ORIGINAL ARTICLE
Screening of bioemulsifier-producing micro-organisms isolated
from oil-contaminated sites
Neha Panjiar&Shashwati Ghosh Sachan&Ashish Sachan
Received: 25 September 2013 / Accepted: 6 May 2014 / Published online: 1 June 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014
Abstract Eighty-eight micro-organisms were isolated from oil-contaminated soils and checked for their extracellular bioemulsifier producing potential. The micro-organisms were screened on the basis of oil spread, drop collapse and emulsi-fication index. Most efficient strains were characterized as Lysinibacillus sp. SP1025 and Bacillus cereus SP1035. In Lysinibacillus sp. SP1025, the E-24 index, surface tension and production of crude bioemulsifier were found to be 83.3 % with diesel, 34.20±0.03 mN/m and 3.07±0.62 g/L, respectively, whereas in the case of Bacillus cereus SP1035, the E-24 index, surface tension and production of crude bioemulsifier recorded were 76.5 % with diesel, 43.42 ± 0.03 mN/m and 3.90±0.3 g/L. Crude biomemulsifier pro-duced by selected micro-organisms was stable, withstanding a wide temperature and pH range with an E-24 Index value greater than 50 %. All emulsions formed were oil-in-water type. Emulsions formed with tested aliphatic and aromatic hydrocarbons, except those formed with ester based oils, were 100 % stable with the entire organic layer converted into emulsion. To the best of our knowledge this is the first report for bioemulsifier production from the genus Lysinibacillus. Keywords Bioemulsifier . Emulsion stability . Lysinibacillus sp. SP1025 . Bacillus cereus SP1035 . Droplet size
measurement
Introduction
The non-biodegradable and toxic nature of chemical surfac-tants has led researchers to focus on better alternatives
(Mawgoud et al. 2010; Panjiar et al. 2013). Biosurfactants have properties similar to chemical surfactants but are of microbial origin. Their advantages over chemical surfactants include their biodegradable nature, biocompatibility, low tox-icity and high speciftox-icity towards substrates (Bognolo1999; Dastgheib et al.2008). Their various functions include emul-sification, foaming, wetting, cleansing, dispersion, phase sep-aration, reduction in viscosity and surface activity (Kosaric
1992). The functional diversity of biosurfactants has resulted in their applications in spheres as diverse as the petroleum, agriculture, food processing, leather, textile, paper, cosmetic and pharmaceutical industries (Kosaric 1992; Batista et al.
2006). They can also be used to enhance oil recovery from wells, reduce heavy oil viscosity, clean oil storage tanks, increase flow though pipelines and stabilize fuel water–oil emulsions (Makkar and Cameotra1997; Bognolo1999).
Oil contamination of terrestrial and aquatic ecosystems has resulted in an enriched microbial community capable of pro-ducing biosurfactant to solubilize and utilize petroleum hy-drocarbons as carbon sources, thus opening ways for the isolation of novel bioemulsifier-producing strains from oil-contaminated sites (Iwabuchi et al.2002; Chikere et al.2009). Various micro-organisms have been reported for their poten-tial capability to produce biosurfactant into the medium. These are Acinetobacter, Pseudomonas, Bacillus, Rhodococcus, Arthrobacter, Halomonas, Enterobacter, and yeast (Desai and Banat 1997; Maneerat 2005; Das et al.
2008). Several authors have documented the isolation of micro-organisms of Pseudomonas sp., Bacillus sp., Serratia sp., fungi and yeasts from oil/hydrocarbon contaminated en-vironment (Batista et al.2006; Kumar et al.2007; Ghojavand et al.2008; Ganesh and Lin2009; Nishanthi et al.2010; Liu et al. 2011; Dhail and Jasuja2012; Granzotto et al. 2012; Saimmai et al.2012).
