Yarrowia lipolytica and pollutants: Interactions and applications.

JBA-06811; No of Pages 14 Biotechnology Advances xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

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Yarrowia lipolytica and pollutants: Interactions and applications

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Smita Zinjarde ⁎, Mugdha Apte, Pallavi Mohite, Ameeta Ravi Kumar

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Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411 007, India

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Article history: Received 24 December 2013 Received in revised form 21 February 2014 Accepted 18 April 2014 Available online xxxx

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Keywords: Yarrowia lipolytica Natural occurrence Hydrocarbons Oil degradation Surfactants Emulsifiers Biosorption Nanoparticles Waste degradation Value-added products

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Yarrowia lipolytica is a dimorphic, non-pathogenic, ascomycetous yeast species with distinctive physiological features and biochemical characteristics that are significant in environment-related matters. Strains naturally present in soils, sea water, sediments and waste waters have inherent abilities to degrade hydrocarbons such as alkanes (short and medium chain) and aromatic compounds (biphenyl and dibenzofuran). With the application of slow release fertilizers, design of immobilization techniques and development of microbial consortia, scale-up studies and in situ applications have been possible. In general, hydrocarbon uptake in this yeast is mediated by attachment to large droplets (via hydrophobic cell surfaces) or is aided by surfactants and emulsifiers. Subsequently, the internalized hydrocarbons are degraded by relevant enzymes innately present in the yeast. Some wild-type or recombinant strains also detoxify nitroaromatic (2,4,6-trinitrotoluene), halogenated (chlorinated and brominated hydrocarbons) and organophosphate (methyl parathion) compounds. The yeast can tolerate some metals and detoxify them via different biomolecules. The biomass (unmodified, in combination with sludge, magneticallymodified and in the biofilm form) has been employed in the biosorption of hexavalent chromium ions from aqueous solutions. Yeast cells have also been applied in protocols related to nanoparticle synthesis. The treatment of oily and solid wastes with this yeast reduces chemical oxygen demand or value-added products (single cell oil, single cell protein, surfactants, organic acids and polyalcohols) are obtained. On account of all these features, the microorganism has established a place for itself and is of considerable value in environment-related applications. © 2014 Published by Elsevier Inc.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Occurrence of different strains in nature and their role therein . . . . . . . . . . . . Isolation of strains from soil . . . . . . . . . . . . . . . . . . . . . . Marine strains of Y. lipolytica . . . . . . . . . . . . . . . . . . . . . . Y. lipolytica from sediment samples and waste waters . . . . . . . . . . . Strategies used to enhance oil degradation abilities of Y. lipolytica . . . . . Scale-up studies and use of mixed cultures . . . . . . . . . . . . . . . In situ remediation by Y. lipolytica, tracking of isolates and other applications Surface interactions with hydrocarbons for their effective utilization . . . . . . . . . . Attachment to large droplets . . . . . . . . . . . . . . . . . . . . . . Production of surfactants and emulsifiers by Y. lipolytica . . . . . . . . . . Morphological changes during growth on hydrophobic substrates . . . . . Degradation of nitro, halogenated and organophosphate compounds . . . . . . . . . . Detoxification and reduction of metals . . . . . . . . . . . . . . . . . . . . . . . Metal tolerance in Y. lipolytica due to inherent properties . . . . . . . . . Use of Y. lipolytica biomass as a biosorbent . . . . . . . . . . . . . . . . Y. lipolytica cells as factories for nanoparticle synthesis . . . . . . . . . . Treatment of wastewaters by strains of Y. lipolytica . . . . . . . . . . . . . . . . . . Degradation of oily wastes . . . . . . . . . . . . . . . . . . . . . . . Modification of solid wastes . . . . . . . . . . . . . . . . . . . . . . Use of wastes as alternative substrates for obtaining value-added products .

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⁎ Corresponding author. Tel.: +91 20 25601385; fax: +91 20 25690087. E-mail address: [email protected] (S. Zinjarde).

http://dx.doi.org/10.1016/j.biotechadv.2014.04.008 0734-9750/© 2014 Published by Elsevier Inc.

Please cite this article as: Zinjarde S, et al, Yarrowia lipolytica and pollutants: Interactions and applications, Biotechnol Adv (2014), http:// dx.doi.org/10.1016/j.biotechadv.2014.04.008

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Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Occurrence of different strains in nature and their role therein

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Y. lipolytica strains are indigenous to contaminated environments where they may mediate natural detoxification of pollutants. Since such strains are regularly exposed to toxic compounds, they have developed the essential enzymatic make-up for their degradation. In particular, the hydrocarbon degradative abilities of Y. lipolytica strains from different sources have been evaluated by several investigators and are summarized in Table 1. They have been isolated from soils, marine environments, sediments and effluents.

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Isolation of strains from soil

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A psychrotrophic strain of Y. lipolytica was isolated from an alpine soil sample. This cold-adapted strain grew best between 15 °C to 20 °C although it survived from 0 °C to 30 °C. This strain degraded 68% of the provided diesel oil in ten days (Margesin and Schinner, 1997a). This psychrotrophic strain (Y. lipolytica RM7/11) was further evaluated for its ability to degrade pure hydrocarbons at low temperatures (Margesin et al., 2003). RM7/11 effectively degraded 40% of the provided n-hexadecane and 35% of n-dodecane within 8 days at 10 °C. Better degradation (50% and 73%, respectively) in a shorter period of time (5 days) was observed at 15 °C. Another strain (Y. lipolytica 180) was

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Yarrowia lipolytica (earlier referred to as Candida lipolytica) is an ascomycetous dimorphic yeast species that has been subjected to exhaustive studies. The organism exhibits unusual physiological, metabolic and genomic features that differentiate it from other yeasts. On account of these characters, several research groups have been projecting it as a model system for basic and applied research (Barth and Gaillardin, 1997; Nicaud, 2012). To investigate fundamental biological aspects, this system has been used to study (i) dimorphism (Dominguez et al., 2000) (ii) the mitochondrial complex (Kerscher et al., 2002) (iii) peroxisomes (van der Klei and Veenhuis, 2006) (iv) lipid accumulation (Beopoulos et al., 2009a) and (v) lipase production (Fickers et al., 2011). With regard to applications, the following aspects have been considered (i) proposed as a model system for biotransformation of hydroxy fatty acids (Nicaud et al., 1998) (ii) a popular tool for the heterologous expression of proteins (Beckerich et al., 1998; Madzak et al., 2004) (iii) manufacturing of organic acids (Finogenova et al., 2005) (iv) production of bio-oil (Beopoulos et al., 2009b) (v) for several industrial and environmental applications (Bankar et al., 2009a; Coelho et al., 2010) (vi) for cell surface display techniques related to bio-refinery issues (Tanaka et al., 2012) and (vii) food-related uses (Zinjarde, 2014). The “safe status” of the organism offers an additional advantage (Groenewald et al., 2014). The yeast is usually encountered in environments that contain hydrophobic substrates such as oily-wastes, foods (dairy and poultry products) and oil-contaminated sites. This feature is due to the inherent presence of several multi-gene families that mediate the efficient degradation of triglycerides and hydrocarbons (Fickers et al., 2005, 2011; Thevenieau et al., 2010). The production of extracellular enzymes such as proteases, lipases, esterases, phosphatases, and RNases also favor growth of this yeast under different conditions. Emulsifiers, surfactants and organic acids are other relevant substances that the organism produces. On the basis of these features, the yeast is able to interact with pollutants such as hydrocarbons (nitro, halogenated and organophosphates), metals and different types of wastes. In addition, strains of this yeast display tolerance towards physical parameters such as low temperatures, presence of salt, acidic and alkaline conditions that may be significant in different remedial applications. For example, its psychrotolerant nature is important in such processes involving cold

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soils and sea water. The salt tolerant property may be advantageous when the yeast is used for marine remedial purposes. Tolerance towards pH (acid and alkaline) would be relevant in handling shock loads during waste treatment procedures. The physiological and õbiochemical properties exhibited by this yeast that are relevant in the degradation of different pollutants are summarized in Fig. 1. From the foregoing discussion it is evident that there are published articles on different basic and applied aspects of this yeast. However, an exclusive review on how this non-conventional yeast interacts with pollutants of different kinds is lacking. This article attempts to provide a recent update on the topic and also discusses its role in developing remedial applications. The literature on this topic has been broadly categorized as follows (i) occurrence of different strains in nature and their role therein (ii) surface interactions with hydrocarbons (iii) degradation of nitro, halogenated and organophosphate compounds (iv) detoxification and reduction of metals and (v) treatment of waste waters by strains of Y. lipolytica.

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Fig. 1. Summary of the important attributes of Yarrowia lipolytica for interaction with different pollutants.


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Table 1 Hydrocarbon degradation abilities of Y. lipolytica strains isolated from different environments.

