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Many Candida species are not ‘generally regarded as safe’ (GRAS) to be handled as baker's yeast (Benjamin and Pandey, 1998). Candida tropicalis is a typical inhabitant of the human body which is not normally considered to cause health issues. It has been noticed that in tropical countries like India, C. tropicalis isolates are more prevalent in clinical materials such as blood, urine and sputum (Eggiman et al., 2003). Humans are the natural but not the exclusive habitat of C. tropicalis. It has been isolated from animal sources such as the intestinal contents of marine mammals, birds and bovine, porcine, canine, murine and equine species (Chengappa et al., 1984), abomasums of cattle (Foley and Schlaf, 1987) and bovine milk (Lagneau et al., 1996). Moreover, the inhabitance of Candida spp. (C. albicans, C. tropicalis, C. krusei, C. rugosa, C. parapsilosis) and Trichosporon (T. cutaneum, T. sericeum) in the guts of ruminants, especially cow and sheep, has been reported (Lund, 1974). However, no report is yet available in the literature regarding the inhabitance of C. tropicalis in the goat (Capra hircus Lin.), an entirely different genus from the sheep (Ovis aries Lin.).
Apart from the clinical significance, only a few studies have been reported on the exploration of C. tropicalis as a microbial source for the production of industrially significant biomolecules such as biosurfactants (Ashish et al., 2011). Biosurfactants are amphipathic molecules with both hydrophilic and hydrophobic moieties that partition preferentially at the interface between fluid phases that have different degrees of polarity and hydrogen bonding, such as oil–water or air–water interfaces (Rodrigues et al., 2006). Some species of Candida, such as C. antarctica (Kitamoto et al., 1992), C. bombicola (Gobbert et al., 1984), C. glabrata (de Luna et al., 2009) and C. lipolytica (Rufino et al., 2011) are known to produce surface-active biomolecules. Microbial surfactants constitute a diverse group of surface-active molecules known to occur in a variety of chemical conjugates, such as glycolipids, lipopeptides, lipoproteins, fatty acids, neutral lipids, phospholipids and polymeric/particulate structures (Mukherjee et al., 2006).
The increasing environmental concern about various chemical pollutants has triggered attention to microbial-derived compounds, essentially due to their low toxicity and biodegradability. In response to problems associated with plastic wastes and their lingering effect on the environment (Pradeep and Benjamin, 2012; Pradeep et al., 2012; Sarath Josh et al., 2012), there has been considerable interest in the development and production of biodegradable plastics, which include polyhydroxyalkanoates (PHAs), polylactides, aliphatic polyesters, polysaccharides and copolymers and/or proper blends of these. Among the candidates for biodegradable plastics, PHAs have been drawing much attention because their characteristic properties, such as crystallinity and tensile strength, are comparable to those of conventional plastics (Lee, 1996). Polyhydroxybutyrate (PHB), the predominant group of PHA, has been well characterized from microflora and established as a natural bioplastic (Lee, 1996). PHB is synthesized and accumulated as granules in the cytoplasm of numerous microbes, which serve as intracellular carbon and energy storage compounds, or as a sink for redundant reducing power in response to limiting nutrients in the presence of excess carbon (Byrom, 1994). Many species of Bacillus, Pseudomonas, Streptomyces, Rhodococcus and Micrococcus are well known to produce PHB granules, whereas eukaryotic microbes are not documented in detail for PHB production (Anderson and Dawes, 1990; Wang and Bakken, 1998; Verlinden et al., 2007). However, a few yeasts, such as Saccharomyces cerevisiae, Candida krusei, Kloeckera apiculata and Kluyveromyces africans, were found to accumulate PHB in their cytoplasm (Leaf et al., 1996; Safak et al., 2002).
Under these circumstances, we introduce in this study a new strain of yeast capable of dual production of biosurfactant and PHB with potential for industrial exploitation. Thus, the specific objectives of this study were: (a) to isolate, enrich and characterize a novel strain of C. tropicalis from the rumen of the Malabari goat; (b) to design a cultivation strategy to adapt it to aerobic growth; (c) to explore its potential for biosurfactant production; and (d) to explore its potential for PHB production.
