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Production, characterization and emulsifying property of exopolysaccharide produced by marine isolate of Pseudomonas fluorescens
Author’s Accepted Manuscript Production, Characterization and Emulsifying property of exopolysaccharide produced by marine isolate of Pseudomonas fluorescens R. Vidhyalakshmi, C. Valli Nachiyar, G. Narendra Kumar, Swetha Sunkar, Iffath Badsha www.elsevier.com/locate/bab
PII: DOI: Reference:
S1878-8181(18)30138-5 https://doi.org/10.1016/j.bcab.2018.08.023 BCAB856
To appear in: Biocatalysis and Agricultural Biotechnology Received date: 9 February 2018 Revised date: 17 August 2018 Accepted date: 30 August 2018 Cite this article as: R. Vidhyalakshmi, C. Valli Nachiyar, G. Narendra Kumar, Swetha Sunkar and Iffath Badsha, Production, Characterization and Emulsifying property of exopolysaccharide produced by marine isolate of Pseudomonas fluorescens, Biocatalysis and Agricultural Biotechnology, https://doi.org/10.1016/j.bcab.2018.08.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Production, Characterization and Emulsifying property of exopolysaccharide produced by marine isolate of Pseudomonas fluorescens R. Vidhyalakshmi1, C. Valli Nachiyar2*, G. Narendra Kumar2, Swetha Sunkar2, Iffath Badsha2 1
2
Department of Microbiology, Dr. MGR Janaki college of Arts and Science, Chennai 28
Department of Biotechnology, Sathyabama Institute of Science and Technology, Chennai 119
*Corresponding Author. Dr. C. Valli Nachiyar , Professor, Department of Biotechnology, Sathyabama
University,
[email protected]
Sholinganallur,
Chennai
600
119.
Ph.
+91-44-24503145.
ABSTRACT Most of the natural emulsifiers are obtained from pig fat and rarely from oils like palm oil and coconut oil. Hence, an attempt has been made to isolate and identify the bacteria capable of producing exopolysaccharides which can serve as good emulsifier and can be used in the preparation of safe creams and lotions. Pseudomonas fluorescens isolated from the biofilm formed on catamaran was found to synthesize 2.9 mg/ml of exopolysaccharide. Box Behnken Optimization method predicted the maximum EPS production of 2.86 mg/ml with sucrose, yeast extract, calcium chloride and casamino acid concentration of 2.5, 0.35, 0.35 and 0.75 g /l respectively. FTIR analysis of the extracted polymer showed the presence of protein which might have helped in the formation of stable emulsion. HPLC report indicated the presence glucose, fructose, galactose, rhamnose and mannose and NMR analysis confirmed the presence of glucose, galactose and mannose. The GC analysis identified palmitic, myristic and stearic acid in the EPS. The emulsification activity was almost found to be similar in hydrocarbon with fatty acid compared to hydrocarbon without fatty acid. This ability qualifies P. fluorescens as a potential candidate for bioremediation processes and the EPS produced as a substitute for synthetic emulsifiers. Key words: P. fluorescens; optimization, Characterization; Emulsification 1. Introduction Microbial polymers are considered as important sources of polymeric materials that have great potential for commercialization. Due to their diversity in structure and unique properties they
have wide range of applications with ease to modify.
They are employed in food,
pharmaceutical and various other industrial applications. These compounds are used to modify flow characteristics of fluids, stabilize suspensions, flocculate particle, encapsulate materials and
produce emulsion (Ye et al., 2012; Saravanan and Shetty, 2016; Adesulu-Dahunsi et al., 2018). Recently, increasing attention has been paid to microbial polysaccharides and a great number of microbial strains have been shown to produce polymer with varying composition and functionalities.
Among them are Xanthomonas, Pseudomonas, Rhizobium, Erwinia,
Leuconostac and Vibrio spp. and number of Lactobacilli spp. (Ates 2015; Castellane et al., 2015). Number of EPS producing bacteria has been reported to date. Only few have been documented for their compounds with emulsifying property (Gutierrez et al., 2008; Peele et al., 2016). Bio emulsifier-producing microorganisms can be divided into three categories (Phetrong et al., 2008]: those producing bio emulsifiers exclusively with alkanes as carbon source, such as Corynebacterium sp.; those producing biosurfactants only with water soluble substrates as the carbon source, such as Bacillus sp.; and those producing bio surfactants with alkanes and water soluble substrates as carbon sources, such as Pseudomonas sp.
Xanthan from Xanthomonas
campestries, Emulsan by Acinetobacter calcoaceticus RAG-1 (Rosenberg and Rosenberg, 1981) are the few in this list that have been commercialized.
Bioemulsifiers of microbial origin are
with various advantages over synthetic emulsifiers; their biodegradability being one of their most important assets (Caruso et al., 2017). Bacteria can develop biofilm on a number of different surfaces, such as natural aquatic and soil environments, living tissues, medical devices or industrial or potable water piping systems. Biofilms have been found to protect the microbial community from environmental stresses (Bramhachari and Dubey, 2006). Microorganisms have been found to attach more rapidly to hydrophobic and non-polar surfaces than hydrophilic surfaces with its biofilm forming ability. Cell surface hydrophobicity, the presence of fimbriae and flagella and the degree of EPS production are the other main factors that have been shown to profoundly influence the rate and
degree of attachment of microbial cells to different surfaces (Sutherland, 2001). These EPS are known as surface active agents and function along with other compounds to reduce the surface tension and help the bacteria to feed on water immiscible materials. These biosurfactants adsorb, emulsify, wet and disperse, solubilize and make the nutrients available for the microbes (Iyer et al., 2005). Surfactants and emulsifiers from bacterial sources create an attention because of their biodegradability and possible production from renewable resources (Viramontes-Ramos et al., 2010; Shekhar et al., 2015; Peele et al., 2016; Santos et al., 2016). Commercially these emulsifiers can be used in oil recovery, emulsify milk products and sausages and the best trade material of recent years the cosmetic products. To make a cream or lotion it is necessary to use an Emulsifier. Mixing oil with water is not possible without using Emulsifiers. The emulsifiers enable two usually non-mixable ingredients to mix together to produce creams and lotions; or in the food industry, margarine and mayonnaise. An Emulsifier should be sourced from natural ingredients such as vegetable oils and other vegetable-based raw materials. Unfortunately, nature provides us with only a few emulsifiers such as lecithin and egg yolk and their performance as emulsifiers is not consistent to be used for commercially available cosmetics products. Most of the natural emulsifiers are obtained from pig fat and rarely from oils like palm oil and coconut oil. Hence, an attempt has been made to isolate and identify the bacteria capable of producing exopolysaccharides which can serve as good emulsifier and can be used in the preparation of safe creams and lotions. 2. Materials and methods 2.1.Microorganism and Culture Conditions
The bacterial samples for EPS production were obtained by scraping and swabbing the wet sides of catamaran. The swabs were kept inside a 25 ml sterile nutrient broth with a pH of 8. After overnight incubation the cultures were isolated by pour plate techniques. The mucoidal colonies were selected and used for the production of biopolymer. The bacterium that produces appreciable amount of exopolysaccharides was identified using microscopic and biochemical characteristics. The bacterium selected for EPS production was grown in a basal salt solution (BSS) having the following composition (g/100 ml): sucrose 1.0, yeast extract 0.5, sodium di hydrogen phosphate 0.3 g and casamino acid 1 ml/100 ml. pH of the medium was adjusted to 7.5 with 1 N NaOH. The medium was sterilized by autoclaving for 20 min at 1210C and was inoculated with 2% (v/v) of 18 h old culture grown in the same medium at room temperature on a rotary shaker at 150 rpm. 2.2.Biopolymer production and extraction Selected and identified colony was inoculated on nutrient broth with 1% glucose and incubated for 48 hours. After incubation the cell pellets were removed by centrifugation at 10,000 rpm for 10 min and the supernatant was treated with twice its volume of ethanol. The set up was left over night at 4˚C. A fine network that appeared in the upper layer of solvent was collected by centrifugation at 15,000rpm for 15 min. The collected pellets were purified by by dialysis using membrane with a pore size of 14,000 K Dal MW against distilled water. Extracted, purified EPS were lyophilized and stored for future use (Vidhyalakshmi et al., 2016). The EPS was estimated by the phenol-sulphuric acid method of Dubois et al., (1956). A sample of 1 ml of the diluted solution was taken and 1 ml of conc. H2SO4 phenol mixture was added and placed in an ice bath for 5–15 min and the absorbance of the samples at 494 nm was determined using spectrophotometer.