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RM7/11 180 NCIM 3589 IMUFRJ 50682 PG-20 LPS 605 – Y103

Alpine soil Oil-contaminated industrial area soil Dockyard sea water Estuarine water Sediment/seawater Refinery sediment Oil-polluted sediment Industrial waste water

Psychrotrophic; degraded diesel oil and n-alkanes (C12 and C16) Degraded aliphatic fraction of crude oil; reduced surface tension Degraded n-alkanes (C10–C18); aliphatic fraction of crude oil Assimilated n-alkanes (C11–C19); isoprenoids; aromatics Degraded crude oil; n-alkanes (C9–C16); produced emulsifiers Hydroxylated and degraded biphenyl Degraded dibenzofuran Hydroxylated and degraded phenol, 4-chlorophenol

Margesin and Schinner (1997a), Margesin et al. (2003) Kim et al. (1999) Zinjarde et al. (1998), Zinjarde and Pant (2002a) Ferreira et al. (2009) Hassanshahian et al. (2012) Romero et al. (2001) Romero et al. (2002) Lee et al. (2001)

Y. lipolytica from sediment samples and waste waters

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Some aromatic hydrocarbon-degrading strains of Y. lipolytica have also been reported. Biphenyl (BP) degrading yeasts (ten isolates) including one strain of Y. lipolytica (LPS 605) have been isolated from refinery sediment samples (Romero et al., 2001). This isolate was capable of degrading and hydroxylating BP. Two products (4-hydroxy biphenyl and 3,4-dihydroxy biphenyl) were observed within 24 h. On further incubation, 4-phenyl-2 pyrone-6 carboxylic acid was also obtained as a result of ring cleavage. The role of cytochrome P450 in mediating this aromatic hydroxylation has been implicated. In another study,

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Strategies used to enhance oil degradation abilities of Y. lipolytica

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In aquatic bodies due to the dilution effect, microorganisms may not be able to encounter pollutants and effectively degrade them. Two main strategies have been employed to address this issue. Firstly, slow release fertilizers (SRF) or oleophilic supplements (that inherently associate at the oil–water interfaces) have been employed. Secondly, hydrocarbondegrading microorganisms have been immobilized on appropriate surfaces. In the event of extensive hydrocarbon pollution, carbon is in excess and nitrogen and phosphorus are limiting. To overcome this imbalance, there is a need to externally add oleophilic fertilizers. Such additives get associated with oil layers and promote the development of oil-degrading microbial populations. In one such study carried on a laboratory scale, it was observed that paraffinized magnesium ammonium phosphate enhanced oil degradation by Y. lipolytica NCIM 3589 (Zinjarde and Pant, 2002a). With the addition of this oleophilic fertilizer, oil degradation was improved by 10–15%. Y. lipolytica 180-mediated biodegradation of oil in sand was also evaluated (Oh et al., 2000a). Biometer flasks with sand were homogeneously contaminated with Arabian light crude oil (0.5%, w/w). The effect of supplementation of Y. lipolytica strain 180 with a slow release fertilizer, SRF (Chobi Co. Ltd. Korea), oleophilic fertilizer (Inipol EAP 22, Elf Aquitaine) or water-soluble nutrients was studied. Among the tested forms of nutrients, Inipol EAP 22 was the best in enhancing the CO2 production rate. In a further attempt to simulate natural conditions, Oh et al. (2001) have conducted a microcosm study to determine the effect of SRF (83.3 mg g−1 urea–nitrogen, and 26.4 mg phosphate–phosphorous g−1 of the fertilizer with silica and latex added as support material) on nutrient availability and oil biodegradation by a consortium (Yarrowia lipolytica 180, Pseudomonas sp. K12-5 and Moraxella sp. K12-7). High (microcosm I) and low (microcosm II) doses of the fertilizer were applied and results were recorded. During the early phase (0 to 21 days), the rates of aliphatic and aromatic hydrocarbon degradation were significantly higher in the former microcosm than in the latter. After 91 days, the biodegradation efficiencies of aliphatic,

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Sea water, particularly near dockyards is often contaminated with pollutants such as hydrocarbons. Routine shipping operations, oil transport in tankers, and ballast water account for chronic pollution. Mishaps at off-shore oil platforms and oil tanker accidents contribute towards acute oil pollution. Oil-degrading tropical marine microorganisms were isolated from a contaminated dockyard water sample near Mumbai, India (Zinjarde and Pant, 2002a). Although several bacterial and yeast cultures were obtained, as a result of the enrichment procedure, one yeast isolate identified as Y. lipolytica and designated as strain NCIM 3589 was most effective. This strain mainly acted on the aliphatic fraction of the Bombay High crude oil and within 5 days at 30 °C with shaking at 200 rpm, 78% of this fraction was degraded. NCIM 3589 also utilized short and middle chain (C10, C12, C14, C16 and C18) alkanes within 24 h (Zinjarde et al., 1998). Another tropical strain (Y. lipolytica IMUFRJ 50682) was obtained from estuarine water in Rio de Janeiro, Brazil (Ferreira et al., 2009). The strain assimilated n-alkanes (C11–C19), isoprenoids (pristane and phytane), and aromatic compounds (naphthalene, phenanthrene and their derivatives). Complex hydrocarbons such as tricyclic terpanes were also used. This strain was projected as a potential candidate for use in bioremediation processes. Crude oil degrading yeasts were isolated from sediment and sea water samples that were collected from the Persian Gulf in Iran (Hassanshahian et al., 2012). Among the six yeasts that were obtained, two strains (PG-20 and PG-32) grew well on crude oil. These were identified as Y. lipolytica on the basis of biochemical tests and 18S rRNA gene sequence analysis. Y. lipolytica PG-20 under optimal conditions degraded 80% for the provided crude oil. This strain also grew on short and middle chain n-alkanes (C9–C16) effectively. The yeast was not able to grow well on aromatic hydrocarbons such as naphtalene, phenanterne or pyrene. Y. lipolytica PG-20 also displayed good cell surface hydrophobicity and produced emulsifiers. The frequent isolation of Y. lipolytica strains from the marine and estuarine environments may be a reflection of their inherent salt tolerant properties.

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Marine strains of Y. lipolytica

Y. lipolytica was isolated when indigenous populations from heavily oil-polluted sediments were analyzed for their dibenzofuran degrading abilities (Romero et al., 2002). The strain preferentially hydroxylated this aromatic hydrocarbon at the third position and 3-hydroxy dibenzofuran was the major product. The hydroxylation at the second or fourth position was to a lesser extent and a ring cleavage intermediate was also identified. Hydroxylation is a major primary step in the degradation of some pollutants and Y. lipolytica strains are efficient in this ability. A strain (Y. lipolytica Y103) isolated from industrial waste waters was capable of degrading some water soluble aromatic compounds such as phenol and 4-chlorophenol (Lee et al., 2001). These compounds were initially hydroxylated to catechol and a subsequent meta-cleavage resulted in the formation of 2-hydroxymuconic semialdehyde. From the foregoing discussion, it is evident that Y. lipolytica strains (sometimes with other yeasts) may be major players in the degradation of hydrocarbon pollutants.

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obtained from an oil-contaminated industrial area soil sample (Kim et al., 1999). This strain degraded 94% of the aliphatic fraction of Arabian light crude oil (supplied at 0.2% v/v) in minimal media within 3 days at 25 °C. It reduced the surface tension of the medium and the cells also displayed inherent surface hydrophobicity.

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In situ remediation by Y. lipolytica, tracking of isolates and other applications

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There are also a few attempts on the field application of this yeast. For example, a psychrotrophic strain (RM7/11) was evaluated for its ability to remediate polluted alpine soils. It was noticed that bioaugmentation with this strain was less effective in remediating the oilcontaminated soils. On the other hand, biostimulation of indigenous soil microorganisms by inorganic fertilizers was more effective (Margesin and Schinner, 1997b). Unlike the abovementioned strain that was ineffective, a strain (A-101) was successful in soil bioremediation

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The investigations related to the degradation of hydrocarbons by Y. lipolytica are generally done at the laboratory-scale. However, a scale-up study on the use of this yeast in alkane degradation has also been attempted. Schmitz et al. (2000) have set up sandy oil microcosms to simulate natural conditions and study relative competitiveness among microorganisms with respect to alkane degradation. During this investigation, the two yeasts, Y. lipolytica and Candida maltosa were found to play a dominant role. These yeasts degraded about 96% of the n-tetradecane that was supplied. They co-existed in nearly constant and equal proportions under the experimental conditions. This study also highlighted the significance of such yeasts in colonizing habitats such as deserts, sandy shores or sand fillings surrounding oil tanks. The yeast in combination with other bacteria has also been effective in enhancing oil degradation. In this regard, mixtures of Y. lipolytica and Pseudomonas sp. or strain EH 59 and Bacillus subtilis have been competent (Horakova and Nemec, 2000; Kaczorek et al., 2008). Kim et al. (2000) have also observed that the emulsifiers produced by the yeast enhanced oil degradation potentials of Moraxella sp. The yeast thus appears to be a potential candidate for the development of effective consortia for bioremediation purposes.

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Scale-up studies and use of mixed cultures

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under “in situ” conditions (Żogała et al., 2005). Petroleum-contaminated soils at a fuel base were seeded with the yeast. The progress of bioremediation was monitored by different techniques (electromagnetic, resistivity, and culture-based methods). The electrical conductivity of soils in the vicinity of was significantly increased. This indicated the potential of Y. lipolytica in bioremediation of petrol-contaminated soils. The authors have concluded that the detoxification was due to some distinctive features of the environmental microsystem that was being developed. Y. lipolytica contributed positively towards this by (i) aiding the development of plant rhizosphere-associated flora and (ii) promoting growth of hydrocarbon-degrading bacterial populations. Environmental applications involving microorganisms are often associated with a need for monitoring them at later stages. Some strains of Y. lipolytica have been genetically modified to assist their detection in nature after release experiments. For example, the β-galactosidase gene has been cloned in Y. lipolytica NCIM 3589 with such an intention (Iyer and Deobagkar, 1998). Collecting samples after treatment procedures and exposing them to X-gal would help in determining the fate of such organisms. Other markers that have been introduced are green fluorescent protein (GFP) and hemolysin (Yue et al., 2008). The presence of GFP or detection of hemolytic activity on blood agar plates would help in differentiating these strains from the wild-type ones. Restriction fragment length polymorphism (RFLP) analysis has also been used to monitor the distribution of inoculated oil-degrading microorganisms including Y. lipolytica (180) during field studies (Young et al., 2005). Tracking would be possible by detecting the presence of signature patterns. The yeast has also been used for the development of a biosensor for monitoring middle chain alkanes. In general, alkanes are detected by using high-end liquid chromatography or gas chromatography–mass spectrometry techniques. These methods are not effective in rapid ‘onsite’ or ‘large area monitoring’ studies. In an attempt to overcome these drawbacks, a biosensor based on a psychrotrophic strain of Y. lipolytica has been developed (Alkasrawi et al., 1999). Cells of the yeast (immobilized on glass beads through polyethylenimine) were packed in a jacketed glass column. For on-line monitoring studies, the rate of oxygen utilization (during the alkane assimilation process) was co-related to the alkane concentrations. The device showed a linear relationship up to 100 mM at 20 °C and the minimum concentration detected was 3 mM. The authors have implicated the use of this sensor in monitoring the success of bioremediation processes in cold climates and for the in situ analysis of ground water samples. This report thus signifies the relevance of Y. lipolytica in developing a promising on-site application. The hydrophobic nature of the yeast has been exploited for the immobilization of Y. lipolytica into patterned films. A protocol for making hydrophobic patterns of a lipid (octadecylamine) has been described (Gole et al., 2002). On these patterns, yeast cells could be effectively immobilized. The authors have suggested that such patterned cells could find potential applications in tissue engineering and enzymebased biotransformations. Although most of the reports on Y. lipolytica are related to the ‘degradation of alkanes’, recently, a recombinant strain has been used to ‘synthesize’ n-pentane (Blazeck et al., 2013). The soybean lipoxygenase protein was expressed in the yeast and it cleaved linoleic acid to pentane and tridecadienoic acid. The initial yield (1.56 mg l−1) of pentane could be improved in a three-fold manner (4.98 mg l−1) by increasing substrate availability and by improving the overexpression of the lipoxygenase. Further work on the metabolic engineering of Y. lipolytica for the production of short-chain n-alkanes could lead to the development of a technology that could provide a possible alternative to petroleum products.

