Isolation and Molecular Characterization of Biosurfactant-Producing Yeasts from Saps of Elaeis guineensis and Raphia africana

Main Article Content

I. V. Nwaguma
C. B. Chikere
G. C. Okpokwasili

Abstract

Aim: This study investigated the screening and molecular characterization of biosurfactant-producing yeasts from saps of Elaeis guineensis (oil palm) and Raphia Africana (Raphia palm).

Methodology: Physicochemical characteristics (pH, temperature, alcohol contents, and reducing sugars) of the saps of Elaeis guineensis and Raphia africana were determined. The capacity of the yeast isolates from both samples to produce biosurfactant was evaluated using emulsification index (E24), emulsification assay, haemolytic assay, oil displacement test, and tilted glass slide. The yeast isolates were identified based on their phenotypic, microscopic, biochemical, and molecular characteristics.

Results: Chemical analysis of the palm wine saps revealed respective pH, temperature, alcohol, and reducing sugars contents of 5.68, 17.1°C, 0.943% and 1.090 mg/mL for Elaeis guineensis and 5.26, 16.9°C, 0.884% and 2.099 mg/mL for Raphia africana. Six isolates (SA-2, SA-5, SB-3, SB-5, SB-6 and SB-8) out of sixteen isolates (16) distributed within both samples were found to produce biosurfactant. Phylogenetic analysis based on the internally transcribed spacer (ITS) genes classified the six isolates as Candida haemulonis SA2, Pichia kudriavzevii SA5, Pichia kudriavzevii SB3, Pichia kudriavzevii SB5, Pichia kudriavzevii SB6, and Pichia kudriavzevii SB8. The sequences obtained from the study have been deposited in GenBank under the accession numbers MN007219.1-MN007224.1. The result obtained from the study revealed high biosurfactant activity with a maximum E24 of 64.5% compared to E24 of 72% by sodium dodecyl sulphate (SDS).

Conclusion: The study demonstrated that saps from Elaeis guineensis and Raphia africana were suitable sources of biosurfactant-producing yeasts with high capacity for hydrocarbon emulsification. The main six biosurfactant-producing yeasts were found to belong to the genera Candida and Pichia.

Keywords:
Biosurfactant, yeasts, molecular characterization, Elaeis guineensis, Raphia africana.

Article Details

How to Cite
Nwaguma, I. V., Chikere, C. B., & Okpokwasili, G. C. (2019). Isolation and Molecular Characterization of Biosurfactant-Producing Yeasts from Saps of Elaeis guineensis and Raphia africana. Microbiology Research Journal International, 29(4), 1-12. https://doi.org/10.9734/mrji/2019/v29i430169
Section
Original Research Article

References

Gudina EJ, Rangarajan V, Sen R, Rodrigues LR. Potential therapeutic applications of biosurfactants. Trends Pharmacol Sci. 2013;34(12):667–675.

Banat IM, Makkar IM, Cameotra SS. Potential commercial applications of microbial surfactants. Appl Microbiol Biotechnol. 2000;53:495-508.

Amaral PFF, Coelho MAZ, Marrucho IMJ, Coutinho JAP. Biosurfactants from yeasts: Characteristics, production and application. In: Sen R. (Eds) Biosurfactants: Adv Exp Med Biol. Springer, New York. 2010;672.

Felse PA, Shah V, Chan J, Rao KJ, Gross RA. Sophorolipid biosynthesis by Candida bombicola from industrial fatty acid residues. Enzyme Microb Technol. 2007;40:316–323.

Accorsini FR, Mutton MJR, Lemos EGM, Benincasa M. Biosurfactants production by yeasts using soybean oil and glycerol as low cost substrate. Braz. J. Microbiol. 2012;43:116-125.

Padmapriya B, Suganthi S, Anishya RS. Screening, optimization and production of biosurfactants by Candida species isolated from oil polluted soils. Biointerfaces. 2013;79:174-183.

Thaniyavarn J, Chianguthal T, Sangvanich P, Roongsawang N, Washio K, Morikawa M, Thaniyavaran S. Production of sophorolipid biosurfactant by Pichia anomala. Biosci Biotechnol Biochem. 2008;72(8):2061–2068.

Johny JM, Saravanakumari P. Comparative study: Different recovery techniques of biosurfactant produced from Pichia fermentans isolated from fermented dairy whey waste. J Pharm Pharm Sci. 2013;3(1):567–582.

