Evaluation of Laboratory-Produced Biosurfactant by Rhodotorula Species and Its Antifungal Activity

AUTHORS

Maral Gharaghani 1 , 2 , Ali Zarei Mahmoudabadi ORCID 1 , 2 , * , Marzieh Halvaeezadeh 2

1 Infectious and Tropical Diseases Research Centre, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

2 Department of Medical Mycology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

How to Cite: Gharaghani M, Zarei Mahmoudabadi A, Halvaeezadeh M . Evaluation of Laboratory-Produced Biosurfactant by Rhodotorula Species and Its Antifungal Activity, Jundishapur J Nat Pharm Prod. Online ahead of Print ; In Press(In Press):e11846. doi: 10.5812/jjnpp.11846.

ARTICLE INFORMATION

Jundishapur Journal of Natural Pharmaceutical Products: In Press (In Press); e11846
Published Online: December 23, 2019
Article Type: Research Article
Received: April 18, 2017
Revised: May 22, 2018
Accepted: May 30, 2018
Uncorrected Proof scheduled for 15 (1)
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Abstract

Background: Biosurfactants are amphiphilic surface-active compounds that are produced by several microorganisms, including bacteria and fungi. Biodegradability, low toxicity, application diversity, and functionality under extreme conditions characterize them from chemical biosurfactants. It is found that Rhodotorula species, read yeasts, have high potency for biosurfactant production. Recently, antimicrobial activities of biosurfactants have been subjected to new antibiotic therapy.

Objectives: The aim of the present study was to evaluate the biosurfactant production by the different strains of Rhodotorula species in laboratory conditions. In addition, the antifungal activity of produced biosurfactant was assessed against several saprophytic fungi.

Methods: In the present study, 54 strains of Rhodotorula including R. glutinis (48 strains), R. minuta (two strains), R. mucilaginosa (two strains), and Rhodotorula species (two strains) were screened for biosurfactant production. The biosurfactant was produced in Sabouraud dextrose broth medium and confirmed by specific tests. The antifungal assay was carried out by a disk diffusion method using serial dilutions of biosurfactant.

Results: In the present study, although all tested strains were capable of producing biosurfactant in vitro, the degree of biosurfactant production varied among the strains. 7.4% of the strains had the highest (+5) biosurfactant activity while 16.7%, 29.5%, 25.8%, and 20.4% had +4, +3, +2, and +1, respectively. In the present study, all tested fungi were inhibited at 40 µL of the biosurfactant.

Conclusions: Rhodotorula species are appropriate organisms for the production of biosurfactants and R. glutinis strains have the greatest ability to produce biosurfactant among other species. Furthermore, our results demonstrated that the produced biosurfactant by R. glutinis presents a valuable potential for biopharmaceutical applications.

Keywords

Biosurfactant Rhodotorula glutinis Antifungal Activity Saprophytic Fungi

Copyright © 2019, Jundishapur Journal of Natural Pharmaceutical Products. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

1. Background

Biosurfactants are surface-active compounds with extracellular (secondary metabolites) or cell wall-associated sources that are produced by several microorganisms such as bacteria and fungi (1-4). In contrast to synthetic surfactants that are not environmentally friendly, biosurfactants have several advantages including biodegradability, low toxicity, diversity of application, and functionality under extreme conditions (5, 6). In addition, biosurfactants are emerging as potential nanoparticle stabilizing agents and used for the treatment of wastewaters containing heavy metals (Cd++ ions) and biodegradation of model hydrocarbons and crude oil in soil (7, 8). Moreover, production of biosurfactants by microorganisms from renewable and cheaper substrates is another reason for its increased use as a green alternative to synthetic surfactants.

Several reports indicated that bacterial species such as Acinetobacter (9), Pseudomonas (10, 11), Bacillus (12), Lactococcus (13), and Nocardia mediterranei (14) are applicable biosurfactants sources. Much research has shown that bacterial biosurfactants possess antimicrobial properties (9, 10, 15, 16). Mostafapour et al. have shown that a biosurfactant produced by Acinetobacter species has anti-Gram positive and negative bacteria effects in vitro (9). In addition, Gomaa examined the biological properties of biosurfactant produced by B. licheniformis and found that it has great potential for the biotechnological and biopharmaceutical applications (17). Moreover, a synergistic effect was found against plant pathogenic fungi and pathogenic bacteria when silver nanoparticles were used with biosurfactants (18). Furthermore, Basit et al. recommended lipopeptide biosurfactant produced by B. cereus as a safe antimicrobial and antioxidant agent (19).

