Promising biosurfactant produced by a new Candida tropicalis UCP 1613 strain using substrates from renewable-resources

1 Post-graduation Program in Biological Sciences, Federal University of Pernambuco, Brazil 50670-420, Recife, Pernambuco, Brazil. 2 Nucleus of Research in Environmental Sciences and Biotechnology, Catholic University of Pernambuco, Brazil 50050590, Recife, Pernambuco, Brazil. 3 CEUMA University, São Luís, 65075120, Maranhão, Brazil. 4 Northeast Network for Biotechnology, Federal Rural University of Pernambuco, 52171-900 Recife, PE, Brazil. 5 Institute for Biological Sciences, Federal University of Minas Gerais, 31270-010, Belo Horizonte, Minas Gerais, Brazil.


INTRODUCTION
Nowadays the presence of surfactants in the daily activities of man makes life easier.These compounds are mainly obtained through chemical synthesis from oil and possess in their composition both hydrophilic and hydrophobic structural moieties.Thus, this duality gives them the ability to lower the surface tension, critical micelle concentration, and interfacial tension between liquid-liquid/liquid-solid systems (Marchant et al., 2012;Sharma et al., 2014).In contrast, biosurfactants are the main classes of natural surfactants produced by fungi, yeast and bacteria.
According to their chemical structures, they are grouped into five major classes of lipopeptides, glycolipids, phospholipids, neutral lipids, and polymeric compounds (Desai and Banat, 1997;Khopade et al., 2012).Microbial surfactants as also known, have numerous advantages when compared to their chemically synthesized counterparts due to their lower toxicity, higher biodegradability, and better environmental compatibility, ability to be synthesized from renewable resources, higher foaming, higher selectivity and specific activity at extreme temperature, pH and salinity (Zheng et al., 2012;Rufino et al., 2014).These properties confer them commercial importance evidenced through the biotechnological applications in pharmaceutical, biomedical, cosmetic, petroleum, and food industries (Nitschke and Costa, 2007;Banat et al., 2010).
However, the benefit of these molecules contrasts with its high costs of the production associated with inefficient methods of recovery turning with distant reality for industry (Makkar et al., 2011).In this context, biocircular economy brings an innovative approach as a demand for global sustainability.Thus, efforts toward the recovery and reuse of waste from agro industrial origin through bioprocesses allow in valorizing the microbial synthesis of biobased products as biosurfactants (Mohan et al., 2016).
In addition, other significant aspects to be explored are the absence of toxicity and antimicrobial activity.These properties are suitable for the biotechnological applications of biomolecules such as biosurfactants (Sobrinho et al., 2013).With this in mind, the present paper was focused on the characterization of the biosurfactant produced by Candida tropicalis UCP 1613 isolated from mangrove sediment in Northeast of Brazil.The biosurfactant was extracted and characterized through compositional analyze, ion charge and Fourier transform infrared spectroscopy (FTIR).The stability of the biosurfactant under different conditions of pH, temperature and salinity was also studied and verified as its efficacy in the phytotoxicity assay and antimicrobial activity.

Mangrove sediments collection
The samples isolated from mangrove sediments of Rio Formoso (localized at 08°39' 50" S 35°09' 32"), Pernambuco state, Brazil, were collected in July, 2015.The samples collected from mangrove ecosystem were previously marked in the area, collected in sterilized bottle, and immediately conducted to the Nucleus of Research in Environmental Sciences and Biotechnology (NPCIAMB), of the Catholic University of Pernambuco.

Agro-industrial substrates
The production medium was composed by agro-industrial wastes: cassava wastewater, a waste from cassava (Manihot esculenta Crantz) processing, if released directly into the environment before proper treatment, which could be a source of pollution.The whey was kindly supplied from the dairy industry of São Bento do Una, PE, Brazil, and waste cooking oil from informal commerce in Recife, PE, Brazil.
A 200 mg/L of chloramphenicol was supplemented after sterilization to minimize bacterial growth.The flasks were incubated at 150 rpm and 28°C for 48 h.After this period, serial dilutions from 10 -1 to 10 -5 were prepared and aliquots of 0.1 mL of each dilution were added to Petri dishes containing Yeast Mold Agar (YMA) with the following composition (w/v): yeast extract (0.3%), malt extract (0.3%), tryptone (0.5%), d-glucose (1.0%) and agar (5%).These plates were then incubated at 30°C for 72 h.