2.3.Optimization of medium—Response Surface Methodology using Box Behnken design Based on prior experiments sucrose, yeast extract, calcium chloride and casamino acid were found to be major variables in polymer production when temperature and pH were kept constant (Vidhyalakshmi and Valli Nachiyar 2012; Vidhyalakshmi et al., 2016). The concentration of each component required to increase the yield of EPS production by P. fluorescens was optimized by statistical experimental design using Design expert version 7.0, Stat-Ease, Minneapolis. This method involves number of empirical techniques to evaluate the correlation of experimental factors and predict the critical concentration of dependent and independent variables. A Box Behnken design was applied to obtain the experimental data that fits in full Quadratic polynomial model representing the response surface over a relatively broad range of parameters. The range and the levels of experimental variables investigated are presented in the Table 1. The quadratic equation Y= γo+ γ1A + γ2B + γ3C + γ4D + γ5A2 + γ6B2 + γ7C2 + γ8D2 + γ9AB + γ10AC + γ11AD + γ12BD + γ13BD + γ14CD
(1)
Where Y is the measured response, A, B and C are the coded independent input variables, γo is the intercept term, γ1, γ2, γ3 and γ4 are the coefficients showing the linear effects, γ5, γ6, γ7 and γ8 are the quadratic coefficients showing the squared effects and, γ9, γ10, γ11, γ12, γ13 and γ14 are the cross product coefficients showing the interaction effects. 2.4.Characterization of Biopolymer
The major functional groups of the polymer were identified using Fourier Transform Infra Red spectrum. 0.5 mg of dried sample was ground with 150 mg of KBr crystals. The mixture was pressed using hydraulic press. The discs were subjected to FTIR analysis using Perkin Elmer IR spectrophotometer. For High Pressure Liquid Chromatographic analysis, 1.00 mg of the test Sample was mixed with 2 ml of water and 2.5 ml of acetonitrile with gentle heating. 0.5 ml of water was added to the sample to form the test solution. Reference solution was prepared with glucose, fructose, galactose, rhamnose and verbecose in 20 ml of water and 25 ml of acetonitrile with gentle heating. Columns used were: stainless steel column of 0.05 m long and 4.6 mm internal diameter followed by a stainless steel column of 0.15 m long and 4.6 mm internal diameter, both packed with amino propylsilyl silica gel for chromatography [3µm] which were maintained at 38⁰C. Mobile phase was prepared by dissolving 0.253 g of sodium di hydrogen phosphate in 220 ml of water and 780 ml of acetonitrile and the mobile phase moved at a flow rate of 1.0 ml/min. Refractometer maintained at a constant temperature was used as detector. (LACHROM L-7000, Series 3456). The Nuclear Magnetic Resonance spectra were obtained on a Bruker AMX-500 instrument (500.13 MHz for 1H NMR) at 700C. and Chemical shifts were reported in PPM relative to sodium-d 4-trimethylsilyl propionate. Gas Chromatography – Mass Spectroscopic analysis (GCMS-QP2010 Ultra, Shimadzu) was carried out to find the fatty acid composition of the exopolysaccharide by the procedure explained by Zheng et al. (2012). 2.5.Emulsifying Property
The polymer obtained was dissolved in 5 ml distilled water (1mg/ml) and mixed with 5 ml each of hydrophobic substances in test tube. The tubes were vortexed to homogeneity and left to stand for 24 hours at 4o C. Emulsifying activity was expressed as the percentage of total height occupied by the emulsion after 24 hours (Prasad et al., 2013). Commercial brands of sunflower oil, olive oil, groundnut oil, Soya bean oil, kerosene, diesel, lubricant oil and grease were the hydrophobic substances used. Tween 80 was used as positive control. 3. Results and discussion A bacterium with the ability to produce exopolysaccharide was isolated from the biofilms formed on the catamaran. The organism was found to be gram negative, rod shaped bacterium formed yellowish pigment which fluoresces under UV. Biochemical characterization identified the organism as P. fluorescens belonging to the genus Pseudomonas (data not shown). Extracted Polymer produced by P. fluorescens was found to be partially soluble in water. Number of Pseudomonas spp. has been reported to form polymers with emulsifying property (Czaczyk and Myszka, 2007; Colin et al., 2011). Initial optimization procedure by conventional method identified sucrose, yeast extract casamino acid and calcium chloride as the good nutrient for P.fluorescens for enhanced the EPS production. The effect of four variables on EPS production was studied by the Response surface methodology. Table 2 gives the Box Behnken design matrix with experimental and predicted values for EPS production. The regression equation shows the EPS production as an empirical function in terms of coded factors as Actual yield of EPS (Y) = 2.86 – 0.0333 A – 0.0083 B + 0 .01 C – 0.1083 D + 0 AB + 0.025 AC + 0.075 AD + 0.025 BC – 0.1 BD + 0 CD – 0.4258 A2 – 0.6633 B2 – 0. 6508 C2 – 0.3380 D2
ANOVA for response surface quadratic model gave F-value 58.6768, with P-values of the model (P < 0.0001), implying its significance. The coefficient of variation of the model was (CV = 4.072%). The goodness of fit of the model was examined by determination coefficient (R2= 0.9832) which implied that sample variation of more than 98.32% was attributed to the variables and only 1.68% of total variance could not be explained by the model. The adjusted determination coefficient (Adj R2 = 0.9665) was also satisfactory to confirm the significance of the model. The results of the response surface quadratic model in the form of analysis of variance (ANOVA) with significance of each coefficient and P-values are listed in Table 3. The larger the magnitude of ‘t’ in t test and smaller the P-value, the more significant is the corresponding coefficient. The model predicted the maximum EPS production of 2.86 mg/ml with sucrose, yeast exract, calcium chloride