Producing biosurfactants by fungi is limited to some species of Candida, Pseudozyma, Yarrowia, Penicillium and Aspergillus species (5, 20-23). Recently, the production of biosurfactants from Rhodotorula glutinis (24), R. mucilaginosa (25), and R. paludigena (26) has been investigated by researchers. It seems that Rhodotorula species are the main producers of biosurfactants; hence, they have new possibilities for industrial application. The antibacterial, antifungal, and antiviral activities of several biosurfactants have been reported by researchers and used for new antibiotic therapy (10, 12, 17, 20).

Although many types of biosurfactants including, glycolipids, rhamnolipids, sophorolipids, mannosylerythritol lipids, phospholipids, polymeric compounds, mycolic acids, and lipopolysaccharides are distinguished (27), it seems that lipopeptides represent remarkable biological activities, such as antibacterial, antifungal, antitumor, antiviral, and antiadhesive activities (15, 17). Environmental applications of biosurfactants were focused by many researchers and during the last decades, the biomedical field applications have been carried out. New more-active antifungal agents with fewer side effects are usually demanded by researchers, clinicians, and patients.

2. Objectives

In the present study, we evaluated the biosurfactant production ability of different strains of Rhodotorula species in laboratory conditions. Furthermore, the antifungal activity of produced biosurfactant was assessed against several fungi (molds and yeasts).

3. Methods

3.1. Organisms

In the present study, 54 strains of Rhodotorula species including, R. glutinis (48 strains), R. mucilaginosa (two strains), R. minuta (two strains), and Rhodotorula species (two strains) were examined for biosurfactant production. All strains had been already collected from different sources, identified, and kept in distilled water in the Department of Medical Mycology affiliated to Ahvaz Jundishapur University of Medical Sciences, Iran (28). All the tested strains were cultured on Sabouraud dextrose agar (SDA, Merck, Germany) slants and incubated at room temperature for four to five days for strains recovery.

3.2. Screening for Biosurfactant Production

Each of the 54 strains of four species of Rhodotorula was separately inoculated into 5 mL portions of Sabouraud dextrose broth (SDB, Merck, Germany) in test tubes and incubated at ambient temperature in a shaker incubator for four to six days. Then, the cultures were centrifuged at 3000 g for 10 minutes to obtain the cell-free broth supernatant containing biosurfactant. Biosurfactant production was confirmed by various standard methods including oil displacement (21), drop collapse (29), and haemolysin tests (30).

3.3. Biosurfactant Production on a Laboratory Scale

At this stage, only was one strain of R. glutinis selected with high ability to produce biosurfactant. The isolate was inoculated into several Erlenmeyer flasks (250 mL volume) containing 100 mL of SDB and incubated in the shaking incubator at 29ºC for one week. The free cell supernatant was removed using centrifugation at 3000 g for 10 minutes. The same volume of supernatant (crude biosurfactant) and chloroform-methanol (2:1 v/v) were mixed, and then the biosurfactant was extracted by centrifugation (26, 31). The gather biosurfactant was dried and kept at -20ºC until use.

3.4. Antifungal Assay

The antifungal activity of the biosurfactant was examined against different strains of Candida albicans (seven isolates), R. glutinis (two isolates), Aspergillus niger (one isolate), Alternaria sp. (one isolate), Rhizopus sp. (one isolate), and Syncephalastrum sp. (one isolate). The antifungal activity was evaluated by the disk diffusion method. Briefly, a standard suspension of each strain was prepared in sterile distilled water and then, 10 µL was spread on the surface of SDA plates in duplicate. Subsequently, 50 mg of the crude biosurfactant was dissolved in 1 mL of DMSO/ethanol completely. Finally, three blank disks were put on each plate and an aliquot of diluted biosurfactant (20, 40, 60, 80, 100, and 120 µL) was added into each disk. The plates were incubated at 29 and 35ºC for molds and yeasts, respectively. The hyaline haloes (without fungal growth) around the disks were measured and the minimum inhibitory concentration (MIC) for each strain was calculated (Figure 1).