Identification of the isolated yeast
The colonial morphology of yeast was identified according to Accorsini et al. (2012) by color, borders and size of the colonies, texture, surface appearance and elevation.The molecular characterization was modified from the methodology performed by White (1990).DNA extraction was performed using Wizard ®Genomic DNA Purification Kit (Promega).
Then, amplicons were purified and sequenced by capillary electrophoresis on the ABI3130 platform (Life Technologies) which allows sequencing to 900 pb quality by the company Myleus Biotechnology.The chromatograms generated were subjected to *Corresponding author.E-mail: galba_takaki@yahoo.com.br.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Phred-Phrap software to read quality check of the base, during the sequencing step.The consensus sequences were submitted for analysis of similarity with sequences deposited in Gene Bank (Genbank) from the National Center for Biotechnology Information (NCBI), using the online tool basic local alignment search nucleotides (Blastn).The hits that had the highest percentage of similarity with the sequence under study, considering the best combination of "score" and "e-value" were regarded as identical.

Culture conditions
Cell grown of yeast on a slant were transferred to 50 mL of YMB.The culture was incubated in an orbital shaker at 150 rpm and 28°C for 24 h.Erlenmeyer's flask containing medium with the following composition: MgSO4 0.2 g/L, K2HPO4 1.0 g/L, KH2PO4 1.0 g/L, CaCl2 0.02 g/L, FeCl3 0.05 g/L and NH4NO3 1.0 g/L, supplemented with whey (3%), cassava wastewater (7%) and soybean post frying oil (10%) were adjusted to pH 5.3.The medium was sterilized by autoclaving at 121°C for 20 min.
After this period, 0.5% of YMB culture containing 10 4 cells/mL was used to initiate the growth.The culture was incubated at 28°C in an orbital shaker at 150 rpm for 96 h in triplicate and at regular intervals, every 24 h samples was taken to determinate surface tension and biomass.

Biomass determination
For biomass determination, 5 mL samples were mixed in preweighed tubes with chilled distilled water and centrifuged at 5000 rpm for 20 min.After two washing cycles, the cell pellet was dried in an oven 90°C for 24 h.All the assays were carried out in triplicate (Rufino et al., 2014).

Determination of surface tension
The determination of surface tension was carried out in the cell-free broth, obtained by centrifugation of the cultures at 5000 rpm for 20 min at room temperature, using a Sigma 700 digital surface tensiometer (KSV Instruments LTD, Finland), working on the principle of the Du Nuoy ring method (Gilanyi et al., 1976).

Isolation of biosurfactant
The biosurfactant was isolated by the precipitation method using the cell-free metabolic liquid with acetone 1:1 (v/v).The precipitate was allowed to stand for 24 h at 4°C, and after this period it was centrifuged at 5000 rpm for 20 h, at 5°C.
The supernatant was discarded and the isolated biosurfactant was submitted to dialysis against deionized water, which was changed every 3 h, for 96 h at 5°C.The yield of the isolated product was calculated as g/L and the analyses were performed in triplicate.The biosurfactant was collected and freeze-dried (Shavandi et al., 2011).

Critical micelle concentration (CMC)
The concentration at which micelles began to form was defined Critical Micelle Concentration (CMC).From known amounts of crude precipitate resuspended in distilled water, these determine the critical micelle concentration (CMC).
These measurements were realized using DuNouy Tensiometer model Sigma 70 (KSV Instruments LTD, Finland) at room  temperature.The CMC was reached by measuring the surface tension until observing a constant value of the surface tension.All experiments were performed in triplicate (Rufino et al., 2014).

Compositional analysis
The protein content in the isolated biosurfactant was determined using the total protein test kit from Labtest Diagnóstica S.A., Brazil.
The phenol-sulphuric acid method allowed in determinating the total carbohydrate content (Dubois et al., 1956).In the case of the lipid content 0.5 g of the isolated material was extracted with chloroform: methanol in different proportions (1:1 and 1:2, v/v).The organic extracts were then evaporated under vacuum and the lipid content was determined by gravimetric estimation (Manocha et al., 1980).

