Isolation and Characterization of Biosurfactant from Bacillus amyloliquefaciens VITANS6 Isolated from Oil Contaminated Soil collected from an Automobile Workshop in Bangalore, India

 

Akshatha N, Niki T, Sripradha S, Veena S, Bhaskara Rao K.V.*

 

ABSTRACT:

The present study focused on the isolation and extraction of biosurfactant producing bacteria. Bacterial colonies were isolated from oil-soaked soil sample collected from an automobile work shop in Bangalore, India on Nutrient Agar medium. A total of 8 bacterial colonies were isolated and were designated as ANS-1 to ANS-8. All isolates were then screened for biosurfactant production. The primary screening consisted of haemolytic assay, where isolates ANS1 and ANS6 showed β-haemolysis and isolates ANS2 and ANS5 showed α-haemolysis. For secondary screening, oil displacement test and drop collapse test and emulsification index calculation were conducted. Isolate ANS6 showed positive for drop collapse test and oil displacement test. ANS6 demonstrated highest emulsification index of 63.333%. Extracted biosurfactant was characterized using FTIR which showed glycerine as a cyclic lipopeptides. The potential isolate with high biosurfactant producing ability was identified using 16s rRNA sequencing as Bacillus amyloliquefaciens VITANS6.

 

INTRODUCTION:

The use of microorganisms in the presence of required and sufficient nutrients along with the optimum conditions to carry-out break down of contaminants such as heavy metals and petroleum hydrocarbons is called as bioremediation. The need for Bioremediation arises from the rate of degradation of the environment in the world which is increasing exponentially. Heavy metals play a vital role in the metabolic processes of the biota and any fluctuation in their amounts will have a negative impact on the environment¹. Chemically produced surfactants are known as synthetic surfactants. Their polar component may contain a quaternary ammonium compound, carboxylate group or sulphate group, while their hydrophobic component can be made of alcohols, olefins, paraffins, alkyl benzenes, etc ².

 

The production of chemical surfactants in industrial scale however results in emissions that add to pollution. Even the persistence of these compounds after their use has proven to be toxic. Phosphate containing surfactants mixed into water bodies encourage the growth of algal blooms, leading to eutrophication. In remediation projects, it has seen that chemical surfactants sometimes form toxic compounds with the pollutants, proving to be counterproductive³. Thus, despite the fact that chemical surfactants currently prove to be the cheaper option, interest has turned onto biosurfactants.

 

Biosurfactants are biological entities which have potential applications in petroleum industry. These are biological surface acting agents whom are capable of reducing interfacial tension between liquids, solids and gases and also amphiphilic molecules consisting both hydrophobic and hydrophilic moieties that interact with phase boundary in a heterogeneous system ^{4, 5}. Due to their amphipathic nature, biosurfactants increases the surface area of water insoluble particles such as oil, kerosene, petroleum and increases water bioavailability of such substances, thus leading to changes on the bacterial cell surface. Therefore this makes them excellent natural emulsifiers, dispersing agents, foaming agents. They have potential applications in bioremediation that is it helps in degrading the hydrocarbons and the contaminants in the petroleum industry. These contaminants can decrease the efficiency of the biofuels. These biosurfactants can be used to rectify the oil spills by the industries into the lakes, and oceans. They have potential application in households and domestic sections⁶. Biosurfactants are preferred over chemical surfactants since they are less hazardous to the environment, degrade pollutants easily and can be active at extreme temperatures, pH and salinity unlike chemical surfactants, which can be decomposed at extreme conditions. Microorganisms growing in oil contaminated soil would have to produce certain substances that enable their thriving in these regions. Many microbes are capable of producing surface active amphoteric agents which in a growth medium helps in emulsification of hydrocarbons and also reduces the interfacial and surface tension, which in turn increase the solubility and emulsification of two immiscible phases and also provides an insoluble substrate for microorganisms, which are known as biosurfactants⁷. Thus, with the assumption that biosurfactant producing microbes are mostly present near soils of petroleum industries, and places where oil is used extensively, organisms have been isolated from such regions and various methods have been opted for extraction for potential use.

