC1 and negative for the
KBSB1 indicating that the isolate PBSC1 could produce a glycolipid type of
biosurfactant.
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5.7. Stability characterization
The morphology of biosurfactants can be significantly affected by changes in
pH, which in turn affects the degree of solubility enhancement. Previously, Shin et al.
(2006) have demonstrated that the effect of a rhamnolipidbiosurfactant on the surface
tension and dispersion of hydrocarbon was a function of pH.
For anionic biosurfactants, it has been shown that the presence of electrolytes
causes a decrease in CMC and therefore an increase in solubility of hydrocarbons
(Wang et al., 2007). When an electrolyte (NaCl) is added to the ionic biosurfactant
solution, it reduces the electrical repulsion between the ionic head groups, causing a
system net curvature and further alterations to micelle formation (Ochoa-Loza
et al., 2001). Addition of solvent such as n -butanol decreases interfacial tension
between biosurfactant solution and hydrocarbons (Xie et al., 2007)
The surface tension and the E24( per cent) activity were stable even at a high
temperature, in contrast to synthetic surfactants such as Sodium Dodecyl Sulphate,
which exhibits a significant loss of emulsification activity above 70°C (Kim et al.
1997). Similar findings were reported for P. aeruginosa isolate Bs20, which exhibited
excellent stability at high temperature (heating at 100°C for 1 h and autoclaving at
121°C for 10 min), salinities up to 6 per cent NaCl and pH values up to pH 13 (Abdel-
Mawgoud et al. 2009).
The special ionic strength tolerance offers the biosurfactants more suitability for
oil related applications, most of which are in highly saline conditions (Shaverdi et al.,
2011).
In the present study, when compared with pH and temperature, the sodium
chloride concentration did not produce any major differences in the emulsification
activity. Five per cent concentration showed the highest emulsification activity
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followed by 10 and 15 per cent. From these results, the biosurfactant was stable in the
different pH levels with various temperatures and various concentration of sodium
chloride. These results were similar for both P.aeruginosa PBSC1 and B. cereus
KBSB1.
Desai and Banat (1997) reported that heat treatment to some biosurfactants
caused no appreciable change in their properties, even after autoclaving at 120◦C
for 15 min. Similarly, Borodoloi and Konwar (2008) reported the biosurfactant
produced by Pseudomonas aeruginosa strains to be stable at temperature of
100◦C for different time periods of 5–60 min with respect to surface tension
changes. Joshi et al. (2008) reported that biosurfactants produced by four Bacillus
strains were stable at 80◦C for 9 days. Khopade et al. (2012) also reported the
stability of biosurfactants under extreme conditions of temperature. Al-Wahaibi et
al. (2014) reported that the biosurfactants produced from ‘CG’ medium or ‘MDM’
were also stable in pH range of 6–12 and salt concentration up to 5 per cent
NaCl. Under highly acidic pH (pH 2.0 and 4.0) biosurfactants showed much less
activity, since the biosurfactant is not soluble under highly acidic conditions and
tends to precipitate.
This higher instability of biosurfactants produced from some Lactobacilli
in acidic conditions was described by some researchers to be related to the
presence of negative charged groups at the polar ends of the molecules (Batista
et al., 2005). Several reports confirmed the stability of biosurfactant at different
pH values, mostly in the alkaline medium (Batista et al., 2006; Pornsunthorntawee
et al., 2008; Joshi et al., 2008; Al-Sulaimani et al., 2011; Darvishi et al., 2011;
Khopade et al., 2012). Bacillus B30 biosurfactant showed stability under various
extreme conditions as reported by other researchers.
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5.8. Thin Layer Chromatography
Sarafin et al. (2014) reported that the thin layer chromatography (TLC) data
revealed a single spot with Rf value of 0.65 under UV detection. Based on the Rf value,
the spot was concluded as a lipid moiety containing the compound of lipopeptides. This
preliminary result suggests that the partially purified biosurfactant produced by
K. marina BS-15 should contain a lipopeptide. Anyanwu et al. (2011) confirmed in
their studies, the TLC data with the R f value of 0.68 and 0.70 after iodine treatment as
lipopeptide. Donio et al. (2013) also confirmed that the biosurfactant extracted from
halophilic Bacillus BS-3 had the Rf value of 0.68 as lipopeptide type. Study conducted
by Vater et al. (2002) also substantiated one surfactant with the Rf values of 0.62 as
lipopeptide.
Silva et al. (2014) reported that the biosurfactant extracted from the cell-free
broth was analyzed using TLC and visualized with specific reagents. A spot
was produced with a retention factor (Rf) of 0.9, which demonstrated positive
reactions for sugars with Molish reagents and for lipids with iodine vapours, but
negative reactions for amino groups with ninhydrin. The presence of both
glycosyl units and lipid moieties on the same spot suggests that the sample was
a glycolipid. These results are similar to the profiles described for a biosurfactant
from Pseudomonas aeruginosa grown in glycerol, for which the Rf for
rhaminolipids was 0.85 (Silva et al., 2010).
Similar results were obtained in the present study. The biosurfactant produced
by P. aeruginosa PBSC1 resulted with a spot having Rf value of 0.81 corresponding to
a rhamnolipid. The biosurfactant produced by Bacillus cereus KBSB1 detected a spot
with iodine spray showed Rf value of 0.6 and 0.8 classified to a lipopeptide class.
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5.9. FTIR analysis of biosurfactant produced form PBSC1 and KBSB1
The FT-IR spectrum produced by the isolate Pseudomonas aeruginosa PBSC1
suggested that, the functional group present were of glycolipid type. The spectra were
recorded and analyzed using the standard methods described by the earlier authors (Lin
et al., 1994; Yin et al., 2008; Pornsunthorntawee et al., 2009). Thavasi et al. (2010)
reported that the biosurfactant produced by B. megaterium was classified as a
glycolipid with carbohydrate and lipid combination of 28:70 per cent. The FTIR
analysis of the biosurfactant revealed that, the most important bands were located at
2929 cm-1
(for the CH aliphatic stretching), 1700 cm-1
(for the C=O ester bond), 1066
cm-1
(PII band: polysaccharides) and 764, 699 cm-1
(for the CH2 group) and 3342 cm-1
(for O–H bonds) confirming the presence of glycolipid moieties. In addition, the mass
spectrometric analysis of the biosurfactant also confirmed the above results with peaks
observed at m/z = 326.5, 413.3, 429.3 for lipids and at 663.4 for carbohydrate moieties.