and casamino acid concentration of 2.5, 0.35, 0.35 and 0.75 g /l respectively. Response surface contours plots and three dimensional graphs help to understand the relationship between the response and experimental levels of each variable. These plots also show the type of interaction between test variables and help to obtain the optimum conditions (Haaland 1989). A total of six response surfaces were shown by considering all the possible combinations (Fig. 1). These plots show the type of interaction between the tested variables and hence allow us obtain the optimum conditions (Myers and Montgomery, 1995). A circular contour plot represents a negligible interaction between the independent variables, while perfect interactions were indicated by the elliptical contours. The maximum predicted value is represented by the surface confined in the smallest ellipse in the contour diagram (Muralidhar et al., 2001). The optimum value of each variable was identified based on the hump in the threedimensional plot, or from the central point of the corresponding contour plot. Each contour curve
represents an infinite number of combination of the two tested variables, with the other two maintained at zero levels. Fig.1 shows the EPS production as a result of interaction between the variables taking two variables at varied concentration and keeping the concentrations of other two variables constant. A high value of carbon source (2.5 g/l), optimum value of nitrogen source (0.35 g/l) and casamino acid (0.75 g/l) and calcium chloride (0.35 g/l) were seen to enhance the EPS production. Response surface plots obtained from the data of the present study clearly show the significance of the mutual interactions between the variables. Final optimized conditions are obtained by solving inverse matrix (Equation given above) and through statistical analysis of the constraints. This optimization has enhanced the EPS production from 0.740 mg/ml to 2.9 mg/ml. Verification of the calculated conditions for EPS production by P. fluorescens was done by carrying out the experiments in Erlenmeyer flasks containing 500 ml medium under conditions predicted by the model. The experimental values were found to be close to the predicted values and hence, the model was successfully validated. Validation of the statistical model was carried out by taking A (2.5), B (0.35), C (0.35) and D (0.75) in the validation experiments. Under these conditions, 2.9 mg/ml EPS was produced, in agreement with the model predicted amount of 2.86 mg/ml FTIR reports (Fig. 2a) on the Polymer showed the presence of carboxyl and protein groups around the regions of 4000 cm-1 to 3200 cm-1. Typical Peaks around the regions of 1541.10 cm-1 clearly shows the presence of Amide groups. The absorption peaks at regions of 1640 cm-1
further ensures the presence of amides. Transmission peak around 1645.32 cm-1
shows the presence of anionic carbohydrate.
Reports of HPLC (Fig. 2b) clearly indicated the presence of mannose, rhamnose, fructose, galactose and glucose in varying concentrations with galactose followed by glucose and fructose being the predominant sugars.
Number of Pseudomonas is known to synthesize
rhamnolipids that have emulsifying property (Chayabutra et al., 2001). The NMR signals for proton correlate with HPLC reports and confirms the presence of glucose, mannose and galactose. The 1H (Fig. 2c) spectra of extracted EPS enabled the extrapolation of some information. 1H NMR was used for identifying the type of interested compound polysaccharide, number of residues in repeated units and also for checking the purity of the polysaccharide. NMR analysis of the present EPS and its spectrum represent the protons from glycosidic group of carbohydrates. H chemical shifts observed in 3-5 PPM indicate the presence of α/γ Glucose;
5.2-5.27 ppm indicate H6α, H3α, H5α, H6γ and 3.35-3.5 ppm indicate α/γ Galactose and 4.15 ppm indicates the presence of γ mannose (Badertscher et al., 2009) . Reports of Gas Chromatography analysis (Fig. 2d) clearly showed the presence of palmitic acid, stearic acid and myristic acid. Palmitic Acid is a saturated fatty acid found naturally in skin and in many vegetable oils. These compounds are often derived from coconuts or other palms. It is an emollient and helps to reinforce the skin’s healthy barrier function, providing occlusive layer protection. Presence of palmitic acid like compound in the extracted polymer ensures its utility in cosmetic formulation. The body makes palmitic acid out of excess carbohydrates and excess protein (Web reference 1). Stearic Acid is a fatty acid found primarily in animal derivatives, but in vegetable fats as well. It is used in a variety of cosmetics and personal care products, as a fragrance ingredient, surfactant and emulsifier. It is also used as the base for the manufacture of other fatty acid ingredients which are used as emulsifers, emollients and lubricants (Web reference 2). Myristic acid is used in the food industry as a flavoring agent.
Myristic acid is found widely distributed in fats throughout the plant and animal kingdom, including common human foodstuffs, such as nutmeg.
Myristic acid, also known as
Tetradecanoic Acid is an important fatty acid, which the body uses to stabilize many different proteins, including proteins used in the immune system and also those that fight against tumors (Kim and Ross, 2013). It has a variety of uses in the beauty industry, including as a: Fragrance Ingredient; Opacifying Agent; Surfactant; Cleansing Agent; and Emulsifier. One of its primary properties is as a lubricant, due to its high rate of absorption by the skin (Web reference 3). Zheng et al. (2012) have reported hexadecanoic acid and octadecanoic acid as the major fatty acids in the lipidic fraction of Aeribacillus pallidus YM-1 bioemulsifier. The emulsification activity was almost found to be similar in hydrocarbon with fatty acid compared to hydrocarbon without fatty acid (Table 4).