 

MATERIALS AND METHODS:

Chemicals and Medias:

All the Chemicals and Media used in this study were purchased from Hi-Media Laboratory Pvt. Ltd., Mumbai.

 

Sample Collection:

Soil with the presence of oil was collected from an auto-mobile repair shop in Bangalore, India (Latitude and Longitude: 12°59'54.7"N 77°40'20.1"E). The sample was collected in sterile plastic bags and was immediately transported aseptically to Molecular and Microbiology Research Laboratory, VIT University, Vellore, and refrigerated at 4⁰C for further use.

 

Isolation of Microorganisms:

Serial dilution was done with one gram of soil and dilutions of 10^-3, 10^-4, 10^-5 and 10^-6 were plated on Nutrient Agar medium by Spread Plate method. Duplicates were made and incubated at 37⁰C for 24 hours. From these plates 8 morphologically distinct colonies were selected and sub-cultured onto nutrient agar plates. The isolated cultures were inoculated into nutrient broth tubes in an orbital shaker and after 24 hours, the culture broth was centrifuged at 14000 rpm 15 min. The supernatant was collected and used for screening tests.

 

Primary Screening of isolates for surfactant production:

Haemolysis test:

The isolated cultures were streaked onto blood agar plates and incubated at 37°C for 24 hours. The plates were observed for haemolysis, characterized by a clear zone around the grown streak ⁸.

 

Secondary Screening of bacteria for surfactant production:

Oil Displacement Test:

Test was carried out by taking a petri plate onto which 20 ml of water was dispensed. About 20µl of coconut oil was dropped onto the distilled water layer with the help of the micropipette and allowed to disperse. Cell free supernatant (10µl) was added, and the oil layer checked for dispersion ⁹.

 

Emulsification Test:

A set of 8 test tubes were taken and into that equal volumes of kerosene and supernatant of bacterial culture were taken. The tubes were subjected to vigorous vortexing and were allowed to settle. The level of the kerosene layer and the total level of the liquids in the tube were measured after half an hour and again after 24 hours after vortexing, and the emulsification index was measured¹⁰.

 

𝑒𝑚𝑢𝑙𝑠𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛𝑖𝑛𝑑𝑒𝑥=

 

𝑢𝑝𝑝𝑒𝑟 ℎ𝑒𝑖𝑔ℎ𝑡 𝑜𝑓𝑡ℎ𝑒 𝑜𝑖𝑙 𝑙𝑎𝑦𝑒𝑟 − ℎ𝑒𝑖𝑔ℎ𝑡 𝑜𝑓𝑡𝑒𝑠𝑡 𝑐𝑢𝑙𝑡𝑢𝑟𝑒 × 100

𝑡𝑜𝑡𝑎𝑙 ℎ𝑒𝑖𝑔ℎ𝑡𝑜𝑓𝑡ℎ𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑡ℎ𝑒 𝑡𝑒𝑠𝑡 𝑡𝑢𝑏𝑒

 

Drop-Collapse Test:

Drop Collapse method is an extremely rapid method which is utilized for the screening of biosurfactants. This was performed in a 96 well micro-titre plate. Oil (10µl) was first added to the well and 10µl of the cell suspension was placed on the oil coated surface. The droplets which contained bio-surfactants collapse into the oil, whilst the culture without bio-surfactants showed no noticeable change in the shape of the droplet ¹¹.

 

Extraction and partial purification of Biosurfactants:

The extraction of bio-surfactant was carried out in optimized conditions; the culture was centrifuged for 20 min at 10,000 rpm in a cooling centrifuge to facilitate the complete removal of bacterial cells. The supernatant was then collected after the discarding of the pellet obtained. The pH of the supernatant was brought down to 2.0 by the addition of concentrated HCl and kept at 4⁰C overnight. This was done to precipitate the proteins and the lipids present in the supernatant. This was centrifuged again for 20 min at 10,000 rpm and the pellet was collected. Ten milliliter of a solution consisting of chloroform and methanol in a 2:1 v/v ratio was added to this pellet and incubated in a shaker at 250 rpm for 30 min. This content was later centrifuged in a cooling centrifuge for 20 min at 10,000 rpm. The resulting supernatant was evaporated by air drying and to the precipitate sodium phosphate buffer was added and then stored at 4 ⁰C^{10, 12}.