Rahman et al. (2010) studied the molecular structure of the rhamnolipids with
the help of FTIR spectroscopy. Strong and broad bands of the hydroxyl group free
(-OH) stretch due to hydrogen bonding were observed in the region (3368 cm-1
). The
presence of carboxylic acid functional group in the molecule was confirmed by the
bending of the hydroxyl (O-H) of medium intensity bands in the region of 1455-
1380cm-1
. The aliphatic bonds CH3, CH2 and C-H stretching with strong bands are
shown in region of 2925 -2856 and 1455-1380 cm–1
. The carbonyl (C=O) stretching
was found in the region of 173 7cm–1
with strong intensity bands. Two other strong
peaks between 1300 and 1033 in the region due to C-O stretch are characteristic
of an ester functional group in the molecule. The peak in the range of 1121–1033 cm−1
was also reported as C–O–C stretching in the rhamnose. Moreover, we noticed stronger
bands of pyranyl I sorption band in region at 918 – 940cm-1
and α- pyranyl II sorption
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band in region of 838 – 844 cm-1
that showed the presence of di-rhamnolipid in the
mixture.
Sriram et al. (2011) reported that the FTIR revealed the presence of carboxyl
group and peptide component in the biosurfacant. The compound showed the C-H
strtching vibrations in the transmittance range 2930cm-1
indicating the aliphatic chain.
The distinct peak values observed at 1540 cm-1
and 3420 cm-1
corresponded to the
deformed and strong N-H bond respectively. The transmittance at 1400 cm-1
referred to
the aliphatic chain of C-H group and he confirmed that the biosurfactant was
lipopeptide in nature.
Rikalovic et al. (2012) studied the IR spectrum of rhamnolipid from
P. aeruginosasan-ai organism. The study revealed that the fingerprint areas between
400–1500 cm–1
showed the deformation C–OH band at 1384 cm–1
, the O–H in plane
deformation at 1315 cm–1
, the O–C–O symmetric band at 1047 cm–1
, the C–O
stretching at 1168, 1127 and 1047 cm–1
, C–H deformations at 1451, 1238 and 808 cm–1
and CH3 rocking at 983 cm–1
for rhamnolipid. There are also the typical stretching
vibrations of the COO– group. The strong symmetric stretching C=O band of the
carboxylate group of rhamnolipid was at 1739 cm–1
. The IR spectra of rhamnolipid
gave absorption bands at 3360 cm−1
for symmetric O–H stretching. The spectrum also
showed vibrations at 2928 cm−1
and 2856 cm−1
typical for the C–H stretching
vibrations of CH2 and CH3 groups. The results are in a good agreement with a typical
IR spectrum of rhamnolipids.
The FT-IR spectrum produced by the isolate Bacillus cereus KBSB1 suggested
that, the functional group present were of lipopeptide type. Ismail et al. (2013)
observed the peaks are those commonly found in the IR spectra of lipopeptide
biosurfactants produced by several Bacillus species. The broad strong band in the range
240
of 3000 to 3700 cm-1
with a maximum at 3417 cm-1
represents –OH, –CH and –NH
stretching vibrations. This is characteristic of carbon-containing compounds with amino
groups. Another strong sharp band was observed at 1659 cm-1
, which signifies CO–N
stretching vibration. Moreover, absorption in the region 1600 -1700
cm-1
is characteristic for amide I vibrations in proteins, thus indicating the presence of
peptide groups in the biosurfactant. The present study also observed with the major
functional group related to the lipopetide biosurfactant and similar findings were
observed with the various authors (Donio et al., 2013; Saraffin et al., 2014; Al-Wahaibi
et al., 2014).
5.10. Factors influencing the biosurfactant production
5.10.1. Effect of Carbon source
The carbon source plays an important role in thebiosurfactant production (Itoh
and Suzuki, 1974). Glucose, fructose and sucrose lipids are formed by Arthrobacter
paraffineus and several species of Corynebacterium, Nocardia and Brevibacterium
during growth on the corresponding sugar (Suzuki et al., 1974). Slight differences in
the maximum cell biomass and biosurfactant production could be observed as the initial
glucose concentration increased above the optimum level (Guerra Santos et al., 1986).
Hydrocarbons added to the fermentation medium are known to induce the production of
biosurfactant (Bento and Gaylarde, 1996). The carbon source was found to affect the
cell mass to a great extent. As the biosurfactant is cell-wall associated, high cell density
is desirable (Bicca et al., 1999).
Several medium components influenced the formation of biosurfactant by the
cells. One of the goals of this investigation was to use the cheapest materials for
production. Hence, we studied different commercial oils as a carbon source instead of
n-Hexadecane, olive oil was considered as the best carbon source based on surface
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tension not on weight; also different concentrations of olive oil were studied and found
that 30 ml/l of olive oil was designed as the best concentration for biosurfactant
production. Biosurfactant production has been demonstrated in the presence of water-
soluble substrates, hydrocarbons and oils. The type of surfactant formed when growing
on these carbon sources can be influenced (Makkar and Cameotra, 1999; Duvnjak and
Kosaric, 1985; Robert et al., 1989).
A concentration of 11 g/l of rhamnolipids was found when P. aeruginosa UW-1
was grown in Canola oil (Sim et al., 1997) and isolate of P. aeruginosa DS10-129
produced 4.3 and 2.9 g/l of rhamnolipids using soybean and safflower oil, respectively
(Rahman et al., 2002). Rhamnolipid concentration of 4.9, 5.4 and 4.8 g/l when
sunflower, olive and soybean oils, respectively were used as carbon sources by
Pseudomonas aeruginosa LB1 (Benincasa et al., 2002). The carbon source, particularly
the carbohydrate, has a major effect on the type of glycolipids formed. The type of
carbon substrate used for production has been reported to influence both the quality and
quantity of biosurfactants (Abouseoud et al., 2008).
In the present study, among the different carbon sources tested, the isolate
B. cereus KBSB1 produced maximum biosurfactant using glucose as a sole carbon
source (5.23 g/l), followed by glycerol with 3.96 g/l. The maximum surface tension
recorded for the isolate was 31.32 mN/m when glucose was used as a carbon source.
The isolate P. aeruginosa PBSC1 utilized glycerol as a sole carbon source and
produced higher amount of biosurfactant 5.14 g/l with the highest surface tension
reduction ability and emulsification activity observed was 30.25 mN/m and 79.65 per
cent respectively by the isolate. The initial surface tension of the different carbon
sources substituted media were significantly reduced after 72 h revealing that all the
carbon sources supported biosurfactant production.