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aromatic and asphaltic hydrocarbons in microcosm I were 92.1 ± 8.5, 80.4 ± 1.7 and 55.1 ± 2.9%, respectively. It was thus concluded that nutrient amendment in higher doses accelerated the initial oil degradation rates and shortened the treatment period involved in the clean-up of contaminated environments. Another approach used to enhance the hydrocarbon-degrading capabilities of microorganisms involves their immobilization on different surfaces. In this regard, agar–alginate composite beads were used for immobilization of Y. lipolytica NCIM 3589. The alginate was selectively leached out yielding a matrix with higher porosity that was suitable for the degradation of hydrocarbons. Immobilized cells of NCIM 3589 showed a twofold advantage (i) they degraded the aliphatic fraction of crude oil more effectively and (ii) continuous use was possible (Zinjarde and Pant, 2000). In another study, Y. lipolytica 180 immobilized on polyurethane (PU) foam has been employed for degrading oil films from surface waters (Oh et al., 2000b). Different methods such as direct attachment, freezedrying, prior immobilization on chitin and effect of slow release fertilizer (SRF) were tested. Chitin immobilized cells incorporated into PU foam were particularly efficient. The foam mediated the adsorption and the cells brought about oil degradation. Increasing foam porosity resulted in enhanced oil absorbance and the maximum absorbency obtained was 7–9 g g− 1 of foam. The yeast degraded 50% of the aliphatic fraction and 20–25% of the aromatic fraction of the Arabian light oil that was used. Thus, a variety of strains isolated from oil-polluted environments have the necessary metabolic capabilities for degrading different hydrocarbons. Such strains may be playing a major role in the detoxification of different pollutants even under natural conditions. Moreover, adding slow release oleophilic fertilizers or by immobilizing cells this ability may be further enhanced.

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Surface interactions with hydrocarbons for their effective utilization 372 As stated earlier, Y. lipolytica uses hydrophobic substrates (triglycer- 373 ides and hydrocarbons) effectively since it possess the metabolic 374



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Attachment to large droplets

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Some strains of Y. lipolytica adhere to large droplets of hydrophobic substrates such as n-alkanes (Zinjarde et al., 1998), methyl ricinoleate (Aguedo et al., 2003), oils (Krzyczkowska, 2012; Zinjarde et al., 2008), and brominated alkanes (Vatsal et al., 2009). Fig. 2 shows the microscopic observations of a strain of Y. lipolytica (NICM 3589) adhering to n-hexadecane. Initially, large droplets of the alkanes are observed. The yeast cells attach to such droplets towards the periphery (Fig. 2a and b). On longer incubation periods under aerobic conditions, the alkane droplets decrease in size and the yeast cells tend to cover the droplets (Fig. 2c and d). There are some reports indicating that the surface of this yeast is hydrophobic in nature. For example, strain 180 was found to be inherently hydrophobic (Kim et al., 2000). When yeast cells are subjected to the MATH (microbial adhesion to hydrocarbon) assay, they are rapidly partitioned to the organic phase. Such observations on the effective adherence of the yeast cells to non-polar solvents have also been made with strain IMUFRJ50682 (Amaral et al.,

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This yeast also produces surfactants and emulsifiers when growing on hydrophobic substrates. About thirty years ago, a report on the production of an emulsifier by C. lipolytica ATCC 8662 in the presence of n-hexadecane was published (Cirigliano and Carman, 1984). Maximum activity was observed after 130 h of incubation. The emulsifier was active in the acidic range and was heat stable (up to 70 °C). The emulsifier named liposan was principally made up of carbohydrate and protein (Cirigliano and Carman, 1985). Later, there has been a report on the tropical marine strain (NCIM 3589) producing an emulsifier in the presence of alkanes. Alkane uptake in this organism

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Production of surfactants and emulsifiers by Y. lipolytica

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2006a). These authors have studied the cell surface characteristics of this strain of Y. lipolytica by different methods and have concluded that certain cell wall proteins contribute towards the hydrophobic nature of the cells. Vatsal et al. (2011) have shown that strain NCIM 3589 displayed high microbial adhesion to solvents. Moreover, cells grown on glucose were hydrophilic and those grown on 1-bromodecane had hydrophobic cell surfaces. There is a report on the characterization of the hydrophobic binding proteins in Y. lipolytica strain H222 cultivated on n-alkanes. Some of these proteins were thought to be responsible for the formation of protrusions (channel-like connections between the cell wall and the interior) that were probably involved in transport of hydrocarbons (Lasserre et al., 2010). The protein profiles of cells grown on n-alkanes were separated and identified by two dimensional blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis and nanoliquid chromatography coupled to tandem mass spectrometry techniques, respectively. The data revealed the presence of forty protein complexes (11 heteromultimeric and 29 homomultimeric) that could interact with alkanes. Thus it is evident that strains of Y. lipolytica seem to be adapting to the presence of hydrocarbons by modifying cell surfaces.

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pathways required for their breakdown. The chemistry behind the degradation and the enzymes involved in the process have been extensively reviewed (Fickers et al., 2005, 2011; Fukuda, 2013) and will not be considered here. Emphasis will be given to the surface interactions of Y. lipolytica with hydrocarbons since they are considered to be pollutants. An understanding of such interactions would be of significance in designing strategies for scale-up studies and in situ applications. Since hydrocarbons are sparingly soluble in water, microorganisms need to employ specialized mechanisms to contact them and mediate their uptake. In general, there are two ways by which cells seek contact with hydrocarbons (i) attachment to large droplets (via hydrophobic cells surfaces) and (ii) production of surface active agents and emulsifiers.

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Fig. 2. Growth of Yarrowia lipolytica on n-hexadecane stained with STYO 9. Single arrow points to droplet margin and double arrows towards the yeast cells.


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Morphological changes during growth on hydrophobic substrates

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The growth of the organism on different substrates has also been associated with specific morphological features. The fungus is dimorphic in nature and exists in the yeast or the mycelial forms. The organism shows a differential dimorphic behavior in response to the presence of carbon sources and oxygen contents. Some carbon sources such as N-Acetyl glucosamine, glycerol and erythritol favor the mycelial form (Dusane et al., 2008). This form is also observed in the presence of pollutants such as triglycerides containing saturated fatty acids (Zinjarde et al., 2008). With other hydrophobic pollutants such as alkanes, the yeast morphology is predominant. When the mycelial forms of this organism were inoculated into alkane-containing media, a speedy transition to the yeast form was observed (Zinjarde et al., 1998). This rapid change indicated the suitability of the yeast form in alkane degradation. Oxygen is an important parameter that governs the morphological transition. It is well known that the synthesis of economically important products such as citric acid is associated with the aerobic growth of this organism (Rywińska et al., 2012). In a manner similar to Kluyveromyces marxianus, Y. lipolytica exists in the yeast from in the presence of oxygen and in the mycelial from in its absence (Bellou et al., 2014; O'Shea and Walsh, 2000). Mycelial forms are important in scavenging oxygen and enabling the organism to tide over unfavorable anaerobic conditions (Zinjarde et al., 1998). In addition, Y. lipolytica is known to exist in the biofilm mode on different hydrophilic and hydrophobic substrates (Dusane et al., 2008). This biofilm-forming ability may be important under field conditions and during the treatment of different pollutants. It is well-known that microorganisms often exist as biofilms during bioremediation applications and in waste treatment procedures where they have inherent advantages over their planktonic counterparts. Cells in biofilm mode are sheltered within the extracellular polymeric substance and have a better chance of adaptation and endurance during stress periods (Singh et al., 2006). An understanding of the morphological features of this organism in the presence of hydrocarbons is significant in applications of the yeast for remedial purposes. The selection of strains exclusively growing in the yeast form or those developing extensive biofilms may be advantageous in field applications.

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Degradation of nitro, halogenated and organophosphate compounds

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Some wild type strains of Y. lipolytica interact with and degrade nitroaromatic pollutants such as 2,4,6-trinitrotoluene (TNT) and halogenated (chlorinated and brominated) hydrocarbons. In addition, Y. lipolytica has been genetically modified to detoxify an organophosphate insecticide (methyl parathion). The range of such compounds acted upon by the yeast is depicted in Fig. 3.