As the concentration of the EPS
increased, there is a corresponding increase in the emulsification activity. The P. fluorescens EPS exhibited highest emulsification against Diesel and Olive oil. Sifour et al. (2007) have reported the emulsification activity of P. aeruginosa RB28 EPS appreciable emulsification activity against Sunflower oil followed by heptadecane and paraffin indicating that EPS can emulsify both hydrocarbons with and without fatty acid effectively. In contrast, Abouseoud et al., (2007) have reported diesel and kerosene as the best substrate for the EPS from P. fluorescens and sunflower oil as less good substrate. 4. Conclusion The search for bioemulsifier from different sources is a continuous process since the availability of emulsifier for commercial purpose is limited. Instrumental analyses of the cell free EPS of P. fluorescens have confirmed the presence of galactose, glucose, fructose, mannose and rhamnose along with proteins. P. fluorescens EPS showed good emulsifying activity towards vegetable
oils (fatty oils) and also problem causing hydrocarbons (non-fatty oils). Its emulsifying activity towards vegetable oils qualifies this EPS as a good substitute for synthetic emulsifiers in cosmetic products. The ability of P. fluorescence to produce an EPA with emulsifying activity towards hydrocarbons indicates that this bacterium can be a potential candidate for the bioremediation processes. Acknowledgment The authors would like to thank the management of Sathyabama Institute of Science and Technology for providing the infrastructure and facilities to carry out this work. References 1. Abouseoud, M., Maachi, R., Amrane, A. 2007. Biosurfactant Production from olive oil by Pseudomonas fluorescens. Communicating Current Research and Educational Topics and Trends in Applied Microbiology A. Méndez-Vilas (Ed.) ©FORMATEX. 340–348 2. Adesulu-Dahunsi, A.T., A.I., Sanni, K., Jeyaram, J.O., Ojediran, A.O., Ogunsakin, K., Banwo, 2018. Extracellular Polysaccharide from Weissella confusa OF126: Production, Optimization and Characterization. Int. J. Biol. Macromol. 111, 432–442. 3. Ates, O., 2015. Systems Biology of Microbial Exopolysaccharides Production. Front. Bioeng. Biotechnol. 3, 200. 4. Badertscher, M., Bühlmann, P., Pretsch, E., 2009.
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Fig. 1. Response Surface Methodology—Box Behnken design in the production of EPS. Fig. 2. Characterization of P. fluorescens exopolysaccharide (a) FTIR spectrum (b) HPLC analysis (c) 1H NMR spectrum and (d) GC chromatogram
Table 1 Factors and levels of RSM-BBD. Factor A B C D
Name Sucrose Yeast Extract Calcium Chloride Casamino Acid
Actual Low High 0.5 2.5 0.2 0.5 0.2 0.5 0.5 1
Coded Low High -1 1 -1 1 -1 1 -1 1
Mean
Std. Dev.
1.5 0.35 0.35 0.75
0.643267521 0.096490128 0.096490128 0.16081688
Table 2 Experimental and predicted yield of EPS values for RSM-BBD. Factor 1 Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
A:Sucrose 1.5 1.5 1.5 2.5 0.5 2.5 0.5 1.5 1.5 1.5 1.5 0.5 1.5 1.5 1.5 1.5 2.5 0.5 1.5 2.5 0.5 1.5 1.5 2.5 2.5
Factor 2 B:Yeast Extract 0.2 0.35 0.35 0.35 0.5 0.5 0.35 0.35 0.2 0.35 0.2 0.2 0.5 0.5 0.35 0.5 0.35 0.35 0.35 0.35 0.35 0.35 0.5 0.35 0.2
Factor 3 Factor 4 C:Calcium D:Casamino Chloride Acid 0.2 0.75 0.2 0.5 0.35 0.75 0.5 0.75 0.35 0.75 0.35 0.75 0.35 1 0.2 1 0.35 0.5 0.5 0.5 0.35 1 0.35 0.75 0.35 0.5 0.35 1 0.35 0.75 0.5 0.75 0.35 1 0.5 0.75 0.35 0.75 0.2 0.75 0.2 0.75 0.5 1 0.2 0.75 0.35 0.5 0.35 0.75
Response Actual 1.5 1.9 2.9 1.8 1.9 1.8 1.9 1.7 1.9 2.1 1.8 1.8 2.1 1.6 2.9 1.6 2.1 1.9 2.8 1.6 1.8 1.9 1.3 2.1 1.7
Predicted 1.5 1.9 2.9 1.9 1.8 1.7 1.9 1.7 1.9 2.1 1.9 1.8 2.1 1.6 2.9 1.7 2.0 1.9 2.9 1.6 1.7 1.9 1.4 2.1 1.7
26 27 28 29
1.5 1.5 1.5 0.5
0.2 0.35 0.35 0.35
0.5 0.35 0.35 0.35
0.75 0.75 0.75 0.5
1.7 2.9 2.8 2.2
1.6 2.9 2.9 2.3
Table 3 Test of significance for regression coefficient. Sum of Source Squares Model 5.447166667 A-Sucrose 0.013333333 B-Yeast Extract 0.000833333 C-Calcium Chloride 0.12 D-Casamino Acid 0.140833333 AB 0 AC 0.0025 AD 0.0225 BC 0.0025 BD 0.04 CD 0 A^2 1.176220721 B^2 2.854126126 C^2 2.747572072 D^2 0.742504505 Residual 0.092833333 Lack of Fit Pure Error Cor Total Std. Dev. Mean C.V. % PRESS
0.080833333 0.012 5.54
p-value df Mean Square F Value Prob > F 14 0.389083333 58.67684022 < 0.0001 1 0.013333333 2.010771993 0.1781 1 0.000833333 0.12567325 0.7282 1 1 1 1 1 1 1 1 1 1 1 1 14
0.12 0.140833333 0 0.0025 0.0225 0.0025 0.04 0 1.176220721 2.854126126 2.747572072 0.742504505 0.006630952
18.09694794 21.23877917 0 0.377019749 3.393177738 0.377019749 6.032315978 0 177.3833762 430.4247659 414.3555728 111.9755447
10 4 28
0.008083333 0.003
2.694444444 0.1760
0.08143066 2 4.071532998 0.48435
R-Squared Adj R-Squared Pred R-Squared Adeq Precision
significant
0.0008 0.0004 1.0000 0.5491 0.0867 0.5491 0.0277 1.0000 < 0.0001 < 0.0001 < 0.0001 < 0.0001 not significant
0.983243081 0.966486161 0.912572202 24.71633013
Table 4. Emulsification activity of P. fluorescens EPS Oils /
Hydrocarbon without fatty acid
Hydrocarbon with fatty acid
EPS
Kerosen
Dies
Crud Mixed
Soya
Oliv
Sunflow
Groundn n 80 /
concentrati
e
el
e 2t
Engine
Bean
e
er Oil
ut Oil
Oil
Oil
Oil
on
0.05%
Negligib 20%
5%
le 0.1%
5%
Negligib Negligib 50% le
50%
10%
Twee
Diese l
5%
10%
20%
60%
10%
30%
28%
le
Negligib 5% le
1.5%
40%
80%
43%
10%
44%
80%
40%
40%
40%
2%
60%
100
55%
30%
70%
100
65%
64%
78%
%
%
PII: DOI: Reference:
S1878-8181(18)30138-5 https://doi.org/10.1016/j.bcab.2018.08.023 BCAB856
To appear in: Biocatalysis and Agricultural Biotechnology Received date: 9 February 2018 Revised date: 17 August 2018 Accepted date: 30 August 2018 Cite this article as: R. Vidhyalakshmi, C. Valli Nachiyar, G. Narendra Kumar, Swetha Sunkar and Iffath Badsha, Production, Characterization and Emulsifying property of exopolysaccharide produced by marine isolate of Pseudomonas fluorescens, Biocatalysis and Agricultural Biotechnology, https://doi.org/10.1016/j.bcab.2018.08.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Production, Characterization and Emulsifying property of exopolysaccharide produced by marine isolate of Pseudomonas fluorescens R. Vidhyalakshmi1, C. Valli Nachiyar2*, G. Narendra Kumar2, Swetha Sunkar2, Iffath Badsha2 1
2
Department of Microbiology, Dr. MGR Janaki college of Arts and Science, Chennai 28
Department of Biotechnology, Sathyabama Institute of Science and Technology, Chennai 119
*Corresponding Author. Dr. C. Valli Nachiyar , Professor, Department of Biotechnology, Sathyabama
University,
[email protected]
Sholinganallur,
Chennai
600
119.