In recent years, various methods have been utilized for extensive screening of biosurfactant-producing
micro-N. Panjiar
:
S. G. Sachan:
A. Sachan (*)Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi 835215, India
organisms. The screening protocol generally utilized multiple screening methods to rule out false positive and false negative results. Such methods include direct surface or interfacial tension measurements, drop collapse, oil spread, emulsifica-tion assay, emulsificaemulsifica-tion index, bacterial adhesion to different hydrocarbon, hemolysis and cetyltrimethylammonium bro-mide (CTAB) agar plate test (Afshar et al. 2008; Satpute et al.2008; Salihu et al.2009). As the name suggests, in the direct surface or interfacial tension measurement method, the interfacial or surface activity of the culture supernatant is measured. The surface tension decreases with increasing sur-factant concentration until the critical micelle concentration is reached. The drop collapse assay is based on the collapsing of liquid droplets by surfactants. If the liquid contains surfactant, the drops spread or even collapse because the force or inter-facial tension between the liquid drop and the hydrophobic surface is reduced. The stability of drops is dependent on surfactant concentration and correlates with surface and inter-facial tension. The emulsification index test determines whether the biosurfactant has emulsifier property or not by calculating the ratio of the height of the stable emulsion layer and the total height of liquid formed after vortexing and leaving it for 24 h. Bacterial adhesion to hydrocarbons (BATH) is a simple photometric assay for measuring the hydrophobicity of the bacteria by calculating the degree of adherence of cells to various liquid hydrocarbons. The hemo-lysis test is based on the principle that biosurfactants can cause lysis of erythrocytes. Therefore, positive strains will cause lysis of blood cells and exhibit a colorless, transparent ring around the colonies. In the oil spread test, the diameter of clearing zone on the oil surface correlates to surfactant activ-ity, also called oil displacement activity. For pure biosurfactant a linear correlation between quantity of surfactant and clearing zone diameter is given.
The chemical nature of the biosurfactants produced varies amongst the microbial community. Microbial surfactants are either low-molecular-mass molecules or high-molecular-mass polymers (Ron and Rosenberg2001). Low-molecular-mass biosurfactants can lower surface and interfacial tension as their primary activity whereas high-molecular-mass biosurfactants are more effective as emulsion-stabilizing agents and are called bioemulsifiers (Desai and Banat1997; Bognolo1999). Bioemulsifiers produces stable water-in-oil or oil-in-water emulsions having numerous environmental and industrial applications like in food (e.g., mayonnaise), deter-gents (e.g., removal of oily stains), pharmaceuticals (e.g., drug emulsions), agriculture (e.g., pesticides) and cosmetic indus-tries (e.g., shampoos, moisturizers) (Saikia et al. 2012). Despite their diverse applications, they have been less studied as compared to biosurfactants (Dastgheib et al. 2008). Bioemulsifiers or high molecular weight polymeric biosurfactants that have been studied include emulsan, alasan, liposan, lipomannan and mannoprotein, which are produced by
Acinetobacter calcoaceticus RAG-I, A. radioresistens, Candida lipolytica, C. tropicalis and Saccharomyces cerevisiae, respec-tively (Desai and Banat1997; Ron and Rosenberg2001).
Research into bioemulsifier production has increased but industrial scale production is yet to be achieved due to high production cost and low yield (Das et al.2008). This creates a necessity for the isolation of novel, high-yielding bioemulsifier-producing micro-organisms. Therefore, the current study was focused on the screening and charac-terization of micro-organisms with elevated bioemulsifier production. Growth kinetics, bioemulsifier production, stability study of crude bioemulsifier and emulsions formed were also analyzed.
Materials and methods
Isolation of micro-organisms from oil-contaminated sites Oil-contaminated soil (1 g) was serially diluted in sterile 0.9 % (w/v) saline solution and 100μL sample was spread over Petri plates containing solid medium supplemented with filter-sterilized hydrocarbon oil (0.2 μm) as a carbon source. Medium toxicity was observed by inoculating the sample in three different medium compositions. The first medium used was mineral salt medium (Dastgheib et al.2008) with oil (2 %, v/v) as sole carbon source, which represented maximum tox-icity for micro-organisms. The mineral salt medium consisted of sodium chloride (5 g/L), ammonium nitrate (2 g/L), potas-sium dihydrogen phosphate (2 g/L), disodium hydrogen phos-phate (5 g/L) and magnesium sulphos-phate (0.1 g/L). The second medium used was nutrient agar medium with oil (2 %, v/v) as an additional carbon source representing moderate toxicity, while the third medium used for isolation was nutrient agar only medium, providing sufficient nutrients with almost no toxicity for micro-organisms. After 24–48 h incubation, mi-crobial colonies on the Petri plates were counted. Pure cultures of morphologically different bacterial isolates were obtained by the quadrant streaking method on nutrient agar plates, and maintained on nutrient agar slants at 4 °C for future use. Pure cultures of fungal isolates were obtained by simple streaking on plates containing potato dextrose agar (PDA), and main-tained on PDA slants at 37 °C and kept at 4 °C for future use. Stocks were sub-cultured monthly.
