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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.

234

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

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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

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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

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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

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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.