Chandran P, Das N. Biosurfactant production and diesel oil degradation by yeast species Trichosporon asahii isolated from petroleum hydrocarbon contaminated soil. Int J Eng Sci. 2010;2(12):6942–6953.

Csutak O, Stoica I, Vassu T. Evaluation of production, stability and activity of biosurfactants from yeasts with application in bioremediation of oil-polluted environment. Revista de Chimie. 2012;10(63):973–977.

Gizele CF, Amaral PFF, Nele M, Coelho MAZ. Factorial design to optimize biosurfactant production by Yarrowia lipolytica. J Biomed Biotechnol. 2010;8(2): 1306-8. Available:https://doi.org/10.1155/2010/821306

Hassanshahian M, Emtiazi G, Cappello S. Isolation and characterization of crude-oil-degrading bacteria from the Persian Gulf and the Caspian Sea. Mar Pollut Bull. 2012;64:7-12.
DOI: 10.1016/j.marpolbul.2011.11.006

Sari M, Kusharyoto W, Made Artika I. Screening for biosurfactant-producing yeast: Confirmation of biosurfactant production. Biotechnol J (Faisalabad). 2014;3(3):106-111.

DOI: 10.3923/biotech.2014.106.111

Goel PK. Structure of protodioscin. U.S. Patent No. US 2010/0160616 A1. Washington, DC: U.S. Patent and Trademark Office.

Oleszek W, Hamed A. Saponin‐based surfactants. In: Kjellin M, Johansson I, Editors. Surfactants from Renewable Sources Resources. John Wiley & Sons Ltd. Chichester, UK. 2010;239-249.
DOI: 10.1002/9780470686607.ch12

Jespersen L. Occurrence and taxonomic characteristics of strains of Saccharomyces cerevisiae predominant in African indigenous fermented foods and beverages. FEMS Yeast Res. 2003;3:191-200.

Zhi XY, Li WJ, Stackebrandt E. An update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new sub-orders and four new families and amended descriptions of the existing higher taxa. Int J Syst Evol Microbiol. 2009;59:589-608.

Sabate J, Cano J, Esteve-Zarzoso B, Guillamón JM. Isolation and identification of yeasts associated with vineyard and winery by RFLP analysis of ribosomal genes and mitochondrial DNA. Microbiol Res. 2002;57:1-8.

Cadez N, Raspor P, de Cock AWAM, Boekhout T, Smith M. Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera. FEMS Yeast Res. 2002;1(4):279-289. Available:https://doi.org/10.1111/j. 15671364.2002.tb00046.x

Ukwuru MU, Awah JI. Properties of palm wine yeasts and its performance in wine making. Afri J Biotechnol. 2013;12(19): 2670-2677.
DOI: 10.5897/AJB12.2447

Nwachukwu IN, Ibekwe VI, Nwabueze RN, Anyanwu BN. Characterisation of palm wine yeast isolates for industrial utilization. Afr J Biotechnol. 2006;5(19):1725-1728.

Nandhini B, Josephine RM. A study on bacterial and fungal diversity in potted soil. Int J Curr Microbiol App Sci. 2013;2(2):1–5.

Olowonibi OO. Isolation and characterization of palm wine strains of Saccharomyces cerevisiae potentially useful as bakery yeasts. Euro J Exp Bio. 2017;7(2):11.

Nwaguma IV, Chikere CB, Okpokwasili GC. Isolation, screening and identification of biosurfactant-producing bacteria from hydrocarbon-polluted and pristine soils within Ogoniland. Nigeria British Microbioil Res. J. 2016;15(4):1-11. Article no.BMRJ.26294. ISSN: 2231-0886. NLM ID: 101608140.

Plaza GA, Zjawiony I, Banat IM. Use of different methods for detection of thermophilic biosurfactant producing bacteria from hydrocarbon contaminated soils. J Petrol Sci Eng. 2006;50(1):71- 77.
DOI: 10.1016/j.petrol.2005.10.005

Youssef NH, Dunacn KE, Nagle DP, Savage KN, Knapp RM, Mcinerney MJ. Comparison of methods to detect biosurfactant production by diverse microorganism. J Microbiol Meth. 2004;56(3):339–347.