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P. aeruginosa can produce rhamnolipids from substrates including C11 and C12
alkanes, succinate, pyruvate, citrate, fructose, glycerol, olive oil, glucose and mannitol
(Robert et al., 1989).
Glucose as a source of carbon could be an important key to regulating
biosurfactant synthesis. There was much evidence on the importance of carbon and its
connection with the production of surface active compounds by microbes (Desai and
Banat, 1997).
It was suggested that both the lipogenic pathway and the formation of a sugar
would regulate a sugar lipid type of surfactant synthesized from a carbohydrate by
glycolytic metabolism. In the present study glucose and glycerol regulated the lipogenic
pathway and formation of sugar portion to lipopeptide and glycolipid type of
biosurfactant by P. aeruginosa PBSC1 and B. cereus KBSB1. The greater reduction of
surface tension and highest emulsification activity was recorded when the isolates were
grown on glucose. The production of biosurfactant, when grown in glucose was also
common with bacteria from the genus of Bacillus and Pseudomonas (Kluge et al.,
1989, Mata-Sandoval et al., 2000). The present study found supportive evidence from
the earlier reports (Daziel et al., 1996; Bodour et al., 2003; Das et al., 2009b; Xu et al.,
2012).
5.10.2. Effect of Nitrogen source
Reports have shown that rhamnolipid production is more efficient under
nitrogen-limiting conditions (Benincasa et al., 2002; Kim et al., 2006). The choice of
nitrogen source has been reported to affect the biosurfactant production (Abouseoud et
al., 2008).
Ammonium nitrate and yeast extract was the best nitrogen source and the
concentration 0.46 g/l ammonium nitrate and 0.2 g/l yeast extract were the best
243
concentration for biosurfactant production. The type of nitrogen present (Whether NH4
+, NO3-, urea oramino acid) influences the biosurfactant produced (Robert et al., 1989;
Duvnjak et al., 1982; Haba et al., 2000).
Interesting observations relate to the effect of nitrogen limitation that appears to
stimulate biosurfactant production and overproduction by some microorganisms
(Suzuki et al., 1974; Guerra-Santos et al., 1984). Arthrobacter paraffineus showed a
preference of ammonium salts and urea as the nitrogen source (Duvnjak et al., 1982).
Robert et al. (1989) while investigating rhamnolipid production by Pseudomonas 44Ti
on olive oil reported that sodium nitrate was the best nitrogen source. Similar results
have been noted for Pseudomonas aeruginosa (Ramana et al., 1989) and Candida
tropicalis IIP-4 (Singh et al., 1990). Maximum biosurfactant production by N. Amarae
was found after 14 days incubation time.
The nitrogen source in the medium influences the production of biosurfactant
(Desai et al., 1994). In the present study, the highest biomass production was obtained using
ammonium nitrate as the sole nitrogen source for isolates B. cereus KBSB1. The highest
biosurfactant production, surface tension reduction and emulsification activity were recorded
as 4.93 g/l , 30.08 mN/m and 79.32 per cent respectively when ammonium nitrate was used
as sole nitrogen source by the isolate KBSB1. Whereas sodium nitrate was found to be the
best source of nitrogen for the growth and biosurfactant production of isolate P. aeruginosa
PBSC1. At 144 h of growth the isolate PBSC1 recorded higher biomass (4.34 g/l),
biosurfactant production (4.96 g/l), maximum emulsification activity (78.52 per cent) and
better reduction in the surface tension (30.28 mN/m).
5.10.3. Effect of pH
The pH played an important role in affecting biosurfactant production through
their effect on cell growth and metabolic activity (Desai and Banat, 1997). The pH of
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7.0 has shown significant influence on cell biomass, production and activity of
biosurfactant by the isolates. The maximum cell dry biomass of 4.87 g/l was achieved
at a pH 7.0 by B. cereus KBSB1. The highest surface tension reduction and
emulsification activity were recorded at a pH 7.0 for B. cereus KBSB1 (29.45 mN/m,
78.34 per cent respectively). The highest biosurfactant production was recorded for the
isolate B. cereus KBSB1 was 5.32 g/l at pH 7.0 and in case of P. aeruginosa PBSC1
5.13 g/l at pH 7. The pH above 8.0 did not support the surface tension or emulsification
activity as reported by Guerra-Santos et al (1986). This might be due to the reason that
at higher pH, microbial metabolism could have been regulated for its survival and more
energy would be channelled for biomass production, thus, the reduction of biosurfactant
production occurred.
The controlled pH condition would cause accumulation of significant amounts
of organic acid from glucose catabolism throughout the fermentation period (Guerra-
Santos et al., 1986). This might result in alteration of membrane permeability of the cell
which could also have led to toxicity in relation with the accumulated organic acid.
Therefore, the microbial population of biosurfactant would be suppressed significantly
(Hommel and Ratledge, 1993).
The difference in surface tension reduction of a medium at various pH by the
selected isolate might be due to the initial pH and the highest reduction of surface and
interfacial tension observed in the culture at pH 7.0 was also in correlation with the
highest concentration of biosurfactant obtained. This observation was well supported by
the report of Hua et al. (2003).
Extremes of pH could possibly transform less surface-active species into more
active emulsifiers by denaturation of proteinaceous components or by increased
ionization. The effectiveness of liposan from C. lipolytica as an emulsifier was also
245
limited to the acid to neutral pH (Cirigliano and Carman, 1984) whereas the
emulsification activity of the biosurfactant produced by Bacillus subtillis was pH stable
(Makkar and Cameotra, 1998). Recently, the bioemulsifier Yansan from Yarrowiali
polytica cultivated on glucose as the substrate displayed a shallow maximum activity
between pH 5 and 7 (Amaral et al., 2006).
5.10.4. Effect of Temperature
Biosurfactants have gained numerous industrial and environmental applications
which frequently involve exposure to extreme conditions (Cameotra and Makkar,
1998). Likewise, similar work, as the present, was reported by Guerra-Santos et al.
(1986). The maximum rhamnolipid production by P. aeruginosa cultivated in mannitol
at 34.5ºC with a higher reduction at temperatures above 36ºC.
The extreme incubation temperatures also led to some changes in the microbial
metabolism as expressed by lower production of biosurfactant (Guera-Santos et al.,
1986). At lower temperature, the rate of protein/enzyme denaturation was negligible;
however cells were affected by the diffusional limitation of solutes such as substrates
into and within the cell (Scragg, 1988). As a result, the biomass and biosurfactant yield
changes at lower or higher temperatures than the optimum.