 

FTIR Analysis:

Infra red absorption spectra were obtained in FT-IR spectroscopy (AVATAR300 FT-IR, Thermo Nicolet, USA) in a dry atmosphere. Absorption spectra were plotted using a built in plotter. IR spectra were collected from 400–4000 wave numbers (cm^-1) with resolution of 2 wave numbers per wave number. Samples were prepared by dispersing the solid uniformly in a matrix of potassium bromide ¹³.

 

Identification of the potent bacterial isolates:

A series of biochemical tests were performed in order to identify the bacterial isolates showing the highest biosurfactant production. Based on the biochemical test results the bacterial isolates were identified using Bergey’s Manual of Determinative Microbiology. Finally the complete identification was done by using molecular characterization based on 16s rRNA sequencing. Bacterial genomic DNA was isolated using the InstaGeneTM Matrix Genomic DNA isolation Kit. The fragments were amplified bi-directionally using the forward (5′AGAGTTTGATCMTGGCTCAG-3′) and reverse (5′-TACGGYTACCTTGTTACGACTT-3′) primers (MJ Research Peltier Thermal Cycler). Analysis of the sequence was done by ABI 3730xl capillary DNA sequencer (ABI Prism 310 Genetic Analyzer, Tokyo, Japan). Finally, the sequence was observed for similarity using NCBI BLAST similarity tool.

 

RESULTS AND DISCUSSION:

Isolation of Microorganisms:

The collected sample was oil-contaminated soil, thus it was assumed that the bacteria present in the sample would have properties that would help them survive in their environment. A total of eight morphologically dissimilar colonies were selected after spread-plating and directly screened for biosurfactant production (Figure 1). Other studies have used either selected specific organisms for the study of biosurfactant production ¹⁴ and have screened a large number of isolates from specific environments such as water and terrestrial samples from areas with a history of oil spills ¹⁵.

 

Haemolysis test:

Primary screening of biosurfactant producing bacteria was carried out by isolation on blood agar and checking for haemolysis. Out of eight isolates, few showed beta-haemolysis and few showed alpha haemolysis and gamma-haemolysis.

 

 

Figure 1: Pure Cultures of the Isolated Colonies on Nutrient Agar

Primary Screening of isolates for surfactant production

 

The isolates ANS 1, 2, 3,4,5,6, showed haemolysis. The haemolysis pattern is illustrated in figure 2. Haemolysis has been observed to be a characteristic of biosurfactants, and thus checking for haemolysis on blood agar serves as an effective primary screening method⁷. In another report Bacillus thuringiensis isolated from oil contaminated region of Vellore, Tamil Nadu showed similar results with good zone of clearance (10-12mm) of β haemolysis¹⁶. Three isolates, ANS 1, 4 and 6 showed β haemolysis with a complete zone of clearance, while three other isolates, ANS 2, 3 and 5 showed α haemolysis with partial zones of clearance.

 

 

Figure 2: Haemolysis observed by the Isolated Colonies on Blood Agar

 

Secondary Screening of isolates for surfactant production:

Oil displacement test:

The isolates ANS 1 to 8 were subjected to the screening method known as oil displacement test for checking for biosurfactant production by the isolate. The results showed that isolates ANS 1 and 6 showed biosurfactant activity by spreading the coconut oil showing a dispersed zone (Table 1). Isolate ANS 1 and 6 showed a maximum dispersal of coconut oil as compared to other isolates. From previous studies, the oil spreading activity was observed higher in gingelly oil by the biosurfactant producing bacteria as compared to coconut oil ¹⁷. In another study on oil displacement study using Bacillus sp MTCC 5877 showed a displacement of 6 cm in soybean oil¹⁸. In our study also a displacement of 6 cm was shown by active isolate ANS-6.