Materials and methods
Rumen content of both male and female Malabari goats was collected aseptically from the local slaughter house at Chelari, near the University of Calicut campus (11.18189600 N, 75.82206300 E), as described by Prive et al. (2010). Initial cultures were done on de Man, Rogosa and Sharpe (MRS) medium (HiMedia, India) and subcultured on potato dextrose agar (PDA) medium (HiMedia, India), incubated at 37 °C. The isolated colonies obtained were examined for the presence of yeasts and yeast-like organisms. The purity of the isolate was confirmed by subculture.
The isolate was macromorphologically characterized by observing colony characteristics such as colour, texture and topography of the surface and edges, according to the method of Larone (2002), and micromorphologically by employing a conventional lactophenol cotton blue staining technique.
Fermentation reactions in media containing glucose, lactose, maltose, sucrose, dextrose or cellulose as the sole source of carbon source were performed to identify the yeast to species level (Hugh and Leifson, 1953). Nitrogen assimilation test was carried out by the modified auxanographic technique (Kurtzman and Fell, 1999). In brief, agar medium containing 2% glucose, 0.1% KH2PO4 and 0.05% MgSO4 was poured into petri dishes and allowed to solidify. Yeast suspension in sterilized double-distilled water was swabbed onto the surface of the medium. Sterilized filter paper saturated with the nitrogen compounds was placed on the solid surface of the inoculated agar medium and incubated at 37 °C. An area of growth was produced around those compounds that were assimilated. In addition, the growth of the strain at 45 °C was evaluated in PDA medium for 48 h.
The yeast isolate was confirmed by PCR amplification and sequencing of the D1/D2 region of the larger subunit of the 28S rDNA gene from the isolated genomic DNA. Forward and reverse DNA sequencing reaction of the PCR amplicon was carried out with DF and DR primers, using a BDT v 3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI 3730xl Genetic Analyser. The gene sequence was used to carry out BLAST with the non-reductant (NR) database of NCBI Genbank. Based on maximum identity score, the first 10 sequences were selected for construction of the phylogenetic tree, using MEGA4 software (Tamura et al., 2007).
Cultivation strategy and media
Initially, the novel isolate was cultured under anaerobic condition in an anaerobic chamber (KIM Microsystems, India) saturated with mixed gas (80% N2, 10% CO2 and 10% H2). The pure culture in liquid medium was gradually adapted to the aerobic system using a conical flask specially designed by us (Figure 1), designated a ‘Benjamin flask’. The pure culture so obtained was further cultivated in liquid basal mineral salt medium (BSM), supplied with the required volume of vegetable (groundnut) oil as the sole source of carbon and incubated at 37 °C for 3–7 days at 140 rpm in an environmental orbital shaker (Scigenics Biotech, India). The composition of the BSM was as follows: 0.2% K2HPO4, 0.2% NaCl, 0.4% (NH4)2SO4, 0.5% NH4NO3, 0.01% MgSO4, 0.0001% FeCl3, 0.0001% MnSO4, 0.0001% CaCl2, 0.0001% CuSO4, 0.0001% COCl2 and 0.05% Cysteine–HCl, with initial pH 6.8 (Pradeep and Benjamin, 2012). Initially, 0.05% groundnut oil was added to the BSM and then subcultured into fresh BSM containing higher concentrations of oil, and gradually adapted to aerobic conditions. By repeated aerobic subculture, the oil consumption was enhanced to 0.3%, which was utilized completely in 6–7 days of growth.
Characterization of biosurfactant
Extraction of crude biosurfactant
The 6 day-old culture in BSM supplemented with 0.3% groundnut oil was centrifuged at 8000 × g for 10 min at 4 °C. The supernatant was acidified to pH 2.0 using 6 m HCl, and was left overnight at 4 °C for the complete precipitation of the biosurfactant. The precipitate was dissolved in water and adjusted to pH 7 using 1 m NaOH. The biosurfactant was extracted with a solvent system containing chloroform:methanol (2:1) in a separating funnel at 26 °C. The organic phase was concentrated using a rotary evaporator.
Biochemical characterization by TLC
Silica gel thin-layer chromatographic (TLC) plates were spotted with 5 µl sample and separated using chloroform:methanol:water (65:25:4) as the solvent system, and then visualized with ninhydrin, anthrone or Sudan 3 spray to detect the presence of protein, carbohydrate and lipid, respectively.
