Screening of Biosurfactant-Producing Bacteria as a Potential Biological Control Agent for Fungal Orchid Pathogens in Thailand


  • Chanika Saenge Chooklin Faculty of Science and Fisheries Technology, Rajamangala University of Technology Srivijaya Trang Campus, Trang 92150, Thailand
  • Natthaporn Rattanapan Faculty of Agricultural Technology, Phuket Rajabhat University, Phuket 83000, Thailand
  • Atipan Saimmai Faculty of Agricultural Technology, Phuket Rajabhat University, Phuket 83000, Thailand, Andaman Halal Science Center, Phuket Rajabhat University, Phuket 83000, Thailand
  • Wiboon Riansa-ngawong Department of Agro-Industry and Management, Faculty of Digital Agro-Industry, King Mongkut’s University of Technology North Bangkok, Prachinburi 25230, Thailand
  • Suppasil Maneerat Center of Excellence in Innovative Biotechnology for Sustainable Utilization of Bioresources, Faculty of Agro-Industry, Prince of Songkla University, Songkhla 90110, Thailand


Acinetobacter calcoaceticus, Biosurfactant, Orchid disease, Phytophthora palmivora


In southern Thailand, biosurfactant-producing bacteria were isolated from palm oil mill facility soil using serial dilution. Palm oil mill effluent (POME) was utilized as the sole carbon source. A total of 231 oil-degrading bacterial isolates were discovered from 40 samples. Based on the examination of surface tension reduction and emulsification activities, 30 isolates showed promising biosurfactant activities; however, only 85 of the isolates tested positive for the formation of biosurfactants based on the oil displacement test. The biosurfactants from 30 bacterial isolates were tested for their antifungal efficacy against Phytophthora palmivora Al2 using an agar well diffusion assay. The strongest antifungal activity was seen in the biosurfactants produced from strain CT03. Analysis of the bacteria's 16S rRNA gene revealed the strain to be Acinetobacter calcoaceticus. It was discovered that MSM containing (NH4)2SO4 and glucose acting as carbon and nitrogen sources, respectively, provided the best conditions for A. calcoaceticus CT 03 to produce biosurfactants. A combination of chloroform: methanol (2:1, vol/vol) was used to extract 1.75 g/L of biosurfactant. The structures of the resulting biosurfactant compounds was determined using three distinct methods of spectroscopy: Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), and mass spectroscopy. Additionally, the biosurfactants created by A.calcoaceticus CT 03 contained molecules of a lipopeptide that resembled surfactin. This study indicates the ability of this biosurfactant mixture to prevent pathogenic fungal growth in orchids.


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Thammasiri K. Current status of orchid production in Thailand. Acta Hortic. 2015; 1078(1078): 25-33.

Christenhusz M.J.M., Byng J.W. The number of known plants species in the world and its annual increase. Phytotaxa (Magnolia Press). 1992; 261(3); 201-217.

Arditi J. Classification and naming of orchids in The Fundamentals of Orchid Biology. New York: John Wiley and Sons Press. 1992: pp. 51-101.

Kuehnle A.R. Chapter 20: Orchids, Dendrobium in Flower Breeding and Genetics, N.O. Anderson, Ed. Springer. 2007: pp. 539- 560.

Srivastava S., Kadooka C., Uchida J.Y. Fusarium species as pathogen on orchids. Microbiol. Res. 2018; 207: 188-195.

Hossain M.M. Orchid mycorrhiza: Isolation, culture, characterization and application. S. Afr. J. Bot. 2022; 151: 365-384.

Li J., Wang R., Wang Z., Kuang P. The phylogenetic relationship and non-specific symbiotic habit of mycorrhiza fungi from a terrestrial orchid (Cymbidium). Nord. J. Bot. 2016; 34: 343-348.

Sowanpreecha R., Rerngsamran P. Biocontrol of orchid-pathogenic mold, Phytophthora palmivora, by antifungal proteins from Pseudomonas aeruginosa RS. Micobiol. 2018; 46(2): 129-137.

Maketon C., Tongjib Y., Patipong T. Greenhouse evaluations of harpin protein and microbial fungicides in controlling Curvularia lunata, Fusarium moniliforme, and Phythopthora palmivora, major causes of orchid diseases in Thailand. Life Sci. 2015; 12: 125-132.

Streda T., Kredl Z., Pokorny R. Effect of wetting period on infection of orchid flowers by Alternaria alternata and Curvularia eragrostidis. N Z J Crop Hortic. Sci. 2013; 41: 1-8.