Screening for bioemulsifier producing micro-organisms Preparation of inoculum culture
Isolated bacterial cultures were screened for their capacity to produce bioemulsifier. Inoculum cultures of isolated bacteria were prepared by transferring a loopful of culture from slants into 6 mL sterile nutrient broth followed by incubation in
aerobic condition at 37 °C and 120 rpm in a shaker incubator (New Brunswick Scientific, Edison, NJ) for 10–15 h. Homogenous inoculum culture [1 % (v/v) of OD600~1] was
inoculated in 100 mL conical flasks containing 25 mL produc-tion medium supplemented with 2 % (v/v) oil as an addiproduc-tional carbon source, followed by incubation at 37 °C and 120 rpm. Detection of bioemulsifier in the culture broth
Biomeulsifier in the culture broth was detected using the tests described below (Gandhimathi et al.2009). These were per-formed at 24-h intervals for 6 consecutive days. For this, the inoculated medium was centrifuged at 5,000 rpm for 20 min at 25 °C and screening tests for biosurfactant production were performed on the culture supernatant. Further confirmation of biosurfactant-producing bacterial isolates were done by blood hemolysis test and surface tension measurement of the culture supernatant.
The oil spread test was performed by adding 20 μL diesel to the surface of distilled water (40 mL) placed in a Petri dish (15 cm diameter) and then 10 μL of the culture supernatant was gently inoculated (Morikawa et al. 2000). The immediate appearance of a clear zone
confirms the presence of biosurfactant in the culture supernatant. Sodium dodecyl sulphate (SDS) and phos-phate buffer saline (PBS) were used as positive and negative controls, respectively.
The drop collapse test was done by mixing culture super-natant (20μL) methylene blue (5 μL) and placing a dopr of the mixture onto a Parafilm surface. The shape of the drop on the surface was inspected after 10 s. If the drop collapses it confirms the presence of biosurfactant in the culture superna-tant (Jain et al.1991). Methylene blue was added solely for the purpose of visualization. SDS and PBS were used as positive and negative controls, respectively.
The drop collapse and oil spread tests are very sensitive methods, and can be used together for primary screening of biosurfactant-producing isolates (Satpute et al.2008).
The emulsification index (E-24) test was performed to evaluate the emulsifying ability of culture supernatant. The E-24 was determined by mixing 6 mL test oil with 4 mL culture supernatant in a test tube, vortexing at high speed for 2 min and allowing to stand for 24 h (Cooper and Goldenberg
1987). The percentage of emulsification index was calculated using Eq.1. SDS and PBS were used as positive and negative controls respectively.
E‐24 Index; % ¼ Height of emulsion formed=Total height of solutionð Þ 100 ð1Þ
The surface tension of the culture supernatant and entire culture broth was measured by dynamic contact angle tensi-ometer DCAT21 (Dataphysics, Filderstadt, Germany) using the Wilhemy plate method. Sterile nutrient broth was used as a control (Nitschke et al.2004).
Phenotypic and genotypic studies of selected micro-organisms
Significant bioemulsifier-producing micro-organisms were identified by morphological, biochemical and molecular char-acterization. Morphological characterization was done on the basis of Gram reaction, shape, motility and endospore staining (Breed et al.1957). Biochemical tests performed were lipase, gelatinase, amylase, protease, catalase, carbohydrate fermen-tation, indole production, methyl red, Voges-Proskauer, citrate utilization, hydrogen sulfide production, urease, nitrate reduc-tion and oxidase tests. Genus was identified according to Bergey’s Manual of Determinative Bacteriology (Breed et al.