In the present study, the optimum temperature for the biosurfactant production
by KBSB1 and PBSC1 was found to be 30 °C (4.98 g/l and 5.12 g/l respectively). The
highest emulsification activity was measured as 78.98 per cent and 78.45 per cent for
the isolate KBSB1 and PBSC1 respectively at 30 °C temperature. This study supports
the previous work of Pornsunthorntawee et al. (2008).
5.10.5. Effect of Trace elements
In the present study, the effect of trace elements on the maximum biomass,
higher surface tension reduction and the maximum emulsification activity was observed
246
in the presence of all the three elements in the media composition. The highest
biosurfactant production recorded for the isolate B. cereus KBSB1 was 5.42 g/l, the
surface tension reduction was 30.13 mN/m with the treatment that contains all the
appropriate trace elements. The same trend was also noticed in the isolate
P. aeruginosa PBSC1.This result supports the report suggested by Wei et al. (2007),
that the trace elements Mg2+
, K+, Mn
2+and Fe
2+were found to be more significant
factors affecting surfactin production by B. subtilis.
The carbon, nitrogen sources and trace elements are found to play a crucial role
in the efficiency of biosurfactant production (Sen, 1997; Desai and Banat, 1997). Wei
and Chu (1998) recommended raising iron concentrations from the micromolar to the
millimolar level to greatly enhance the surfactin production from B. Subtilis ATCC
21332.
In particular, trace elements are shown to be extremely critical to biosurfactant
production. Earlier studies have shown supplementation of iron (Wei and Cha, 1998,
2004; Wei et al., 2003) and manganese (Wei and Chu, 2002) resulted in drastic
enhancement of surfactin production as observed in the present study.
5.10.6. Effect of Hydrocarbons
Petroleum hydrocarbons and vegetable oils have been used widely to improve
the production of biosurfactants and bioemulsifiers from microbial cultures (Banat,
1995; Randhir, 1999). Some bacteria use the petroleum hydrocarbons (liquid paraffin,
hexadecane, n-tetradecane and crude oil) as their sole source of carbon during the
production of cell wall associated biodemulsifier (Duvnjak and Kosaric, 1987; Huang,
2009).
In this study, rhamnolipid mixture efficiently emulsified n-hexadecane, up to 68
per cent suggesting that the addition of such rhamnolipids into a remediation process
247
may enhance the availability of the recalcitrant hydrocarbon (Banat, 1995). A similar
degree of emulsification of kerosene (74 per cent) and diesel (75 per cent) has been
reported by Wei et al. (2005). The rhamnolipid mixture produced by P. aeruginosa
AT10 emulsified 50 per cent and 100 per cent of the kerosene when added at
concentrations of 5 per cent and 15 per cent, respectively (Abalos et al., 2004). The
production of biosurfactant by a psychrophillic strain Arthrobacter protophormiae during the
growth on an immisible carbon source, n-hexadecane has been reported by Pruthi and
Cameotra (1997). These earlier reports strongly supported the present findings.
In the present study, the isolate KBSB1 when grown on the crude motor oil at
one per cent significantly influenced higher by cell biomass (5. 18 g/l), greater surface
tension reduction ability (31.25 mN/m) and maximum emulsification activity (75.86
per cent). The isolate P. aeruginosa PBSC1 when grown on the crude motor oil
enhanced the biosurfactant production (4.99 g/l) but produced statistically on par results
with the n-hexadecane (4.76 g/l). The present result showed that, the biosurfactant
could emulsify different hydrocarbons, which confirmed their applicability against
different hydrocarbon pollution such that it enhanced the availability of the recalcitrant
hydrocarbons (Banat, 1995; Maier and Soberon-Chavez, 2000).
5.11. Use of agro industrial wastes for the production of biosurfactant
The options to produce biosurfactants from cost-free or cost-credit substrates
like industrial and domestic wastes having an appropriate balance in the nutrient
content is the present need of the day. For this purpose the agroindustrial by products
(Robert et al., 1989; Mercade et al., 1996; Mercade and Manresa, 1994) and soil bean
oil wastes (Abalos et al., 2001). A variety of cheap raw materials, including plant-
derived oils, oil wastes, starchy substances and lactic whey, have been reported to
support biosurfactant production (Rahman et al., 2002b; Haba et al., 2000; Nitschke
248
et al., 2006; Dubey and Juwarkar, 2004; Parthasarathi and Sivakumaar, 2010).
Accordingly, in the present study, an attempt were made to synthesize biosurfactant
using agro industrial waste such as Cashew Apple Juice and Cassava Waste Water.
The commercial value product is the seed (cashew nut) and in India only 12 per
cent of the total peduncle is consumed “in natura” or processed industrially to produce
a wide variety of products from concentrated juice to desserts. Furthermore, the
majority of cashew apples rot in the soil (Azevedo and Rodrigues, 2000).
Rocha et al., (2007) reported cashew apple juice (CAJ) as a complex medium
for Acinetobacter calcoaceticus growth and production of biosurfactant. CAJ
supplemented with peptone in an adequate medium for growth and biosurfactant
production by P. aeruginosa (Rocha et al., 2007). The Cashew Apple Juice contained
21.00 g of total carbohydrate per 100 g of juice. The percentage of reducing sugars was
found to be 11.12 per cent and the non-reducing sugars was 0.37 per cent. The cashew
apple juice was rich in starch (8.34 per cent) and ascorbic acid (267 mg/100g). The pH of
the cashew apple juice was highly acidic. It also contained calcium, phosphorous and
iron. These results are in accordance with the previous works of Rocha et al. (2007).
The maximum biosurfactant was achieved with PBSC1 (9.14 g/l) at 72 h in cashew
apple juice as such incorporated medium. The surface tension reduction of cashew
apple juice medium was maximum in P. aeruginosa PBSC1 (31.33 mN/m).
Another prospective agroindustrial waste is cassava waste water. The
constituent analysis of cassava waste water revealed the presence of 35.34 g/l of total
carbohydrate and 14.24 g/l of reducing sugar. The pH of the cassava waste water was
found to be 4.6. Similar observations on the nutrient constituents were observed by the
earlier works (Sandrin et al., 1990; Lin, 1996). The moisture content was found to be
10.58 per cent, Total solids 14.35 g/l content and the pH is 4.6. The surface tension of
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the medium was reduced to 31.35 mN/m and the maximum level of biosurfactant (9.56 g/l)
was recorded with cassava waste water as medium. The presence of important nutrients in
adequate quantity in cassava waste water might be attributed as the reason for the
enhanced biosurfactant production by KBSB1 and PBSC1.
