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A statistical approach on optimization of exopolymeric substance production by Halomonas sp. S19 and its emulsification activity
© Karuppiah et al. 2015
Received: 15 July 2015
Accepted: 27 November 2015
Published: 21 December 2015
An exopolymer producing bacterial strain was identified as Halomonas sp. S19 by 16S rRNA gene sequencing isolated from Mandapam, Southeast coast of India. Strain S19 produces a significant amount of exopolymer (320 mg L−1) in a medium optimized with 2.5 % glucose, 0.6 % peptone, 7.5 % salt and pH 7.5 at 35 °C. The exopolymer consists of total sugars (65 %), proteins (4.07 %), uronic acids (8.08 %) and sulphur contents (6.39 %). FT-IR and 1H NMR analysis revealed the presence of functional groups corresponding to carbohydrates, proteins and sulphates. The exopolymer of Halomonas sp. S19 emulsifies different oils. However, 10 % exopolymer shows 55.18, 55.18, 49.81 and 24.62 % of emulsifying activity for sesame oil, coconut oil, paraffin and kerosene. The present study was focused on optimisation of exopolymer production using Box–Behnken experimental design and its possibility for potential emulsification index.
The genus Halomonas consists of a broad range of taxonomically and physiologically differed organisms growing at a diverse range of salinities (Kushner and Kamekura 1988). In marine environment, most of the bacteria exclusively secrete exopolymeric substances outside the cell, which may be tightly or loosely bounded on the cell surface and assist the cell to survive (Sutherland 2001). The exopolymeric substances are composed of sugars and non-sugar components (proteins, uronic acids, sulphates and acetyl group) (Llamas et al. 2012).
The presence of proteins, sulphates and uronic acids in bacterial exopolymeric substance confers anticancer, immune modulatory and emulsification activity (Ruiz Ruiz et al. 2011; Perez Fernandez et al. 2000; Bouchotroch et al. 2000). Bio-emulsifiers are higher molecular weight compounds consisting complex mixtures of heteropolysaccharides, lipopolysaccharides, lipoproteins and proteins (Perfumo et al. 2009). Bio-emulsifiers efficiently emulsify two immiscible liquids even at low concentrations, but in contrast is less effective at reduced surface tension. In an oil-polluted environment, the emulsifier plays a significant role in dispersing the hydrocarbons by binding and preventing from merging. It has been attributed to the presence of high number of reactive groups exposed in their structures.
Many microbial polysaccharides serve as emulsifiers due to their ability to stabilise emulsions between water and hydrophobic compounds. When compared with the chemically derived compounds, the bacterial exopolymeric substances were found to be stable in extreme conditions like temperature, pH and salinity (Banat et al. 2000). Hence, an interest has been focused towards the production of biologically derived compounds. Bio-emulsifiers are essential in the formation and stabilisation of emulsion. It reduces the surface tension of oil and water interface by forming a protective layer around emulsion droplets and blocks coalescence by adsorbing with the oil/water interface.
Most of the research fields are bound with factorial assessments. Box–Behnken experimental design is used to assess the optimal level of variables. Response surface methodology (RSM) is a vital one in research field and a combination of mathematical and statistical approaches are gainful for analysing and modelling experiments. This technique is a resourceful one to optimise various parameters resulting in response surface (Kadirgama et al. 2007; Abou-El-Hossein et al. 2007; Nguyena and Borkowskib 2008). The present study was focused to optimise the exopolymer production by Halomonas sp. S19 using Box–Behnken model and its significant feature of emulsification activity on various oils.
Isolation and characterisation of the strain
The exopolymer producing bacteria was isolated from soil sample collected from Southeast Coastal area of Mandapam, India. The bacteria were screened based on the mucoidal appearance on zobell marine agar plates and by staining with Sudan Black B. The DNA samples were collected and amplified in PCR. The 16S rRNA sequencing was performed using 20 µL of purified PCR products (50 ng/µL), the primers 518F (CCAGCAGCCGCGGTAATACG) and 800R (TACCAGGGTATCTAATCC) and Big Dye terminator cycle sequencing kit (Applied BioSystems, USA) (Neefs et al. 1990). The most similar sequences to the sequence of the isolated strain were obtained using BLAST similarity search tool, and the multiple sequence alignment was performed using the program MUSCLE by progressive alignment method. A computer-based program, Gblocks, was used for alignment refinement. The phylogenetic tree was constructed using the programme PhyML 3.0 approximate likelihood-ratio test (aLRT) with HKY85 substitution model for the neighbour-joining method.