Screening for PHB production
Microscopic visualization and viable colony methods were employed for the screening. Under viable colony methods, Nile blue sulphate, Sudan black B and Sudan 3 staining techniques were employed.
Sudan black stain was prepared as a 0.3% w/v solution in 60% ethanol. Six day-old culture in BSM using groundnut oil as the sole carbon source was smeared onto a glass slide and stained with Sudan black B solution for 10 min, rinsed with xylene and counterstained with 0.5% safranin (BD Sciences) for 5 s. Stained samples were observed under oil immersion at ×100 magnification with direct brightfield illumination (Burdon et al., 1942).
Nile blue sulphate
Nile blue sulphate solution was prepared by dissolving 0.05 g Nile blue sulphate in l00 ml ethanol. Colonies on PDA were stained with 5 ml staining solution and shaken gently at 26 °C. After 20 min, excess stain was drained off and the plate was air-dried. PHB producing colonies were detected by irradiating the plate with a short-wave ultraviolet (UV) light at a distance of 254 nm (about 10 cm) from the UV lamp (Kitamura and Doi, 1994).
Sudan black B
A 0.02% alcoholic solution of Sudan black B was prepared and colonies grown on PDA plates were stained with 5 ml of the solution. The plates were kept undisturbed for 30 min. The dye was then decanted and the plates were gently rinsed by pouring on 100% ethanol. PHB-producing colonies appeared bluish black, whereas non-producers appeared white (Juan et al., 1998).
A staining solution was prepared by dissolving 0.2 g Sudan 3 in 25 ml alcohol with gentle warming and then cooled; 25 ml glycerin was added to remove undissolved stain. Colonies on PDA were stained with the solution for 20 min. Excess stain was removed, followed by washing three times with distilled water. PHB-producing colonies appeared dark pink.
The isolate was identified as Candida tropicalis strain BPU1 (Genbank Accession No. JQ353488), the first-ever report from a goat. The pure colonies were smooth, white-to-cream in colour and were generally spherical (Figure 2). The cell size was 4–9 µm in length × 3–5.5 µm in diameter.
Tests of fermentation and nitrogen assimilation showed that C. tropicalis BPU1 efficiently utilized glucose, sucrose, maltose and dextrose, but not lactose and cellulose. It also assimilated (NH4)2SO4, arginine, peptone, glycine and vegetable oil, but not NaNO3. Interestingly, C. tropicalis BPU1 efficiently utilized groundnut oil as the sole source of carbon in a simple synthetic salt medium. In addition, growth at 45 °C was positive, which is typical of the Candida spp. (C. albicans, C. glabrata, C. kefyr and C. tropicalis) (Larone, 2002). These results are summarized in Table 1.
Table 1. Biochemical characteristics of C. tropicalis strain BPU1
Utilization of vegetable oil
The isolate was further confirmed as C. tropicalis based on nucleotide homology and phylogenetic analysis. A consensus sequence of 603 bp of the D1/D2 region of the 28S rDNA gene was generated to carry out BLAST with the NR database of NCBI Genbank. Based on maximum identity score, the first 11 sequences were selected and a phylogenetic tree was constructed, using MEGA 4. Analysis of the evolutionary relationship of 11 taxa showed that C. tropicalis strain BPU1 is as a recently evolved strain (Figure 3).
Characterization of biosurfactant
C. tropicalis was able to utilize vegetable oil as the sole source of carbon within 6–7 days of incubation. The pattern of utilization is shown in Figure 4. Initial large oil droplets became dispersed and fragmented within 2 days of incubation and completely exhausted by 6–7 days. The biosurfactant extracted was biochemically characterized by TLC. On spraying with Sudan 3 and anthrone, it showed visible spots indicating the presence of lipid and carbohydrate, respectively; whereas with ninhydrin a negative result was obtained (Figure 5), indicating no protein. Since the separated sample spots for the presence of lipid and carbohydrate were detected at the similar Rf value of 0.88, we were able to conclude that the surfactant is a complex of carbohydrate and lipid.