5.12. Response Surface Methodology for the optimization of biosurfactant
production from agroindustrial waste
The availability of raw materials for scaled-up production processes and
acceptable production economics has widened the scope of biosurfactants. Most of the
biosurfactants are produced from agricultural residues and from the industrial waste
products. The main problem related to use of alternative substrates as culture medium is
to find a waste with the right balance of nutrients that permits cell growth and product
accumulation (Makkar and Cameotra, 1999a).
A variety of cheap raw materials, including plant-derived oils, oil wastes,
starchy substances and lactic whey, have been reported to support biosurfactant
production (Rahman et al., 2002b; Haba et al., 2000; Nitschke et al., 2006; Dubey and
Juwarkar, 2004; Parthasarathi and Sivakumaar, 2010).
In statistical-based approaches, response surface methodology (RSM) has
beenextensively used in fermentation media optimization. RSM is a collection of
statistical techniques for designing experiments, building models, evaluating the effects
of factors and searching for the optimum conditions. It is a statistically designed
experimental protocol in which several factors are simultaneouslyvaried. In RSM, the
experimental responses to design of experiments (DOEs) are fitted to quadratic
function. The number of successful applications of RSM suggests that second-order
relation can reasonably approximate many of the fermentation systems.
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In the present study the Response Surface methodology results of P. aeruginosa
PBSC1 revealed that the R2
value of 0.9935 which was closer to 1 shows the model to
be stronger which can better predict the response and model could explain 99 per cent
of the variability in the reduction of surface tension. The ‘Pred R-Squared’ of 0.9641
was in reasonable agreement with the “Adj R-squared “ of 0.9875. During the RSM
experiment, nitrogen source i.e. sodium nitrate was found to be limiting nutrient for the
reduction of surface tension of the medium. Three dimensional response surface curves
were plotted to study the interaction of substrates on the surface tension reduction.The
maximum surface tension reduction predicted was 32.2 mN/m and the actual reduction
obtained with optimized medium condition i.e. glycerol 2.5 g/l , sodium nitrate
concentration 4.5 g, pH 7.0 at temperature 30 ºC, was 31.1 mN/m, which was a closer
agreement to be model prediction and from the response study, it was obvious that all
variables have a significant impact on the surface tension (ST) reduction.
In case of the isolate B. cereus KBSB1 the R2
value of 0.9975 which was closer
to 1 shows the model to be stronger which can better predict the response and model
could explain 99 per cent of the variability in the reduction of surface tension. The
‘Pred R-Squared’ of 0.9881 was in reasonable agreement with the “Adj R-squared “ of
0.9951. During the RSM experiment, nitrogen source i.e. ammonium nitrate was found
to be limiting nutrient for the reduction of surface tension of the medium. The elliptical
shape of the curve indicated good interaction of the two variables and circular shape
indicated no interaction between the variables. The elliptical nature of the contour in
graphs depicted that the mutual interactions of all the variables the similar reports were
studied by the following authors (Rodrigues et al., 2006; Desai et al., 2008; Mutalik et
al., 2008; Kiran et al., 2010; Najafi et al., 2011; Arutchelvi et al., 2011).
251
Abalos et al. (2002) reported the utilization of response surface methodology to
optimize the culture media for the production of rhamnolipids by Pseudomonas
aeruginosa AT10. Similarly, Joshi et al. (2007) also reported the statistical
optimization of medium components for the production of biosurfactant by Bacillus
licheniformis K51.
5.13. Biosurfactant mediated synthesis of Silver nanoparticles (SNPs)
Considering the need of greener bioprocess and novel enhancers for the
synthesis using microbial processes, biosurfactants and/or biosurfactant producing
microbes are emerging as an alternate source of rapid synthesis of nanoparticles (Xie et
al., 2006; Kasture et al., 2008; Reddy et al., 2009). A micro-emulsion technique using
oil–water–surfactant mixture was shown to be a promising approach for nanoparticles
synthesis (Xie et al., 2006). Although chemical surfactants are highly promising, these
chemicals could be toxic to the environment. Recently, the focus on biosurfactant-
mediated processes is steeply increasing due to their potential implications on the
synthesis of silver nanoparticles (Palanisamy and Raichur, 2009; Reddy et al., 2009).
Xie et al., (2006) reported that rhamnolipid biosurfactant could be used as a stabilized
agent for silver nanoparticles. In the present study, revealed the possibilities’ of using
glycolipid and lipopeptide mediated synthesis of silver nanoparticles would be effective
and advantageous over chemical surfactants.
The green synthesis of silver nanoparticles involves three main steps, which
must be evaluated based on green chemistry perspectives, including (1) selection of
solvent medium, (2) selection of environmentally benign reducing agent and (3)
selection of nontoxic substances for the silver nanoparticle stability (Barnickel et al.,
1992).
252
Micro-emulsion techniques using oil-water surfactant mixtures were shown to
be a promising approach for nanoparticle synthesis, as described by Xie et al. (2006);
Kasture et al. (2008) and Reddy et al. (2009). According to these literatures, in the
present study the silver nanoparticles were synthesized and stabilized.
5.14. SNPs synthesized using biosurfactant produced using agro industrial waste
as substrate
Kiran et al. (2010) studied glycolipid biosurfactant produced from sponge-
associated marine Brevibacteriumcasei MSA19 using the agro-industrial and industrial
waste as substrate to synthesize silver nanoparticles. In our present study the agro
industrial wastes such as Cashew Apple Juice and Cassava Waste Water was used to
synthesize biosurfactant from P. aeruginosa PBSC1 and B. cereus KBSB1 respectively.
The recovered biosurfactant was used to synthesize the silver nanoparticles by reverse
micelles method.
Farias et al. (2014) reported that the synthesis of silver nanoparticles from a
laboratory biosurfactant produced from agro-industrial waste are promising since the
majority of reports describing the use of biosurfactants in the synthesis of silver
nanoparticles are already published in the literature used commercial rhamnolipids.
5.15. UV spectroscopy
UV–visible absorption spectrum is sensitive to the formation of silver
nanoparticles because silver particles can show an intense absorption peak around 400
nm originating from the surface plasmon absorption of nanosized silver particles (Petit
et al., 1992; Barnickel et al., 1992; Huang et al., 1996; Kapoor, 1998; Ji et al., 1999).
Decrease in the intensity is due to a change in the free electron density. Particle
aggregation was studied with change in yields, a variation of the width and the red-shift
of the maximum in the absorption spectrum (Limin et al., 1999). Metal nanoparticles
253
have a surface Plasmon resonance absorption in the UV– visible region. This result
evidenced that the Nano-scale silver can be synthesized in reverse micelles using
glycolipid as stabilizer (Petit et al., 1992 Huang et al., 1996 Ji et al., 1999 Kitamoto et
al., 2002).This result indicates that the nano-scale silver can be synthesized in reverse
micelles using the low-cost biosurfactant as stabilizer. Decrease in the intensity is due
to a change in the free electron density.
