Increasing in the Extraction Yield of Environmentally Friendly Antifouling Agent from Pseudomonas Aeruginosa MUT3 by Response Surface Methodology (RSM)

Document Type: Research Article


1 Chemical Engineering-Biotechnology Group, Malek Ashtar University of Technology, Tehran, I.R. IRAN

2 Microbiology Group, Shahed University, Tehran, I.R. IRAN

3 Polymer Engineering Group, Amir Kabir University of Technology, Tehran, I.R.


In the present study, the solvent-solvent extraction of phenazine 1-carboxylic acid (PCA) as an environmentally friendly antifouling agent from pseudomonas aeruginosa MUT3 culture was investigated. Accordingly, after screening the extraction ability of various solvents, the combined effects of operating parameters such as solvent type (ethyl acetate, dichloromethane, and n-hexane), solvent percent and mixing time on the PCA extraction process were analyzed using response surface methodology (RSM). As a consequence, ethyl acetate showed higher extraction yield (68%) and the optimum condition for PCA extraction were identified as 150% of solvent and 120 min mixing time. Meanwhile, the extraction yields for dichloromethane and n-hexane
were measured by HPLC assay around 48.75 and 25.2%, respectively. The accuracy
of the obtained model was proved by 99.90% R2 and 99.84% Adj R2. In addition, the disk diffusion test showed 9.2, 8 and 7.3 mm inhibition zone for ethyl acetate, dichloromethane and n-hexane, respectively. Consequently, the present study provided a great insight into the solvent-solvent extraction of antibiotics from the fermentation broth.


Main Subjects

[1] Schultz M.P., Bendick J.A., Holm E.R., Hertel W.M., Economic Impact of Biofouling on a Naval Surface Ship, Biofouling, 27(1): 87-98 (2011).

[2] Schultz M.P., Effects of Coating Roughness and Biofouling on Ship Resistance and Powering, Biofouling, 23(5): 331-341 (2007).

[3] Schultz M. P., Bendick J.A., Holm E.R., Hertel W.M., Frictional Resistance of Antifouling Coating Systems, J. Fluids Eng., 126(6): 1039-1047 (2004).

[4] Chambers L.D., Stokes K.R., Walsh F.C., Wood R.J., Modern Approaches to Marine Antifouling Coatings, Surf. Coat. Tech., 201(6): 3642-3652 (2006).

[5] Aguila-Ramírez R.N., Hernández-Guerrero C.J., González-Acosta B., Id-Daoud G., Hewitt S.,
Pope J., Hellio C., Antifouling Activity of Symbiotic Bacteria from Sponge Aplysina Gerardogreeni,
Int. Biodet. Biodeg., 90: 64-70 (2014).

[6] qing Feng D., Qiu Y., Wang W., Wang X., Gang Ouyang P., Huan Ke, C., Antifouling Activities of Hymenialdisine and Debromohymenialdisine from the Sponge Axinella sp.,Int. Biodet. Biodeg., 85: 359-364 (2013).

[7] Mol V.L., Raveendran T.V., Abhilash K.R., Parameswaran P.S., Inhibitory Effect of Indian Sponge Extracts on Bacterial Strains and Larval Settlement of the Barnacle, Balanus Amphitrite,
Int. Biodet. Biodeg., 64(6): 506-510 (2010).

[8] Xu Y., Li N., Jiao W.H., Wang R.P., Peng Y., Qi S.H., Lin H.W., Antifouling and Cytotoxic Constituents from the South China Sea sponge Acanthella Cavernosa, Tetrahedron, 68(13): 2876-2883 (2012).

[9] Chambers L.D., Hellio C., Stokes K.R., Dennington S.P., Goodes L.R., Wood R.J.K., Walsh F.C., Investigation of Chondrus Crispus as a Potential Source of New Antifouling Agents, Int. Biodet. Biodeg., 65(7): 939-946 (2011).

[10] Silkina A., Bazes A., Mouget J.L., Bourgougnon N., Comparative Efficiency of Macroalgal Extracts and Booster Biocides as Antifouling Agents to Control Growth of Three Diatom Species, Mar. Poll. Bulletin, 64(10): 2039-2046 (2012).