Morikawa M, Hirata Y, Imanaka T. A study on the structure-function relationship of the lipopeptide biosurfactant. Biochim. Biophys. Acta. 2000;1488:211-218.

Nitschke M, Pastore GM. Biosurfactant production by Bacillus subtilis using cassava-processing effluent. Appl Biochem Biotechnol. 2004;112(3):163-72.

Patil JR, Chopade BA. Distribution and in vitro antimicrobial susceptibility of Acinetobacter species on the skin of healthy humans. Natl Med J India. 2001;14(4):204-8. PMID: 11547525.

Persson A, Molin G. Capacity for biosurfactant production of environmental Pseudomonas and Vibrionaceae growing on carbohydrates. Appl Microbiol Biotechnol. 1987;26(5):439-442.

Shaikh RA, Chowdhury BR, Mondal P, Rajak S. Screening and characterization of biosurfactants producing microorganism from natural environment (whey spilled soil). J. Nat. Sci. Res. 2013;3:13.

Banat IM. The isolation of a thermophilic biosurfactant producing Bacillus sp. Biotechnol Lett. 1993;15(6):591–594.

Carrillo PG, Mardaraz C, Pitta-Alvarez SI, Giuliett AM. Isolation and selection of biosurfactant-producing bacteria. World J Microbiol Biotechnol. 1996;12:82-84.

Kumar M, Shukla PK. Use of PCR targeting of internal transcribed spacer regions and single-stranded conformation polymorphism analysis of sequence variation in different regions of rRNA genes in fungi for rapid diagnosis of mycotic keratitis. J Clinical Microbiol. 2005;43(2): 662-668.

Hall TA. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 1999;41:95-98.

Katemai W, Maneerat S, Kawai F, Kanzaki H, Nitoda T, H-Kittikun A. Purification and characterization of a biosurfactant produced by Issatchenkia orientalis SR4. The J Gen Appl Microbiol. 2008;54(1)79-82.

Romero MC, Chiaravalli JC, Reinoso EH. Sorbed anthracene degradation by sophorolipid producing yeasts. IJWI. 2016;5:25-31.

Yalcin HT, Ergin-Tepebasi G, Uyar E, Isolation and molecular characterization of biosurfactant producing yeasts from the soil samples contaminated with petroleum derivatives. J Basic Microbiol. 2018;58(9): 782-792.

Konishi M, Hatada Y, Horiuchi JI. Draft genome sequence of the basidiomycetous yeast-like fungus Pseudozyma hubeiensis SY62, which produces an abundant amount of the biosurfactant mannosylerythritol lipids. Genome Announc. 2013;1(4):e00409-13.
DOI: 10.1128/genomeA.00409-13

Ahmed ME, Zeinat K, Nermeen H. Isolation and genetic identification of yeast producing biosurfactants, evaluated by different screening methods. Microc J. 2019;146.
Available:https://doi.org/10.1016/j.microc.2019.01.020

Santos APP, Silva MDS, Costa EVL, Rifino RD, Santos VA, Ramos CS, Sarrubbo LA, Porto ALF. Production and characterization of a biosurfactant produced by Streptomyces sp. DPUA 1559 isolated from lichen of the Amazon region. Braz J Med Biol Res. 2018;51(2):6657.

Satpute SK, Bhawsar BD, Dhakephalkar PK, Chopade BA. Assessment of different screening methods for selecting biosurfactant producing marine bacteria. Indian J Mar Sci. 2008;37(3):243–250.

Ndibe TO, Eugene WC, Usman JJ. Screening of biosurfactant-producing bacteria isolated from River Rido, Kaduna State, Nigeria. J. Appl Sci Environ Manage. 2018;22(11):1855–1861.
DOI:https://dx.doi.org/10.4314/jasem.v22i11.22

Bodour AA, Miller-Maier R. Application of a modified drops collapse technique for surfactant quantitation and screening of biosurfactant producing microorganisms. 1998;32:273–280.

Cooper DG, Goldenberg BG. Surface-active agents from two Bacillus species. App-l Environ Microbiol. 1987;53(2):224–229.

Sarrubo LA, Moura de Luna J, Maria de Campos-Takaki G. Production and stability studies of the bio-emulsifiers obtained from a new strain of Candida glabrata UCP 1002. Elect. J. Biotechnol. 2006;9:1–7.