Xu et al. (2006) studied that the UV–visible absorption spectrum of silver
nanoparticles in n-heptane. A strong absorption peak at approximately 406 nm
originates from the surface plasmon absorption of nanosized silver particles. Similar
results were recorded in our study with the absorption spectrum of 432 nm for the SNPs
synthesized using biosurfactant from PBSC1 and 405 nm for SNPs synthesized using
biosurfactant from KBSB1. The good symmetric absorption peak implies that the size
distribution of the nanoparticles is narrow (He et al., 2001). Xu et al. (2006) further
reported, to detect the stability of the 18-3(OH)-18-capped silver nanoparticles in n-
heptane at room temperature and in air ambient, the absorption spectra of the system
were re-recorded after 2 months. No obvious variation in the shape, position and
symmetry of the absorption peak is observed, which indicates that the asprepared silver
nanoparticles can remain stable for at least 2 months.
5.16. Dynamic Light Scattering (DLS)
The mean particle size observed in the DLS analysis was larger due to pH,
temperature, light scattering etc., when a pH starts decreasing in a solution, the
significant part of nanoparticles starts precipitating and aggregating, which may
contribute to increase the particle size mean value measured by DLS.
The other reason for the increased mean particle size was the light scatter from
bigger SNPs was so intense that the scatter light coming from smaller SNPs was
254
concealed. Consequently it is not possible to detect the signal coming from 95 per cent
of smaller SNPs in the presence of 5per cent bigger SNPs (Poda et al., 2010;
Tomaszewska et al., 2013; Jannathul and Lalitha, 2013).
Temperature also played a vital role in the average particle size of the prepared
nanoparticles. For the range of temperatures under consideration, at lower temperatures
the particles are smaller and with increasing temperatures, the average particle size goes
through a maximum and becomes smaller again towards higher temperature. As the
reaction rate increases the silver ions are consumed faster thus leaving less possibility
for particle size growth and hence smaller particles and narrower size distributions at
higher temperatures (Patel et al., 2008).
5.17. High Resolution Transmission Electron Microscopy (HR-TEM)
The typical TEM micrographs of the silver nanoparticles (Limin et al., 1999;
Lin et al., 2001) were obtained in this study. This indicates that the distribution of silver
nanoparti-cles stabilized by rhamnolipid is rather uniform. However, some larger
particles on the films are observed. Two possibilities are concerned. One is that the
nanometer-sized water layers limit the packing of the particles in the direction
perpendicular to the water layers when the particles are growing in reverse micelles, the
absorption of surfactant molecules cannot totally prevent particles from aggregating
and the thickness of the water layers cannot absolutely restrict the particle size due to
the flexibility of the surfactant bilayers (Limin et al., 1999). The other is that during the
extraction and redispersion process a part of particles impact each other and
aggregation.
The structure of the biosurfactant plays an important role in determining the
morphology of the synthesized nanoparticles. These micelles are spherical in shape and
favoured the formation of spherical nanoparticles during synthesis. As biosurfactants
255
are natural surfactants derived from microbial origin composed mostly of sugars and
fatty acids moieties, they have higher biodegradability, lower toxicity and excellent
biological activities. Since the biosurfactants reduce the formation of aggregates due to
electrostatic force of attraction they facilitate uniform morphology and stability of
nanoparticles (Xie et al., 2006)
Some larger particles on the films are alsoobserved. Two possibilities are of
concern. One is that the nanometer-sized water layers limit the packing of the particles
in the direction perpendicular to the water layers when the particles are growing in
reverse micelles, the absorption of surfactant molecules cannot totally prevent particles
from aggregating and the thickness of the water layers cannot absolutely restrict the
particle size due to the flexibility of the surfactant bilayers (Kiran et al., 2010). The
other is that during the extraction and re-dispersion process many particles impact each
other promoting aggregation between them. The stability of silver nanoparticles
synthesized through the biosurfactant, were stable for 3 months. The biosurfactant
would have acted as stabilizing agent and prevented the formation of aggregates and
favoured the production and stability of the nanoparticles under the experimental
conditions (Farias et al., 2014).
5.18. X-ray Diffraction (XRD)
There were five well-defined characteristic diffraction peaks at 38.3°,
44.5°, 64.7°, 77.6° and 81.8°, respectively, corresponding to (111), (200), (220),
(311) and (222) planes of face centred cubic (fcc) crystal structure of metallic
silver. Theinterplanar spacing values (dh k l ) values (2.348, 2.034, 1.439, 1.229
and 1.176 ˚A) and the lattice constant (4.065 ˚A) calculated from the XRD
spectrum of silver nanoparticles are in agreement with the standard silver values
(JCPDS PDF card 04-0783). It is clear that for the synthesized silver
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nanoparticles the (111) lattice plane is the preferred orientation which is also
known for their high antibacterial activity (Kora et al., 2012).
In our study, SNPs synthesized using biosurfactant form P. aeruginosa PBSC1
Showed well defined peaks around 23.45°, 29.1°, 32.07°, (110, 111, 200) and the SNPs
synthesized using biosurfactant form B. cereus KBSB1 23.05° , 26.2° 20° belonged to
(110, 111, 110) plane was found to be the prominent peak which showed the material
was more oriented towards that plane. The peaks observed for various angles strongly
possessed the anatase formation. Similar results were reported by
El-Shanshoury et al. (2011) and Nagajyothi and Lee (2011).
5.19. Stability studies of silver nanoparticles
Xie et al. (2006) reported that on increasing the time from 1 to 60 days, the
Plasmon absorption bands are quite similar. They have no obvious changes in the
position and symmetry of the absorption peak except for the decrease of the
absorbance, indicating a little aggregation of silver nanoparticles upon storage. The
silver nanoparticles solution prepared in reverse micelles can remain relatively stable
for at least 2 months. The remnant rhamnolipid in the solution is regarded as the
stabilizer, which form a steric hindrance around the particles to preventing them
aggregation greatly by electrostatic interactions.
Kiran et al. (2010) used a glycolipid biosurfactant produced from sponge-
associated marine Brevibacterium casei MSA19 synthesized silver nanopartilcles were
uniform and stable for 2 months.
Farias et al. (2014) reported that the silver nanoparticles solution prepared in
such proportional reverse micelles can remain relatively stable for at least three months.