Ionic charge
The ionic charge of the biosurfactant was determined by using a Zeta potentiometer model ZM3-D-G, Zeta Meter System 3.0+, with direct images to the video of the Zeta Meter, San Francisco, CA, USA.
The top row was filled with a pure compound of a known ionic charge.The substance is known as anionic sodium dodecyl sulfate (SDS) at a concentration of (0.02 M) cationic substance and barium chloride (0.05 M).Petri dish was kept at room temperature for 48 h.The result was calculated when the precipitation lines appear as per Meylheuc et al. (2001) (Silva et al., 2014).

Fourier transform infra-red (FTIR) analysis
The identification of functional groups in the isolated biosurfactant was determined using a FTIR spectrophotometer (Bruker IFS 66) with KBr in the pestle.The FTIR spectrum was obtained at a frequency range of 4000 -400 cm -1 (Smith, 1996).

Stability assay
In order to demonstrate biosurfactant stability under different conditions, the superficial tension of its partially purified solution (1 g/L) was examined.Samples were heated at 0, 5, 28, 70, and 100°C and cooled to room temperature, after which the surface tension was measured.
In the case of pH, this was adjusted to various pH values (2 to 12) by adding HCl or NaOH to a solution at room temperature and the surface was measured.The effect of NaCl concentrations (2 to 12%) was also a determinate.The tests were performed in triplicate (Tados, 2005).

Phytotoxicity assay
The phytotoxicity of the biosurfactant was evaluated based on seed germination and root elongation of cabbage (Brassica oleracea), lettuce (Lactuca sativa L.) and tomato (Solanum lycopersicum), according to Tiquia et al. (1996).
Solutions of the isolated biosurfactant were prepared with distilled water at concentrations of 1, 1.5 and 2%.Toxicity was determined in sterilized Petri dishes (1 × 10 cm) containing Whatman N°1 filter paper.The seeds were pre-treated with sodium hypochlorite.Ten (10) seeds were inoculated in each Petri dish with 5 mL of the test solution at 27°C.After five days of incubation in the dark, seed germination, root elongation (≥5 mm) and the

Antimicrobial assay
The isolated biosurfactant was evaluated by the agar disc diffusion method (Bauer et al., 1996).Sterile discs (0.6 cm) soaked with the biosurfactant solution in methanol was assayed on the surface of and nutrient agar and malt extract medium for bacteria and yeast, respectively inoculated with the tested microorganism.
After incubation period for 24 h at 37± 2°C and 48 h at 25 ± 2°C for bacteria and yeast, respectively, the diameter of inhibition zones was measured (Bradshaw, 1992).Negative controls were prepared using the same solvents as employed to obtain the extract.Positive controls, ofloxacin (5 µg, Oxoid) was used for Gram-positive bacteria, cefaperazone-sulbactam (10 µg, Oxoid) for Gramnegative bacteria and amphotericin B (30 µg, Sigma) for Candida albicans.All the experiments were conducted in triplicates.

Morphological and molecular identification of the yeast producer of biosurfactant isolated from mangrove sediment
According to morphological observations of strain, yeast showed a characteristic of cream, smooth, glossy colonies whereas microscopic analysis revealed cells with small and medium sized with budding and no filaments characteristic.These traits allow presumptively the classifying of the isolate as Candida sp.(Figure 1).
Additionally, the molecular analyses of the sequences of ITS region were compared with the nucleotide database using the NCBI-blast tool.Candida sp.showed highest genetic agreement with C. tropicalis with a similarity of 99%.Thus the isolated was identified as C. tropicalis UCP 1613.