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Explosives such as TNT are a major cause of concern with respect to the pollution of soils, surface water, and groundwater (Khan et al., 2013). There are four reports on the detoxification of TNT by strains of Y. lipolytica. In general, it is observed that the transformation of this explosive occurs via two pathways (i) nitro group reduction and (ii) direct aromatic ring reduction. The tropical marine isolate NCIM 3589 could not use TNT as a source of carbon or nitrogen (Jain et al., 2004). However in a complete medium containing glucose and ammonium sulfate, the culture transformed TNT. When the major pathway (reduction of nitro groups) was ensued, amino derivatives were accumulated in the medium. However, inclusion of glucose (as an external reductant) shunted the pathway to the ring reduction and denitration mode. As an outcome of this shunt, relatively non-toxic products such as 2,4-dinitrotoluene and 2-nitrotoluene were produced which in turn could be metabolized by other microorganisms. The role of inherent reductases in the biotransformation process was suggested. There is another report on strain AN-L15 bringing about TNT transformation (Ziganshin et al., 2007). Direct aromatic ring reduction was the main way by which transformation occurred in this strain. Nitro group reduction resulting in hydroxylamino or amino derivatives was observed albeit, to a lesser extent. The ring reduction step yielded eight distinct mono and dihydride complexes of TNT. In a later study, Ziganshin et al. (2010) have shown that this strain was acid-tolerant and produced organic acids. TNT transformation in the yeast was dependent on its ability to acidify the culture medium. In media with lower buffering capacities and with lesser aeration, growth of the culture was associated with rapid acidification of the medium. This influenced the rate and extent of TNT transformation and under such conditions, nitrate was the major product. Since iron is one of the most abundant elements and iron-containing minerals are likely to influence the transformation of nitroaromatic compounds, further studies on this strain were carried out (Khilyas et al., 2013). The authors have investigated the effect of ferrihydrite on TNT transformation. Although the inclusion of ferrihydrite decreased the rate of TNT biotransformation, metabolites such as hydride-Meisenheimer complexes, hydroxylaminoand amino-dinitrotoluenes were observed. The flux towards ring reduction and subsequent cleavage (rather than accumulation of potentially toxic nitro group reduction products) in the presence of ferrihydrite was considered a environmentally favorable feature. The ability of Y. lipolytica (then referred to as Candida lipolytica) in assimilating halogenated compounds was recognized about thirty years ago (Murphy and Perry, 1984). This yeast and two filamentous fungi (Cunninghamella elegans and Penicillium zonatum) assimilated chlorinated alkanes (1-chlorohexadecane or 1-chlorooctadecane) as sole carbon and energy sources. After growth on these substrates,

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Fig. 3. The range of nitro, halogenated and organophosphate compounds degraded by Y. lipolytica.

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was mediated by attachment to large droplets (Fig. 2). A lipidcarbohydrate–protein complex was present in association with the cell wall. This increased the cell surface hydrophobicity during the log phase. The extracellular synthesis of this emulsifier was observed in the stationary phase when nitrogen was limiting and carbon was in excess. Other parameters required for extracellular synthesis were an initial pH of 8.0 and sodium chloride at a concentration of 2 to 3%. Both the emulsifiers (cell-associated and extracellular) had similar properties (Zinjarde and Pant, 2002b; Zinjarde et al., 1997). Thereafter, a variety of emulsifiers have been reported from different strains. For example strain IA 1055 produced a polysaccharide–protein–lipid complex, IMUFRJ 50682 yielded Yansan, and UCP 0988 formed Rufisan (Amaral et al., 2006b; Sarubbo et al., 1999, 2007). Such information on the mode of attachment and uptake of hydrocarbons by different strains would play a role in designing strategies for their effective degradation.

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Metal tolerance in Y. lipolytica due to inherent properties

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Y. lipolytica has the natural capacity to tolerate metal ions and has in-built mechanisms for detoxifying them. In general, copper in high

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Heavy metals such as cobalt, nickel, chromium, mercury, cadmium and lead are toxic to living forms. Contamination of soil and water with metal species can directly or indirectly affect human health, cause economic losses and alter species diversity (Machado et al., 2010; Soares et al., 2003). Several microorganisms tolerate metal ions and respond to them in different ways. Strains of Y. lipolytica also interact with a variety of metal ions and mediate differential responses as discussed in the following sections and as depicted in Fig. 4. In general, studies on such interactions can be categorized into the following headings (i) metal tolerance of Y. lipolytica due to inherent properties (ii) use of Y. lipolytica biomass as biosorbents (iii) Y. lipolytica cells as factories for nanoparticle synthesis.

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Detoxification and reduction of metals

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concentrations is known to affect growth by inhibiting enzymes (Samarelli and Campbell, 1983). However, some strains of Y. lipolytica are resistant to this metal and the underlying mechanism has been investigated (García et al., 2002). The presence of the gene “YlCRF1” that encodes for a copper responsive transcription factor was demonstrated in this organism. Although this factor was transported into the nucleus after interaction with copper, its role in enhancing the production of metal binding proteins such as metallothioneins or superoxide dismutase (SOD) could not be established. The authors have therefore suggested the possibility of an unidentified novel role of this transcription factor in metal detoxification. In a later study, Ito et al. (2007) have shown the involvement of melanin and SOD in copper tolerance. Y. lipolytica cells retained viability even in the presence of 6 mM of copper (although the lag phase was prolonged). Moreover, the concentration of copper in the cytosol was found to be low indicating the presence of an inherent metal efflux system that protected the cells. The brown pigment, melanin, also played a significant role in the binding and accumulation of copper. Though there was no increase in the melanin content in response to increasing copper concentrations in the medium, the metal was localized in the periplasm where melanin was present. It prevented the copper from entering the cytosol and thus protected the cells. The cell wall also acted as a major physical barrier for the entry of the metal ions into the cells. Besides the above mechanisms, the levels of SOD (an enzyme involved in protecting cells against damage from toxic reactive oxygen species generated by metal ions) were also found to be higher. The impact of four metals on the growth of Y. lipolytica (strain CCM 451) has been studied (Strouhal et al., 2003). While zinc and cobalt were classified as essential, nickel and cadmium were toxic. The sites of metal-interaction were the cell wall and cell membrane debris. When the concentration of Cd was increased, an increase in the level of metallothioneins (heat stable, cysteine-rich proteins binding to heavy metals) was observed. This response was not noted with increasing concentrations of the essential metals. This investigation highlighted the importance of metallothioneins in the detoxification of toxic heavy metals in Y. lipolytica. During a study on the effect of aluminum on the growth of Y. lipolytica, it was observed that the lag and log phases of growth were not altered in the presence of 0.5 to 1.0 mM aluminum potassium sulfate although the cells failed to form filaments (Lobao et al., 2007). Additionally, the pH was found to be lowered (probably due to the action of an effective H+ ATPase system). This was postulated to be reason for the tolerance of the cells towards the metal and also for the inhibition of mycelium formation. Golubev and Golubev (2002) have studied selenium tolerance in yeasts belonging to ascomycetes and

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approximately 50% of the cellular fatty acids were found to be in the chlorinated form. There is also a more recent report on the growth of the tropical marine strain of Y. lipolytica (NCIM 3589) on bromoalkanes (Vatsal et al., 2011). The strain was capable of growing aerobically on 2-bromopropane, 1-bromobutane, 1,5-dibromopentane and 1bromodecane. With 1-bromodecane (that was best utilized) growth rate was maximal (0.055 h− 1 ). Unlike the earlier report (Murphy and Perry, 1984) wherein the fatty acids were found to be chlorinated, the growth of the tropical marine strain on brominated substrates was associated with bromide release. An increase in cell size and surface hydrophobicity along with debromination were the strategies employed by this strain. Organophosphate pesticides such as methyl parathion are also major pollutants that have hazardous effects (Jaga and Dharmani, 2006). A recombinant strain of Y. lipolytica harboring the ‘mph gene’ from Pseudomonas sp. WBC-3 (encoding for methyl parathion hydrolase) was generated (Wang et al., 2012). A transformant (Z51) produced 59.5 U of enzyme activity per mg of dry cell weight. Under optimal conditions (pH: 9.5 and temperature: 40 °C), 90.8% of methyl parathion was hydrolyzed within half an hour. The organism is routinely isolated from polluted dockyards and sediments and laboratory studies have shown that it tolerates high contents of crude oil (Zinjarde and Pant, 2000). On account of such exposures to large quantities of crude oil, some isolates may have evolved methods of detoxifying nitro, halogenated and phosphate containing compounds as well.

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Fig. 4. Summary of the interactions of Yarrowia lipolytica with metal pollutants.


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Y. lipolytica cells as factories for nanoparticle synthesis

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Biosorption has been described as non-directed physicochemical interaction between the sorbent (biological matter) and the sorbate such as metal ions until equilibrium is achieved (Shumate and Stranberg, 1985). Yeasts are effective biosorbents on account of their efficient metal biosorption capacities and specificity (Bankar et al., 2012a). The biomass of Y. lipolytica has been employed as a valuable biosorbent. This capability was applied in the biosorption of hexavalent chromium [Cr (VI)] ions (Bankar et al., 2009b). Two Y. lipolytica strains (NCIM 3589 and 3590) were used for achieving this. The influence of different factors such as pH, temperature, biomass, metal salt content and contact time were checked. By optimizing conditions (pH 1.0, 35 °C with agitation at 130 rpm) equilibrium was reached within 2 h. In order to increase the biosorption capacities of Y. lipolytica biomass, three strategies have been employed (i) use in combination with sludge (ii) magnetic modification of cells and (iii) use of cells in the biofilm form. Combinations of Candida lipolytica (1.0 g l− 1) and dewatered sewage sludge (3.0 g l− 1) have been evaluated for the removal of Cr (VI) ions from electroplating waste waters (Ye et al., 2010). The mixtures reduced more than 96% of Cr (VI) within a pH range of 1.0 to 2.0. In addition, the mixture was more efficient in adapting to changes in pH when compared to C. lipolytica or the sewage sludge alone. The removal followed the second-order kinetics and the mixture also adsorbed other metal ions such as copper and zinc. Fourier transform infrared spectral analysis suggested the role of hydroxyl and secondary amide groups in the biosorption process. In a recent study, cells of Y. lipolytica, NCIM 3589 and NCIM 3590 have been magnetically-modified and evaluated for the removal of the Cr (VI) ions. The magnetic modification was mediated by phytoinspired Fe0/Fe3O4 nanoparticles (Rao et al., 2013). Fe0/Fe3O4 nanoparticles were selected on the basis of an earlier report on their effectiveness in the removal of Cr (VI) ions (Wu et al., 2009a). The optimum conditions for the removal of Cr (VI) ions by the magnetically modified cells were pH 2.0 and agitation at 100 rpm. Equilibrium with such cells was reached within 1 h. The specific uptake values observed with the modified cells were three times more than the unmodified ones. The nanocomposites were able to detoxify Cr (VI) ions to the less toxic Cr (III) ions. The role of specific functional groups on the yeast cells and the Fenton chemistry associated with the Fe0/Fe3O4 nanostructures was thought to be responsible for the more efficient removal of Cr (VI) ions. As described earlier, Y. lipolytica is known to form biofilms when grown on different substrates (Dusane et al., 2008). In general, biofilms are more effective in removing heavy metals from waste waters than their planktonic counterparts (Quintelas et al., 2008). Two strains of Y. lipolytica (NCIM 3589 and 3590) formed biofilms in the presence of Cr (VI) ions (Bankar et al., 2012b). Although biofilm formation was initially retarded, with longer incubation times, such growth patterns were observed. When compared to planktonic cells, biofilms were more effective in removing Cr (VI) ions. The specific uptake by both the strains was almost five times more than the free cells. Nickel is a divalent metal species that displays toxicity to biological forms. Since Y. lipolytica was effective in removing Cr (VI) ions, its role