1957). Species identification of selected bacterial isolates were done on the basis of 16S rRNA gene sequencing (Xcelris Labs, Ahmedabad, India). Consensus sequences thus obtained were aligned using Clustal W along with the se-quence of type strains (Thompson et al.1997). Homology of
the sequences was examined by BLAST of National Center for Biotechnology Information (NCBI). Molecular Evolutionary Genetics Analysis (MEGA) software version 5 (Hall 2013) was used to construct phylogenetic trees using a neighbor-joining (NJ) method (Saitou and Nei
1987). Nearly complete 16S rDNA sequences of isolates SP1025 and SP1035 have been submitted to GenBank under accession numbers KC879304 and KC879305, respectively.
Growth kinetic study and bioemulsifier production of selected micro-organisms
The growth kinetics of selected bacterial cultures were studied both in the presence and absence of diesel, by transferring an aliquot of 1 % (v/v) of homogeneous inoculum culture (OD600
~1) to 250 mL Erlenmeyer flasks containing 62.5 mL steril-ized nutrient broth (control) and nutrient broth supplemented with filter sterilized (0.2μm) diesel (2 %, v/v), incubated in an orbital shaker (120 rpm) at 37 °C. Samples were taken at different time intervals to measure microbial growth (OD600 nm) and its ability to emulsify diesel by measuring
Bioemulsifier extraction from liquid medium
The culture supernatant obtained after centrifugation at 5,000 rpm for 20 min at 25 °C was acid hydrolyzed with concentrated hydrochloric acid to precipitate bioemulsifier. During acidifica-tion, bioemulsifier present in protonated form become less soluble in aqueous solution. It was then allowed to stand overnight at 4 °C and precipitated bioemulsifier was collected by centrifugation at 5,000 rpm for 20 min at 4 °C. Collected bioemulsifier was washed twice with distilled water, dried at 65 °C and weighed to calculate its production (Nitschke et al.2004).
Stability study of extracellular bioemulsifier and emulsions produced by selected isolates
Effect of temperature and pH on the stability of crude bioemulsifier
Culture supernatant obtained after centrifugation at 5,000 rpm for 20 min at 25 °C was considered as the source of crude bioemulsifier. Its stability was determined by subjecting the culture supernatant to various pH and temperature ranges and thereafter studying the emulsification index. pH values of 2– 10 was adjusted by hydrochloric acid (1 N)/sodium hydroxide (1 N). Temperatures considered for study were 4, 10, 20, 40, 60 and 80 °C (Khopade et al.2012).
Emulsifying and stabilizing capacity of extracellular bioemulsifiers
Emulsification was performed according to the method proposed by Cooper and Goldenberg (1987) with
different hydrophobic substrates (hexane, heptane, oc-tane, hexadecane, dodecane, benzene, toluene, xylene, diesel, kerosene, soyabean, mustard, groundnut, olive and coconut oil). The tube containing hydrophobic sub-strates and culture supernatant was vortexed and left to stand for 1 h, which was considered as starting time, 0 h. Emulsification Index (E-24 Index, %), relative emulsion volume (EV, %), emulsion stability (ES, %) and emulsified organic phase (EOP, %) were calculated at 24-h intervals up to 72 h, 1 month and then after 3 months intervals from Eqs. 1, 2,3 and 4, respectively (Batista et al. 2006; Portilla-Rivera et al. 2010):
EV; % ¼ emulsion height; cm cross section area; cm2 100=total liquid volume; cm3 ð2Þ
ES; % ¼ EV at time h 100ð Þ=EV at 0 h ð3Þ
EOP; % ¼ TOP; cm 3− NEOP; cm cross section area; cm2 100=vol TOP; cm3 ð4Þ
where TOP is total volume of organic phase and NEOP is non-emulsified organic phase.
The emulsions stabilized by the culture supernatant were also compared with those formed by 1 % (w/v) solution of the chemical surfactant SDS in deionized water.
Droplet size distribution study of emulsions formed
Droplet size measurement studies were also conducted by using an optical microscope (Portilla-Rivera et al.