Laurence M.H., Howard C., Summerell B.A. Identification of Fusarium solani f. sp. phalaenopsis in Australia. Australas. Plant Dis. Notes. 2016; 11: 3.

Meera T., Louis V., Beena S. Diseases of Phalaenopsis: symptoms, etiology and management. Int. J. Agric. Innov. Res. 2016; 5: 296-300.

Cating R., Palmateer A., Stiles C. Black rot of orchids caused by Phytophthora cactorum and Phytophthora palmivora in Florida. Plant Health Prog. 2009. DOI:10.1094/ PHP-2010-0614-01-DG

Pane A.S. Cacciola O. Badala F. Martini P., Magnano di San Lio G. Il marciume radicale da Phytophthora nei vivai di piante ornamentali. Terra e Vita. 2006; 18: 24-28.

Uchida J.Y. Diseases of Orchids in Hawaii. Plant Dis. 1994; 78(3): 220-224.

Cacciola S., Pane O.A., Martini P.G., Agosteo E., Raudino F., Magnano di San Lio G. Recovery of Phytophthora species from potted ornamentals in commercial nurseries in Italy. Plant Pathol. J. 2008; 90: 185.

Orlikowski L.B., Szkuta G. Phytophthora rot of some orchids -new disease in Poland. Phytopathol. Pol. 2006; 40: 57-61.

Cating R.A., Palmateer A.J., Stiles C.M., Rayside P.A. Black rot of orchids caused by Phytophthora cactorum and Phytophthora palmivora in Florida. Online. Plant Health Prog.2010. doi:10.1094/PHP-2010-0614-01--DG

Zin N.A., Badaluddin N.A. Biological functions of Trichoderma spp. for agriculture applications. Ann. Agric. Sci. 2020; 65(2): 168-178.

Banat I.M., Franzetti A., Gandolfi I., Bestetti G., Martinotti M.G., Fracchia L., Smyth T.J., Marchant R. Microbial biosurfactants production, applications and future potential. Appl. Microbiol. Biotechnol. 2010; 87: 427-444.

Pathak K.V., Keharia H. Identification of surfactins and iturins produced by potent fungal antagonist, Bacillus subtilis K1 isolated from aerial roots of banyan (Ficus benghalensis) tree using mass spectrometry. 3 Biotech. 2014; 4(3): 283-295.

Meena K.R., Shamar A., Kanwar S.S. Lipopeptides: a distinct class of antibiotics with diverse applications. Adv. Biotechnol. Microbiol. 2017; 7(2): 26-32.

Meena K.R., Kanwar S.S. Lipopeptides as the antifungal and antibacterial agents: applications in food safety and therapeutics. BioMed Res. Int. 2015; 473050: 9

Rani M., Weadge J.T., Jabaji S. Isolation and characterization of biosurfactant-producing bacteria from oil well batteries with antimicrobial activities against food-borne and plant pathogens. Front. Microbiol. 2020; 27: 1-9.

Kannurin A., Sudarslal S., Arunan C., Sreejith K. A novel lipopeptide from Bacillus cereus strain AK1: isolation, structural evaluation and antifungal activities. J. Appl. Microbiol. 2013; 115(6): 1287-1296.

Deleu M., Paquot M., Nylander T. Effect of Fengycin, a Lipopeptide Produced by Bacillus subtilis, on model biomembranes. Biophys. J. 2008; 94(7): 2667-79.

Sharma J., Sundar D., Srivastava P. Biosurfactants: Potential agents for controlling cellular communication, motility, and antagonism. Front. Mol. Biosci. 2021; 8: 727070.

Özyilmaz U., Benlioglu K. Enhanced biological control of phytophthora blight of pepper by biosurfactant-producing Pseudomonas. Plant Pathol. J. 2013; 29(4): 418-426.

Saimmai A., Tani A., Sobhon V., Maneerat S. Mangrove sediment, a new source of potential biosurfactant producing bacteria. Ann. Microbiol. 2012; 62: 1669-1679.

Saimmai A., Rukadee O., Onlamool T., Sobhon V., Maneerat S. An efficient biosurfactant-producing bacterium Selenomonas ruminantium CT2, isolated from mangrove sediment in south of Thailand. World J. Microbiol Biotechnol. 2012; 29: 87-102.

Saimmai A., Sobhon V., Maneerat S. Molasses a whole medium for bosurfactants production by Bacillus strains and their application. Appl. Biochem. Biotech. 2011; 165: 315-335.