Kinetics of growth, biosurfactant production and biosurfactant yield
In this study, C. tropicalis UCP 1613 was cultivated in medium containing the agro-industrial substrates whey, cassava wastewater and soybean post frying oil during 96 h at 28°C.As shown in Figure 2, the growth of microorganism started rapidly after inoculation and remained increasing to 96 h of cultivation, when biomass reached 7.7 g/L.The surface tension dropped rapidly from 70 to 32.9 mN/m in the first 24 h and continued decreasing to 28.5 mN/m at 48 h of growth, indicating excellent surface-active properties.Biosurfactant production started in the early stages of the exponential growth phase, simultaneously to the surface tension reduction which increased significantly until the end of cultivation, which attained the maximum biomass yield.The yield of the crude biosurfactant produced by C. tropicalis UCP 1613 was 4.9 g/L after 96 h of cultivation in medium containing 3% whey, 7% cassava wastewater and 10% soybean post frying oil (Figure 3).

Critical micelle concentration (CMC)
Thus in this study the biosurfactant obtained from the use of agro-industrial wastes showed a great surface tension reduction capacity, since the water surface tension was reduced from 70 to 28.5 mN/m with the increase of the biosurfactant concentration up to CMC of 1.5%, and then remained constant (Figure 4).

Preliminary characterization of biosurfactant
The determination of biochemical composition of the biosurfactant analyzed revealed the presence of 51% proteins, 37% lipids and 11% carbohydrates, suggesting its polymeric nature.Furthermore, an anionic profile with −57.4 ZPmv, 3, 4 μS/cm at 25.3°C was detected.In addition, the FTIR analysis was also used as complementary in this study.
According to the spectrum showed in Figure 4, stretching bonds of functional groups N-H between 3,514-3,257cm -1 indicated the presence of a peptide component.Also, a complex sequence of peaks due to the stretching vibration mainly C-C and C-O-P of oligo and polysaccharides (starches) were detected at an interval of 1200 to 1000 cm -1 .In addition, functional  groups -CH=CH 2 and CH 3 were identified at 1448.27 cm -1 corresponding to fatty acids (Figure 5).Bands at approximately 1756.31 cm -1 showed the presence C=O consistent with ester functional group in lipids (Verma et al., 2015).

Biosurfactant stability
The behavior of the biosurfactant produced by C. tropicalis UCP 1613 was tested in different conditions of temperature, pH and NaCl concentrations.As it is shown in Figure 6, it was noticeable how the surface tension of the biosurfactant remained stable over a wide range of temperature, pH and NaCl concentrations.

Biosurfactant phytotoxicity
The phytotoxicity effect was evaluated through the germination index (GI) which combines measures of relative seed germination and relative root elongation.Seeds of vegetables Brassica oleracea, Lactuca sativa L. and Solanum gilo were used with different concentrations  In this regard, the Table 1 which displays that this metabolite did not show a phytotoxic effect against seeds tested.Also, it was interesting, the proportional relation was observed between the different germination index (GI) and the concentrations of biosurfactant.

Antimicrobial activity
In the present study, the polymeric biosurfactant produced by C. tropicalis UCP 1613 exhibited interesting antimicrobial activities.Table 2 shows that, Gram-positive bacteria tested mainly Lactobacillus sp.displayed susceptibility to the biosurfactant, albeit to different degrees.In contrast, Gram-negative bacteria were less sensitive as well as yeast.

DISCUSSION
The interest on Candida species has been increased in recent years due to its diverse biotechnological role.Specifically, C. tropicalis has been recovered from seawater, sediments, mangrove plants, mud flats, marine algae and shrimp, indicating its wide distribution in tropical and subtropical marine environment (Luna et al., 2011;Yadav et al., 2012).The use of agro-industrial residues for the production of biosurfactant by members of the genus Candida have been previously described by several researchers (Luna et al., 2011;Rufino et al., 2014;Brasileiro et al., 2015).The ability of these microorganisms to grow and produce biosurfactants in wastes-based medium has been used to reduce the high costs of production of surface active compounds of biotechnological interest.