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Use of Y. lipolytica biomass as a biosorbent

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in removing divalent Ni ions was also evaluated (Shinde et al., 2012). In this study, both the marine strains (NCIM 3589 and 3590) displayed maximum uptake of 95.33 mg−1 and 85.44 mg−1, respectively under optimum conditions (pH 7.5, agitation rate of 200 rpm) and equilibrium was established within 2 h. Another study has shown the effectiveness of Y. lipolytica NCIM 3589 in bioleaching metals from fly ash, a by-product of thermal power plants (Bankar et al., 2012c). Fly ash is known to contain metals such as Al, Fe, Cd, Cr, Mn, As, Cu, Zn and Mo that pose environmental hazards. When Y. lipolytica cells were incubated with fly ash, different metals were leached. In addition, metal ions were also taken up by the cells by a process of bioaccumulation. Both these factors contributed to the leaching of the metals from the fly ash. The accumulation was confirmed by transmission electron microscope analysis. Zinc was accumulated to the maximum extent. This was followed by Ni, Cu, Cr, Al and Si. The cells also effectively formed biofilms on the surface of fly ash. The role of citric acid, extracellular proteins and bioaccumulation by cells in the bioleaching process was implicated. From the above studies it can be concluded that Y. lipolytica biomass is an effective biosorbent. It must be understood that large quantities of yeast biomass is generated during the waste treatment procedures. This biomass could be used for the removal of heavy metals from waste waters and in the bioleaching of metals from solid wastes such as fly ash.

From the foregoing discussion it is evident that Y. lipolytica tolerates metal ions. In order to overcome the toxicity of metal ions, microorganisms often sequester them by reductive processes (Pawar et al., 2012). Strains of Y. lipolytica isolated from polluted areas containing toxic and hazardous metals have been employed for this purpose. Most of the work has been focused on a tropical marine isolate of Y. lipolytica (NCIM 3589). This strain has been obtained from oil-polluted sea water and tolerates high contents of different pollutants including TNT and metals. It produces reductases and other enzymes that may play a role in nanoparticle formation. On account of these features, the isolate was evaluated for the synthesis of nanoparticles. During an initial study, gold nanoparticles were synthesized when cells were incubated with chloroauric acid at 30 °C for 72 h (Agnihotri et al., 2009). The presence of gold nanoparticles (associated with the cell wall) was confirmed by transmission electron microscopy. Both the forms of this dimorphic fungus synthesized the nanostructures. The synthesis depended on the pH of the reaction mixtures. At low pH (2.0), large triangular and hexagonal plates were observed and at neutral or alkaline pH the size was around 15 nm. Extending the studies further, the same strain was used for custom-designing gold nanoparticles with respect to size (Pimprikar et al., 2009). The biomass content (109, 1010 or 1011 cells ml−1) and gold salt concentrations (0.5 to 5.0 mM) were varied. With increasing cell numbers and constant concentration of the gold salt, the particle size was found to decrease. On the other hand, with increasing concentration of the gold salt and the same number of cells, the particle size increased. These cell-associated nanostructures were easily dislodged into the medium by incubation at low temperature (20 °C). In order to identify the biomolecules involved in the nanoparticle synthetic process, further studies were carried out. It is known that Y. lipolytica produces melanin, the dark pigment that plays an important role in metal resistance (Ito et al., 2007). It must be noted that Y. lipolytica produces two types of melanins, pyomelanin or eumelanin (Carreira et al., 2001a, 2001b). Since the structure of melanin includes a large number of phenolic groups, it was postulated to be involved in the synthesis of nanoparticles. Melanin is known to be associated with cells and can be extracted by standard protocols. In addition, cells may be induced to over-produce the pigment by incubating resting cells with precursors such as L-tyrosine or L-DOPA. In a recent report, both cell associated (extracted melanins) as well as L-DOPA-induced melanin

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basiodiomycetes groups. Amongst the ascomycetes, 19 strains of Y. lipolytica could tolerate sodium selenate (up to 10− 1 M). The detoxification was attributed to the presence of an agent that brought about the reduction of Se ions to their elemental form (evident from the appearance of pink colored colonies in presence of high quantities of sodium selenate in the medium). From the foregoing discussion it is evident that Y. lipolytica tolerates and detoxifies metals by (i) sequestration at the level of the cell wall and cell membrane (ii) flushing out via efflux systems (iii) production of metal binding biomolecules such as melanin and cysteine-rich metallothioneins and (iv) enhanced production of enzymes such as SOD and H+ATPase as depicted in Fig. 4.

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Different strains of Y. lipolytica have been used for the treatment of a 795 variety of wastes waters. The wastes that have been effectively treated 796 can be categorized as oily wastes and solid wastes. 797

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Treatment of wastewaters by strains of Y. lipolytica

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incubating cells of NCIM 3590 with L-DOPA (Apte et al., 2013c). Successful synthesis of gold and silver nanostructures was possible with the induced melanin (characterized to be pyomelanin). The silver nanoparticles thus produced were effective in controlling the growth of a wall-disfigurement-causing fungus. From the above discussion it is evident that both the strains of Y. lipolytica (NCIM 3589 and 3590) investigated with respect to nanoparticle synthesis were of marine origin. The dynamic conditions in the sea often force microorganisms to rapidly adapt to such changes and produce bioactive molecules. It is thus evident that strains of Y. lipolytica interact with a variety of metals (Fig. 4). The role of different biomolecules in detoxification, sequestration in different cellular compartments, use as effective biosorbents and their ability to reduce metal ions into nanostructures are some aspects that are significant.

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obtained from NCIM 3589 mediated the reduction of chloroauric acid to elemental nanostructures (Apte et al., 2013a). These structures were characterized and used as effective antibiofilm agents. There is also a report on the synthesis of cadmium oxide and cadmium sulfide nanostructures by Y. lipolytica NCIM 3589 in the extracellular and cellassociated manner (Pawar et al., 2010). There appears to be a strain-dependent response with respect to gold nanoparticle synthesis. A psychrotrophic marine strain (NCYC 789 or NCIM 3590) also synthesized gold nanoparticles in a cellassociated manner (Nair et al., 2013). A variety of nanostructures were observed when the cell numbers and salt contents in the reaction mixtures were varied as indicated by the visual observations depicted in Fig. 5a. The scanning electron microscope images of Y. lipolytica NCIM 3590 not exposed to gold salt and cells with gold nanostructures are shown in Fig. 5b, c, d and e. Spherical (white arrows), triangular and hexagonal (black arrows) structures were obtained. The melanin associated with cells was isolated and shown to be involved in nanoparticle synthesis. This strain also produced silver nanoparticles in a cellassociated mode (Apte et al., 2013b). Melanin extracted from cells reduced silver ions into nanostructures. Since the quantity of cellassociated melanin is often less, its production was improved by

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Fig. 5. Synthesis of gold nanoparticles by Yarrowia lipolytica NCIM 3590. (a) Visual observations of cells exposed to chloroauric acid for 120 h [Row 1: 109; Row 2: 1010; Row 3: 1011 cells ml−1; Tubes 1 to 7: Cell control, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mM HAuCl4, respectively]. Scanning electron micrographs of (b) control cells (c) and (d) cells with gold nanoparticles on the surface magnified 10,000 times (e) magnified 20,000 times. White arrows point to spherical nanostructures and black arrows indicate hexagonal and triangular nanostructures.



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Y. lipolytica has been employed for treating and modifying some solid wastes as well. This yeast has been effective in up-grading fishmeal. When this material has higher lipid contents it is regarded to be of low-grade. To decrease the lipid content in fish waste, minced fish were subjected to solid-state fermentation (Yano et al., 2008). Among all the microorganisms that were evaluated, Y. lipolytica MBRC-10073 was most competent. After 96 h, with intermittent mixing, the lipids were reduced by 46%. The quality of fishmeal derived from fish waste could thus be improved after treatment with Y. lipolytica. Another solid waste (crude coconut fat) was subjected to solid-state fermentation with Y. lipolytica (RO13). The effect of water activity and time on hydrolysis was determined (Parfene et al., 2013). As a result of fermentation, the hydrolysates contained high contents (70%) of lauric acid. The fatty acid containing hydrolysates displayed antimicrobial activity against some food-borne pathogens. Y. lipolytica could thus upgrade the coconut fat hydrolysates to a product with preservative effects. There are a few reports on the utilization and modification of animal fat by Y. lipolytica. For example, when strain (ACA-DC 50109) was cultivated on stearin (an industrial derivative of animal fat containing mixtures of saturated free fatty acids) along with glycerol, lipidenriched biomass and citric acid were obtained (Papanikolaou et al., 2003). The single-cell oil thus produced [enriched in stearic acid (50–70%, wt/wt) with lower contents of palmitic (15–20%, wt/wt), oleic (7–20%, wt/wt), and linoleic (2–7%, wt/wt) acid] had a composition that mimicked cocoa-butter. Later on, these authors have employed a modeling approach to determine the kinetic behavior of the strain on stearin and hydrolyzed oleic rapeseed oil (Papanikolaou and Aggelis, 2003a). The biomass and cellular lipid evolution were successfully simulated and the results were experimentally validated. The study revealed that the specific rate of fat-free biomass formation was

