biosurfactants are amphiphilic molecules which have the ability to depict a wide variety of surface activity .
they comprise both a hydrophobic and a hydrophilic group that aid in its accumulation between fluid phases .
because of this property , they allow easy accessibility to nonpolar hydrocarbons so that microorganisms in oil - rich ecological niches can easily degrade them .
a number of high molecular weight biosurfactants and bioemulsifiers are produced by both bacteria and fungi .
biosurfactants of bacterial origin belong to most classes of compounds including polysaccharides , proteins , lipopolysaccharides , lipoproteins and combinations of many of these structural types .
almost all classes of microorganisms ( table 1 ) produce biosurfactants ( finnerty   and healy et al . ) .   owing to their xenobiotic nature , synthetic surfactants have the potential disadvantage of persisting in the environment , long after they are applied for a remedial measure .
also , some of the synthetic surfactants are comparatively more toxic to human health ( dehghan - noudeh et al . ) .
as biosurfactants are of microbial origin , they have been under the active scrutiny of researchers for more than a decade .
biosurfactants have the potential to be considered as a viable alternative to the chemically synthesized surfactants for environmental cleanup . according to habe and omori ,
the biological treatment of pah - contaminated soil should be an economically viable and efficient process .
the biological approach has a lot of advantages including complete degradation of the pollutants , lower treatment cost , greater safety , and lesser soil disturbance .
kosaric   enlisted an exhaustive list of advantages in favour of biosurfactants : biodegradability , low toxicity , biocompatibility and digestibility , availability of raw materials for production , acceptable production economics , environmental control , specificity , and effectiveness .
biodegradation of the hydrocarbons at a specific contaminated site depends on the indigenous soil microbial population , type and concentration of hydrocarbons present , soil characteristics , and availability of nutrient and oxygen .
the genera of soil microorganisms that are known to degrade hydrocarbons include pseudomonas , flavobacterium , achromobacter , arthrobacter , micrococcus , and acinetobacter ( kosaric ) .
the present work attempts to investigate the use of a novel biosurfactant - producing bacterial strain , isolated from oil - contaminated sites in manipal ( karnataka , india ) for enhanced oil recovery operations .
oil - soaked soil samples were collected from a local automobile workshop in manipal ( karnataka , india ) .
the samples were enriched by inoculating 1  g of the soil sample into 50  ml of sterile bushnell haas broth ( himedia , mumbai ) , taken in a 250  ml conical flask at 30c in a shaker incubator ( rotek , india ) , & set at 150  rpm .
the medium was also constituted with filter - sterilized 1% ( v / v ) n - hexadecane as the sole carbon source . serial dilution of the sample was performed after 48  h of incubation and plated onto sterile bushnell haas agar plates .
after incubation for 48  h at 30c , morphologically distinct colonies were reisolated by transfer to fresh agar plates thrice to obtain pure cultures .
the chosen isolates were further screened for the production of biosurfactants using multiple screening methods .
48-hour - old cultures of the isolates grown in bushnell haas broth were taken to perform the screening tests .
all the screening tests were performed in triplicate . in the oil spreading technique developed by morikawa et al .
, 30  ml of distilled water was taken in a petri dish to which 1  ml of coconut / sesame oil was added to the centre .
20  l of the culture supernatant from the broth was added on top of the oil layer .
the petri dishes were closely observed for a zone of displacement in the oil , and the diameter of displacement was measured .
blood agar hemolysis test was performed to check the hemolytic activity of the microbial isolates , as described by mulligan et al . .
the isolates were streaked onto sheep blood agar plates ( himedia , mumbai ) in a sterile environment .
the bacterial colonies were visually examined for the presence of clear zones around the streaks .
the extent of clearing was classified into 4 categories , as described by rodrigues et al . : no hemolysis ; incomplete to partial hemolysis with a clearing < 1  cm ; complete hemolysis with a clearing > 1  cm but <3  cm ; complete hemolysis with a clearing > 3  cm .  
sterile glass slides were coated with commercially available engine oil ( sg sae 20w-40 grade ) and fully covered to allow equilibration for 24 hours at room temperature .
