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Go Back       IAR Journal of Agriculture Research and Life Sciences | IAR J Agri Res Life Sci, 2(4), | Volume:2 Issue:4 ( Aug. 31, 2021 ) : 30-35.
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DOI : 10.47310/iarjals.2021.v02i04.005       Download PDF       HTML       XML

Biosurfactant Activity of Candida tropicalis and Aspergillus clavatus Isolated from Oil-Polluted Soil

Article History

Received: 31.07. 2021 Revision: 10. 08. 2021 Accepted: 20. 08 .2021 Published: 31. 08. 2021

Author Details

Mbachu, A.E*1, Mbachu, N.A2 and Chukwura, E.I1

Authors Affiliations

1Department of Applied Microbiology & Brewing, Faculty of Biosciences, Nnamdi Azikiwe University, Awka, Nigeria

2Department of Human Biochemistry, Faculty of Basic Medical Sciences, Nnamdi Azikiwe University, Nnewi Campus, Nigeria


Abstract: Biosurfactants are surface-active molecules synthesized by microorganisms which have both hydrophobic and hydrophilic domains and are capable of lowering the surface tension and the interfacial tension of the growth medium. They are more active and less toxic than the chemical surfactants which are more difficult to degrade from the environment. The aim of this study was to determine the biosurfactant activity of fungi: Candida tropicalis and Aspergillus clavatus isolated from oil polluted soil. C. tropicalis and A. clavatus were isolated from soil samples obtained from three automobile workshops at Old Motor Spare Parts, Nkpor, Nigeria, using the spread plate technique. They were identified using the standard methods and confirmed using 18S rRNA gene sequence. Biosurfactant activities of the isolates were determined using the emulsification index (E24), oil spreading technique and microbial cell surface hydrophobicity in three different oils: used engine oil (UEO), diesel and petrol. Data were analyzed using analysis of variance (ANOVA) at P = 0.05. The biosurfactant activity of the isolates showed high emulsification index (> 50%), oil displacement area (ODA) (> 4.5 cm2) and cell surface hydrophobicity (> 50%) in UEO, diesel and petrol. However, the E24 of the isolates were significantly higher in petrol (P < 0.05) compared with UEO and diesel. The broth culture containing A. clavatus showed significantly higher ODA in petrol (P < 0.05) when compared with UEO and diesel. The isolates C. tropicalis and A. clavatus showed biosurfactant activity in UEO, diesel and petrol, thus have promising potential against different hydrocarbon pollution.


Keywords: Emulsification index, oil displacement area, cell surface hydrophobicity.

Introduction

Microbial surfactants (biosurfactants) are structurally diverse group of microbial surface active molecules that are produced by a range of microbial communities (Banat et al., 2014). They can be produced as part of the cell membrane by a variety of yeast, bacteria and filamentous fungi (Chen et al., 2007). Microorganisms synthesize biosurfactants, which have both hydrophobic and hydrophilic domains and are capable of lowering the surface tension and the interfacial tension of the growth medium. Biosurfactants are synthesized during microbial growth period on water-immiscible substrates, providing an alternative to chemically prepared conventional surfactants. They are more active and less toxic than chemical surfactants which are more difficult to degrade in the environment.


Many biosurfactant-producing microorganisms have been isolated and identified to belong to the genus: Bacillus, Agrobacterium, Streptomyces, Pseudomonas, and Thiobacillus as producers of amino acids-containing biosurfactants; Pseudomonas, Torulopsis, Candida, Mycobacterium, Micromonospora, Rhodococcus, Arthrobacter, Mycobacterium, Corynebacterium, Mycobacterium, and Arthrobacter as producers of glycolipids; Thiobacillus, Aspergillus, Candida, Corynebacterium, Micrococcus, and Acinetobacter as producers of phospholipids and fatty acids (Liu et al., 2015, Abdulsalam et al., 2016). In the case of the microbial degradation of hydrocarbons, microbial cells synthesize biosurfactants that solublizes oil droplets into the aqueous phase making easier the oil uptake by microbial cells (Ganesh and Lin, 2009).


Generally petroleum hydrocarbon compounds bind to soil components and are difficult to remove or degrade (Calvo et al., 2008). Biosurfactants can emulsify hydrocarbons, thus enhancing their water solubility, decreasing surface tension and increasing the displacement of oily substances from the soil (Calvo et al., 2008). Biosurfactant producing microorganisms are naturally present in oil-contaminated soils. Oil-contaminated soils and water contain large amount of hydrocarbons (both aliphatic and aromatic hydrocarbons). Microorganisms exhibit emulsifying activity by producing biosurfactants often mineralizing the hydrocarbons or converting them into harmless constituents (Priya and Usharanti, 2009).