Ph.
+91-44-24503145.
ABSTRACT Most of the natural emulsifiers are obtained from pig fat and rarely from oils like palm oil and coconut oil. Hence, an attempt has been made to isolate and identify the bacteria capable of producing exopolysaccharides which can serve as good emulsifier and can be used in the preparation of safe creams and lotions. Pseudomonas fluorescens isolated from the biofilm formed on catamaran was found to synthesize 2.9 mg/ml of exopolysaccharide. Box Behnken Optimization method predicted the maximum EPS production of 2.86 mg/ml with sucrose, yeast extract, calcium chloride and casamino acid concentration of 2.5, 0.35, 0.35 and 0.75 g /l respectively. FTIR analysis of the extracted polymer showed the presence of protein which might have helped in the formation of stable emulsion. HPLC report indicated the presence glucose, fructose, galactose, rhamnose and mannose and NMR analysis confirmed the presence of glucose, galactose and mannose. The GC analysis identified palmitic, myristic and stearic acid in the EPS. The emulsification activity was almost found to be similar in hydrocarbon with fatty acid compared to hydrocarbon without fatty acid. This ability qualifies P. fluorescens as a potential candidate for bioremediation processes and the EPS produced as a substitute for synthetic emulsifiers. Key words: P. fluorescens; optimization, Characterization; Emulsification 1. Introduction Microbial polymers are considered as important sources of polymeric materials that have great potential for commercialization. Due to their diversity in structure and unique properties they
have wide range of applications with ease to modify.
They are employed in food,
pharmaceutical and various other industrial applications. These compounds are used to modify flow characteristics of fluids, stabilize suspensions, flocculate particle, encapsulate materials and
produce emulsion (Ye et al., 2012; Saravanan and Shetty, 2016; Adesulu-Dahunsi et al., 2018). Recently, increasing attention has been paid to microbial polysaccharides and a great number of microbial strains have been shown to produce polymer with varying composition and functionalities.
Among them are Xanthomonas, Pseudomonas, Rhizobium, Erwinia,
Leuconostac and Vibrio spp. and number of Lactobacilli spp. (Ates 2015; Castellane et al., 2015). Number of EPS producing bacteria has been reported to date. Only few have been documented for their compounds with emulsifying property (Gutierrez et al., 2008; Peele et al., 2016). Bio emulsifier-producing microorganisms can be divided into three categories (Phetrong et al., 2008]: those producing bio emulsifiers exclusively with alkanes as carbon source, such as Corynebacterium sp.; those producing biosurfactants only with water soluble substrates as the carbon source, such as Bacillus sp.; and those producing bio surfactants with alkanes and water soluble substrates as carbon sources, such as Pseudomonas sp.
Xanthan from Xanthomonas
campestries, Emulsan by Acinetobacter calcoaceticus RAG-1 (Rosenberg and Rosenberg, 1981) are the few in this list that have been commercialized.
Bioemulsifiers of microbial origin are
with various advantages over synthetic emulsifiers; their biodegradability being one of their most important assets (Caruso et al., 2017). Bacteria can develop biofilm on a number of different surfaces, such as natural aquatic and soil environments, living tissues, medical devices or industrial or potable water piping systems. Biofilms have been found to protect the microbial community from environmental stresses (Bramhachari and Dubey, 2006). Microorganisms have been found to attach more rapidly to hydrophobic and non-polar surfaces than hydrophilic surfaces with its biofilm forming ability. Cell surface hydrophobicity, the presence of fimbriae and flagella and the degree of EPS production are the other main factors that have been shown to profoundly influence the rate and
degree of attachment of microbial cells to different surfaces (Sutherland, 2001). These EPS are known as surface active agents and function along with other compounds to reduce the surface tension and help the bacteria to feed on water immiscible materials. These biosurfactants adsorb, emulsify, wet and disperse, solubilize and make the nutrients available for the microbes (Iyer et al., 2005). Surfactants and emulsifiers from bacterial sources create an attention because of their biodegradability and possible production from renewable resources (Viramontes-Ramos et al., 2010; Shekhar et al., 2015; Peele et al., 2016; Santos et al., 2016). Commercially these emulsifiers can be used in oil recovery, emulsify milk products and sausages and the best trade material of recent years the cosmetic products. To make a cream or lotion it is necessary to use an Emulsifier. Mixing oil with water is not possible without using Emulsifiers. The emulsifiers enable two usually non-mixable ingredients to mix together to produce creams and lotions; or in the food industry, margarine and mayonnaise. An Emulsifier should be sourced from natural ingredients such as vegetable oils and other vegetable-based raw materials. Unfortunately, nature provides us with only a few emulsifiers such as lecithin and egg yolk and their performance as emulsifiers is not consistent to be used for commercially available cosmetics products. Most of the natural emulsifiers are obtained from pig fat and rarely from oils like palm oil and coconut oil. Hence, an attempt has been made to isolate and identify the bacteria capable of producing exopolysaccharides which can serve as good emulsifier and can be used in the preparation of safe creams and lotions. 2. Materials and methods 2.1.Microorganism and Culture Conditions
The bacterial samples for EPS production were obtained by scraping and swabbing the wet sides of catamaran. The swabs were kept inside a 25 ml sterile nutrient broth with a pH of 8. After overnight incubation the cultures were isolated by pour plate techniques. The mucoidal colonies were selected and used for the production of biopolymer. The bacterium that produces appreciable amount of exopolysaccharides was identified using microscopic and biochemical characteristics. The bacterium selected for EPS production was grown in a basal salt solution (BSS) having the following composition (g/100 ml): sucrose 1.0, yeast extract 0.5, sodium di hydrogen phosphate 0.3 g and casamino acid 1 ml/100 ml. pH of the medium was adjusted to 7.5 with 1 N NaOH. The medium was sterilized by autoclaving for 20 min at 1210C and was inoculated with 2% (v/v) of 18 h old culture grown in the same medium at room temperature on a rotary shaker at 150 rpm. 2.2.Biopolymer production and extraction Selected and identified colony was inoculated on nutrient broth with 1% glucose and incubated for 48 hours. After incubation the cell pellets were removed by centrifugation at 10,000 rpm for 10 min and the supernatant was treated with twice its volume of ethanol. The set up was left over night at 4˚C. A fine network that appeared in the upper layer of solvent was collected by centrifugation at 15,000rpm for 15 min. The collected pellets were purified by by dialysis using membrane with a pore size of 14,000 K Dal MW against distilled water. Extracted, purified EPS were lyophilized and stored for future use (Vidhyalakshmi et al., 2016). The EPS was estimated by the phenol-sulphuric acid method of Dubois et al., (1956). A sample of 1 ml of the diluted solution was taken and 1 ml of conc. H2SO4 phenol mixture was added and placed in an ice bath for 5–15 min and the absorbance of the samples at 494 nm was determined using spectrophotometer.