0.01  ml of the culture supernatant was dropped on the surface of the equilibrated glass slides .
the shape of the drops was observed for activity of the culture supernatant on the oil after an hour .
depending on the concentration of the crude biosurfactant , the drop collapses to varying degrees .
the extent of drop collapse was assessed as follows : no collapse ; partial collapse if diameter after collapse < 1  cm ; near complete collapse if diameter after collapse > 1  cm and < 1.5  cm ; complete collapse if diameter after collapse > 1.5  cm .
ctab agar plate test , developed by siegmund and wagner , was performed for detection of anionic surfactants .
mineral salts agar ( himedia , mumbai ) was supplemented with 2% ( w / v ) glucose as carbon source , 0.5  mg / ml cetyltrimethylammonium bromide ( himedia , mumbai ) , and 0.2  mg / ml methylene blue ( himedia , mumbai ) as performed by satpute et al . .
a well was punctured into the plate using a sterile cork borer and filled with 50  l of the culture supernatant .
the plates were incubated for 4872 hours at 30c and observed for the appearance of bluish / greenish halos around the wells to imply biosurfactant production . in the tilting glass slide test , developed by persson and molin ,
a single colony is picked up from the bushnell haas agar plate and transferred on the surface of a sterile glass slide near one of the edges .
the slide is gradually tilted to the other side and was examined for flow of a water droplet over its surface .
the surface tension of the 48-hour - old culture broth was measured using a digital surface tensiometer ( described in section 2.7.2 ) .
the microbial isolate rt10 was identified based on its morphological and biochemical characteristics as per bergey 's manual of determinative bacteriology .
gene sequencing ( 16s rrna method ) was performed at agharkar research institute ( pune , india ) to identify the bacterial strain .
genomic dna was isolated from the culture by using a commercial kit ( genelute bacterial genomic dna kit , sigma , usa ) .
a polymerase chain reaction was carried out using the universal primers for 1.5  kb fragment amplification for eubacteria .
the 20  l master mix for the pcr was composed of 3  l of template dna ( 10  ng ) , 2  l each of 200  m dntp mix and 10x pcr buffer , 0.4  l each of forward and reverse primers , and 0.2  l of taq dna polymerase ( bangalore genei , bangalore ) in 12  l of double distilled water .
the pcr was performed using gradient mastercycler system ( eppendorf , germany ) with the following cycle program : 94c for 5  min ; 30 cycles of 94c , 60c , and 72c for 1  min each , and final extension at 72c for 10  min followed by a final sample hold at 4c .
the pcr product was precipitated using 8.5% polyethylene glycol6000 , washed thrice using 70% ethanol , and dissolved in 10  mm tris - hcl ( ph 8) .
the abi prism bigdye terminator cycle sequencing ready reaction kit ( applied biosystems , usa ) was used for the sequencing reaction .
the sequencing output was analyzed using the dna sequence analyzer software ( applied biosystems ) .
the sequence was compared with national center for biotechnology information ( ncbi ) genbank entries by using the blast algorithm .
samples of the identified microbial isolate were obtained both from bushnell haas broth and nutrient broth ( himedia , mumbai ) .
the extracted plasmid dna samples and the control were subjected to agarose gel electrophoresis ( 0.7% agarose ) as described by sambrouk and russell .
the bacterial isolate rt10 was inoculated in 25  ml sterile nutrient broth in a shaking incubator set at 30c and 150  rpm until od600  nm reaches 0.8 - 0.9 .
this seed culture was used in the production medium at 2% ( v / v ) . as formulated by makkar and cameotra ,
biosurfactant production was carried out in 250  ml conical flasks containing 50  ml of a mineral salt medium with the following composition : kno3 ( 0.3% ) , na2hpo4 ( 0.22% ) , kh2po4 ( 0.014% ) , nacl ( 0.001% ) , mgso4 ( 0.06% ) , cacl2 ( 0.004% ) , feso4 ( 0.002% ) , and 0.1  ml of trace element solution containing ( g / l ) : 2.32  g znso47h2o , 1.78  g mnso44h2o , 0.56  g h3bo3 , 1.0  g cuso45h2o , 0.39  g na2moo42h2o , 0.42  g cocl26h2o , 1.0  g edta , 0.004  g nicl26h2o , and 0.66  g ki ( all chemicals were of analytical grade from merck , usa ) .
crude oil ( from a local refinery ) was used as the sole source of carbon at 2% ( v / v ) concentration .