Optimization of exopolymer production
The carbon (glucose, sucrose, lactose and galactose) and nitrogen sources (peptone) were separately provided in the basal salt medium at different concentrations and the best source was selected for further studies. The optimum carbon-to-nitrogen ratio was determined by providing the nitrogen at different concentrations (0.5–1 %) and with constant concentration of carbon source (2.5 %) in 250-mL Erlenmeyer flasks containing 100 mL of basal salt medium (BSM). After determining the optimum carbon-to-nitrogen ratio, the medium was statistically optimised by Box–Behnken model with salt concentration, pH and growth temperature as variables; totally, seventeen runs were studied at low and high levels of the variables. The low and high levels of sea salt were 2.5 and 7.5 %. In the case of pH, 6.5 and 7.5 were observed as low and high levels. In the case of temperature, the low and high values were 25 and 45 °C, respectively. 2 ml aliquots of 24 h isolated strain was used as inoculum and allowed to grow for 72 h on a rotary shaker at 110 rpm. The experimental design, statistical and graphical analysis of the data were performed using ‘Design Expert’ software (version 9, Stat-Ease, Inc., Minneapolis, MN, USA).
Extraction and characterization of exopolymer
The exopolymeric substance was extracted (Parthiban et al. 2014), and the total sugar (Dubois et al. 1956), proteins (Lowry et al. 1951), uronic acids (Filisetti-Cozzzi and Carpaita 1991) and sulphates (Dodgson 1961) were determined spectrophotometrically. The monosaccharide constituent was determined by hydrolysing the exopolymer in 4 mol∙L−1 Trifluoroacetic acid (TFA) and heated at 100 °C for 10 min and filtered through 0.45-µm syringe filter. Distillation using methanol removed residual acid from the filtrate. Finally, the sugars were analysed by HPLC (Thermo Scientific, Model Accela) and compared with the standard (Freitas et al. 2009). The dried sample was mixed with potassium bromide and analysed at spectral range of 4000–400 cm−1 using Fourier transform infrared spectrophotometer (FT-IR Bruker IFS 85). 1H NMR spectrum (Bruker AVANCE III 500 MHz AV 500) was recorded by dissolving 20 mg of pure exopolymer in 1 mL of D2O at room temperature. The thermal properties of bacterial exopolymer were studied by differential scanning colorimetric (DSC) (Mettler Toledo DSC 822e) analysis at 40–450 °C (10 °C/min) under nitrogen atmosphere.
Emulsification index (EI24)
Results and discussion
Identification of exopolymer producing strain
Optimization of exopolymer production by Box–Behnken model
Effect of various carbon sources on exopolymer production
Exopolymer production for different concentrations of sugars (mg L−1)
29.63 ± 0.11
35.43 ± 0.11
41.40 ± 0.17
43.43 ± 0.11
51.53 ± 0.05
28.40 ± 0.10
34.10 ± 0.10
40.16 ± 0.15
42.16 ± 0.15
47.50 ± 0.10
28.13 ± 0.15
31.33 ± 0.15
35.43 ± 0.11
39.13 ± 0.15
42.43 ± 0.15
27.60 ± 0.17
30.10 ± 0.10
33.46 ± 0.05
35.36 ± 0.11
37.15 ± 0.15
Effect of carbon:nitrogen ratio on exopolymer production
Sucrose (carbon source) g L−1
Peptone (nitrogen source) g L−1
Exopolymer (mg L−1)
218.66 ± 0.57
204.86 ± 0.23
185.10 ± 0.17
114.13 ± 0.15
72.10 ± 0.10
65.36 ± 0.11
The interaction between carbon and nitrogen sources (peptone) plays a significant role in exopolymer production. The higher concentration of peptone was found to play an inhibitory role on exopolymer production. However, the carbon:nitrogen ratio has a vital role in exopolymer production. The high amount of nutrients might somehow reduce the exopolymer production. Apart from nutritional conditions, exopolysaccharide synthesis and yields largely depend on the environmental condition like pH, temperature, aeration, etc. However, higher or lower the optimal range resulted in decreasing of exopolymeric substances (EPS) yield (Kumar et al. 2007; Gandhi et al. 1997).
Experimental design generated with Design Expert 9.0: the predicted and actual values of production of exopolymer by Halomonas sp. S19
A: sea salt (%)
C: Temp °C
Predicted value exopolymer (mg)
Actual value exopolymer (mg)
ANOVA for response surface quadratic model
Sum of squares
p value Prob > F
Lack of fit
In the present study, Halomonas sp. S19 produced 110 mg L−1 exopolymer in basal salt medium containing 2.5 % glucose, and 320 mg L−1 exopolymer in a medium optimised with glucose: peptone (14.28:1.00), 7.5 % salt and pH 7.5 at 35 °C. These results corroborated with the production by H. ventosae and H. anticariensis, which produced 283.5 and 289.5 mg L−1 exopolymer in a medium containing 7.5 % sea salt, 1 % glucose (Mata et al. 2006). However, H. almeriensis produces 1.7 g L−1 exopolymer in MY complex medium containing 1 % glucose and 7.5 % total salts at 32 °C (Llamas et al. 2012). As far as production is concerned, Halomonas maura gives the highest yield (4.28 g L−1) at a salt concentration of 5 % in MY medium containing 1 % glucose and 0.3 % yeast extract at pH 7 (Arias et al. 2003).