Germination index (%)
Biosurfactant concentration (%) Experiments were performed in triplicate and the results represent means ± standard deviations of the three independent experiments.
In this study, the biosurfactant showed a growthassociate production, similar to the ones produced for Candida sphaerica cultivated in distilled water supplemented with ground-nut oil refinery residue and corn steep liquor as substrates (Sobrinho et al., 2008) and for Candida lipolytica cultivated in soybean oil refinery residue and glutamic acid (Rufino et al., 2014).
Although the biosurfactants produced by bacteria are more effective in reducing the surface tension, with values up to 25 to 26 mN/m, several yeasts biosurfactants described in the last few decades have shown similar values to those obtained by bacteria (Luna et al., 2011).One example is the biosurfactant produced by C. tropicalis in this study, which reduced the surface tension of medium to 28.5 mN/m after 48 h of cultivation.This value is lower than other biosurfactants produced by C. tropicalis: 35 mN/m (Haba et al., 2000); 35.44 mN/m (Coimbra et al., 2009), 37.86 mN/m (Campos et al., 2014) and 29.98 mN/m (Almeida et al., 2017).
By other side, the biosurfactant yield of C. tropicalis UCP 1613 was 4.9 g/L, which was better than others, previously reported for C. tropicalis strains: 3.0 g/L by C. tropicalis UCP 0996 cultured for 120 h in medium containing molasses, corn steep liquor and waste canola oil (Almeida et al., 2015) and 3.61 g/L by C. tropicalis cultivated in waste frying oil (Batista et al., 2010).However, other Candida members showed highest biosurfactant yields, for example: C. lipolytica (8.0 g/L) cultivated during 72 h in optimized medium containing soybean oil refinery residue and glutamic acid (Rufino et al., 2014) and C. sphaerica (9.0 g/L) cultured during 144 h in medium composed by ground-nut oil refinery residue and corn steep liquor (Luna et al., 2016).
In addition, Candida utilis achieved 12.52 g/L after 88 h of cultivation in waste canola frying oil and ammonium nitrate (Campos et al., 2014).These results demonstrate that the medium composition and culture conditions influence on yields of biosurfactants is produced by Candida strains.Also, the suitability of whey, cassava wastewater and soybean post frying oil was confirmed as alternative substrates for biosurfactant production (Silva  .5 et al., 2014;Andrade et al., 2015).Another important characteristic of biosurfactants is the critical micelle concentration (CMC) which is defined as the minimal concentration of the compound, required to yield maximal surface tension reduction of water which initiate micelle formation (Santos et al., 2013).The biosurfactant produced showed a greater capacity to reduce surface tension in comparison to biosurfactants from Candida glabrata (31 mN/m) (Luna et al., 2009), and C. lipolytica (32 mN/m) (Verma et al., 2015) (33 mN/m) (Santos et al., 2013).But also, this displayed a lower value of CMC than other biosurfactants reported, considering rates of 2.5% such as the biosurfactant from C. glabrata (Luna et al., 2009) and C. lipolytica (Sarubbo et al., 2007).The compositional information of microbial surfactants allows the determining of the heterogeneous nature of these compounds.Thus, these are classified according to their molecular structure into glycolipids, lipopeptides, phospholipids, fatty acids, lipopolysaccharides, protein complexes, neutral lipids and polymers (Aparna et al., 2012).The analytical evidence confirmed the presence of carbohydrates and proteins in hydrophilic region of the molecule and also, the existence of lipids in the region hydrophobic of the biosurfactant.Similarly to the biosurfactant obtained in this study from C. tropicalis UCP 1613, C. lipolytica UCP 1002 has been also reported as producer of a heteropolymer composed 45% protein, 20% lipid and 10% carbohydrate (Sarubbo et al., 2006).However, surfactants produced by this genus can differ widely from one species to another (Rufino et al., 2014).In addition, the zeta potential determines the function of the surface charge of the particle which serves to predict and control the stability of colloidal suspensions and emulsions.The higher values obtained indicate good stability by repulsion between hydrophilic particles, as per the literature (Pornsunthorntawee et al., 2008;Satpute et al., 2010).Other biosurfactants produced by Candida species also show an anionic character (Sobrinho et al., 2008;Andrade et al., 2015;Luna et al., 2013).
Different environmental factors such as pH, salinity and temperature affect biosurfactants activity and stability.Therefore, it is important to study the influence of these parameters when considering specific applications for these compounds (Velmurugan et al., 2015).These results obtained suggest the feasibility of application in industries which works under extreme conditions of salinity, temperature and pH, as it was informed for other biosurfactants from Candida species (Gusmão et al., 2010;Luna et al., 2014) In this regard, the absence of toxicity is also fundamental for possible application of biosurfactant in the environment.Eco-toxicity bioassays are analytical methods which evaluate toxic effect of the chemical substances.
The exposure of living organisms employed as bioindicators to these substances constitutes a valuable tool (Fletcher, 1991).The use of plants in toxicity tests offers several advantages among them is low maintenance cost and rapid results, with a special benefit assessment of the potential eco-toxic compounds in terrestrial environments (Farré and Barceló, 2003).In general, in this study was observed that from the different solutions of biosurfactant tested there was a positive effect on the growth seeds (Table 1).Also from the observation of leaves and the elongation of secondary, it was suggested a growth stimulating effect which was verified considering that GI higher than 80%, indicate the absence of phytotoxicity (Tiquia et al., 1996).Similar results were observed by Luna et al. (2013) and Rufino et al. (2014), who investigated the phytotoxic potential of the biosurfactant produced by C. sphaerica UCP 0995 and C. lipolytica UCP 0988, respectively.The same observations were confirmed by Krawczyńska et al. ( 2012), Alsohim et al. (2014) and Silva et al. (2015) which detected the positive effect of biosurfactants in the seedling development.
Alternatively one useful property of many biosurfactants that has been reviewed recently is their antimicrobial activity (antibacterial, antifungal and antiviral) (Banat et al., 2014;Rienzo et al., 2015;Borsanyiova et al., 2016).In this study, the yeasts were less sensitive than Gramnegative bacteria.Some studies confirm that biosurfactants, even in low concentrations, may destabilize the microorganism's membranes and finally inhibit their growth (Carrillo et al., 2003;Calvo et al., 2009).In this regard, it has been observed that Grampositive bacteria are more susceptible to biosurfactants than Gram-negative bacteria (Elving et al., 2000), which are not inhibited at all or present a slight inhibition.This evidence is in agreement with the results obtained in this study.Similarly, biosurfactants produced by C. sphaerica and C. lipolytica have also demonstrated antimicrobial activity against different species of fungi and bacteria, suggesting the use of these biomolecules as an alternative antimicrobial agents in the medical field (Luna et al., 2011;Rufino et al., 2011).