2.3.Optimization of medium—Response Surface Methodology using Box Behnken design Based on prior experiments sucrose, yeast extract, calcium chloride and casamino acid were found to be major variables in polymer production when temperature and pH were kept constant (Vidhyalakshmi and Valli Nachiyar 2012; Vidhyalakshmi et al., 2016). The concentration of each component required to increase the yield of EPS production by P. fluorescens was optimized by statistical experimental design using Design expert version 7.0, Stat-Ease, Minneapolis. This method involves number of empirical techniques to evaluate the correlation of experimental factors and predict the critical concentration of dependent and independent variables. A Box Behnken design was applied to obtain the experimental data that fits in full Quadratic polynomial model representing the response surface over a relatively broad range of parameters. The range and the levels of experimental variables investigated are presented in the Table 1. The quadratic equation Y= γo+ γ1A + γ2B + γ3C + γ4D + γ5A2 + γ6B2 + γ7C2 + γ8D2 + γ9AB + γ10AC + γ11AD + γ12BD + γ13BD + γ14CD
(1)
Where Y is the measured response, A, B and C are the coded independent input variables, γo is the intercept term, γ1, γ2, γ3 and γ4 are the coefficients showing the linear effects, γ5, γ6, γ7 and γ8 are the quadratic coefficients showing the squared effects and, γ9, γ10, γ11, γ12, γ13 and γ14 are the cross product coefficients showing the interaction effects. 2.4.Characterization of Biopolymer
The major functional groups of the polymer were identified using Fourier Transform Infra Red spectrum. 0.5 mg of dried sample was ground with 150 mg of KBr crystals. The mixture was pressed using hydraulic press. The discs were subjected to FTIR analysis using Perkin Elmer IR spectrophotometer. For High Pressure Liquid Chromatographic analysis, 1.00 mg of the test Sample was mixed with 2 ml of water and 2.5 ml of acetonitrile with gentle heating. 0.5 ml of water was added to the sample to form the test solution. Reference solution was prepared with glucose, fructose, galactose, rhamnose and verbecose in 20 ml of water and 25 ml of acetonitrile with gentle heating. Columns used were: stainless steel column of 0.05 m long and 4.6 mm internal diameter followed by a stainless steel column of 0.15 m long and 4.6 mm internal diameter, both packed with amino propylsilyl silica gel for chromatography [3µm] which were maintained at 38⁰C. Mobile phase was prepared by dissolving 0.253 g of sodium di hydrogen phosphate in 220 ml of water and 780 ml of acetonitrile and the mobile phase moved at a flow rate of 1.0 ml/min. Refractometer maintained at a constant temperature was used as detector. (LACHROM L-7000, Series 3456). The Nuclear Magnetic Resonance spectra were obtained on a Bruker AMX-500 instrument (500.13 MHz for 1H NMR) at 700C. and Chemical shifts were reported in PPM relative to sodium-d 4-trimethylsilyl propionate. Gas Chromatography – Mass Spectroscopic analysis (GCMS-QP2010 Ultra, Shimadzu) was carried out to find the fatty acid composition of the exopolysaccharide by the procedure explained by Zheng et al. (2012). 2.5.Emulsifying Property
The polymer obtained was dissolved in 5 ml distilled water (1mg/ml) and mixed with 5 ml each of hydrophobic substances in test tube. The tubes were vortexed to homogeneity and left to stand for 24 hours at 4o C. Emulsifying activity was expressed as the percentage of total height occupied by the emulsion after 24 hours (Prasad et al., 2013). Commercial brands of sunflower oil, olive oil, groundnut oil, Soya bean oil, kerosene, diesel, lubricant oil and grease were the hydrophobic substances used. Tween 80 was used as positive control. 3. Results and discussion A bacterium with the ability to produce exopolysaccharide was isolated from the biofilms formed on the catamaran. The organism was found to be gram negative, rod shaped bacterium formed yellowish pigment which fluoresces under UV. Biochemical characterization identified the organism as P. fluorescens belonging to the genus Pseudomonas (data not shown). Extracted Polymer produced by P. fluorescens was found to be partially soluble in water. Number of Pseudomonas spp. has been reported to form polymers with emulsifying property (Czaczyk and Myszka, 2007; Colin et al., 2011). Initial optimization procedure by conventional method identified sucrose, yeast extract casamino acid and calcium chloride as the good nutrient for P.fluorescens for enhanced the EPS production. The effect of four variables on EPS production was studied by the Response surface methodology. Table 2 gives the Box Behnken design matrix with experimental and predicted values for EPS production. The regression equation shows the EPS production as an empirical function in terms of coded factors as Actual yield of EPS (Y) = 2.86 – 0.0333 A – 0.0083 B + 0 .01 C – 0.1083 D + 0 AB + 0.025 AC + 0.075 AD + 0.025 BC – 0.1 BD + 0 CD – 0.4258 A2 – 0.6633 B2 – 0. 6508 C2 – 0.3380 D2
ANOVA for response surface quadratic model gave F-value 58.6768, with P-values of the model (P < 0.0001), implying its significance. The coefficient of variation of the model was (CV = 4.072%). The goodness of fit of the model was examined by determination coefficient (R2= 0.9832) which implied that sample variation of more than 98.32% was attributed to the variables and only 1.68% of total variance could not be explained by the model. The adjusted determination coefficient (Adj R2 = 0.9665) was also satisfactory to confirm the significance of the model. The results of the response surface quadratic model in the form of analysis of variance (ANOVA) with significance of each coefficient and P-values are listed in Table 3. The larger the magnitude of ‘t’ in t test and smaller the P-value, the more significant is the corresponding coefficient. The model predicted the maximum EPS production of 2.86 mg/ml with sucrose, yeast exract, calcium chloride