The salt concentration and temperature have a significant role in exopolymer production by halophilic bacteria. I. fontislapidosi F23Tl showed the best growth and high yield of exopolymer (1.50 g L−1) at sea salt concentrations of 7.5 % and 32 °C, whereas the exopolymer production decreased (1.25 g L−1) at lower salt concentration (2.5 %) (Mata et al. 2008).
Characterization of exopolymeric substance
FT-IR spectroscopy of exopolymer
1H NMR study of exopolymer
Differential scanning calorimetric analysis
Emulsification index (EI24)
Emulsification index of exopolymer and Tween 20 upon different oils
Emulsification index %
49.44 ± 0.5
55.18 ± 0.6
67.03 ± 0.6
95.37 ± 1.6
49.81 ± 0.3
55.18 ± 0.3
72.03 ± 0.3
88.51 ± 0.6
38.51 ± 0.6
49.81 ± 0.3
72.22 ± 0.5
92.22 ± 0.9
16.48 ± 0.3
24.62 ± 0.3
27.40 ± 0.6
78.70 ± 1.6
The results of monomeric sugar composition of Halomonas sp. S19 are similar to exopolymer of H. almeriensis that contains glucose and mannose, and small quantities of rhamnose (Llamas et al. 2012). However, in Halomonas sp HE67, the detected monosaccharides were glucuronic acid, glucosamine, mannose, rhamnose, galactose, galactosamine and glucose (Gutierrez et al. 2007). The exopolymer produced by H. anticarinesis consists of glucose (17 %), mannose (43 %), rhamnose (1.5 %), xylose (1.5 %) and galacturonic acid (37.5 %) (Mata et al. 2006).
A considerable emulsification index observed for the exopolymeric substance produced by Halomonas sp. S19 comprised protein (4.1 %), sulphate (6.39 %) and uronic acid (8.08 %). The presence of various groups in the exopolymer allows good adhesion to oil droplets during emulsification and providing steric stability to the emulsion. Such interactions play a significant role in the formation, structure and stabilisation of emulsions (Bach and Gutnick 2005).
The presence of considerable amount of proteins and uronic acids in the exopolymer makes it to play an eminent role in the emulsification of various oils (Mata et al. 2006; Bramhachari et al. 2007). The emulsification index of exopolymer produced by Halomonas sp. TG39 (Gutierrez et al. 2008), and H. almeriensis M8T (Llamas et al. 2012) was mainly influenced by its proteins, sulphates and uronic acids. The exopolymer produced by H. almeriensis effectively emulsifies sunflower (65 %) and mineral oil (67.5 %) (Llamas et al. 2012). The exopolymer producing H. eurihalina showed 57.59 % emulsification activity on mineral oil at 0.5 % concentration, which contains proteins (7.27 %) and sulphates (7.15 %) in the exopolymer (Martinez Checa et al. 2007). Mauran, an exopolysaccharide produced by H. maura, consists of 2.57 % proteins and 8.14 % uronic acids, reported to produce up to 78 % activity against hexadecane (Bouchotroch et al. 2000). The exopolymeric substance produced by H. eurihalina H96 consists of proteins (7 %), uronic acids (7 %) and sulphates (17.6 %); the emulsification index was observed as 73 % in crude oil and 19 % in light oil at 0.5 % concentration (Perfumo et al. 2009).
A large number of bacteria produce polymers that are emulsifying efficiently at low concentrations and exhibiting considerable substrate specificity. They are composed of polysaccharides, proteins, lipopolysaccharides, lipoproteins, etc., (Banat et al. 2000). The exopolymer reported for the emulsification in the present study can be classified under polysaccharide–protein complex (polymeric microbial surfactant). The presence of carboxyl group and sulphates provides overall negative charge to the polymer, thereby imparting binding and adsorptive properties for divalent cation by electrostatic interactions.
Biopolymers are promising invariably and exploring in the space of synthetic emulsifiers. Chemical analysis revealed that the exopolymeric substance of Halomonas sp. S19 in the present study consists of different sugars and non-sugar components that can be used as a safe alternative to chemical emulsifiers. Property of the exopolymer emulsifying edible oils makes as lucrative emulsifier and exploited owing to their advantages against synthetic products.
RT and KP carried out the synthesis and characterisation of the exopolymer. VV and NV carried out the computational experiments and drafted the manuscript. All authors read and approved the final manuscript.
The authors are grateful to the management of H.K.R.H College, Uthamapalayam, Theni District, DST-FIST and UGC-SAP DRS-II for instrumentation facilities in the Department of Animal Science, Centre of Excellence in Life Sciences, Bharathidasan University, Tiruchirappalli, SAIF-IITM, Chennai for NMR analysis and STIC cochin for DSC analysis.
The authors declare that they have no competing interests.
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