Conclusions
In the present study with the new yeast isolated from mangrove, sediments were identified as C. tropicalis UCP 1613.The optimal carbon and nitrogen sources was a combination of whey (3%), cassava wastewater (7%) and soybean post frying oil (10%) resulting significantly reduction of the surface tension.
The biosurfactant produced was non-cytotoxic which showed stable surface tension to high temperature, acid to alkaline pH, and all concentrations of electrolyte (up to 12%).Besides, the new biosurfactant is characterized as anionic and polymeric molecule.The tension-active molecule showed properties to effective antimicrobial activity, Gram-positive and negative bacteria, and yeasts.This study strongly suggested the biosurfactant produced by C. tropicalis UCP 1613 which play a promising role in biomedical application due to its non-toxicity, stability and higher antimicrobial activity.The use of renewable resources as agro-industrial waste from circular bioeconomy approach allowed the obtaining of biosurfactant as a value added product.

Figure 2 .
Figure 2. Phylogenetic tree based on the ITS1 primer used for the identification of Candida tropicalis UCP 1613.

Figure 3 .
Figure 3. Growth, surface tension and yield of biosurfactant isolated from C. tropicalis UCP 1613 cultured in mineral medium supplemented with 3% whey, 7% cassava wastewater and 10% soybean post frying oil.

Figure 4 .
Figure 4. Surface tension versus concentration of the biosurfactant isolated from C. tropicalis UCP 1613 cultured in mineral medium supplemented with 3% whey, 7% cassava wastewater and 10% soybean post frying oil.

Figure 6 .
Figure 6.Stability of surface tension of biosurfactant produced by Candida tropicalis UCP 1613 in mineral medium supplemented with 3% whey, 7% cassava wastewater and 10% soybean post frying oil.Influence of temperature (A), pH (B) and sodium chloride concentrations (C) on surface tension stability.

Table 2 .
Antimicrobial activity of the biosurfactant produced by Candida tropicalis UCP 1613.