and casamino acid concentration of 2.5, 0.35, 0.35 and 0.75 g /l respectively. Response surface contours plots and three dimensional graphs help to understand the relationship between the response and experimental levels of each variable. These plots also show the type of interaction between test variables and help to obtain the optimum conditions (Haaland 1989). A total of six response surfaces were shown by considering all the possible combinations (Fig. 1). These plots show the type of interaction between the tested variables and hence allow us obtain the optimum conditions (Myers and Montgomery, 1995). A circular contour plot represents a negligible interaction between the independent variables, while perfect interactions were indicated by the elliptical contours. The maximum predicted value is represented by the surface confined in the smallest ellipse in the contour diagram (Muralidhar et al., 2001). The optimum value of each variable was identified based on the hump in the threedimensional plot, or from the central point of the corresponding contour plot. Each contour curve
represents an infinite number of combination of the two tested variables, with the other two maintained at zero levels. Fig.1 shows the EPS production as a result of interaction between the variables taking two variables at varied concentration and keeping the concentrations of other two variables constant. A high value of carbon source (2.5 g/l), optimum value of nitrogen source (0.35 g/l) and casamino acid (0.75 g/l) and calcium chloride (0.35 g/l) were seen to enhance the EPS production. Response surface plots obtained from the data of the present study clearly show the significance of the mutual interactions between the variables. Final optimized conditions are obtained by solving inverse matrix (Equation given above) and through statistical analysis of the constraints. This optimization has enhanced the EPS production from 0.740 mg/ml to 2.9 mg/ml. Verification of the calculated conditions for EPS production by P. fluorescens was done by carrying out the experiments in Erlenmeyer flasks containing 500 ml medium under conditions predicted by the model. The experimental values were found to be close to the predicted values and hence, the model was successfully validated. Validation of the statistical model was carried out by taking A (2.5), B (0.35), C (0.35) and D (0.75) in the validation experiments. Under these conditions, 2.9 mg/ml EPS was produced, in agreement with the model predicted amount of 2.86 mg/ml FTIR reports (Fig. 2a) on the Polymer showed the presence of carboxyl and protein groups around the regions of 4000 cm-1 to 3200 cm-1. Typical Peaks around the regions of 1541.10 cm-1 clearly shows the presence of Amide groups. The absorption peaks at regions of 1640 cm-1
further ensures the presence of amides. Transmission peak around 1645.32 cm-1
shows the presence of anionic carbohydrate.
Reports of HPLC (Fig. 2b) clearly indicated the presence of mannose, rhamnose, fructose, galactose and glucose in varying concentrations with galactose followed by glucose and fructose being the predominant sugars.
Number of Pseudomonas is known to synthesize
rhamnolipids that have emulsifying property (Chayabutra et al., 2001). The NMR signals for proton correlate with HPLC reports and confirms the presence of glucose, mannose and galactose. The 1H (Fig. 2c) spectra of extracted EPS enabled the extrapolation of some information. 1H NMR was used for identifying the type of interested compound polysaccharide, number of residues in repeated units and also for checking the purity of the polysaccharide. NMR analysis of the present EPS and its spectrum represent the protons from glycosidic group of carbohydrates. H chemical shifts observed in 3-5 PPM indicate the presence of α/γ Glucose;
5.2-5.27 ppm indicate H6α, H3α, H5α, H6γ and 3.35-3.5 ppm indicate α/γ Galactose and 4.15 ppm indicates the presence of γ mannose (Badertscher et al., 2009) . Reports of Gas Chromatography analysis (Fig. 2d) clearly showed the presence of palmitic acid, stearic acid and myristic acid. Palmitic Acid is a saturated fatty acid found naturally in skin and in many vegetable oils. These compounds are often derived from coconuts or other palms. It is an emollient and helps to reinforce the skin’s healthy barrier function, providing occlusive layer protection. Presence of palmitic acid like compound in the extracted polymer ensures its utility in cosmetic formulation. The body makes palmitic acid out of excess carbohydrates and excess protein (Web reference 1). Stearic Acid is a fatty acid found primarily in animal derivatives, but in vegetable fats as well. It is used in a variety of cosmetics and personal care products, as a fragrance ingredient, surfactant and emulsifier. It is also used as the base for the manufacture of other fatty acid ingredients which are used as emulsifers, emollients and lubricants (Web reference 2). Myristic acid is used in the food industry as a flavoring agent.
Myristic acid is found widely distributed in fats throughout the plant and animal kingdom, including common human foodstuffs, such as nutmeg.
Myristic acid, also known as
Tetradecanoic Acid is an important fatty acid, which the body uses to stabilize many different proteins, including proteins used in the immune system and also those that fight against tumors (Kim and Ross, 2013). It has a variety of uses in the beauty industry, including as a: Fragrance Ingredient; Opacifying Agent; Surfactant; Cleansing Agent; and Emulsifier. One of its primary properties is as a lubricant, due to its high rate of absorption by the skin (Web reference 3). Zheng et al. (2012) have reported hexadecanoic acid and octadecanoic acid as the major fatty acids in the lipidic fraction of Aeribacillus pallidus YM-1 bioemulsifier. The emulsification activity was almost found to be similar in hydrocarbon with fatty acid compared to hydrocarbon without fatty acid (Table 4).