The antifungal activity of biosurfactant against <i>Aspergillus niger</i> after 48 h
Figure 1. The antifungal activity of biosurfactant against Aspergillus niger after 48 h

4. Results and Discussion

In the present study, although all the tested strains were capable of producing biosurfactant in vitro, the degree of biosurfactant was different among the strains. Out of 54 Rhodotorula strains, only could four strains of R. glutinis comparatively show higher zones in the oil displacement test (2.1 - 2.5 cm) confirmed by drop collapse and haemolysin tests (Table 1). These results indicated that they had a high potential for biosurfactant production. Mahalingam and Sampath believe that the oil displacement technique is very sensitive for detecting biosurfactants even at low levels (29). In this test, a larger diameter represents a higher surface activity of the biosurfactant (32). Rhodotorula species are new sources for producing different biosurfactants. Extracellular glycoprotein biosurfactants from R. glutinis (24) and astaxanthin from R. mucilaginosa (25) are two types of biosurfactants that have been recently detected. Previous studies have shown that the biosurfactants produced by R. glutinis are composed of lipids (5).

Table 1. Biosurfactant Production by Rhodotorula Species Using Oil Displacement Test
Halo Diameter (cm)Rhodotorula
R. glutinisR. minutaR. mucilaginosaRhodotorula SpeciesTotal
> 0.5 (+1)11 (20.4%)0.00.00.011 (20.4%)
0.6 - 1 (+2)10 (18.5%)2 (3.7%)1 (1.8%)1 (1.8%)14 (25.8%)
1.1 - 1.5 (+3)14 (25.9%)0.01 (1.8%)1 (1.8%)16 (29.5%)
1.6 - 2 (+4)9 (16.7%)0.00.00.09 (16.7%)
2.1 - 2.5 (+5)4 (7.4%)0.00.00.04 (7.4%)
Total48 (88.9%)2 (3.7%)2 (3.6%)2 (3.6%)54 (100%)

Due to developing fungal resistance to antifungal agents, a lot of attention has been paid to new natural compounds with antifungal properties. In the present study, we found that the growth of all tested fungi including yeasts and molds strains completely was inhibited by 40 µL of the biosurfactant. Despite their potential for biomedical fields, only have a few studies been carried out on the antifungal activity of biosurfactants. Antifungal activities of biosurfactants produced by Lactobacillus lactis (13), B. subtilis (12), and P. aeruginosa (10) were investigated by several researchers. Furthermore, Ceresa et al. have shown that the biosurfactant produced by L. brevis has antibiofilm formation activity in C. albicans (33). A synergistic effect of surfactin with ketoconazole against C. albicans was also considered by Liu et al. (27). Furthermore, Halvaeezadeh and Zarei Mahmoudabadi showed that the produced biosurfactant by R. paludigena in combination with caspofungin have synergistic effects against C. albicans strains (26).

Anti-adhesive activity, permeabilizing ability, and cellular damaging ability are reported as the possible effective mechanisms of biosurfactants (13, 16, 20). Furthermore, biosurfactants could inhibit the adhesion of Candida to the silicon surface (33).

4.1. Conclusions

In conclusion, Rhodotorula species are applicable organisms for the production of biosurfactants and R. glutinis strains have the greatest ability to produce biosurfactants among other species. Furthermore, our results demonstrated that the biosurfactant produced by R. glutinis had a valuable potential for biopharmaceutical applications.

Acknowledgements

Footnotes

References

  • 1.

    Fontes GC, Amaral PFF, Coelho MAZ. Produção de biossurfactante por levedura. Química Nova. 2008;31(8):2091-9. doi: 10.1590/s0100-40422008000800033.

  • 2.

    Aparna A, Srinikethan G, Hegde S. Isolation, screening and production of biosurfactant by Bacillus clausii 5B. Res Biotechnol. 2012;3(2):49-56.

  • 3.

    Elshafie AE, Joshi SJ, Al-Wahaibi YM, Al-Bemani AS, Al-Bahry SN, Al-Maqbali D, et al. Sophorolipids production by Candida bombicola ATCC 22214 and its potential application in microbial enhanced oil recovery. Front Microbiol. 2015;6:1324. doi: 10.3389/fmicb.2015.01324. [PubMed: 26635782]. [PubMed Central: PMC4659913].

  • 4.

    Rodrigues L, Banat IM, Teixeira J, Oliveira R. Biosurfactants: Potential applications in medicine. J Antimicrob Chemother. 2006;57(4):609-18. doi: 10.1093/jac/dkl024. [PubMed: 16469849].