Screening for PHB production
Colony staining with the specific stains for PHB showed positive results. After treating with an ethanol solution of Nile blue sulphate, colonies on the agar plate were stained blue. Initially there was no difference between the colonies with and without PHB granules under visible light. When the stained colonies were irradiated with a short-wave (254 nm) UV light in a dark room, the colonies with PHB fluoresced bright orange but the other colonies without PHB remained unchanged (Figure 6a). On staining with Sudan black B and Sudan 3, PHB-producing colonies were stained dark blue and red, respectively, whereas non-producing colonies remained colourless (Figure 6b, c). Microscopic visualization showed PHB granules as dark blue-black in safranin-stained cells (Figure 6d).
Although C. tropicalis has been isolated from clinical samples and various parts of animals (Eggiman et al., 2003; Chengappa et al., 1984), no report is yet available in the literature showing the inhabitance of Candida sp. in the rumen of any goat, especially the Malabari goat. The rumen is the first chamber of the alimentary canal in cattle and is the primary site of microbial fermentation, where it meets the bulk of hydrocarbons as a part of daily animal feed. Therefore, the rumen provides a suitable site for the isolation of hydrocarbon-utilizing microbes, which are harmless to mankind compared to the microbes that grow in harsh extreme environments. It is therefore expected that many industrially useful hydrocarbon-utilizing microbes (bacteria, fungi, yeast) have evolved in this environment by continuous exposure to hydrocarbon-based substrates. Moreover, adaptation with aerobic conditions and a high concentration of vegetable oil enables the culture to grow under limited nutrient conditions, which essentially is of industrial significance.
In recent years, greater emphasis has been placed on the environmental impacts of chemical surfactants and many new surfactants have been developed to be used in the pharmaceutical and biomedical industries. A variety of microorganisms, such as many spp. of bacteria, especially Bacillus, Pseudomonas, Micrococcus, Neisseria (Hamed et al., 2012) and yeast (Kappell and Fiechter, 1976), are known to produce surface-active biomolecules. We showed that C. tropicalis is able to assimilate vegetable oil as the sole source of carbon, coupled with the production of biosurfactant. It has been reported previously that the yeast C. tropicalis assimilates a variety of carbon sources, such as n-alkanes and free fatty acids (Ueda et al., 1985; Kurihara et al., 1992), but still vegetable oil is an untapped carbon source for the cultivation of C. tropicalis. Moreover, the simple mineral salt medium we designed for cultivation allows further manipulations for the enhancement of biosurfactant production. Our study showed that the biosurfactant produced by C. tropicalis is a complex of carbohydrate and lipid when vegetable oil is used as the carbon source. Similar results were obtained in the studies of Kappell and Fiechter (1976), using hydrocarbons as the carbon source. Further elucidation of the physical, chemical and functional characteristics of the biosurfactant produced would facilitate the development of the useful forms of this bioemulsifier to meet future needs. Moreover, this study opens up a new way of exploiting our oil resources in an economic way.
PHB is an important biopolymer produced as an intracellular storage compound in many microorganisms. PHB is a biodegradable, biocompatible thermoplastic and its physical properties are similar to those of polypropylene (Shahhosseini, 2004). Generally, bacteria are known to accumulate PHB, but only a few studies have been reported in yeasts (Anderson and Dawes, 1990; Wang and Bakken, 1998; Verlinden et al., 2007; Leaf et al., 1996; Safak et al., 2002). Among them, C. tropicalis is not well documented as a PHB producer. We have employed all the possible staining techniques to show its ability to produce PHB.
As we described, the use of cheaper substrates (such as vegetable oil) to lower the initial raw material costs for the production of various biomolecules from C. tropicalis would provide a significant impact on the current bio-industry with a thrust for eco-friendly molecules.
This study physically elucidates the potential of C. tropicalis BPU1 to produce biosurfactant and biopolymer in a simple mineral salt medium, using vegetable oil as the sole carbon source. Exploitation of eukaryotes, especially unicellular microbes such as yeast, for the production of industrially significant molecules is of great significance, possibly with simple genetic manipulations to enhance the production to meet future needs.
The authors gratefully acknowledge the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, for a research grant (No. BT/PR-12714/FNS/20/411/2009).