[11] Prabhakaran S., Rajaram R., Balasubramanian V., Mathivanan K., Antifouling Potentials of Extracts from Seaweeds, Seagrasses and Mangroves Against Primary Biofilm Forming Bacteria, Asian Pac. J. Trop. Biomed., 2(1): S316-S322 (2012).

[12] Rajan R., Selvaraj M., Palraj S., Subramanian G., Studies on the Anticorrosive & Antifouling Properties of the Gracilaria Edulis Extract Incorporated Epoxy Paint in the Gulf of Mannar Coast, Mandapam, India, Prog. Org. Coat., 90: 448-454 (2016.).

[13] Soliman Y.A., Mohamed A.S., NaserGomaa M., Antifouling Activity of Crude Extracts Isolated from Two Red Sea puffer Fishes, Egyp. J. Aqua.Res., 40(1): 1-7 (2014).

[14] Satheesh S., Ba-akdah M.A., Al-Sofyani A.A., Natural Antifouling Compound Production by Microbes Associated with Marine Macroorganisms—A Review, Elect. J. Biotech., 21: 26-35 (2016).

[15] Gatenholm P., Holmström C., Maki J.S., Kjelleberg S., Toward Biological Antifouling Surface Coatings: Marine Bacteria Immobilized in Hydrogel Inhibit Barnacle Larvae, Biofouling, 8(4): 293-301 (1995).

[17] Kharchenko U., Beleneva I., Evaluation of Coatings Corrosion Resistance with Biocomponents as Antifouling Additives, Corr. Sci., 72: 47-53 (2013).

[18] Peres R.S., Armelin E., Moreno-Martínez J.A., Alemán C., Ferreira C.A., Transport and Antifouling Properties of Papain-Based Antifouling Coatings, App. Sur. Sci., 341: 75-85 (2015).

[19] Olsen S.M., Kristensen J.B., Laursen B.S., Pedersen L.T., Dam-Johansen K., Kiil S., Antifouling Effect of Hydrogen Peroxide Release from Enzymatic Marine Coatings: Exposure Testing under Equatorial and Mediterranean Conditions, Prog. Org. Coat., 68(3): 248-257 (2010).

[20] Olsen S.M., Pedersen L.T., Dam-Johansen K., Kristensen J.B.,  Kiil S., Replacement of Traditional Seawater-Soluble Pigments by Starch and Hydrolytic Enzymes in Polishing Antifouling Coatings, J. Coat. Tech. Res., 7(3): 355-363 (2010).

[21] Wang H., Jiang Y., Zhou L., Gao J., Bienzyme System Immobilized in Biomimetic Silica for Application in Antifouling Coatings, Chin. J. Chem. Eng., 23(8): 1384-1388 (2015).

[22] Ozupek N.M., Cavas L., Triterpene Glycosides Associated Antifouling Activity from Holothuria Tubulosa and H. Polii, Region. Stud. Mar. Sci., 13: 32-41 (2017).

[23] Clare A.S., Marine Natural Product Antifoulants: Status and Potential, Biofouling, 9(3): 211-229 (1996).

[24] Guezennec J., Herry J.M., Kouzayha A., Bachere E., Mittelman M.W., Fontaine MN., Exopolysaccharides from Unusual Marine Environments Inhibit Early Stages of Biofouling, Int. Biodet. Biodeg., 66(1): 1-7 (2012).

[25] Kharchenko U., Beleneva I., Dmitrieva E., Antifouling Potential of a Marine Strain, Pseudomonas Aeruginosa 1242, Isolated from Brass Microfouling in Vietnam, Int. Biodet. Biodeg., 75: 68-74 (2012).

[26] Ghafari M.D., Bahrami A., Rasooli I., Arabian D., Ghafari F., Bacterial Exopolymeric Inhibition of Carbon Steel Corrosion, Int. Biodet. Biodeg., 80: 29-33 (2013).

[27] Burgess J.G., Boyd K.G., Armstrong E., Jiang Z., Yan L., Berggren M., Adams D.R., The Development of a Marine Natural Product-Based Antifouling Paint, Biofouling, 19(S1): 197-205 (2003).