the temperature of the medium was maintained at 30c with shaking at 150  rpm .
culture medium samples were drawn for estimation of biomass , biosurfactant production , and surface tension , once every 24 hours for five days .
bacterial cell growth was monitored by measuring the dry cell weight , as described by cooper and goldenberg .
the culture broth was centrifuged in a refrigerated centrifuge ( plastocraft model superspin r - v / fm , mumbai ) at 10000  rpm for 20  min at 4c to obtain a cell - free supernatant .
the ph of the supernatant was adjusted to 2 using 6n hcl and was subjected to acid precipitation by placing it at 4c overnight .
the off - white precipitate was separated by centrifugation at 10000  rpm for 30  min at 4c .
the precipitate was extracted thrice with a 2  :  1 chloroform - ethanol mixture .
the organic phase was removed , and the biosurfactant was concentrated using a rotary evaporator ( superfit model supervac , mumbai ) at 40c .
the solvents were evaporated leaving behind relatively pure biosurfactant as a viscous light brown matter . at periodic time intervals ,
1  ml samples of culture broth were collected in a sterile manner and centrifuged at 10000  rpm for 20  min .
the paste was dried by heating in a hot air oven set at 50c70c until constant weight was attained , without allowing the cells to be charred .  
the surface tension property was studied by taking a sample of the culture broth and centrifuging at 10000  rpm for 20  min .
the cell pellet was discarded , and the surface tension of the supernatant was measured by the wilhelmy plate method   using a sigma model 702 digital surface tensiometer ( ksv instruments ltd . , helsinki , finland ) .
initially , the plate and glassware were cleaned with chromic acid , milli - q water , and acetone .
all the measurements were taken in triplicate .   the ability of the biosurfactant to emulsify hydrocarbons was determined by the addition of 2  ml sample of the culture supernatant and 2  ml of a hydrocarbon ( hexadecane ) , taken in a glass test tube .
the emulsification activity was checked after being allowed to settle for 24  h , and the emulsification index ( e24 ) was calculated by measuring the emulsion layer , expressed as a percentage of the total height of the mixture in the tube , as described by cooper and goldenberg .
the emulsification power of a mixture of equal volumes of 1  mg / ml sds and the hydrocarbon was used as the control .
the biosurfactant sample was spotted on precoated silica gel 60 f254 plate ( merck , usa ) and subjected to thin layer chromatography ( tlc ) , as described by das et al . .
the plate was developed with a solvent system consisting of chloroform , methanol , and water ( 65  :  25  :  4 ) .
further , the plate was sprayed with 0.2% ninhydrin ( in absolute alcohol ) and heated to 110c . to understand the overall chemical nature of the extracted biosurfactant , fourier transform infrared spectroscopy ( ftir ) was employed .
the technique helps to explore the functional groups and the chemical bonds present in the crude extract .
samples were prepared by homogeneous dispersal of 1  mg of the biosurfactant sample in pellets of potassium bromide ( merck , usa ) .
ir spectra were collected over the range of 4504500  cm with a resolution of 4  cm .
the spectral data were the average of 50 scans over the entire range covered by the instrument .
the stability studies were carried out with respect to the effect of temperature , ph , and salinity on surface tension and emulsification capacity of the biosurfactant .
the analysis was done using the 24-hour cell - free culture broth obtained by centrifuging the culture sample at 10000  rpm for 15 minutes .
, 10  ml of the cell - free broth was incubated at temperatures ranging from 4c to 121c for 30 minutes .
the effect of ph was determined by estimating the variation of surface activity by adjusting the ph of 10  ml of cell - free broth from 2 to 12 with 6n hcl or 6n naoh solutions .
the effect of salinity was checked by varying the concentration of sodium chloride ( 0% to 20% w / v ) , added to 10  ml samples of cell - free broth .
in all the three studies , the samples were allowed to stand at room temperature for 6 hours after the respective treatments , before making the measurements of surface tension and emulsification index .
microbial enhanced oil recovery ( meor ) is a unique residual oil extraction technology making use of microorganisms .
the suitability of the biosurfactant for meor was investigated by employing a jacketed glass column as described by abu - ruwaida et al . .
the performance of the biosurfactant in terms of oil recovery was also compared against sodium dodecyl sulphate ( sds ) ( merck , usa ) , an amphiphilic surfactant and a common ingredient of many commercial detergents .