Vasdinyei R., Deák T. Characterization of yeast isolates originating from Hungarian dairy products using traditional and molecular identification techniques. Int. J. Food Microbiol. 2003; 86(1-2): 123-130.

Dlauchy D., Tornai-Lehoczki J., Péter G. Restriction enzyme analysis of PCR amplified rDNA as a taxonomic tool in yeast identification. Syst. Appl. Microbiol. 1999; 22(3): 445-53.

Zampolli J., De Giani A., Di Canito A., Sello G., Di Gennaro P. Identification of a novel biosurfactant with antimicrobial activity produced by Rhodococcus opacus R7. Microorganisms. 2022; 10: 475.

Tamura K., Stecher G., Peterson D., Filipski A., Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Bio. Evol. http://dx. doi. org/10.1093/molbev/mst197. 2013.

Saisa-ard K., Maneerat S., Saimmai A. Isolation and characterization of biosurfactants-producing bacteria isolated from palm oil industry and evaluation for biosurfactants production using low-cost substrates. Biotechnol. 2013; 94(3): 275-284.

Nilsson W.B., Strom M.S. Detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction length polymorphism (T-RFLP) analysis of 16S rRNA genes. Dis. Aquat. Org. 2002; 48: 175-185.

Bodour A.A., Drees K.P., Maier R.M. Distribution of biosurfactant- producing bacteria in undisturbed and contaminated arid southwestern soils. Appl. Environ. Microbiol. 2003; 69(6): 3280-3287.

Batista S.B., Mounteer A.H., Amorim F.R., Totola M.R. Isolation and characterization of biosurfactant/bioemulsifier-producing bacteria from petroleum contaminated sites. Biores. Technol. 2006; 97: 868-875.

Cai Q., Zhang B., Chen B., Zhu Z., Lin W., Cao T. Screening of biosurfactant producers from petroleum hydrocarbon contaminated sources in cold marine environments. Mar. Pollut. Bull. 2014; 86: 402-410.

Shahi A., Aydin S., Ince B. Ince O. Evaluation of microbial population and functional genes during the bioremediation of petroleum-contaminated soil as an effective monitoring approach. Ecotoxicol. Environ. Saf. 2016; 125: 153-160.

Batista S.B., Mounteer A.H., Amorim F.R., Totola M.R. Isolation and characterization of biosurfactant/bioemulsifier-producing bacteria from petroleum contaminated sites. Biores. Technol. 2006; 97(6): 868-875.

Saimmai A., Rukadee O., Onlamool T., Sobhon V., Maneerat S. Characterization and phylogenetic analysis of microbial surface-active compounds-producing bacteria. Appl. Biochem. Biotechnol. 2012; 168: 1003-1018.

Edosa T.T., Jo Y.H., Keshavarz M., Han Y.S. Biosurfactants: Production and potential application in insect pest management. Trends Entomol. 2018; 14: 79-87.

Markande A.R, Patel D., Varjani S. A review on biosurfactants: properties, applications and current developments, Biores. Technol. 2021; 330: 124963.

Montoneri E., Fabbri G., Quagliotto P.L., Baglieri A., Padoan E., Boero V., Negre M. High molecular weight biosurfactants from mild chemical reactions of fermented municipal biowastes. ChemistrySelect. 2020; 5: 2564-2576.

Xi W., Ping Y., Alikhani M.A. A Review on Biosurfactant applications in the petroleum industry. Int. J. Chem. Eng. 2021; 1-10.

Bagheri H., Mohebbi A., Amani F.S., Naderi M. Application of low molecular weight and high molecular weight biosurfactant in medicine/biomedical/pharmaceutical industries. Green Sust. Proc. Chem. Environ Eng Sci. 2022; 1-60.

Mohanty S.S., Koul Y., Varjani S. A critical review on various feedstocks as sustainable substrates for biosurfactants production: a way towards cleaner production. Microb. Cell Fact. 2021; 20: 120.

Gautam K.K., Tyagi V.K. Microbial surfactants: a review. J. Oleo Sci. 2006; 55(4): 155-166.

Sharma N., Lavania M., Lal B. Biosurfactant: a next-generation tool for sustainable remediation of organic pollutants. Front. Microbiol. 2022; 1-6.

Tsai H.L., Huang L.C., Ann P.J., Liou R.F. Detection of orchid phytophthora disease by nested PCR. Bot. Stud. 2006; 47: 379-387.