[28] Stead P., Rudd B.A., Bradshaw H., Noble D., Dawson M.J., Induction of Phenazine Biosynthesis in Cultures of Pseudomonas Aeruginosa by LN-(3-oxohexanoyl) Homoserine Lactone, FEMS Microb. Lett., 140(1): 15-22 (1996).

[29] Luo Q., Hu H., Peng H., Zhang X., Wang W., Isolation and Structural Identification of Two Bioactive Phenazines from Streptomyces Griseoluteus P510, Chin. J. Chem. Eng., 23(4): 699-703 (2015).

[30] Raio A., Reveglia P., Puopolo G., Cimmino A., Danti R., Evidente A., Involvement of Phenazine-1-Carboxylic Acid in the Interaction between Pseudomonas Chlororaphis Subsp. Aureofaciens Strain M71 and Seiridium Cardinale in Vivo, Microb. Res., 199: 49-56 (2017).

[33] Jain R., Pandey A., A Phenazine-1-Carboxylic Acid Producing Polyextremophilic Pseudomonas Chlororaphis (MCC2693) Strain, Isolated from Mountain Ecosystem Possesses Biocontrol and Plant Growth Promotion Abilities, Microb. Res., 190: 63-71 (2016).

[36] Hu H.B., Xu Y.Q., Feng C., Xue H.Z.,  Hur B.K., Isolation and Characterization of a New Fluorescent Pseudomonas Strain that Produces Both Phenazine 1-carboxylic Acid and Pyoluteorin, J. Microb. Biotech., 15(1): 86-90 (2005).

[37] Chi X., Wang Y., Miao J., Feng Z., Zhang H., Zhai J., Huang R., Development and Characterization of
a Fusion Mutant with the Truncated lacZ to Screen Regulatory Genes for Phenazine Biosynthesis in Pseudomonas Chlororaphis G05
, Biologic. Control, 108: 70-76 (2017).

[38] Xie K., Peng H., Hu H., Wang W., Zhang X., OxyR, an Important Oxidative Stress Regulator to Phenazines Production and Hydrogen Peroxide Resistance in Pseudomonas Chlororaphis GP72, Microbiol. Res., 168(10): 646-653 (2013).

[39] Raio A., Puopolo G., Cimmino A., Danti R., Della Rocca G., Evidente A., Biocontrol of Cypress Canker by the Phenazine Producer Pseudomonas Chlororaphis Subsp. Aureofaciens Strain M71, Biologic. Cont., 58(2): 133-138 (2011).

[40] Gorantla J.N., Kumar S.N., Nisha G.V., Sumandu A.S., Dileep C., Sudaresan A., Kumar B.D., Purification and Characterization of Antifungal Phenazines from a Fluorescent Pseudomonas Strain FPO4 Against Medically Important Fungi, J. Medic. Mycolog., 24(3): 185-192 (2014).

[41] Ramos I., Dietrich L.E., Price-Whelan A., Newman D.K., Phenazines affect Biofilm Formation by Pseudomonas Aeruginosa in Similar Ways at Various Scales, Res. Microb., 161(3): 187-191 (2010).

[42] Chen Y., Shen X., Peng H., Hu H., Wang W., Zhang X., Comparative Genomic Analysis and Phenazine Production of Pseudomonas Chlororaphis, a Plant Growth-Promoting Rhizobacterium, Genom. Data, 4: 33-42 (2015).

[44] Su J.J., Zhou Q., Zhang H.Y., Li Y.Q., Huang X.Q., Xu Y.Q., Medium Optimization for Phenazine-1-Carboxylic Acid Production by a gacA qscR Double Mutant of Pseudomonas sp. M18 using Response Surface Methodology, Biores. Tech., 101(11): 4089-4095 (2010).

[45] Yuan L.L., Li Y.Q., Wang Y., Zhang X.H.,  Xu Y.Q. al., Optimization of Critical Medium Components Using Response Surface Methodology for Phenazine-1-carboxylic Acid Production by Pseudomonas sp.
, J. Biosci. Bioeng., 105(3): 232-237 (2008).

[46] Ye L., Zhang H., Xu H., Zou Q., Cheng C., Dong D., Xu Y., Li R., Phenazine-1-carboxylic Acid Derivatives: Design, Synthesis and Biological Evaluation Against Rhizoctonia Solani Kuhn, Bioorgan. Medicin. Chem. Lett., 20(24): 7369-7371 (2010).