2010). The emulsion was observed through 10×
0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 SP1025 SP1035 E 24 I n dex (%)
Incubation period (days)
Fig. 1 Emulsification index test performed with the culture supernatant of selected micro-organisms and diesel, inoculated in nutrient broth with 2 % diesel as an additional carbon source at 120 rpm, 37 °C. Error bars Standard deviation
objective lens by placing a drop of emulsion on a glass slide and measuring the radii of the droplets through a standard micrometric procedure. Images of several re-gions from each slide were taken in order to capture a representative structure of the emulsion.
Results and discussion Isolation of micro-organisms
A diversity of micro-organisms was present in the oil-contaminated samples. A minimum number of micro-organisms was isolated from minimal medium supplemented with hydrocarbon oil as sole carbon source (data not shown). It was reported earlier that there could be an increase to 25 % from 2 to 3 % biosurfactant producers amongst the screened popula-tion in hydrocarbon-impacted soil as compared to uncontami-nated soil (Saimmai et al.2013). Eighty-eight pure cultures of bacteria were isolated. Results indicated that Gram negative bacteria were prevalent in the oil-contaminated site (42.41 %) followed by Gram positive bacteria (35.34 %), fungi (15.14 %) and streptomyces (11.11 %). The result obtained is in accordance
Table 1 Distribution of the number of micro-or-ganisms on the basis of the maximum E-24 In-dex test (%) performed with diesel and culture supernatant
Group E-24 index Number of micro-organisms A 0–49 76 B 50–59 2 C 60–69 8 D 70–79 1 E 80–90 1
Fig. 2 Phylogenetic tree of a SP1025 and b SP1035 as constructed by MEGA 5.05 software using the neighbour joining (NJ) method. Numbers at nodes indicate levels of bootstrap values (%) based on a NJ analysis of 1,000 replicates; only values >50 % are given
with a previous study by Batista et al. (2006), who found the presence of more Gram negative than Gram positive bacteria due to the presence of outer membranes, which acts as biosurfactants. Screening of bioemulsifier-producing micro-organisms Bacterial isolates were subsequently screened for bioemulsifier production by using multiple screening tests at fixed time intervals. The emulsification index test (Fig.1) was included to determine emulsifying ability (Dhail and Jasuja
2012). The drop collapse test of all the screened micro-organisms was positive, collapsing the culture supernatant drop within 10 s. The oil spread test performed with diesel sorted all the screened micro-organisms into two major
groups: A (diameter of clear zone in the range of 0–4.9 cm) and B (diameter of clear zone≥5 cm) with 63 and 25 micro-organisms, respectively, in each group.
On the basis of E-24 Index results, micro-organisms were grouped into the five categories listed in Table1. According to Willumsen and Karlson (1997), a promising bioemulsifier has an E-24 Index greater than 50 %. Therefore, significant bioemulsifier producers with E-24 Index≥50 % fall into the category B, C, D and E consisting of 12 isolates, namely SP1001, SP1002, SP1003, SP1004, SP1014, SP1017, SP1025, SP1035, SP2058, SP2061, SP2065 and SP3092 with substantial results for drop collapse test (drop collapsed within 10 s) and oil spread tests (diameter≥5 cm). Of these 12, 8 are Gram negative coccus-shaped, 2 are Gram negative
Table 2 Morphological and biochemical characterization in selected isolates according to Bergey’s Manual of Determi-native Bacteriology
Strain SP1025 Strain SP1035 Morphological characterization
Gram stain (G+/G−) G (+) G (+)
Motility Motile Motile
Spore formation Spore forming Spore forming
Colony characteristics on agar plates
Cell shape Bacillus Bacillus
Size Large Large
Pigmentation White White
Form Irregular Circular
Margin Serrate Undulate
Elevation Convex Convex
Growth in broth media Sediment Sediment
Growth on agar slants
Abundance of growth Large Large
Pigmentation White White
Optical characteristics Opaque Opaque
Form Filiform Echinulate
Biochemical tests
Starch hydrolysis Negative Positive
Lipid hydrolysis Positive Positive
Gelatin hydrolysis Negative Positive
Glucose fermentation Negative, no gas Acid production, no gas Lactose fermentation Negative, no gas Negative, no gas Sucrose fermentation Negative, no gas Negative, no gas
Catalase activity Positive Positive
Nitrate reduction Negative Positive (nitrite is further reduced to ammonia or molecular nitrogen)
Indole test Negative Negative
Methyl red test Negative Negative
Voges-Proskauer test Negative Positive
Citrate utilization Negative Negative
Hydrogen sulphide production Negative Negative
bacillus-shaped and 2 are Gram positive bacillus-shaped bacteria. Two Gram positive bacilli, SP1025 and SP1035, with maximum E-24 Index of 80.3 % and 76.47 %, respectively, were selected for further studies. Bioemulsifier production with respect to incubation pe-riod (6 days) is presented in Fig.2. In both the studied strains it was observed that values of the E-24 Index test increased initially then decreased with further incubation.