Violeta O., Oana S., Matilda C., Maria C.D., Catalina V., Gheorghe C., Petruta C. Production of biosurfactants and antifungal compounds by new strains of Bacillus spp. isolated from different sources. Rom. Biotechnol. Lett. 2011; 16: 84-91.

Mnif I., Mnif S., Sahnoun R., Maktouf S., Ayedi S., Ellouze-Chaabouni S. Biodegradation of diesel oil by a novel microbial consortium: comparison between co-inoculation with biosurfactant-producing strain and exogenously added biosurfactants. Environ. Sci. Poll. Res. 2015; 22: 14852-14861.

Joshi S., Bharucha C., Desai A.J. Production of biosurfactant and antifungal compound by fermented food isolate Bacillus subtilis 20B. Biores. Technol. 2008; 99(11): 4603-4608.

Santos J.R.A., Rocha A.A., Macedo A.T., Santana A.A., da Silva J.B., Souza M.E.P., Holanda R.A., Cru G. Antifungal activity of biosurfactant against profound mycosis. Green Sust. Proc. Chem. Environ Eng Sci.: Biome. Appl. Biosur. Med. Sec. 2022; 257-287.

Sen S., Borah S.N., Bora A., Deka S. Production, characterization, and antifungal activity of a biosurfactant produced by Rhodotorula babjevae YS3. Microb. Cell Fact. 2017; 16: 95.

Baruah R., Mishra S.K., Kalita D.J., Silla Y., Chauhan P.S., Singh A.K., Deka Boruah HP. Assessment of bacterial diversity associated with crude oil-contaminated soil samples from Assam. Int. J. Environ. Sci. Technol. 2017; 14: 2155-2172.

Dahal R.H., Chaudhary D.K., Kim J. Acinetobacter halotolerans sp. nov., a novel halotolerant, alkalitolerant, and hydrocarbon degrading bacterium, isolated from soil. Arch. Microbiol. 2017; 199: 701-710.

Hamzah M., Manikan V., Abd Aziz N.A.F. Biodegradation of tapis crude oil using consortium of bacteria and fungi: optimization of crude oil concentration and duration of incubation by response surface methodology. Sains Malays. 2017; 46: 43-50.

Sazykin I., Sazykina M., Khmelevtsova L., Khammami M., Karchava S., Zhuravleva M., Kudeevskaya E. Expression of SOD and production of reactive oxygen species in Acinetobacter calcoaceticus caused by hydrocarbon oxidation. Ann. Microbiol. 2016; 66: 1039-1045.

Pirog T.P., Konon A.D., Sofilkanich A.P., Skochko A.B. Effect of biosurfactants Acinetobacter calcoaceticus K-4 and Rhodococcus erythropolis EK-1 on some microorganisms. Mikrobiol. Zhurnal. 2011; 73(3): 14-20.

Wu J.Y., Yeh K.L., Lu W.B., Lin C.L., Chang J.S. Rhamnolipid production with indigenous Pseudomonas aeruginosa EM1 isolated from oil-contaminated site. Biores. Technol. 2008; 99: 1157-1164.

Saimmai A., Sobhon V., Maneerat S. Production of biosurfactant from a new and promising strain of Leucobacter komagatae 183. Ann. Microbiol. 2012; 62: 391-402.

Hegazy G.E., Abu-Serie M.M., Abou-elela G.M., Ghozlan H., Sabry S.A., Soliman N.A., Teleb M., Abdel-Fattah Y.R. Bioprocess development for biosurfactant production by Natrialba sp. M6 with effective direct virucidal and anti-replicative potential against HCV and HSV. Sci Rep. 2022; 12: 16577.

Maqsood M.I., Jamal A. Factors affecting rhamnolipid biosurfactant production. Pakistan. J. Biotechnol. 2011; 8: 1-5.

Saikia R.R., Deka S., Deka M., Banat I.M. Isolation of biosurfactant-producing Pseudomonas aeruginosa RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production. Ann Microbiol. 2011; 62: 753-763.

Kumari K., Behera H.T., Nayak P.P., Sinha A., Nandi A., Ghosh A., Saha U., Suar M., Panda P.K., Verma S.K., Raina V. Amelioration of lipopeptide biosurfactants for enhanced antibacterial and biocompatibility through molecular antioxidant property by methoxy and carboxyl moieties. Biomed. Pharmaco. 2023; 161: 1-16.