75  mg of sand was pretreated by washing with 1n hcl in a conical flask , rotated at 150  rpm for 1 hour .
it was dried completely in a hot air oven ( rotek , india ) set at 100c for 12 hours . a glass column ( 45  cm  2  cm i.d . )
the column was saturated with 50  ml of commercially available engine oil ( sl 20w-40 ; jaso m 345 grade ) .
an aqueous solution of 25  mg biosurfactant in 50  ml distilled water was applied to the column .
the temperature of the column was maintained at 30c by passing water through the jacket using a peristaltic pump coupled to a water bath maintained at 30c .
the leachate from the column was collected over a 36-hour period , and the volume of engine oil released from the column was measured .
the impact of temperature on biosurfactant - mediated oil recovery was observed by conducting similar runs at 50c and 70c . in a separate set of studies , an aqueous solution of 25  mg sds in 50  ml sterile water
was poured onto the column and the oil recovery was determined at 30c , 50c , and 70c .
a total of 11 different bacterial specimens were isolated from the oil contaminated soil samples .
the isolates were chosen based on their distinct colony morphology , obtained by serial dilution and streak plating techniques .
the eleven isolates were then subjected to screening for biosurfactant production by multiple methods like oil spreading technique , blood agar haemolysis , drop collapse test , ctab agar plate test , and tilting glass slide test ( table 2 ) . in the oil spreading technique , morikawa et al
.   showed that the extent of oil displacement is directly proportional to the concentration of the biosurfactant produced .
of the eleven isolates , four samples significantly displaced the oil layer and started to spread in the water , showing a zone of displacement . in the blood agar technique , mulligan et al .  
however , it is not necessary that all biosurfactants have a hemolytic activity ( carrillo et al . ) .
accordingly , in the present study , three of the isolates displayed excellent hemolytic activity .
described the drop collapse test according to which the degree of collapse of the culture supernatant describes the surfactant concentration .
of the eleven isolates , three strains showed near - complete collapse , while for two other samples the drops turned absolutely flat . in the ctab test designed by siegmund and wagner , two isolates showed greenish halos around the colonies on ctab methylene blue agar medium .
the tilted glass slide test , developed by persson and molin , was positive for four isolates .
but in the case of rt10 , the rate of drop collapse was very rapid and exhibited the lowest surface tension of 29.8  mn / m .
it satisfied four of the six tests , and hence , this isolate was picked up as a potential candidate for further studies .
the current report , henceforth , discusses the potential use of the strain rt10 for enhanced oil recovery .  
the results of screening procedures consistently showed the biosurfactant - producing property of the isolate .
the results of almost all the tests led to the selection of the bacterial isolate , rt10 .
the reduction of surface tension was the greatest in case of rt10 . based on morphological and biochemical analysis , in accordance with bergey 's manual of determinative bacteriology
, the best isolate belonged to the genus bacillus ( tables 3 and 4 ) .
the 16s rrna analysis revealed that the isolate rt10 showed 99.8% similarity to bacillus siamensis .
the neighbor - joining tree based on the 16s rrna sequence for the strain has been shown in figure 1 .
the 16s rrna sequence alignment shows that the strain rt10 was closely related to the species in genus bacillus .
the results of the plasmid extraction process did not reveal the presence of any plasmid in the isolate , irrespective of the media in which the cells were cultured .
the study infers that the ability of the isolate to produce biosurfactant was not conferred upon due to the presence of any plasmid
. the property should be the result of a chromosomally - mediated mechanism of the bacterium .
since the isolate was enriched from an oil - contaminated site , preliminary batch fermentation studies were performed in mineral salt medium , makkar and cameotra , supplemented with 2% ( v / v ) crude oil as the sole carbon substrate .