 

Table 1: Oil displacement shown by eight isolates

 

Oil Drop Collapse test:

Out of eight isolates, only isolate ANS-6 showed collapsing activity towards the drop of olive oil. This method significantly implies that isolate ANS-6 is a potential biosurfactant producing bacteria. The drop collapse method gives the bio emulsification activity of the isolates, thus serving as a qualitative screening method for biosurfactant production. Only isolate ANS-6 showed a first change in the shape of the drop of the cell-free supernatant and then collapsing of the drop, when added to the a micro-titre well containing olive oil ⁷. Only isolate ANS-6 showed a positive result for all the screening methods, indicating strong biosurfactant activity and thus was taken forward for the extraction of biosurfactant. Bacillus sp MTCC 5877 isolated from soil contaminated regions showed positive results in oil drop collapse activity with soybean oil along with all different carbon source ¹⁸.

 

Emulsification index test:

Emulsification index Measurement was carried out for the eight isolates ANS 1-8 with kerosene. The emulsification measurement was conducted based on the method described by Cooper and Goldenberg 1987. In this test, the efficiency of emulsification activity by bio-surfactant producing bacteria was examined and the Emulsification Index (EI) was determined. The Emulsification Index differs based on the organism and the biosurfactant’s nature. The isolates ANS 4 and ANS 6 had the highest Emulsification Index of 46.875 % and 63.333 % respectively as shown in table 2. Our study is in accordance with a recent study reported where Bacillus sp isolated from oil contaminated soil samples of china showed emulsification index of 64.035 % ¹⁹. In another study bio-surfactant producing Bacillus subtilis showed emulsification of 59.03% ²⁰.

 

Table 2: Emulsification Index shown by isolated cultures

 

Extraction and purification of Biosurfactant:

FTIR analysis:

FTIR spectra obtained from purified bio-surfactant reveals that biosurfactant produced consist of the main characteristic group of surfactin molecules. This indicates the presence of aliphatic and well as peptic fractions. Most important absorption band was assigned by comparing with spectra obtained in different literatures ^{21, 22}. In the spectra obtained in figure 3, four main can be seen. The peak with a maximum of 3304 cm^-1 corresponds to the N-H stretch which represents the peptide residues. Another band with maximum of 1635 cm^-1can be realted to the absorption of C=O group from lactonization. The bands at 1078 cm^-1 indicate aliphatic chains (-CH2-CH2). These results suggest that the biosurfactant produced by ANS6 in a medium containing glycerine as a cyclic lipopeptide, mainly surfactin. Purification study is in accordance with previous study where biosurfactant production was reported using Bacillus sp with presence of similar functional groups when characterized using FTIR analysis²³. A recent report also showed similar results in which biosurfactant isolated from Bacillus sp ZG0427 was partially purified and characterized using FTIR as lipopeptides ¹⁹.

 

 

Figure 3: FTIR spectra shown by purified biosurfactant producing isolate ANS6

Identification of the Bacterial isolate:

Bacterial isolate ANS6 was selected for complete identification as it showed the highest activity of surfactant production in both primary and secondary screening. Various biochemical tests including different carbohydrate fermentation tests were conducted for the potential isolate. Isolate ANS6 was identified as Bacillus sp using Bergey’s manual of Determinative Microbiology with the help of biochemical tests. In species level identification using 16S rRNA sequencing Potential isolate showed 98 % of similarity with Bacillus amyloliquefaciens in the blast search analysis. The 16S rRNA sequencing of potential strains was confirmed that it occupies a distinctive phylogenetic position with the radiation, including representatives of the family using neighbor joining tree. Hence the potential isolate ANS6 was identified as Bacillus amyloliquefaciens VITANS6

 

CONCLUSION:

This study showed biosurfactant production ability of the bacterium Bacillus amyloliquefaciens VITANS6 isolated from oil contaminated soil samples. Our study showed that Bacillus amyloliquefaciens VITANS6 can be used as a potential source for the production of surfactant. Produced biosurfactant can be used in different fields like textile, detergents, paint, cosmetics and agriculture. It can also be used for bioremediation process by cleansing the contamination caused by human activities.

 

ACKNOWLEDGEMENTS:

Authors are very thankful to the management of VIT University for providing necessary facilities to carry out this study.

 

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Received on 14.08.2017         Modified on 26.08.2017

Accepted on 13.09.2017      © RJPT All right reserved