Biosurfactants have many properties including soaping, emulsifying, dispersing; and have gained importance in the fields of enhanced oil recovery, environmental bioremediation, food processing and pharmaceuticals (Priya and Usharanti, 2009). Biosurfactants can also be used in handling industrial emulsions, control of oil spills, biodegradation and detoxification of industrial effluents and in bioremediation of contaminated soil (Padmapriya et al., 2013). The present study deals with the biosurfactant activities of C. tropicalis and A. clavatus isolated from oil polluted soil using their emulsification index, oil displacement area, and microbial cell surface hydrophobicity in three different oils such as used engine oil, diesel and petrol.


Materials and Methods

Isolation of Candida tropicalis and Aspergillus clavatus

Oil contaminated soil samples were collected from three automobile workshops at Old Motor Spare Parts, Nkpor, Nigeria. The homogenized soil samples were serially diluted and spread onto the surface of solidified Sabouraud Dextrose Agar (SDA) plates. Incubation was done at 280C for 7 days. After 7 days incubation, each distinct colony was purified by subculturing onto the surface of a solidified Sabouraud Dextrose Agar plates. The pure cultures were maintained on Sabouraud Dextrose Agar slant.


Identification of the Isolates

The isolates were identified using the wet mount and the microslide culture technique with reference to the Manual of Fungal Atlas (Barnett and Hunter, 2000, Watanabe, 2002). They were also characterized using their 18Sr RNA gene sequence.


Assay for Biosurfactant Activity of the Isolates

A 24 hour pure cultures of the two isolates Candida tropicalis and Aspergillus clavatus were inoculated into 250 ml Erlenmeyer flasks containing 100 ml of Mineral Salt broth and 1% (v/v) sterile used engine oil as carbon source. Uninoculated control flasks were also set up. The flasks were incubated in an Orbital Shaker at 120 rpm for 7days at 28oC. After 7 days incubation, the culture broth containing biosurfactant was separated from the culture media by centrifugation (at 3000rpm for 30 minutes at 4oC) to get the culture supernatant. The supernatant was used for the determination of the emulsification index and oil spreading technique, while the fungal cells were harvested and used for the determination of the cell surface hydrophobicity in three different oils namely used engine oil, diesel and petrol.


Emulsification Index (E24)

Emulsification index (E24)of culture sample was determined by mixing equal volume of substrate (used engine oil) and cell free broth supernatant in a test tube and vortexed for 2 minutes. The mixture was allowed to stand for 24 hour. E24 was calculated by dividing the height of the emulsion layer by the mixture total height and then multiplying by 100 (Techaoel et al., 2011, Priya and Usharanti, 2009). The process was repeated with diesel and petrol.


Oil Spreading Technique

Fifty millilitres of distilled water was added to a large Petri dish (15 cm diameter) followed by addition of 20µL of used engine oil to the water surface and 10µL of culture supernatant (Priya and Usharanti, 2009). A clear zone was visible under light. The area of this circle was measured and calculated for oil displacement area (ODA) using the following equation: ODA = 22/7 (radius)2 cm2


The triplicate assays from the same sample were measured (Techaoel et al., 2011). The process was repeated for diesel and petrol.


Microbial Adhesion to the Hydrocarbon (MATH)

Microbial cell surface hydrophobicity was assessed by microbial adhesion to the hydrocarbon method (MATH) (Padmapriya et al., 2013). The harvested fungal cells were washed twice with PUM buffer (buffer salt solution, pH 7.0). The cells were again suspended in PUM buffer and absorbance (Ao) was measured at 600nm on a Lightwave Diode Array Spectrophotometer. Used engine oil (500µL) was added to 5ml of fungal suspension and vortexed for 2 minutes. The absorbance (A1) of the aqueous phase was measured after 10 minutes. The percentage of the cell surface hydrophobicity (H %) was calculated using the following equation: H% = (Ao – A1/Ao) x 100. The same process was repeated for diesel and petrol.


Statistical Analysis

Data were analysed and presented as mean ± standard deviation (SD) of three replicates. Analysis of variance (ANOVA) was used to test significance of variations within and among the groups. A statistical package for social sciences (SPSS) software was used for statistical analysis in this study and test for significance between means was implied at P = 0.05 level.


Results and Discussion

Isolation and Identification of Candida tropicalis and Aspergillus clavatus

Candida tropicalis and Aspergillus clavatus were isolated from soil samples obtained from oil-contaminated soils at Old Motor Spare Parts, Nkpor, Nigeria. Preliminary identification was done using cultural and microscopic characteristics. Padmapriya et al. (2013) reported the isolation of Klebsiella sp., Bacillus sp., Aspergillus niger, Candida tropicalis, Candida albicans and Pseudomonas aeruginosa from petroleum-contaminated soil. Okonkwo et al. (2020) reported the isolation of Arthrobacter sp., Enterobacter sp., Bacillus sp. and Pseudomonas sp. from crude oil polluted soil. The cultural and microscopic characteristics of the fungal isolates are presented in Table I.