Jagtap S, Yavankar S, Pardesi K, Chopade B. Production of bioemulsifier by Acinetobacter sp. from healthy human skin of tribal population. Ind J Expt Biol. 2010;48:70-76.

Thanomsub B, Watcharachaipong T, Chotelersak K, Arunrattiyakorn P, Nitoda T, Kanzaki H. Monoacylglycerols: Glycolipid biosurfactants produced by a thermotolerant yeast, Candida ishiwadae. J Appl Microbiol. 2004;96(3)588-592.
DOI: 10.1111/j.1365-2672.2004.02202.x

Casas J, Ochoa FG. Sophorolipid production by Candida bombicola: Medium composition and culture methods. J Biosci Bioeng. 1999;88(5):488-494.
DOI: 10.1016/S1389-1723(00)87664-1

Cavalero DA, Cooper DG. The effect of medium composition on the structure and physical state of sophorolipids produced by Candida bombicola ATCC 22214. J. Biotechnol. 2003;103(1):31-41.
DOI: 10.1016/S0168-1656(03)00067-1

Almeida DG, Soares de Silva RCF, Luna JM, Rufino RD, Santos VA, Sarubbo LA. Response surface methodology for optimizing the production of biosurfactant by Candida tropicalis on industrial waste substrates. Front Microbiol. 2017;8:157.

Andrade RFS, Antunes AA, Lima RA, Araújo HWC, Resende-Stoianoff MA, Franco LO, Campos-Takaki GM. Enhanced production of a glycolipid biosurfactant produced by Candida glabrata UCP/WFCC1556 for application in dispersion and removal of petroderivatives. IJMAS. 2015;4(7):563-576.

Hommel RK, Weber L, Weiss U, Himmelreich U, Riike OKHP, Kleber HP. Production of sophorose lipid by Candida (Torulopsis) apicola grown on glucose. J Biotechnol. 1994;33(2):147-155.

Kitamoto D, Yanagishita H, Shinbo T, Makane T, Kamisawa C, Nakahara T. Surface active properties and antimicrobial activities of mannosylerythritol lipids as biosurfactant produced by Candida antractica. J. Biotechnol. 1993;29:91- 96.

Sobrinho HBS, Rufino RD, Luna JM, Salgueiro AA, Campos-Takaki GM, Leite LFC, Sarubbo LA. Utilization of two agro industrial by-products for the production of a surfactant by Candida sphaerica UCP0995. Process Biochem. 2008;43(9): 912-917.
DOI: 10.1016/j.procbio.2008.04.013

Amézcua-Vega C, Poggi-Varaldo HM, Esparza-García F, Ríos-Leal E, Rodríguez-Vázquez R. Effect of culture conditions on fatty acids composition of a biosurfactant produced by Candida ingens and changes of surface tension of culturemedia. Bioresour Technol. 2007;98(1):237-240.
DOI: 10.1016/j.biortech.2005.11.025

Campos JM, Stamford TLM, Sarubbo LA. Characterization and application of a biosurfactant isolated from Candida utilis in salad dressings. Biodegradation. 2019;30(4):313-324.
DOI: 10.1007/s10532-019-09877-8

Sheperd R, Rockey J, Sutherland IW, Roller S. Novel bio-emulsifiers from microorganisms for use in foods. J Biotechnol. 1995;40(3):207-217.

DOI: 10.1016/0168-1656(95)00053-S

Mossa TAA, Ahmed GM, Abdel-Hamid SMS. Optimization of cultural conditions for biosurfactant production from Nocardia amarae. J Appl Sci Res. 2006;2(11)844-850.

Sarubbo LA, Marçal MC, Neves MLC, Silva MPC, Porto ALF, Campos-Takaki GM. Bioemulsifier production in batch culture using glucose as carbon source by Candida lipolytica. Appl Biochem Biotechnol. 2001;95(1):59-67.
DOI: 10.1385/ABAB:95:1:59

Johny JM. Screening, gene sequencing and biosurfactant production from Pichia fermentans isolated from dairy effluents. IOSR J Environ Sci Toxicol Food Technol. 2013;6(5):2319–2402.

Martins SCS, Aragao VO, Martins CM. Pichia spp. yeasts from Brazilian industrial wastewaters: Physiological characteriza-tion and potential for petroleum hydrocarbon utilization and biosurfactant production. Afri J Microbiol Res. 2014;8(7): 664-672.
DOI: 10.5897/AJMR2014.6037