  • 5.

    Amaral PF, Coelho MA, Marrucho IM, Coutinho JA. Biosurfactants from yeasts: Characteristics, production and application. Adv Exp Med Biol. 2010;672:236-49. doi: 10.1007/978-1-4419-5979-9_18. [PubMed: 20545287].

  • 6.

    Mazaheri Assadi M, Tabatabaee MS. Biosurfactants and their use in upgrading petroleum vacuum distillation residue: A review. Int J Environ Res. 2010;4(4):549-72.

  • 7.

    Kang SW, Kim YB, Shin JD, Kim EK. Enhanced biodegradation of hydrocarbons in soil by microbial biosurfactant, sophorolipid. Appl Biochem Biotechnol. 2010;160(3):780-90. doi: 10.1007/s12010-009-8580-5. [PubMed: 19253005].

  • 8.

    Asci Y, Nurbas M, Acikel YS. A comparative study for the sorption of Cd(II) by K-feldspar and sepiolite as soil components, and the recovery of Cd(II) using rhamnolipid biosurfactant. J Environ Manage. 2008;88(3):383-92. doi: 10.1016/j.jenvman.2007.03.006. [PubMed: 17462813].

  • 9.

    Mostafapour MJ, Saffari M. Isolation and identification of biosurfactant-producing strains from the genus Acinetobacter spp and antibacterial effects of biosurfactant produced on some of the negative and gram-positive bacteria in vitro. New Cel Mol Biotechnol J. 2014;4(14):79-91.

  • 10.

    Tomar S, Singh BA, Khan MA, Kumar S, Sharma S, Lal M. Identification of Pseudomonas aeruginosa strain producing biosurfactant with antifungal activity against Phytophthora infestans. Potato J. 2013;40(2).

  • 11.

    Thavasi R, Subramanyam Nambaru VR, Jayalakshmi S, Balasubramanian T, Banat IM. Biosurfactant production by Pseudomonas aeruginosa from renewable resources. Indian J Microbiol. 2011;51(1):30-6. doi: 10.1007/s12088-011-0076-7. [PubMed: 22282625]. [PubMed Central: PMC3209860].

  • 12.

    Olteanu V, Sicuia O, Ciuca M, Carstea DM, Voaides C, Campeanu G, et al. Production of biosurfactants and antifungal compounds by new strains of Bacillus spp. isolated from different sources. Romanian Biotechnol Lett. 2011;16(1 Suppl):84-91.

  • 13.

    Saravanakumari P, Nirosha P. Mechanism of control of Candida albicans by biosurfactant purified from Lactococcus lactis. Int J Curr Microbiol Appl Sci. 2015;4(2):529-42.

  • 14.

    Usharani MV, Sukirtha TH. Production and qualitative analysis of biosurfactant and biodegradation of the organophosphate by nocardia mediterranie. J Bioremed Biodegrad. 2013;4(6). doi: 10.4172/2155-6199.1000198.

  • 15.

    Mukherjee S, Das P, Sivapathasekaran C, Sen R. Antimicrobial biosurfactants from marine Bacillus circulans: Extracellular synthesis and purification. Lett Appl Microbiol. 2009;48(3):281-8. doi: 10.1111/j.1472-765X.2008.02485.x. [PubMed: 19187506].

  • 16.

    Luna JM, Rufino RD, Sarubbo LA, Rodrigues LR, Teixeira JA, de Campos-Takaki GM. Evaluation antimicrobial and antiadhesive properties of the biosurfactant Lunasan produced by Candida sphaerica UCP 0995. Curr Microbiol. 2011;62(5):1527-34. doi: 10.1007/s00284-011-9889-1. [PubMed: 21327556].

  • 17.

    Gomaa EZ. Antimicrobial activity of a biosurfactant produced by Bacillus licheniformis strain M104 grown on whey. Brazilian Arch Biol Technol. 2013;56(2):259-68. doi: 10.1590/s1516-89132013000200011.

  • 18.

    Joanna C, Marcin L, Ewa K, Grazyna P. A nonspecific synergistic effect of biogenic silver nanoparticles and biosurfactant towards environmental bacteria and fungi. Ecotoxicology. 2018;27(3):352-9. doi: 10.1007/s10646-018-1899-3. [PubMed: 29411207]. [PubMed Central: PMC5859040].