Table I: Cultural and Microscopic Characteristics of the Fungal Isolates

Isolates

Colonial Morphology

Microscopic Observation

Suspected Organisms

1




2


White/creamy, smooth, soft and glabrous


Colonies are fast growing, flat and yellow-green to dark green in colour

Spherical to sub-spherical budding yeast-like cells



Distinctive conidial heads with flask-shaped phialides arranged in whorls on vesicle

Candida sp




Aspergillus sp



Molecular Characterization of the Isolates

The 18SrRNA gene sequence wasused to characterize and confirm the isolates. Database comparism using BLAST program revealed that isolate 1 had a high similarity of 98 % with those of strain Candida tropicalis. However, isolate 2 had 100% similarities with that of strainAspergillus clavatus(Table II). The expected values (E-value) for the two isolates are zero.


Table II: Molecular Identification of the Isolates

Isolates

Closest match

Percentage (%) identity

E-value

1

2

Candida tropicalis

Aspergillus clavatus

98

100

0

0


Assay for Biosurfactant Activity of the Isolates

The isolates; Candida tropicalis and Aspergillus clavatus showed biosurfactant activity through emulsification test, oil displacement assay and microbial adhesion to the hydrocarbon (MATH). The result seems quite promising and allows the identification of some adjustments that could be introduced for the scale up of bioremediation process. For instance, selection of biosurfactant producing microorganisms is advocated in order to avoid the introduction of chemical surfactants in the environment, which are more toxic and non-biodegradable.


The biosurfactant activity of the two isolates (Candida tropicalis and Aspergillus clavatus) revealed that the isolates exhibited high emulsification index (E24) (>50%) in UEO, diesel and petrol. However, E24 of the isolates were significantly higher (P<0.05) in petrol, when compared to used engine oil and diesel (Fig. 1). Similar results of E24 values were obtained from biosurfactants isolated from 30oC and 45oC growth culture (Namir et al., 2009). Thavasi et al. (2010) reported that biosurfactant produced from a substrate can emulsify different hydrocarbons to a greater extent, which confirmed its applicability against different hydrocarbon pollution.


In oil spreading technique, Aspergillus clavatus exhibited the highest oil displacement area (ODA) in petrol, followed by diesel and the least ODA was recorded in used engine oil. Similarly, C. tropicalis recorded the highest ODA in petrol, followed by used engine oil, and the least ODA was recorded in diesel (Fig. 2). Moreover, the supernatants obtained on centrifugation of the media in which the organisms (C. tropicalis and A. clavatus) were grown, produced visible clear zones in the hydrocarbon substrates (Figs. 3-4). This indicated that the two organisms have high biosurfactant activity, as well as having bioremediation potential. The ability of Candida species to produce clear zone in oil spreading test has been reported elsewhere (Padmapriya et al., 2013).


The microbial adhesion to the hydrocarbon (MATH) revealed that in the presence of petrol, Candida tropicalis and Aspergillus clavatus exhibited higher cell surface hydrophobicity than in the presence of used engine oil and diesel. However, highest cell surface hydrophobicity was achieved by C. tropicalis in petrol (Fig. 5). The cell surface hydrophobicity (>52%) achieved in this study was an indication of the high affinity of the isolates to the hydrocarbon substrates (used engine oil, diesel and petrol), which confirmed their ability to utilize them as carbon sources. Kaczorek et al. (2011) reported a 31.4% adhesion to the hydrocarbon substrate, diesel oil, with Pseudomonas alcaligens. Padmapriya and Suganthi (2013) reported that C. tropicalis have higher hydrophobicity to hydrocarbon substrates than Pseudomonas aeruginosa.


Figure Image is Available in PDF Format


Fig. 1: Emulsification index (% E24) of the isolates in UEO, diesel and petrol. Bars indicate the average of triplicate samples while the error bars show the Standard Deviation.


Figure Image is Available in PDF Format


Fig. 2: Oil displacement area (ODA cm2) of the isolates in UEO, diesel and petrol.Bars indicate the average of triplicate samples while the error bars show the Standard Deviation.


Figure Image is Available in PDF Format


Fig. 3: Zone formation by biosurfactant producing fungi Candida tropicalis in oil spreading technique.


Figure Image is Available in PDF Format


Fig. 4: Zone formation by biosurfactant producing fungi Aspergillus clavatus in oil spreading technique.

Figure Image is Available in PDF Format


Fig. 5: Microbial adhesion to the hydrocarbon (MATH) assay of the isolates in UEO, diesel and petrol.Bars indicate the average of triplicate samples while the error bars show the Standard Deviation.


Conclusion

The fungi C. tropicalis and Aspergillus clavatus showed high biosurfactant activity in used engine oil, diesel and petrol. This indicates that they have great potential for biosurfactant production as well as potential application against different hydrocarbon pollutants.


Acknowledgements

Authors acknowledge the efforts of the Director and the entire staff of the National Agency for Food and Drug Administration and Control (NAFDAC), Agulu, Nigeria, for providing us with the enabling environment for the conduct of this research.


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