Surface tension was measured by the Wilhelmy plate meth-od. Lowest surface tension values achieved in our study were 34.20±0.03 mN/m and 43.42±0.03 mN/m of culture super-natant of SP1025 and SP1035 respectively. No significant reduction in surface tension was observed but values were around the threshold value of around 40 mN/m or lower (Olivera et al. 2003). This may be due to production of polymeric biosurfactants, which do not reduce surface tension considerably but have emulsification abilities (Willumsen and Karlson 1997; Plaza et al. 2006). Similar surface tension values in both culture broth and culture supernatant indicated the extracellular nature of the biosurfactant.
Characterization of selected micro-organisms by phenotypic and genotypic studies
Selected bacterial isolates SP1025 and SP1035 were provi-sionally identified according to Bergey’s manual of determi-native bacteriology (Table2). For further confirmation, se-quencing of 16S rRNA gene was performed (Xcleris, Ahmedabad, India) and the consensus sequence used for similarity search using the BLAST algorithm with the NCBI GenBank nrdatabase. Based on the maximum identity score, the first ten sequences were selected and aligned using the multiple alignment software Clustal W. A phylogenetic tree was constructed using MEGA 5 software (Fig.2a,b). Analysis of 16S rDNA sequence indicated that isolate SP1025 showed similarity with Lysinibacillus xylanilyticus (AB662958, 99 % similarity according to BLAST, NCBI) whereas isolate SP1036 was identified as Bacillus cereus strain RR8 (HM367740, 99 % similarity according to BLAST, NCBI). SP1035 belongs to genera that have previously been reported and characterized for bioemulsifier production in the literature (Cooper and Goldenberg1987; Itah et al.2009; Sriram et al.
Fig. 3 Growth kinetics and bioemulsifier production profile in terms of emulsification index (%) of a Lysinibacillus sp. SP1025, and b Bacillus cereus SP1035 grown in nutrient broth and nutrient broth supplemented with diesel (2 %) as an additional carbon source at 120 rpm and 37 °C. Error bars Standard deviation
2011; Cerqueira et al. 2012). To the best of our knowledge, this is the first report on the potential of the genus Lysinibacillus to produce bioemulsifier. The 16S rDNA se-quences of SP1025 and SP1035 have been submitted to D D B J / E M B L/ G e nB a nk u n de r a c ce s s i o n nu m b e r s KC879304 (http://www.ncbi.nlm.nih.gov/nuccore/ KC879304.1) and KC879305 (http://www.ncbi.nlm.nih.gov/ nuccore/KC879305.1), respectively.
Growth kinetic study and bioemulsifier production of selected micro-organisms
Growth kinetics and biosurfactant production were studied simultaneously by measuring the OD600 and emulsification
index (E-24) at different time intervals. No bioemulsifier was detected in the control sample. In control medium, growth rate of Lysinibacillus sp. SP1025 and Bacillus cereus SP1035 was found to be 0.63 and 0.98 generations/h. In Lysinibacillus sp. SP1025 (Fig.3a) significant bioemulsifier production started after 12 h of incubation period when grown in production medium, and attained a maximum value after 24 h of incuba-tion (E-24 Index of 83.3 %). However, in the case of Bacillus cereus SP1035, bioemulsifier production started when the micro-organism was in the middle of the log phase of its growth and reached a maximum (E-24 Index of 76.5 %) after 48 h of incubation in production medium (Fig.3b). Lysinibacillus sp. SP1025 and Bacillus cereus SP1035 was found to grow at the rate of 2.25 and 3.83 generations/h in the production medium. Pornsunthorntawee et al. (2008) have described maximum biosurfactant production during stationary phase of the studied bacterial strain Bacillus subtilis PT2. Different authors have also reported biosurfactant production during their stationary phase (Batista et al. 2006; Sriram et al.2011). The amount of bioemulsifier produced, as determined by the acid precip-itation method, was found to be 3.07 ± 0.62 and 3.90 ± 0.3 g/L by Lysinibacillus sp. SP1025 and Bacillus cereus SP1035 respectively. The precipitate was again dissolved in distilled water and pH was adjusted to 7 by adding sodium hydroxide (1N) and the culture checked for the presence of bioemulsifier by emulsification index test. The remaining culture supernatant was also checked and found to give a negative result for emulsification index test.