As the concentration of the EPS
increased, there is a corresponding increase in the emulsification activity. The P. fluorescens EPS exhibited highest emulsification against Diesel and Olive oil. Sifour et al. (2007) have reported the emulsification activity of P. aeruginosa RB28 EPS appreciable emulsification activity against Sunflower oil followed by heptadecane and paraffin indicating that EPS can emulsify both hydrocarbons with and without fatty acid effectively. In contrast, Abouseoud et al., (2007) have reported diesel and kerosene as the best substrate for the EPS from P. fluorescens and sunflower oil as less good substrate. 4. Conclusion The search for bioemulsifier from different sources is a continuous process since the availability of emulsifier for commercial purpose is limited. Instrumental analyses of the cell free EPS of P. fluorescens have confirmed the presence of galactose, glucose, fructose, mannose and rhamnose along with proteins. P. fluorescens EPS showed good emulsifying activity towards vegetable
oils (fatty oils) and also problem causing hydrocarbons (non-fatty oils). Its emulsifying activity towards vegetable oils qualifies this EPS as a good substitute for synthetic emulsifiers in cosmetic products. The ability of P. fluorescence to produce an EPA with emulsifying activity towards hydrocarbons indicates that this bacterium can be a potential candidate for the bioremediation processes. Acknowledgment The authors would like to thank the management of Sathyabama Institute of Science and Technology for providing the infrastructure and facilities to carry out this work. References 1. Abouseoud, M., Maachi, R., Amrane, A. 2007. Biosurfactant Production from olive oil by Pseudomonas fluorescens. Communicating Current Research and Educational Topics and Trends in Applied Microbiology A. Méndez-Vilas (Ed.) ©FORMATEX. 340–348 2. Adesulu-Dahunsi, A.T., A.I., Sanni, K., Jeyaram, J.O., Ojediran, A.O., Ogunsakin, K., Banwo, 2018. Extracellular Polysaccharide from Weissella confusa OF126: Production, Optimization and Characterization. Int. J. Biol. Macromol. 111, 432–442. 3. Ates, O., 2015. Systems Biology of Microbial Exopolysaccharides Production. Front. Bioeng. Biotechnol. 3, 200. 4. Badertscher, M., Bühlmann, P., Pretsch, E., 2009.
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Fig. 1. Response Surface Methodology—Box Behnken design in the production of EPS. Fig. 2. Characterization of P. fluorescens exopolysaccharide (a) FTIR spectrum (b) HPLC analysis (c) 1H NMR spectrum and (d) GC chromatogram
Table 1 Factors and levels of RSM-BBD. Factor A B C D
Name Sucrose Yeast Extract Calcium Chloride Casamino Acid
Actual Low High 0.5 2.5 0.2 0.5 0.2 0.5 0.5 1
Coded Low High -1 1 -1 1 -1 1 -1 1
Mean
Std. Dev.
1.5 0.35 0.35 0.75
0.643267521 0.096490128 0.096490128 0.16081688
Table 2 Experimental and predicted yield of EPS values for RSM-BBD. Factor 1 Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
A:Sucrose 1.5 1.5 1.5 2.5 0.5 2.5 0.5 1.5 1.5 1.5 1.5 0.5 1.5 1.5 1.5 1.5 2.5 0.5 1.5 2.5 0.5 1.5 1.5 2.5 2.5
Factor 2 B:Yeast Extract 0.2 0.35 0.35 0.35 0.5 0.5 0.35 0.35 0.2 0.35 0.2 0.2 0.5 0.5 0.35 0.5 0.35 0.35 0.35 0.35 0.35 0.35 0.5 0.35 0.2
Factor 3 Factor 4 C:Calcium D:Casamino Chloride Acid 0.2 0.75 0.2 0.5 0.35 0.75 0.5 0.75 0.35 0.75 0.35 0.75 0.35 1 0.2 1 0.35 0.5 0.5 0.5 0.35 1 0.35 0.75 0.35 0.5 0.35 1 0.35 0.75 0.5 0.75 0.35 1 0.5 0.75 0.35 0.75 0.2 0.75 0.2 0.75 0.5 1 0.2 0.75 0.35 0.5 0.35 0.75
Response Actual 1.5 1.9 2.9 1.8 1.9 1.8 1.9 1.7 1.9 2.1 1.8 1.8 2.1 1.6 2.9 1.6 2.1 1.9 2.8 1.6 1.8 1.9 1.3 2.1 1.7
Predicted 1.5 1.9 2.9 1.9 1.8 1.7 1.9 1.7 1.9 2.1 1.9 1.8 2.1 1.6 2.9 1.7 2.0 1.9 2.9 1.6 1.7 1.9 1.4 2.1 1.7
26 27 28 29
1.5 1.5 1.5 0.5
0.2 0.35 0.35 0.35
0.5 0.35 0.35 0.35
0.75 0.75 0.75 0.5
1.7 2.9 2.8 2.2
1.6 2.9 2.9 2.3
Table 3 Test of significance for regression coefficient. Sum of Source Squares Model 5.447166667 A-Sucrose 0.013333333 B-Yeast Extract 0.000833333 C-Calcium Chloride 0.12 D-Casamino Acid 0.140833333 AB 0 AC 0.0025 AD 0.0225 BC 0.0025 BD 0.04 CD 0 A^2 1.176220721 B^2 2.854126126 C^2 2.747572072 D^2 0.742504505 Residual 0.092833333 Lack of Fit Pure Error Cor Total Std. Dev. Mean C.V. % PRESS
0.080833333 0.012 5.54
p-value df Mean Square F Value Prob > F 14 0.389083333 58.67684022 < 0.0001 1 0.013333333 2.010771993 0.1781 1 0.000833333 0.12567325 0.7282 1 1 1 1 1 1 1 1 1 1 1 1 14
0.12 0.140833333 0 0.0025 0.0225 0.0025 0.04 0 1.176220721 2.854126126 2.747572072 0.742504505 0.006630952
18.09694794 21.23877917 0 0.377019749 3.393177738 0.377019749 6.032315978 0 177.3833762 430.4247659 414.3555728 111.9755447
10 4 28
0.008083333 0.003
2.694444444 0.1760
0.08143066 2 4.071532998 0.48435
R-Squared Adj R-Squared Pred R-Squared Adeq Precision
significant
0.0008 0.0004 1.0000 0.5491 0.0867 0.5491 0.0277 1.0000 < 0.0001 < 0.0001 < 0.0001 < 0.0001 not significant
0.983243081 0.966486161 0.912572202 24.71633013
Table 4. Emulsification activity of P. fluorescens EPS Oils /
Hydrocarbon without fatty acid
Hydrocarbon with fatty acid
EPS
Kerosen
Dies
Crud Mixed
Soya
Oliv
Sunflow
Groundn n 80 /
concentrati
e
el
e 2t
Engine
Bean
e
er Oil
ut Oil
Oil
Oil
Oil
on
0.05%
Negligib 20%
5%
le 0.1%
5%
Negligib Negligib 50% le
50%
10%
Twee
Diese l
5%
10%
20%
60%
10%
30%
28%
le
Negligib 5% le
1.5%
40%
80%
43%
10%
44%
80%
40%
40%
40%
2%
60%
100
55%
30%
70%
100
65%
64%
78%
%
%