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Modification of solid wastes

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In this category, olive mill wastewater (OMW) and palm oil mill effluents (POME) have been treated. Wastewaters from oil processing units typically have high chemical oxygen demand, COD (De Felice et al., 1997). Y. lipolytica ATCC 20255 has been applied for the treatment of OMW (De Felice et al., 1997; Scioli and Vollaro, 1997). This strain brought about 80% reductions in the COD within 24 h. Lanciotti et al. (2005) have evaluated the ability of different Y. lipolytica strains to grow in undiluted OMW and subsequently reduce the COD levels. One strain (PO1) reduced the COD by 41.22%. In another investigation, wild-type strain of Y. lipolytica (W29) was effective in treating OMW with initial COD loads of 19 g l−1 (Lopes et al., 2008). The strain brought about a reduction in COD content by 80% and the total phenol content by 70%. Gonçalves et al. (2009) have evaluated some strains of Y. lipolytica for their ability to grow on un-diluted OMW. The abovementioned strain (W29) reduced the COD (by 29–37%). The same strain (W29) was immobilized in calcium alginate and used for the degradation of oil waste waters (Wu et al., 2009b). The immobilized cells were effective in degrading 2000 mg l− 1 of oil and reducing 2000 mg l−1 COD after 50 h. These beads could be stored and used effectively several times. Palm oil mill effluents have also been successfully treated by Y. lipolytica. Strain NCIM 3589 resulted in 95% decrease in COD values within two days (Oswal et al., 2002). Y. lipolytica Polg has been genetically manipulated to display the lipRS gene (from Rhizopus stolonifer) on the cell surface by using the flocculation functional domain of Saccharomyces cerevisiae (Flo1p, encoded by FLO) as the protein anchor. This recombinant strain was effective in treating oily waste water (Song et al., 2011). In open activated sludge bioreactors with the recombinant strain after 72 h of treatment, 96.9% of oil and 97.6% of COD were removed. The main effect of the treatment of oily wastes was thus a considerable reduction in COD values.

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unaffected by the substrate fatty acid composition. However, the concentration of lipid inside the yeast cell and the accumulation rate of cellular lipids were maximal when stearic acid contents were higher in the media. The strain also showed a preferential utilization of specific fatty acids when mixtures of these acids were used as substrates (Papanikolaou and Aggelis, 2003b). The yeast removed C12:0, C14:0, Δ9C18:1, Δ9, 12C18:2 and Δ9, 12, 15C18:3 at remarkably greater rates than C16:0 and C18:0. The residual substrate completely lacked C12:0, C14:0 and Δ9, 12, 15C18:3 fatty acids. Other fatty acids (Δ9C18:1 and Δ9, 12C18:2) were decreased by 55-80%. The contents of C18:0 on the other hand, increased (2.1 to 3.5 fold) in the residual substrate. This fatty acid (C18:0) was also selectively accumulated in the storage lipid. The significance of this yeast in the production of tailor-made lipids and new fats with predetermined compositions has been reviewed extensively (Papanikolaou and Aggelis, 2010). The authors have further studied the dynamics of biomass, reserve lipid, and citric acid production on stearin along with glucose (Papanikolaou et al., 2006). The kinetic behavior was simulated by using numerical models and the optimized parameters were validated by experimental procedures. In nitrogen-limited cultures containing glucose as a sole carbon source, satisfactory growth and optimal citric acid production (final concentration 42.9 g l− 1, yield 0.56 g g− 1 of sugar consumed) were observed. When stearin and glucose were used as co-substrates, the cellular lipid contents increased (0.3 to 2.0 g l−1). On the basis of these and other reports, the significance of this promising renewable substrate in the production of Y. lipolytica-mediated lipid, single-cell protein and lipases has been discussed in a review article (Papanikolaou et al., 2007). Some solid agro-wastes have also been treated with Y. lipolytica and some value-added products have been obtained. For example, Imandi et al. (2008) have used strain NCIM 3589 for treating pineapple wastes. After applying statistical experimental designs (Plackett–Burman) the production of citric acid was optimized in solid-state fermentation. When the parameters were standardized, the yield of citric acid obtained was 202 g kg− 1 of dried pineapple waste. Some of these solid agro-wastes have been hydrolyzed and used for obtaining oilenriched biomass (SCO). When Y. lipolytica Po1g was grown on detoxified sugarcane bagasse hydrolysate along with peptone, 11.42 g l−1 of biomass was obtained (Tsigie et al., 2011). In another report from the same laboratory, defatted rice bran hydrolysates were used (Tsigie et al., 2012). The dry biomass and lipid contents of the yeast grown on this hydrolysate under optimum conditions were 10.75 g l− 1 and 48.02%, respectively.

D

Degradation of oily wastes

T

798

E

10

861 862 863 864 865 866 Q8 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903

Use of wastes as alternative substrates for obtaining value-added products 904 Large volumes of wastes are generated by food processing, agricultural management and chemical industries all over the world. Since these wastes are organic in nature, they can be assimilated by microorganisms and a variety of products can be obtained. There is a two-fold advantage in employing this strategy (i) the waste is effectively disposed and (ii) a value-added product is obtained. Some such wastes have been used (as alternative cost-effective substrates) for cultivating strains of Y. lipolytica and obtaining products such as biosurfactants, emulsifiers, enzymes, organic acids and oil-enriched biomass. In this regard, milk factory wastewaters have been used for the production of biosurfactants (Yilmaz et al., 2009). Pretreated (pH 4.5; autoclaved) whey wastewaters were inoculated with Y. lipolytica (MFW5). After 96 h of incubation, the cell-free supernatants were evaluated for surfactant activity by the drop-collapse method, haemolysis, emulsification assay and surface tension reduction. Biochemical analysis and FTIR spectra confirmed the lipopeptide nature of the biosurfactant. This study highlighted the use of whey as an inexpensive substrate for the production of biosurfactants. Strain UCP 0988 has been cultivated on a variety of alternative inexpensive substrates to obtain surfactants as value-added


905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924


Pollutant interaction/process/application

Reference

RM7/11

Degradation of diesel oil In situ remediation Development of middle chain alkane sensor Degradation of n-alkanes (C12 and C16) Degradation of aliphatic fraction of crude oil Contaminated sand studies, effect of SRF Increased degradation after immobilization Microcosm and mixed culture studies Monitoring released microorganisms Isolation of emulsifier Degradation of n-alkanes Monitoring released isolates Degradation of crude oil by immobilized cells Degradation of aliphatic fraction of crude oil Patterned assembly onto hydrophobic films Production of emulsifier Treatment of palm oil mill effluent Transformation of 2,4,6-Trinitro toluene Formation of Biofilms Morphology on hydrophobic substrates Treatment of pineapple wastes Removal of Cr (VI) ions Synthesis of gold nanoparticles Custom designing gold nanoparticles Growth on bromoalkanes Synthesis of cadmium nanostructures Removal of Ni (II) ions Growth on waste oil and use as single cell oil Bioleaching of metals from fly ash Melanin mediated synthesis of nanostructures Cr (VI) removal by magnetized cells Production of emulsifier Yansan Assimilation of n-alkanes, isoprenoids and aromatic compounds Production of emulsifier on agroindustrial residue CCAJ Production of surfactant on vegetable oil refinery residue Production of emulsifier on soybean oil refinery residues Use of surfactant in metal removal Production of biosurfactant on animal fat and corn steep liquor

Margesin and Schinner (1997a) Margesin and Schinner (1997b) Alkasrawi et al. (1999) Margesin et al. (2003) Kim et al. (1999) Oh et al. (2000a) Oh et al. (2000b) Oh et al. (2001) Young et al. (2005) Zinjarde et al. (1997) Zinjarde et al. (1998) Iyer and Deobagkar (1998) Zinjarde and Pant (2000) Zinjarde and Pant (2002a) Gole et al. (2002) Zinjarde and Pant (2002b) Oswal et al. (2002) Jain et al. (2004) Dusane et al. (2008) Zinjarde et al. (2008) Imandi et al. (2008) Bankar et al. (2009b) Agnihotri et al. (2009) Pimprikar et al. (2009) Vatsal et al. (2011) Pawar et al. (2012) Shinde et al. (2012) Katre et al. (2012) Bankar et al. (2012a, 2012b, 2012c) Apte et al. (2013a) Rao et al. (2013) Amaral et al. (2006b) Ferreira et al. (2009) Fontes et al. (2012) Rufino et al. (2007) Rufino et al. (2008) Rufino et al. (2011) Santos et al. (2013)

NCIM 3589

Q3 t2:32

930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953

E

T

C

E

R

928 929

products. For example, in one process, vegetable oil refinery residue was used as an unconventional substrate (Rufino et al., 2007). The cell-free supernatants displayed biosurfactant and emulsification activities. The emulsification activity was not affected by salt although incubation at high temperatures decreased the activity. The concentration of the surfactant was 4.5 g l − 1, and it reduced the surface tension of water from 71 to 32 mN m − 1 . Chemically, the biosurfactant was a protein–lipid–carbohydrate complex (50, 20 and 8%, respectively) with possible applications in oil-recovery. This strain (UCP 0988) has also been cultivated on soybean oil refinery residues (SORR) to obtain the emulsifier (Rufino et al., 2008). Factorial design experiments were used to optimize conditions for maximum activity. The surfactant was stable in a wide range of pH (2 to 12), temperatures (0 to 120 °C) and salinity (2 to 10% NaCl). The use of this industrial waste thus allowed the development of an economically viable method for the production of the biosurfactant. The biosurfactant produced on SORR was subsequently used for detoxifying heavy metals and petroleum derivates in soil (Rufino et al., 2011). Around 96% of Zn and Cu were removed and the concentrations of Pb, Cd, and Fe were also lowered. The biosurfactant was also effective in removing waste oil. Another report discusses the production of biosurfactants by this strain on low-cost media based on animal fat and corn steep liquor with supplements (Santos et al., 2013). After 6 days of incubation, the surface tension was reduced from 50 to 28 mN m− 1. The surfactant (anionic glycolipid) could be effectively used in oil-recovery. The production of a biosurfactant by Y. lipolytica IMUFRJ 50682 on clarified cashew apple juice CCAJ, (an agroindustrial residue) and crude glycerol has also been reported (Fontes et al., 2012). Emulsification indices (68.0 and 70.2%) and maximum variations in surface tension (18.0 and

R

926 927

UCP 0988

N C O

925

IMUFRJ 50682

U

t2:33 t2:34 t2:35 t2:36 t2:37 t2:38 t2:39 t2:40

O

180

F

Strain

R O

t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 Q2 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28 t2:29 t2:30 t2:31

P

t2:3

Table 2 Summary of some selected strains of Y. lipolytica, their interactions with pollutants and applications.