Stability study of extracellular bioemulsifier and emulsions produced by selected isolates
Effect of temperature and pH on the stability of crude bioemulsifier
This study was performed using culture supernatant and sub-jecting it to different environmental conditions. Results
obtained indicated that crude biomemulsifier produced by Lysinibacillus sp. SP1025 is stable at a temperature range of 10–80 °C and pH range of 6–9, giving an E-24 Index value greater than 50 %. However, bioemulsifier from Bacillus cereus SP1035 is more stable, withstanding a temperature range of 4–80 °C and pH of 7–10.5 (Fig.4a, b).
Emulsifying and stabilizing capacity of extracellular bioemulsifiers
The emulsifying and stabilizing capacity of extracellular bioemulsifiers was evaluated by determining the E-24 Index (%), EV (%), ES (%) and EOP (%) with different hydrophobic substrates, namely hexane, heptane, octane, hexadecane, dodecane, benzene, toluene, xylene, diesel, kerosene, soyabean, mustard, groundnut, olive and coconut oil at 0 h, 24-h, 1-month and 3-months intervals. All the emulsions formed during this study were of the oil-in-water type. Bioemulsifier produced by Lysinibacillus sp. SP1025 was able to efficiently emulsify all the tested aliphatic and aromatic hydrocarbons but was less capable of emulsifying ester-based oils. From the Fig.5a–d, it is apparent that emulsions formed
0 10 20 30 40 50 60 70 80 90
a
b
4 10 20 37 40 60 80 Emulsification Index (%) Temperature (°C)Bacillus cereus SP1035 Lysinibacillus sp. SP1025
0 10 20 30 40 50 60 70 80 90 6 7 8 9 10 10.5 Emulsification Index (%) pH
Bacillus cereus SP1035 Lysinibacillus sp. SP1025
Fig. 4 Stability of crude bioemulsifier produced by Lysinibacillus sp. SP1025 and Bacillus cereus SP1035 at different a temperature and b pH. Error bars Standard deviation
with aromatic hydrocarbons had a greater E-24 index (76 %), EV values (75.9 %) after diesel (83.3 % E-24 Index and 77.9 % EV). All the emulsions formed were 100 % stable with the entire organic layer converted into emulsion, except those formed with ester-based oils. It was unable to emulsify soyabean, groundnut and mustard oil.
The bioemulsifier produced by Bacillus cereus SP1035 was more compatible with aromatic and aliphatic hydrocarbons by efficiently emulsifying the entire organic layer into a stable emulsion (Fig.5e–h). Soyabean and groundnut oil were unable to be emulsified by the produced bioemulsifier. Maximum value of E-24 Index (83.3 %) was with diesel. E-24 Index value and EV recorded was 76 and 75.9 % respectively, with benzene, toluene and xylene. Therefore the bioemulsifier pro-duced could also be effective against aromatic hydrocarbons.
E-24 Index and EV of SDS and PBS were 75 and 0 % respectively. ES and EOP of SDS and PBS were found to be 100 and 0 %, respectively. Thus, the emulsifying ability and stability results of the studied bacteria were found to be comparable to the chemical surfactant used.