  • 19.

    Basit M, Rasool MH, Naqvi SAR, Waseem M, Aslam B. Biosurfactants production potential of native strains of Bacillus cereus and their antimicrobial, cytotoxic and antioxidant activities. Pak J Pharm Sci. 2018;31(1(Suppl.)):251-6. [PubMed: 29386151].

  • 20.

    Rufino RD, Luna JM, Sarubbo LA, Rodrigues LR, Teixeira JA, Campos-Takaki GM. Antimicrobial and anti-adhesive potential of a biosurfactant Rufisan produced by Candida lipolytica UCP 0988. Colloids Surf B Biointerfaces. 2011;84(1):1-5. doi: 10.1016/j.colsurfb.2010.10.045. [PubMed: 21247740].

  • 21.

    Chandran P, Das N. Role of sophorolipid biosurfactant in degradation of diesel oil by Candida tropicalis. Bioremed J. 2012;16(1):19-30. doi: 10.1080/10889868.2011.628351.

  • 22.

    Kiran GS, Hema TA, Gandhimathi R, Selvin J, Thomas TA, Rajeetha Ravji T, et al. Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3. Colloids Surf B Biointerfaces. 2009;73(2):250-6. doi: 10.1016/j.colsurfb.2009.05.025. [PubMed: 19570659].

  • 23.

    Gautam G. A cost effective strategy for production of bio-surfactant from locally isolated penicillium chrysogenum SNP5 and its applications. J Bioprocess Biotech. 2014;4(6). doi: 10.4172/2155-9821.1000177.

  • 24.

    Foaad MA. Production of extracellular glycoprotein biosurfactant from Rhodotorula glutinis and its use in elimination of solar pollution. Egyptian J Botany. 2007;47:77-97.

  • 25.

    Kawahara H, Hirai A, Minabe T, Obata H. Stabilization of astaxanthin by a novel biosurfactant produced by Rhodotorula mucilaginosa KUGPP-1. Biocontrol Sci. 2013;18(1):21-8. doi: 10.4265/bio.18.21. [PubMed: 23538848].

  • 26.

    Halvaeezadeh M, Mahmoudabadi AZ. Anti-Candida activity of biosurfactant produced by Rhodotorula paludigena. Curr Enzym Inhib. 2017;13(3). doi: 10.2174/1573408013666161219150524.

  • 27.

    Liu X, Ren B, Gao H, Liu M, Dai H, Song F, et al. Optimization for the production of surfactin with a new synergistic antifungal activity. PLoS One. 2012;7(5). e34430. doi: 10.1371/journal.pone.0034430. [PubMed: 22629294]. [PubMed Central: PMC3356355].

  • 28.

    Seifi Z, Zarei Mahmoudabadi A, Hydrinia S. Isolation, identification and susceptibility profile of Rhodotorula species isolated from two educational hospitals in Ahvaz. Jundishapur J Microbiol. 1970;6(6). doi: 10.5812/jjm.8935.

  • 29.

    Mahalingam PU, Sampath N. Isolation, characterization and identification of bacterial biosurfactant. European J Experiment Biol. 2014;4(6):59-64.

  • 30.

    Youssef NH, Duncan KE, Nagle DP, Savage KN, Knapp RM, McInerney MJ. Comparison of methods to detect biosurfactant production by diverse microorganisms. J Microbiol Methods. 2004;56(3):339-47. doi: 10.1016/j.mimet.2003.11.001. [PubMed: 14967225].

  • 31.

    Chander CRS, Lohitnath T, Kumar DJM, Kalaichelvan PT. Production and characterization of biosurfactant from Bacillus subtilis MTCC441 and its evaluation to use as bioemulsifier for food bio-preservative. Adv Appl Sci Res. 2012;3(3):1827-31.

  • 32.

    Chandran P, Das N. Characterization of sophorolipid biosurfactant produced by yeast species grown on diesel oil. Int J Sci Nat. 2011;2:63-71.

  • 33.

    Ceresa C, Tessarolo F, Caola I, Nollo G, Cavallo M, Rinaldi M, et al. Inhibition of Candida albicans adhesion on medical-grade silicone by a Lactobacillus-derived biosurfactant. J Appl Microbiol. 2015;118(5):1116-25. doi: 10.1111/jam.12760. [PubMed: 25644534].

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