Similar results were obtained in the present study that the silver nanoparticles were
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stable for 2 months in the solution, hence it was proved that the biosurfactant act as a
stabilizing agent and prevented the formation of aggregates.
5.20. Minimum Inhibitory Concentration and Minimum Bactericidal
Concentration of silver nanoparticles
Antibacterial activity of silver nanoparticles has been demonstrated in several
investigations, but the reported MIC values range through a wide extent of variation.
Hence, it is difficult to compare their results, because there is no standard protocol for
evaluation of antimicrobial activity of nanoparticles and different methods have been
used by researchers. In the present study, silver nanoparticles showed good antibacterial
activity against the tested pathogens. The results of MIC and MBC tests revealed a
higher MIC and MBC values were recorded for S. aureus comparing to the E. coli. This
may be due to the differences in bacterial cell walls, since Gram negative bacteria have
thinner cell wall comparing to Gram positive bacteria (Rai et al., 2009).
Silver nanoparticles with size of 1-10 nm have been reported to be most effective
against bacteria through direct interaction with bacterial cells (Morones et al., 2005). In
agreement, Kim et al. (2007) reported that S. aureus was more resistant against
nanosilver than Gram negative E. coli.
Pal et al. (2007) found that interaction of nanoparticles with E. coli was shape-
dependent, since truncated triangular particles showed higher activity compared to
spherical and rod spherical particles. Results of silver nanoparticles exhibited more
growth inhibitory against E. coli bacterium (50 per cent) compared with S. aureus. This
difference suggests that the antimicrobial effects of Ag nanoparticles can be associated
with characteristics of certain bacterial species.
However, in our study, the MBC values were higher for S. aureus for both the
silver nanoparticles tested. It has been previously stated that bactericidal property of
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nanoparticles is dependent on the concentration and size of nanoparticles and also the
initial bacterial concentration (Ruparelia et al., 2008).
5.21. Silver nanoparticles coated textiles
Many works involving silver nanoparticles have been reported to enhance anti-
bacterial activity of textile fabrics. Duran et al. (2007) incorporated silver nanoparticles
synthesized by fungi on cotton fabrics and demonstrated that they show good
antibacterial activity against Staphylococcus aureus.
Perelshtein et al. (2008) deposited silver nanoparticles onto the surface of
different fabrics (nylon, polyester and cotton) by ultrasound irradiation and they
demonstrated that coated fabrics with nanosilver as an antibacterial agent had excellent
antibacterial activity against Escherichia coli and S. aureus. Similarly, in our study, the
silver coated fabrics showed excellent antibacterial activity against E. coli and
S. aureus.
The antibacterial efficacy of nanosized silver colloidal solutions on cellulose
based and synthetic fabrics for S. aureus and Klebsiella pneumoniae was investigated
by Lee et al. (2003). They found that the antibacterial treatment of the textile fabrics
was easily achieved by padding them with nanosized silver colloidal solution and the
antibacterial activity of the fabrics was maintained after many cycles of laundering.
However, so far no method has been developed to give permanent antibacterial activity
to the surfaces using silver or silver derivatives. To overcome this problem and develop
an efficient method or at least to extend the permanency, many methods using silver
nanoparticles have been proposed including pre-treatment of the textile surface,
embedding nanoparticles on the fiber polymeric matrix and coating the surface with a
thin film of polymer containing nanoparticles and even in situ production of silver
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nanoparticles on cotton fabrics (Lee et al., 2005; Ibrahim et al., 2008; Dastjerdi et al.,
2009; El-Shishtawy et al., 2011).
Ibrahim et al. (2008) and Dastjerdi et al. (2009) were coated the antibacterial
agents using trimethylol melamine (TMM) and polysiloxanecrosslinkers on a cotton
surface respectively.
Gulrajani et al. (2008) used poly (vinyl pyrrolidone), PVP, to stabilize silver
nanoparticles during the synthesis, which were then applied to a silk fabric surface by
the exhaust method. Further, they reported the antibacterial activity against the Gram-
positive bacterium S. aureus on silk fabrics as well as the durability to washing.
The others examples of enhanced antibacterial activity are connected with
surface modification of the fabrics and subsequent coating by silver nanoparticle sols
synthesized mostly by the sol–gel technique (Tarimala et al., 2006; Ilic et al., 2009;
Xing et al., 2007).
Many possible mechanisms have been proposed to describe the antibacterial
activity of silver nanoparticles, including attachment to the cell membrane leading to
decreasing membrane permeability and respiration and activity in the cell (Russel and
Hugo, 1994; Morones et al., 2005; Maneerung et al., 2008; Ravindra et al., 2010).
When elemental silver nanoparticles are in contact with water or dissolved
oxygen, silver ions are released from the surface of nanoparticles (Hoskins et al.,
2002).The silver ions might also catalyze the production of oxygen radicals, resulting in
oxidation of the molecular structure of the living organism (Percival et al., 2005). The
radicals formed due to the binding of silver ions to the cell wall and enzyme proteins
might also inhibit many processes in living cells (Percival et al., 2005). Thus,
developing a matrix providing a controlled release of silver ions is of great importance
for long-term antibacterial activity
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According to Dastjerdi et al. (2009&2010) silver ions might destroy and/or pass
through the cell membrane and bond to the –SH groups of cellular enzymes. This
causes a critical decrease in enzymatic activity which might change microorganism
metabolism and inhibit their growth, lead to the death of the cell.The general consensus
is that the antibacterial activity is due to silver ions released from silver nanoparticles
(Maneerung et al., 2008; Ravindra et al., 2010).
Silver ions shows antibacterial activity even at a concentration of 10-7
g/l,
therefore the determination of the number of nanoparticles per unit area and the size of
silver nanoparticles in contact with water at this concentration improved antibacterial
surfaces (Budama et al., 2013).
5.22. Finishing methodology for antimicrobial textiles
Ramachandran et al. (2004) worked on different procedures for the industrial
applications of silver nanoparticles onto the fabrics and one such method tried by them
was pad-dry cure method. Their results demonstrated that the higher the drying
temperature in padding, the better antibacterial properties with 100 per cent bacterial
reduction. This effect could possibly be due to the higher thermal energy that each
particle received during drying at higher temperature causing deeper penetration of
silver nanoparticles inside the cotton fiber with better durability. In addition, it was
possible that, at higher temperatures, the chemical structure of the dispersing agent used
in colloidal solution of silver nanoparticles was decomposed in which the dispersing
agent acted as a surfactant that could help washing off the deposited nanoparticles. Also
due to the removal of the dispersing agent, this could possibly cause better contact
between the nanoparticles and the bacteria with subsequent higher antibacterial
efficiency, because these particles were only effective when they come into contact
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with the microorganisms. Yadav et al. (2006) explained that the pad-dry cure coating
on cotton fabrics resulted in uniform and very thin coating.