D

t2:1 t2:2

11

22.0 mN m−1) were obtained with CCAJ and crude glycerol, respectively. The yield of the biosurfactant was 6.9 g l−1 with CCAJ and 7.9 g l−1 with crude glycerol. Citric acid is another product that has been obtained by cultivating Y. lipolytica on different wastes. Commercially, this is used as an acidity regulator or flavor enhancer and in the manufacture of pharmaceutical products (Vandenberghe et al., 2000). Although the citric acid production by Y. lipolytica on a variety of substrates has been documented for a very long time, in recent years, the use of alternative inexpensive substrates has become popular. Y. lipolytica ACA-DC 50109 when grown on diluted OMW enriched with additional glucose yielded 28.9 g l− 1 of citric acid (Papanikolaou et al., 2008a). The final citric acid concentration and conversion yields were greater when OMW was present. Raw glycerol is another waste product that is generated during the manufacture of biodiesel. This acts as a pollutant if not utilized appropriately. A few reports on the use of this substrate in the synthesis of value added products are discussed here. The production of citric acid by Y. lipolytica (ACA-DC 50109) and 1, 3-propanediol by Clostridium butyricum F2b from raw glycerol has been investigated by a modeling approach. While the kinetic behavior of citric acid production could be simulated by Monod-Verhlust and Williams models, the production of 1, 3-propanediol followed the Contois-type model (Papanikolaou and Aggelis, 2003c). In another report along with the production of the aforementioned two products, single cell oil (SCO) was also obtained when raw glycerol was volarized (Papanikolaou et al., 2008b). In a later report, specific morphological and biochemical features of the yeast in repeated batch cultures were also studied (Makri et al., 2010). The earlier reports on the use of raw glycerol for the synthesis of different products have been discussed extensively (Papanikolaou and


954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 Q9 976 977 978 979 980 981 982

1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048

This yeast with unique physiological traits and enzymatic features has been associated with a vast variety of pollutants. Several laboratories all over the world have been studying this yeast in relation to such interactions and associated applications. The frequent incidence of this yeast in contaminated soils, sea water and wastes implies that it may be playing a major role in detoxifying hydrocarbons even under natural conditions. With the use of slow release oleophilic fertilizers, immobilization techniques and in conjunction with other microorganisms, the degradative capabilities can be enhanced. The natural ability of the yeast to produce surfactants can also help in remedial procedures. Apart from the aliphatic and aromatic hydrocarbons that it effectively breaks down, wild-type and recombinant strains can also degrade nitroaromatic, halogenated and organophosphate compounds. The yeast has an inherent ability to detoxify metal ions and this has been exploited for developing bioremedial applications involving biosorption and for the synthesis of nanoparticles. The organism can act on different kinds of wastes including oily and solid wastes and bring about a reduction in COD or yield value-added products such as citric acid, surfactants, enzymes and single cell oil. Moreover, some strains have been exceptionally versatile with respect to their interactions with pollutants and may be projected as major players in the development of technologies in the future.

1058 1059

Acknowledgments

1080

O

F

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R O

1004 1005

Conclusion

P

1002 1003

1049 1050

D

1000 1001

and corn steep liquor. The biosurfactant thus obtained was used in the removal of heavy metals and petroleum derivates. There is thus considerable diversity among Y. lipolytica strains obtained from different locations. Each strain interacts with pollutants in a characteristic manner and in some instances, interesting applications have been designed. There appears to more scope for exploration of such strains from hitherto unexplored locales so that the range of pollutants addressed can be expanded further.

T

998 999

C

996 997

E

994 995

R

992 993

R

990 991

O

989

C

987 988

N

985 986

Aggelis, 2009). Furthermore, in a recent article, Rywińska et al. (2013) have reviewed the work on Y. lipolytica-mediated conversion of crude glycerol into value-added products such single cell protein (SCP), SCO, organic acids (citric, pyruvic and α-ketoglutaric acid) and sugar alcohols (erythritol, mannitol). The reader is referred to this review for studies on the use of crude glycerol for value-added products. The biomass of Y. lipolytica has also been employed as single cell protein since a long time. Recently, the abilities of some newly isolated strains for biomass production using glycerol wastes were evaluated (Juszczyk et al., 2013). One strain (S6) was most suitable for the purpose and yielded 11.7 and 12.3 g l−1 of biomass with 1.30 and 1.37 g l−1 h−1 productivity, respectively when pure and raw glycerol (25 g l−1) were used. The yeast protein amino acid profiles were also desirable with contents of lysine, threonine and phenylalanine/tyrosine being higher than those of the standard egg proteins. Y. lipolytica belongs to the group of oleaginous microorganisms that can accumulate large quantities of lipid in their biomass (Ageitos et al., 2011; Kosa and Ragauskas, 2011). The biomass of Y. lipolytica has been projected as SCO. Food waste and municipal waste water have been used for the production of microbial lipid (Chi et al., 2011). Different oleaginous yeasts including Y. lipolytica displayed comparable growth on hydrolyzed food waste and glucose (as control). These strains were also grown in municipal primary wastewater. In another report, Katre et al. (2012) have evaluated five strains of Y. lipolytica as potential SCO candidates. The tested strains accumulated more than 20% (w/w) of lipid in their dry biomass. Glucose and wastes (waste cooking oil or waste motor oil) were employed as carbon sources. One strain (NCIM 3589) was most efficient and its biomass was enriched in saturated and monounsaturated fatty acids in a manner similar to conventional vegetable oils. Some other inexpensive renewable material (palm oil mill effluent, serum latex and crude glycerol) were also evaluated for cultivating Y. lipolytica (Louhasakul and Cheirsilp, 2013). With crude glycerol and the effluent as co-substrates, the biomass obtained was 3.21 g l− 1 with the lipid content being 68% of the dry weight. With scale-up and process improvement, the biomass and lipid could be increased to 5.53 and 2.81 g l−1, respectively. Although these processes involving the growth of Y. lipolytica on wastes are effective in bringing about either a considerable reduction in COD or result in the synthesis of value-added products, they have been restricted to lab-scale levels. With the use of appropriate strains (wild type, mutants or recombinants), standardization of process parameters, scale-up and patenting of processes, Y. lipolytica could be a major player in the field of environmental biotechnology in the future. On the basis of the literature survey on the topic and the detailed discussion in the previous sections, it is evident that several strains of Y. lipolytica can interact with and subsequently detoxify different pollutants. From the above sections, it is also clear that some strains have been exhaustively studied in this regard. Their details are included in Table 2 and some highlights are presented here. After assessing the ability of the psychrotrophic strain (RM7/11) in degrading hydrocarbons (diesel and n-alkanes), it was evaluated for in situ applications and also for the development of middle chain alkane sensors. There are multiple reports on another strain of Y. lipolytica (180) that inherently is hydrophobic in nature and degrades aliphatic fraction of crude oil in shake flaks, contaminated sand and microcosm studies. The oil degradation by this strain could be increased by the addition of a slow release fertilizer (Inipol EAP 22) or after immobilization on polyurethane (PU) foam. Of particular interest is the tropical marine strain NCIM 3589. This is known to detoxify more than one kind of pollutants (crude oil, aliphatics, TNT, halogenated compounds, palm oil mill effluents, heavy and noble metals) and also produce value-added products such as citric acid, surfactants, single cell oil and nanoparticles. Strain IMUFRJ 50682 assimilated hydrocarbons (n-alkanes, isoprenoids and aromatic compounds) and produced the emulsifier ‘Yansan’ on different waste residues. UCP 0988 is another strain that has been exploited for the production of biosurfactant on vegetable oil refinery residues, animal fat

U

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1051 1052 1053 1054 1055 1056

1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079

All authors thank University Grants Commission for funding under 1081 UPE Phase II. PM thanks CSIR, India for Senior Research Fellowship. 1082 MA thanks University of Pune for research fellowship. 1083 References

1084

Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG. Oily yeasts as oleaginous cell factories. Appl Microbiol Biotechnol 2011;90:1219–27. Agnihotri M, Joshi S, Kumar AR, Zinjarde S, Kulkarni S. Biosynthesis of gold nanoparticles by the tropical marine yeast Yarrowia lipolytica NCIM 3589. Mater Lett 2009;63: 1231–4. Aguedo M, Wache´ Y, Mazoyer V, Sequeira-LeGrand A, Belin JM. Increased electron-donor and electron-acceptor characters enhance the adhesion between oil droplets and cells of Yarrowia lipolytica as evaluated by a new cytometric assay. J Agric Food Chem 2003;51:3007–11. Alkasrawi M, Nandakumar R, Margesin R, Schinner F, Mattiasson B. A microbial biosensor based on Yarrowia lipolytica for the off-line determination of middle chain alkanes. Biosens Bioelectron 1999;14:723–7. Amaral PFF, Lehocky M, Barros-Timmons AMV, Rocha-Leão MHM, Coelho MAZ, Coutinho JAP. Cell surface characterization of Yarrowia lipolytica IMUFRJ 50682. Yeast 2006a; 23:867–77. Amaral PF, da Silva JM, Lehocky M, Barros-Timmons AMV, Coelho MAZ, Marrucho IM, et al. Production and characterization of a bioemulsifier from Yarrowia lipolytica. Process Biochem 2006b;41:1894–8. Apte M, Girme G, Nair R, Bankar A, Kumar AR, Zinjarde S. Melanin mediated synthesis of gold nanoparticles by Yarrowia lipolytica. Mater Lett 2013a;95:149–52. Apte M, Gaikwad S, Sambre D, Joshi S, Bankar A, Kumar AR, et al. Psychrotrophic yeast Yarrowia lipolytica NCYC 789 mediates the synthesis of antimicrobial silver nanoparticles via cell-associated melanin. AMB Express 2013b;3:32. Apte M, Girme G, Nair R, Bankar A, Kumar AR, Zinjarde S. 3,4-dihydroxy-L-phenylalaninederived melanin from Yarrowia lipolytica mediates the synthesis of silver and gold nanostructures. J Nanobiotechnol 2013c;11:2. Bankar AV, Kumar AR, Zinjarde SS. Environmental and industrial applications of Yarrowia lipolytica. Appl Microbiol Biotechnol 2009a;84:847–65. Bankar AV, Kumar AR, Zinjarde SS. Removal of chromium (VI) ions from aqueous solution by adsorption onto two marine isolates of Yarrowia lipolytica. J Hazard Mater 2009b; 170:487–94.