[47] Chandaliya V.K., Banerjee P., Biswas P., Optimization of Solvent Extraction Process Parameters of Indian Coal, Miner. Process. Extract. Metall. Rev., 33(4): 246-259 (2012).

[48] Jeganathan P.M., Venkatachalam S., Karichappan, T., Ramasamy S., Model Development and Process Optimization for Solvent Extraction of Polyphenols from Red Grapes Using Box–Behnken Design, Prepar. Biochem. Biotech., 44(1): 56-67 (2014).

[50] Nansathit A., PHAOSIRI C., Pongdontri P., Chanthai S., Ruangviriyachai C., Synthesis, isolation of Phenazine Derivatives and Their Antimicrobial Activities. Walailak J. Sci. Tech. (WJST), 6(1): 79-91 (2011).

[52] Levitch M., Stadtman E., A Study of the Biosynthesis of Phenazine-1-Carboxylic Acid, Arch. Biochem Biophys., 106: 194-199 (1964).

[53] Rosales A.M., Thomashow L., Cook R.J.,  Mew T.W., Isolation and Identification of Antifungal Metabolites Produced by Rice-Associated Antagonistic Pseudomonas spp., Phytopathology, 85(9): 1028-1032 (1995).

[54] Mosmeri H., Alaie E., Shavandi M., Dastgheib S.M.M., Tasharrofi S., Bioremediation of Benzene from Groundwater by Calcium Peroxide (CaO2) Nanoparticles Encapsulated in Sodium Alginate,
J. Taiwan. Inst. Chem. Eng.
, 78: 299-306 (2017).

[56] Box G.E, Behnken D.W., Some New Three Level Designs for the Study of Quantitative Variables, Technometrics, 2(4): 455-475 (1960).

[58] Goleij M., Fakhraee H., Response Surface Methodology Optimization of Cobalt (II) and
Lead (II) Removal from Aqueous Solution using MWCNT-Fe3O4 Nanocomposite
, Iran. J. Chem. Chem. Eng. (IJCCE), 36(5): 129-141

[59] Khanahmadi M., Ghaffarzadegan R., Khalighi-Sigaroodi F., Naghdi Badi H., Mehrafarin A., Hajiaghaee R., Optimization of the Glycyrrhizic Acid Extraction from Licorice by Response Surface Methodology, Iran. J. Chem. Chem. Eng. (IJCCE), 37(1): 121-129 (2018).

[60] Mosmeri H., Alaie E., Shavandi M., Dastgheib S.M.M., Tasharrofi S., Benzene-Contaminated Groundwater Remediation using Calcium Peroxide Nanoparticles: Synthesis and Process Optimization, Environ. Monit. Assess., 189(9): 452-462 (2017).

[61] Wani A.A., Sogi D.S., Grover L.,  Saxena D.C., Effect of Temperature, Alkali Concentration, Biosys. Eng., 94(1): 67-73 (2006).

[62] Spigno G., Tramelli L., De Faveri D.M., Effects of Extraction Time, Temperature and Solvent on Concentration and Antioxidant Activity of Grape Marc Phenolics, J. Food Eng., 81(1): 200-208 (2007).

[63] Salamatinia B., Hashemizadeh I., Ahmad Zuhairi A., Alkaline Earth Metal Oxide Catalysts for Biodiesel Production from Palm Oil: Elucidation of Process Behaviors and Modeling Using Response Surface Methodology, Iran. J. Chem. Chem. Eng. (IJCCE), 32(1): 113-126 (2013).

[64] Yousefi N., Pazouki M., Alikhani Hesari F., Alizadeh M., Statistical Evaluation of the Pertinent Parameters in Bio-synthesis of Ag/MWf-CNT Composites Using Plackett-Burman Design and Response Surface Methodology, Iran. J. Chem. Chem. Eng. (IJCCE), 35(2): 51-62 (2016).

[65] Taghavi K., Purkareim S., Pendashteh A., Chaibakhsh N., Optimized Removal of Sodium Dodecylbenzenesulfonate by Fenton-Like Oxidation Using Response Surface Methodology, Iran. J. Chem. Chem. Eng. (IJCCE), 35(4): 113-124 (2016).