The importance of padding has also been detailed by Hong and Sun (2008) and
according to them padding was the most common finishing method for application of
chemical formulation to textile materials in continuous processes and padding consists
of contacting the textile material with the formulation, usually by immersion and
squeezing the formulation out with squeeze rolls. In a research work reported by Anita
et al. (2010), the cotton fabrics coated with copper oxide nanoparticles by pad-dry cure
method exhibited a bacterial reduction of 100 per cent against the test organism E.coli.
Rajendran et al. (2011) coated the fabrics with herbal nanoparticles by pad-dry
cure method and confirmed that the pad-dry cure was efficient for nanoparticles coating
onto the cotton fabrics. In their study herbal plants such as Curcuma longa and
Daturametel were selected and bioactive compounds were extracted and standardized.
Nanoparticles of the medicinal plant extracts were prepared by coacervation method
using bovine serum albumin, cross-linked with gluteraldehyde and finished on100 per
cent pure cotton by pad-dry-cure method.The importance of pad-dry cure process for
the application of nanoparticles onto the cotton fabrics was explained by Khoddami
et al. (2011).
Studying the results of durability indicated that laundering and abrasion
decreased the samples antimicrobial properties. During dip-dry process, the silver
nanoparticles were just being physically absorbed and kept among fibers; therefore, the
durability was not high enough against laundering and abrasion. The results also
showed that wash fastness was better than abrasion fastness due to the sensitivity of the
deposited nanoparticles on the fibers surface to high level of mechanical action. Poor
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wash and abrasion fastness led the authors to pad dry cure procedures having long
lasting antibacterial effect (Khoddami et al., 2013).
From the results obtained, it was evident that, the fabrics finished by pad-dry
cure method demonstrated good degree of antimicrobial activity with enhanced wash
durability when compared to the other two finishing methodologies namely dip dry
method and exhaustion method. Considering the advantages, pad-dry-cure method was
selected in the present study for coating the cotton fabrics with silver nanoparticles and
further optimization studies were carried out with pad-dry-cure method.
5.23. Antifungal activity of silver nanoparticles coated fabrics
It was very clear from the results that the silver naoparticles treatment was
found to enhance the resistance of cotton towards fungal attack when measured in terms
of loss in breaking load and damage of fibres due to soil burial. Chattopadhyay and
Patel (2010) explored the antifungal activity of nanosized colloidal copper on cotton
fabric by soil burial method. They found that the breaking load of untreated control
samples were drastically reduced due to bacterial damage during soil burial test
whereas copper nanoparticle treated sample could not only protect the sample against
bacterial attack but also improved its strength. Simoncic and Tomsic (2010)
investigated on the influence of antimicrobial activity of two contemporary finishes,
specifically a dispersion of colloidal silver (Ag) and 3-(trimethoxysilyl)-
propyldimethyloctadecyl ammonium chloride (Si-QAC), on the degree of
biodeterioration of 100 per cent cotton (CO) fabric and fabric composed of a mixture of
cotton and polyester (CO/PET) by soil burial test after 3, 6 and 12 days of exposure to
soil microflora. Their results reflected the impairment of the mechanical properties of
the fibres due to hydrolytic and oxidative damage during degradation by soil bacteria
and fungi. They proved that the presence of the antimicrobial agent significantly
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increased the breaking strength of the fabrics and the results of their study correlated
with the results of present study in all the antifungal testing methods.
5.24. Wash durability
Wash fastness can be further improved with the formation of covalent bonding
between nanoparticles and the fabrics surface. In these cases the excellent UV blocking
properties are still maintained after fifty five home laundering (Daoud and Xin 2004).
Vigneshwaran et al. (2006) demonstrated wash fastness is a particular
requirement for textile and it was strongly correlated with the nanoparticles adhesion to
the fibers. In order to increase the wash fastness, the nanoparticles can be applied by
dipping the fabrics in a solution containing a specific binder.
The wash durability of the silver nanoparticle coated cotton fabrics was
demonstrated by Raja and Thilagavathi (2009) and they confirmed that the nanoparticle
coating of fabrics retained the antimicrobial activity up to thirty five washing cycles.
The durability of the effect of the self-assembled multilayer films on the cotton
fabric functional properties was analysed after ten andtwenty washing cycles at 40 °C
for 30 min and the results proved that the nanoparticles were durable up to 20 wash
cycles (Ugur et al., 2010). In the present study also, the antibacterial activity was
retained up to 30 washing cycles by both the silver nanoparticles coated fabrics.
5.25. Physical characterization of treated fabrics
Since the actual damage to human skin from UV radiation is a function of
wavelength, with most of the damage done by radiation in the range of 300 to 320 nm,
fabrics must demonstrate effectiveness in these ranges (Schindler and Hauser, 2004).
High UV absorption as a result of dark brown colour of the silver nanoparticles coated
fabric could be a reason for such high UV-blocking (Gorensek and Recelj, 2007).
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The results revealed that the obtained silver nanoparticles fabric could provide
excellent UV-blocking in the mentioned range and also in the UV-A range. The UPF
rating indicates how effective a fabric is at blocking out solar ultraviolet radiation and,
the higher the UPF value, the better the protection of garment would be.
Typically, fabrics with UPF value of more than 40 are considered as providing
excellent protection against UV radiation (Khalilabad et al., 2013). In our present
study, the UPF 31.4 was recorded for silver nanoparticles synthesized using
biosurfactant from P. aeruginosa PBSC1.
Coating on the fibers is composed of silver crystals which imparts high
conductivity to thetextiles with electric resistance as low as 37.0 Ω ± 1.8 Ω
measured using a multimeter. However, in the case of the original textile, the
resistance is infinity due to its insulation (Xue et al., 2012). It was clear from the
present study that the surface resistivity was the highest in the untreated control fabrics
(1.3 x 109
Ω/square). The surface resistivity was the lowest for the fabrics treated with
silver nanoparticles synthesized using biosurfactant from P. aeruginosa PBSC1
(1.1 x108
Ω/square).
The present study is an attempt to synthesize and stabilize the silver
nanoparticles using biosurfactant produced from economically cheaper agro industrial
wastes. Biosurfactant mediated synthesis of silver nanoparticles were coated on to the
cotton fabrics which acted as an excellent antibacterial, antifungal, UV blocking and
with good resistivity properties.