Najafi A.R., Rahimpour M.R., Jahanmiri A.H., Roostaazad R., Arabian D., Ghobadi Z. Enhancing biosurfactant production from an indigenous strain of Bacillus mycoides by optimizing the growth conditions using a response surface methodology. Chem. Eng. J., 2010; 163: 188-194.

Chen J., Huang P.T., Zhang K.Y., Ding F.R. Isolation of biosurfactant producers, optimization and properties of biosurfactant produced by Acinetobacter sp. from petroleum‐contaminated soil. J. Appl. Microbiol. 2012; 112: 660-671.

Suwansukho P., Rukachisirikul V., Kawai F., H-Kittikun A. Production and applications of biosurfactant from Bacillus subtilis MUV4. Songklanakarin J. Sci. Technol. 2008; 30: 87-93.

Ilori M.O., Amobi C.J., Odocha A.C. Factors affecting biosurfactant production by oil degrading Aeromonas spp. isolated from a tropical environment. Chemosphere. 2005; 61: 985-92.

] Moussa T.A.A., Mohamed M.S., Samak N. Production and characterization of di-rhamnolipid produced by Pseudomonas aeruginosa TMN. Braz. J. Chem. Eng. 2014; 31: 867-880.

Yeh M., Wei Y., Chang J. Enhanced production of surfactin from Bacillus subtilis by addition of solid carriers. Biotechnol. Prog. 2005; 21: 1329-1334.

Ghribi D., Ellouze-Chaabouni S. Enhancement of Bacillus subtilis lipopeptide biosurfactants production through optimization of medium composition and adequate control of aeration. Biotechnol. Res. Int. 2011; 653654.

] Fooladi T., Bin A., Hamid A., Mohtar W., Yusoff W. Production of biosurfactant by indigenous isolated bacteria in fermentation system. AIP Conf. Proc. 2013; 197: 197-201.

Tomar G.S., Srinikethan G. Studies on production of biosurfactant from Pseudomonas aeruginosa (MTCC7815) and its application in microbial enhanced oil recovery. Res. J. Chem. Environ. Sci. 2016; 4: 84-91.

Willenbacher J., Rau J.T., Rogalla J., Syldatk C., Hausmann R. Foam-free production of surfactin via anaerobic fermentation of Bacillus subtilis DSM 10T. AMB Express. 2015; 5: 1-9.

Ejike Ogbonna K., Victor Agu C., Okonkwo C.C., Tochukwu Ughamba K., Akor J., Njoku O.U. Use of Spondias Mombin fruit pulp as a substrate for biosurfactant production. Bioeng. 2021; 12: 1-12.

Almansoory A.F., Hasan H.A., Idris M., Abdullah S.R.S., Anuar N. Biosurfactant production by the hydrocarbon-degrading bacteria (HDB) Serratia marcescens: optimization using central composite design (CCD). J. Ind. Eng. Chem. 2017; 47: 272-280.

Sharma R., Singh J., Verma N. Optimization of rhamnolipid production from Pseudomonas aeruginosa PBS towards application for microbial enhanced oil recovery. 3 Biotech. 2018; 8: 20-34.

Paranji S., Swarnalatha S., Sekaran G. Lipoprotein biosurfactant production from an extreme acidophile using fish oil and its immobilization in nanoporous activated carbon for the removal of Ca2+ and Cr3+ in aqueous solution. RSC Adv. 2014; 4(64): 34144-34155.

Jha S., Joshi S., Joshi G.S. Lipopeptide production by Bacillus subtilis R1 and its possible applications. Braz. J. Microbiol. 2016; 47(4): 955-964.

Kiran G.S., Priyadharsini S., Sajayan A., Priyadharsini G.B., Poulose N., Selvin J. Production of lipopeptide biosurfactant by a marine Nesterenkonia sp. and its application in food industry. Front. Microbiol. 2017; 8: 1138

Kumar A.P., Janardhan A., Viswanath B., Monika K., Jung J.Y., Narasimha G. Evaluation of orange peel for biosurfactant production by Bacillus licheniformis and their ability to degrade naphthalene and crude oil. 3 Biotech. 2016; 6(1): 43.




How to Cite

Chanika Saenge Chooklin, Rattanapan, N., Saimmai, A. ., Riansa-ngawong, W. ., & Maneerat, S. (2023). Screening of Biosurfactant-Producing Bacteria as a Potential Biological Control Agent for Fungal Orchid Pathogens in Thailand. Science & Technology Asia, 28(3), 292–312. Retrieved from



Biological sciences