A study by Plaza et al. (2006) reported 100 % E-24 Index with xylene, toluene, diesel and petroleum oil by isolate T-I/2, when grown in crude-oil-containing medi-um. The best studied polymeric biosurfactants with sig-nificant emulsifying ability is emulsan, produced by Acinetobacter calcoaceticus RAG I. Bioemulsifier of RAG-1 efficiently emulsified a mixture of aliphatic and aromatic/cyclic alkane; however, it did not emulsify pure aliphatic, aromatic or cyclic hydrocarbons (Ron and Rosenberg 2001). 0 20 40 60 80 100
a
b
c
E24 Index (%) 24 h 1 month 3 months 0 20 40 60 80 100 EV (%) 0 20 40 60 80 100 120 ES (%) 0 20 40 60 80 100 120d
e
f
EOP (%) 0 20 40 60 80 100 E24 Index (%) 0 20 40 60 80 100 EV (%)Fig. 5 E-24 Index, EV, ES and EOP of Lysinibacillus sp. SP1025 (a, b, c and d respectively) and Bacillus cereus SP1035 (e, f, g and h respectively) with different hydrophobic substrates at 24 h, 1 and 3 months interval. Error bars indicate standard deviation
Droplet size distribution study of emulsions formed
The particle sizes of emulsions were also calculated by a standard micrometric procedure. The distribution of droplet diameter for different emulsions formed is shown in Fig.6a,b in which it can be observed that compact and stable emulsions comprised of more than 80 % of droplets of diameter between 0 and 24.9μm, whereas loose and unstable emulsions contain 18–36 and 7.5–43 % of droplets of diameter 50–99.9 and 100–150 μm, respectively. Optical images of compact emul-sions showed that size of droplets is apparently smaller as compared to less compact emulsions (Fig. 6c). Moreover, compact emulsions formed polydisperse emulsions character-ized by droplets of different diameter whereas loose emulsions are more homogeneous with larger droplet size (Fig. 6d), which is in accordance with the findings of Portilla-Rivera et al. (2010). Compact emulsions were formed by crude bioemulsifiers of Lysinibacillus sp. SP1025 and Bacillus cereus SP1035 with all the aliphatic and aromatic hydrocar-bons tested, whereas emulsions formed with ester-based oils were less compact.
Conclusion
A total of 88 micro-organisms were isolated from oil-contaminated soil and checked for their bioemulsifier producing ability. Of these micro-organisms, Bacillus cereus SP1035 and Lysinibacillus sp. SP1025 were selected for further experiments on the basis of higher E-24 index test results. To the best of our knowledge, this is the first report for bioemulsifier production from Lysinibacillus species. Maximum bioemulsifier accumula-tion into the medium was observed during the staaccumula-tionary phase of growth in both selected micro-organisms. Maximum E-24 index recorded for culture supernatant of Lysinibacillus sp. SP1025 and Bacillus cereus SP1035 were 83.3 % and 76.5 %, respectively, with hydrocarbon oil. A reduction in surface ten-sion was observed up to 34.20±0.03 and 43.42±0.03 mN/m for Lysinibacillus sp. SP1025 and Bacillus cereus SP1035, respec-tively. Production of bioemulsifier obtained was 3.07±0.62 and 3.90±0.27 g/L for Lysinibacillus sp. SP1025 and Bacillus cereus SP1035, respectively. Crude biomemulsifier produced by Lysinibacillus sp. SP1025 and Bacillus cereus SP1035 were found to be stable at a temperature range of 10–80 °C. Efficient
Fig. 5 (continued) 0 20 40 60 80 100 120
g
h
ES (% ) 0 20 40 60 80 100 120 EO P (% ) Hydrophobic substratesemulsion stability and emulsifying activity for tested aliphatic and aromatic hydrocarbons confirms its aptness for the use in the petroleum industry and in environmental applications for min-eralization of aromatic hydrocarbons in contaminated systems. Work is in progress to optimize the production process of these products by using different substrates and to estimate the hydro-carbon degrading ability of the producer micro-organisms.
Acknowledgments N.P. acknowledges Council of Scientific and In-dustrial Research (CSIR), New Delhi for the research fellowship provided [09/554(0023)/2010-EMR-I] and corresponding authors acknowledge the University Grant Commission (UGC), Government of India for the financial support [F. No. 40-160/2011(SR)]. The authors are grateful to Central Instrumentation Facility (CIF) at Birla Institute of Technology, Mesra, Ranchi for providing instrumentation facilities necessary to carry out the work.
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