24-hour cultures were periodically sampled out to monitor the biomass growth , biosurfactant production , and surface tension . as shown in figure 2 , at 96  h of fermentation maximum biosurfactant yield of 0.64 
g / l , maximum biomass yield of 3.2  g / l , and the lowest surface tension of 36.1  mn / m were obtained . the emulsification index ( e24 ) of the culture supernatant against hexadecane reached a maximum of 70% at 72  h. when the plate was sprayed with 0.2% ninhydrin , the biosurfactant component was observed as a single spot on the tlc plate .
the observation implied the presence of amino acids in the sample . as a result of c 
h stretching vibrations and n  h stretching vibrations , a broad absorbance peak ( centred around 3433  cm ) with wave numbers ranging from 3600  cm to 3100  cm was observed ( figure 3 ) .
sharp absorbance peaks are observed at 1463  cm , 1379  cm , 2955  cm , and 2854  cm and are indicative of aliphatic chains ( ch3 and ch2 ) .
a strong band was also observed at 1741  cm , 1726  cm , and 1713  cm .
the presence of c = o bonds causing c = o stretching vibrations leads to absorbance peaks in these regions .
the stability of the biosurfactant was checked by subjecting the fermentation broth to conditions of high stress that included temperature , ph , and salinity .
when the temperature was varied from 0c to 121c , both surface tension and emulsification index ( e24 ) showed little variation and remained nearly constant at around 39 - 40  mn / m ( figure 4(a ) ) .
with respect to ph variation from 2 to 12 , the values of surface tension were centred around 39  mn / m without large deviations ( figure 4(b ) ) .
the emulsification index ( e24 ) dipped to lower values ( 60% ) , when the ph was increased .
variability in surface tension and emulsification index was not profoundly observed in various studies involving changes in broth ph which is in accordance with the literature [ 26 , 27 ] .  
surface tension was similar to the effect of ph with largely no changes ( figure 4(c ) ) .
the literature study in the past also confirms that many biosurfactants have stable surface activity even at high levels of salinity [ 27 , 28 ] .
the applicability of the biosurfactant was verified using the sand pack column test , while maintaining the column at 30c , 50c , and 70c .
with rise in temperature , the recovery also increased to 55% and 60% at 50c and 70c , respectively .
bordoloi and konwar   performed the study at room temperature , 70c , and 90c using a sand pack column .
they reported oil recovery in the range from 35% to 60% across the temperatures , for various bacillus and pseudomonas strains .
in the present study , biosurfactant - producing organisms were enriched and isolated from an oil - contaminated site .
the morphological , biochemical , and 16s rrna analysis were performed to identify the organism .
this is the first report describing the isolation and use of bacillus siamensis as a biosurfactant producer . during fermentation studies ,
the isolate was able to produce a lipopeptide biosurfactant , using crude oil as the sole carbon source .
the biosurfactant was extracted and partially characterized by using tlc and ftir spectroscopy to confirm its chemical nature . a maximum biosurfactant yield of 0.64 
g / l was obtained at 96  h. the biosurfactant had good surface tension reducing capability , reducing the surface tension to 36.1  mn / m .
stability studies were performed to investigate the effect of extreme variations in temperature , ph , and salinity levels on the surface tension and emulsification capacities of the biosurfactant . in order to examine the suitability of the biosurfactant in enhanced oil recovery processes ,
the column studies were performed at higher temperatures , and the percentage of oil recovery was estimated to be 60% .
the organism and its product present a huge potential for use in environmental remediation strategies .
 biosurfactants are surface - active compounds derived from varied microbial sources including bacteria and fungi . 
 they are secreted extracellularly and have a wide range of exciting properties for bioremediation purposes . 
 they also have vast applications in the food and medicine industry . with an objective of isolating microorganisms for enhanced oil recovery ( eor ) operations , the study involved screening of organisms from an oil - contaminated site . 
 morphological , biochemical , and 16s  rrna analysis of the most promising candidate revealed it to be bacillus siamensis , which has been associated with biosurfactant production , for the first time . 
 initial fermentation studies using mineral salt medium supplemented with crude oil resulted in a maximum biosurfactant yield of 0.64  g / l and reduction of surface tension to 36.1  mn / m at 96  h. characterization studies were done using thin layer chromatography and fourier transform infrared spectroscopy . 
 ftir spectra indicated the presence of carbonyl groups , alkyl bonds , and c  h and n  
 h stretching vibrations , typical of peptides . 
 the extracted biosurfactant was stable at extreme temperatures , ph , and salinity . 
 its applicability to eor was further verified by conducting sand pack column studies that yielded up to 60% oil recovery . 
1. Introduction 
2. Materials and Methods
3. Results and Discussion
4. Conclusions