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the production of citric acid from pineapple waste. Bioresour Technol 2008;99: 4445–50. Ito H, Inouche M, Tohoyama H, Joho M. Characteristics of copper tolerance in Yarrowia lipolytica. Biometals 2007;20:773–80. Iyer VR, Deobagkar DD. Expression of exogenously introduced β-galactosidase gene in a tropical, marine, oil-degrading strain of Yarrowia lipolytica NCIM 3589. Curr Sci 1998;75:385–6. Jaga K, Dharmani C. Methyl parathion: an organophosphate insecticide not quite forgotten. Rev Environ Health 2006;21:57–67. Jain MR, Zinjarde SS, Deobagkar DD, Deobagkar DN. 2,4,6-Trinitrotoluene transformation by a tropical marine yeast Yarrowia lipolytica NCIM 3589. Mar Pollut Bull 2004;49: 783–8. Juszczyk P, Tomaszewska L, Kita A, Rymowicz W. Biomass production by novel strains of Yarrowia lipolytica using raw glycerol, derived from biodiesel production. Bioresour Technol 2013;137:124–31. Kaczorek E, Chrzanowski Ł, Pijanowska A, Olszanowski A. Yeast and bacteria cell hydrophobicity and hydrocarbon biodegradation in the presence of natural surfactants: rhamnolipids and saponins. Bioresour Technol 2008;99:4285–91. Katre G, Joshi C, Khot M, Zinjarde S, Kumar AR. Evaluation of single cell oil (SCO) from a tropical marine yeast Yarrowia lipolytica 3589 as a potential feedstock for biodiesel. AMB Express 2012;2:36. Kerscher S, Dröse S, Zwicker K, Zickermann V, Brandt U. Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I. Biochim Biophys Acta 2002; 1555:83–91. Khan MI, Lee J, Park J. A toxicological review on potential microbial degradation intermediates of 2,4,6-trinitrotoluene, and its implications in bioremediation. J Civil Eng 2013;17:1223–31. Khilyas IV, Ziganshin AM, Pannier AJ, Gerlach R. Effect of ferrihydrite on 2,4,6trinitrotoluene biotransformation by an aerobic yeast. Biodegradation 2013;24: 631–44. Kim TH, Lee JH, Oh YS, Bae KS, Kim SJ. Identification and characterization of an oildegrading yeast, Yarrowia lipolytica 180. J Microbiol 1999;37:128–35. Kim TH, Oh YS, Kim SJ. The possible involvement of the cell surface in aliphatic hydrocarbon utilization by oil-degrading yeast, Yarrowia lipolytica 180. J Microbiol Biotechnol 2000;10:333–7. Kosa M, Ragauskas AJ. Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol 2011;29:53–61. Krzyczkowska J. The use of castor oil in the production of γ-decalactone by Yarrowia lipolytica KKP 379. Chem Technol 2012;3:58–61. Lanciotti R, Gianotti A, Baldi D, Angrisani R, Suzzi G, Mastrocola D, et al. Use of Yarrowia lipolytica strains for the treatment of olive mill wastewater. Bioresour Technol 2005;96:317–22. Lasserre JP, Nicaud JM, Pagot Y, Joubert-Caron R, Caron M, Hardouin J. First complexomic study of alkane-binding protein complexes in the yeast Yarrowia lipolytica. Talanta 2010;80:1576–85. Lee JS, Kang EJ, Kim MO, Lee DH, Bae KS, Kim CK. Identification of Yarrowia lipolytica Y103 and its degradability of phenol and 4-chlorophenol. J Microbiol Biotechnol 2001;11: 112–7. Lobao FA, Facanha AR, Okorokov LA, Dutra KR, Okorokova-Facanha AL. Aluminum impairs morphogenic transition and stimulates H+ transport mediated by the plasma membrane ATPase of Yarrowia lipolytica. FEMS Microbiol Lett 2007;274:17–23. Lopes M, Araújo C, Aguedo M, Gomes N, Gonçalves C, Teixeira JA, et al. The use of olive mill wastewater by wild type Yarrowia lipolytica strains: medium supplementation and surfactant presence effect. J Chem Technol Biotechnol 2008;84:533–7. Louhasakul Y, Cheirsilp B. Industrial waste utilization for low-cost production of raw material oil through microbial fermentation. Appl Biochem Biotechnol 2013;169: 110–22. Machado MD, Soares EV, Soares HM. Removal of heavy metals using a brewer's yeast strain of Saccharomyces cerevisiae: chemical speciation as a tool in the prediction and improving of treatment efficiency of real electroplating effluents. J Hazard Mater 2010;180:347–53. Madzak C, Gaillardin C, Beckerich JM. Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol 2004;109: 63–81. Makri A, Fakas S, Aggelis G. Metabolic activities of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures. Bioresour Technol 2010; 101:2351–8. Margesin R, Schinner F. Bioremediation of diesel-oil contaminated alpine soils at low temperatures. Appl Microbiol Biotechnol 1997a;47:462–8. Margesin R, Schinner F. Efficiency if indigenous and inoculated cold-adapted microorganisms for biodegradation of diesel oil in alpine soils. Appl Environ Microbiol 1997b;63: 2660–4. Margesin R, Gander S, Zacke G, Gounot AM, Schinner F. Hydrocarbon degradation and enzyme activities of cold-adapted bacteria and yeasts. Extremophiles 2003;7:451–8. Murphy GL, Perry JJ. Assimilation of chlorinated alkanes by hydrocarbon-utilizing fungi. J Bacteriol 1984;160:1171–4. Nair V, Sambre D, Joshi S, Bankar A, Kumar AR, Zinjarde S. Yeast-derived melanin mediated synthesis of gold nanoparticles. J Bionanosci 2013;7:1–10. Nicaud JM. Yarrowia lipolytica. Yeast 2012;29:409–18. Nicaud JM, Clainche AL, Le Dall MT, Wang H, Gaillardin C. Yarrowia lipolytica, a yeast model for the genetic studies of hydroxy fatty acids biotransformation into lactones. J Mol Catal B: Enzym 1998;5:175–81. Oh YS, Choi WY, Lee YH, Choi SC, Kim SJ. Biological treatment of oil-contaminated sand: comparison of oil degradation based on thin-layer chromatography/flame ionization detector and respirometric analysis. Biotechnol Lett 2000a;22:595–8.

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Int Microbiol 1998;1:123–30. Bellou S, Makri A, Triantaphyllidou IE, Papanikolaou S, Aggelis G. Morphological and metabolic shifts of Yarrowia lipolytica induced by the alteration of the dissolved oxygen concentration in the growth environment. Microbiology 2014. http://dx.doi. org/10.1099/mic.0.074302-0. Beopoulos A, Chardot T, Nicaud JM. Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation. Biochimie 2009a;91:692–6. Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM. Yarrowia lipolytica as a model for bio-oil production. Prog Lipid Res 2009b;48:375–87. Blazeck J, Liu L, Knight R, Alper HS. Heterologous production of pentane in the oleaginous yeast Yarrowia lipolytica. J Biotechnol 2013;165:184–94. Carreira A, Ferreira LM, Loureiro V. Brown pigments produced by Yarrowia lipolytica result from extracellular accumulation of homogentisic acid. Appl Environ Microbiol 2001a; 67:3463–8. 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Food-related applications of Yarrowia lipolytica.

Yarrowia lipolytica and its multiple applications in the biotechnological industry.

Fungemia caused by Yarrowia lipolytica.

Engineering Yarrowia lipolytica for Campesterol Overproduction.

A molecular genetic toolbox for Yarrowia lipolytica.

Development and physiological characterization of cellobiose-consuming Yarrowia lipolytica.

Activating and Elucidating Metabolism of Complex Sugars in Yarrowia lipolytica.

Engineering Yarrowia lipolytica to produce biodiesel from raw starch.

The safety of β-carotene from Yarrowia lipolytica.

Engineering of a high lipid producing Yarrowia lipolytica strain.

Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica.

Role of Pex11p in Lipid Homeostasis in Yarrowia lipolytica.

Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica.

Metabolic Flexibility of Yarrowia lipolytica Growing on Glycerol.

Physiological and biochemical responses of Yarrowia lipolytica to dehydration induced by air-drying and freezing.

A comparative study on glycerol metabolism to erythritol and citric acid in Yarrowia lipolytica yeast cells.

Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering.

Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function.

Reductase.

Biotransformation of acetophenone and its halogen derivatives by Yarrowia lipolytica strains.

Inference and interrogation of a coregulatory network in the context of lipid accumulation in Yarrowia lipolytica.

Isolation and characterisation of a double-stranded RNA virus-like particle from the yeast Yarrowia lipolytica.

Comparison of the phosphate-repressible and-constitutive acid phosphatases of Yarrowia lipolytica.

New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica.