Biodegradation of phenol via meta cleavage pathway triggers de novo TAG biosynthesis pathway in oleaginous yeast

Accepted Manuscript Title: Biodegradation of phenol via meta cleavage pathway triggers de novo TAG biosynthesis pathway in oleaginous yeast Authors: Alok Patel, Km Sartaj, Neha Arora, Vikas Pruthi, Parul A Pruthi PII: DOI: Reference:

S0304-3894(17)30512-5 http://dx.doi.org/doi:10.1016/j.jhazmat.2017.07.013 HAZMAT 18705

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

9-5-2017 4-7-2017 5-7-2017

Please cite this article as: Alok Patel, Km Sartaj, Neha Arora, Vikas Pruthi, Parul A Pruthi, Biodegradation of phenol via meta cleavage pathway triggers de novo TAG biosynthesis pathway in oleaginous yeast, Journal of Hazardous Materialshttp://dx.doi.org/10.1016/j.jhazmat.2017.07.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REVISED MANUSCRIPT (HAZMAT-D-17-02339) Biodegradation of phenol via meta cleavage pathway triggers de novo TAG biosynthesis pathway in oleaginous yeast Alok Patel, Km Sartaj, Neha Arora, Vikas Pruthi, Parul A Pruthi* Molecular Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee (IIT R), Roorkee, Uttarakhand, India- 247667. *Corresponding Author :Dr. (Mrs.) Parul Aggarwal Pruthi, Scientist, (Bio-Care Programme, DBT Govt. of India), Molecular Microbiology Laboratory, Biotechnology Department, Indian Institute of Technology Roorkee (IIT-R), Roorkee, Uttarakhand, India-247667. Phone: 091-1332-285530 (office), 091-1332-285110 (Resi.) Fax: 091-1332-273560 Mobile: 09760214585 email: [email protected] Graphical abstract

Highlights     

Oleaginous yeast of Rhodosporidium spps degrade phenol via meta-cleavage pathway Phenol biodegradation triggers Acetyl-CoA accumulation in the cytosol Products of phenol degradation feed as precursors for de novo TAG synthesis pathway Live Fluorescence images showed accumulated TAG as LD of 6.12 ± 0.78 µm Toxic phenol (1000 mg/l) as feedstock for producing ecofriendly Biodiesel

Abstract Phenol is reported to be one of the most toxic environmental pollutants present in the discharge of various industrial effluents causing a serious threat to the existing biome. Biodegradation of phenol by oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 was found to degrade 1000 mg/l phenol. The pathways for phenol degradation by both ortho and meta-cleavage were proposed by the identification of metabolites and enzymatic assays of ring cleavage enzymes in the cell extracts. Results suggest that this oleaginous yeast degrade phenol via meta-cleavage pathway and accumulates a high quantity of lipid content (64.92%; wt/wt) as compared to control glucose synthetic medium (GSM). Meta-cleavage pathway of phenol degradation leads to formation of pyruvate and acetaldehyde. Both these end products feed as precursors for de novo triacylglycerols (TAG) biosynthesis pathway which causes accumulation of TAG in the lipid droplets (LD) of 6.12 ± 0.78 µm grown on phenol while 2.38 ± 0.52 µm observed on GSM. This was confirmed by fluorescence microscopic images of BODIPY505-515nm stained live yeast cells. GC-MS analysis of extracted total lipid showed enhanced amount of monounsaturated fatty acid (MUFA) which was as 51.87%, 58.33% and 62.98% in presence of 0.5, 0.75 and 1g/l of phenol. Keywords: phenol degradation; oleaginous yeast; lipid accumulation; fatty acid methyl esters; Introduction Phenol is listed as a priority pollutant by the U.S. Environmental Protection Agency [1]. It is the most hazardous industrial effluent with lowest toxicity limits at 1 mg/l relatively set by the World Health Organization (WHO) to control the phenol concentration in drinking water [2]. Thus, the treatment of wastewater containing phenolic effluents is essential [3–5]. Yeasts are found to be the most important phenol biodegraders [6–8]. Yeast species such as Trichosporon spps. [9,10], Saccharomyces cerevisiae [8] and Candida spps. [11–13] were reported to degrade phenol up to 200,

800 and 1000 mg/l, respectively. Fungal species also degrade and utilize phenol as a sole carbon

source for their growth [14–16]. The aerobic biodegradation of aromatic compounds such as phenol involve three common steps (i) triggering of the aromatic ring, (ii) ring division, (iii) breakdown of the divided products to Krebs cycle intermediates [17,18]. The first step of metabolic cleavage of phenol follows the breakup of dehydroxylate, the benzene ring to form a catechol derivative which is catalyzed by phenol hydroxylase (PH) and followed by either ortho (intradiol) or meta (extradiol) fission of the aromatic ring [19]. Catechol is oxidized via ortho-cleavage pathway to cis, cis-muconic acid by catechol 1, 2-dioxygenase, or by meta- pathway to 2hydroxymuconic semialdehyde (2-HMS) by catechol 2, 3-dioxygenase [20,21]. 2-HMS can be further degraded either via the hydrolytic route or the 4-oxalocrotonate (4-OC) route [22]. It can possible that both ortho and meta routes will activate in the same microorganism depending on the provided substrates e.g. in the case of Pseudomonas cepacia, salicylate activates the ortho route while both routes were activated by benzoate [19,21–23]. These pathways depend not only on the substrates but also on its concentration. Pseudomonas putida grown in low of benzoate (200–300 mg/l) activates only the ortho-pathway while both degradation routes are activated at higher concentrations [21]. Yeast strains of the genera Aureobasidium, Rhodotorula and Trichosporon exhibited the activity of catechol 1,2-dioxygenase and phenol hydroxylase in free cell extracts from cells grown on phenol, suggesting that catechol was oxidized by the ortho type of ring fission [7,10]. Since, there is a lacuna in understanding the biodegradation of phenol in oleaginous yeast of Rhodosporidium spps. It is important to observe and identify the intermediates formed during their degradation. Oleaginous yeast can synthesize TAG as lipid droplets which may accounts for 70% of their biomass whereas non-oleaginous yeast such as Saccharomyces cerevisiae and Candida utilis cannot accumulate lipid content more than 10% of their biomass [24]. The

difference in fatty acid metabolism is the key for different neutral lipid storage mechanism in them [24,25]. They are known to utilize a large number of renewable substrates and low-cost materials such as agricultural wastes and industrial effluents for their growth and lipid accumulation [26– 30]. Previous studies demonstrated that glucose is preferred source but it doesn’t constrain the utilization of other sources in its presence as observed in the yeast Rhodosporidium kratochvilovae HIMPA1 [31]. They convert the excess carbon source present in the medium into fatty acids in the form of lipid droplets (LDs) that is mainly composed of triacylglycerols (TAG) [24]. The present investigation deals with the pioneering study to understand the mechanism of phenol degradation in this strain R. kratochvilovae HIMPA1 and proposing the hypothesis of utilization of its end products as precursors for de-novo TAG biosynthesis pathway for enhanced lipid accumulation. The data reveals that in this oleaginous yeast, phenol is degraded via meta-cleavage pathway. Meta-cleavage pathway of phenol degradation leads to pyruvate and acetaldehyde as end products [32]. Pyruvate can be metabolized by Krebs cycle without any modification while acetaldehyde dehydrogenase (AcDH) catalyzed the conversion of acetaldehyde to acetyl-CoA. Acetyl-CoA is the precursor for de novo TAG (Triacylglycerols) biosynthesis pathway also known as Kennedy Pathway. The utilization of de novo pathway which accumulates TAG in the LDs of this oleaginous yeast was confirmed by fluorescence images of BODIPY505-515nm stained live yeast cells by direct cell imaging technique using LED based EVOS FL microscope. The enhanced TAG was transesterified further to produce biodiesel. This study predicts the metabolic route of phenol degradation pathway leading to buildup of acetyl-CoA which is a central building block for fatty acid synthesis. The present study proposes that both the end products of the Meta-cleavage pathway of phenol degradation; pyruvate and acetaldehyde feed as precursors for de novo

triacylglycerols (TAG) biosynthesis pathway which causes higher accumulation of TAG in the lipid droplets (LD) of R. kratochvilovae HIMPA1. 2. Materials and methods 2.1 Chemicals and reagents All reagents, media, and solvents used in this study were of analytical grade. For growth and batch cultivation of oleaginous yeast, media such as glucose, ammonium sulfate, agar, phenol, yeast extract peptone dextrose (YPD), complete supplement mixture (CSM), yeast nitrogen base without ammonium sulfate (YNB), were obtained from Himedia Pvt. Ltd, India. Estimation of residual phenol and determination of phenol degradation pathway chemicals such as 4- amino antipyrine,

potassium

ferricyanide

K3Fe(CN)6,

ammonium

hydroxide,

catechol,

2-

mercaptoethanol and tris-HCl buffer were purchased from Merck, India. Organic solvents and acids such as n-hexane, chloroform, methanol, and H2SO4 for lipid extraction and transesterification were acquired from Rankem, India. Neutral lipid staining green fluorescence dye, boron dipyrromethene (BODIPY505/515) was obtained from Invitrogen (Life Technology, USA). 2.2 Microorganism and growth condition Oleaginous yeast R. kratochvilovae HIMPA1 used in this study was isolated from the permafrost soil samples collected from Tungnath Hill area in the Himalayan Garhwal ranges, Uttarakhand (India). Our earlier studies showed that this yeast can utilize the phenol-containing wastewater such as pulp and paper mill effluent [29]. The seed culture (0.063 OD600nm) was prepared by inoculating 50 ml of YPD broth with this strain and the culture was grown at 30 ºC on a rotary shaker at 180 rpm for 48 h.

2.3 Phenol degradation experiment Sterilization of phenol stock solution (5 g/100 ml distilled water) was done by 0.22 µm filter with the help of a syringe. For the biodegradation of phenol by oleaginous yeast, eleven set of flasks (500 ml) were prepared and labelled from A to K. The components of flask A were glucose (70 g/l) along with minimal medium containing ammonium sulphate (5 g/l), CSM; 1.7 g/l and YNB without ammonium sulphate (0.79 g/l) that were used as negative control for the phenol degradation experiments. Initial phenol concentrations added varies from 0.5 g/l to 1.5 g/l with an interval of 0.25 g/l from the flasks B to F, supplemented with the minimal medium. To determine the influence of the supplementary carbon source on phenol biodegradation by oleaginous yeast, glucose (7%) was added along with the above mentioned initial concentration of phenols and minimal medium in the flasks G to K. The cell density in the culture was detected at OD600. For the optimization of suitable initial cell densities, initially, all flasks were inoculated with three different cell densities (0.063, 0.126 and 0.189; OD at 600nm) of R. kratochvilovae HIMPA1 seed culture in sterile conditions. All the flasks (pH; 6.5) were kept at 30 ºC on a rotary shaker at 180 rpm for 144 h. Samples (1 ml) were withdrawn from the growing culture with every 12 h of intervals and OD were measured at 600nm. All the experiments were performed three times and results were averaged. 2.4 Estimation of residual phenol Estimation of residual phenol in the oleaginous yeast cultures were done by withdrawing the cell free supernatant (1 ml) at different time intervals. The concentrations of residual phenol in collected samples were estimated by using 4-aminoantipyrine colorimetric method [10]. The phenol concentration present in the supernatant depends on the production of red coloured antipyrine dye. Standard solutions (100 ml) containing 0.1 to 0.5 mg phenol were mixed with 2.5

ml of 0.5M NH4OH and pH of the solution was adjusted to 7.9. To this 4-aminoantipyrine (1.0 ml) and K3Fe(CN)6 (1.0 ml) solutions were added and mixed. After 15 min of incubation at room temperature, the absorbance was taken at 500 nm with UV-vis spectrophotometer (Lasany, LI2800, India). Similar procedure was also done for estimation of residual phenol in cells free supernatant samples and OD of samples was observed at 500 nm. 2.5 Enzymatic assay for the estimation of phenol degradation pathway The enzymatic assay were based on the activity of two enzymes, catechol 1, 2-dioxygenase and catechol 2, 3-dioxygenase. Biodegradation of phenol occurs mainly via ortho or meta-cleavage pathway in which these two enzymes play a major role [19]. The enzymatic assay performed to determine the biodegradation pathway in oleaginous yeast was carried out using cell-free extracts prepared by growing the cells at different concentrations of phenol (0.5 g/l to 1.5 g/l). After harvesting, the cells were subjected to sonication at 20 kHz for 5 min. This slurry was centrifuged at 10000 rpm for 5 min. The supernatant obtained (cell-free extract) was kept at 4 °C and assayed for catechol dioxygenase activity [10]. 2.5.1 Catechol 1, 2-Dioxygenase Activity The ortho pathway of phenol degradation was determined by adding 50 mM Tris-HCl buffer (2 ml at pH 8), 100 mM 2-mercaptoethanol (0.1 ml), cell-free extract (0.1 ml) and distilled water (0.7 ml) in a quartz cuvette (3 ml). After mixing by inversion, 1 mM catechol (0.1 ml) was added into the cuvette and absorbance was measured at 260 nm for 5 min. Formation of cis-cis muconic acid was depicted by an increase in absorbance with respect to time [33]. 2.5.2 Catechol 2, 3-Dioxygenase Activity The meta-pathway of phenol degradation was identified by the presence or absence of 2hydroxymuconic semialdehyde which is usually formed after meta-cleavage of catechol by

catechol 2, 3-dioxygenase enzyme activity. The cell-free extract (0.2 ml) was mixed with 2 ml Tris-HCl buffer (pH 7.5) and 0.6 ml distilled water. After gentle agitation, 100 mM catechol was mixed with the solution and formation of 2-hydroxymuconic semialdehyde was measured by an increase in absorbance at 375 nm with respect to time up to maximum 5 min [19]. 2.6 Determination of cell dry biomass (g/l), total lipid (g/l) and TAG accumulation in oleaginous yeast To determine the cell dry biomass, 50 ml culture was harvested by centrifugation and the pellets obtained were kept on preweighed filter paper. The filter paper along with biomass was dried in hot air oven at 60 °C for 24 h and weighed using an analytical balance (Citizen Scale India Private Limited, India). For the total lipid quantification, the harvested cell dry biomass was used for lipid extraction by modified method of Bligh and Dyer, described in the protocol of Patel et al., 2014 [27]. The live fluorescence microscopy for the examination of neutral lipid (TAG) accumulation in the LDs of the cellular compartments of R. kratochvilovae HIMPA1 was carried out by the protocol of Patel et al., 2014 using LED based EVOS FL microscope [27]. 2.7 Transesterification and analysis of fatty acid profile Lipid samples attained were transesterified by using the protocol of Chopra et al., 2016 [34]. Briefly, the extracted lipids were transferred in a round bottom flask and properly mixed with methanol (5:1, v/w; methanol: biomass) along with 2% H2SO4 as a catalyst. The reaction was carried out in a water bath at 65 °C for 4 h. A sample (4 µl) was aspirated at mid of reaction time (2 h) for monitoring the reaction process by TLC. After completion of this reaction, the FAME sample was then extracted from the polar methanol phase with hexane. The products of transesterification reaction were analyzed by GC-MS (Agilent, Santa Clara, CA, USA) using the

protocol of Patel et al., 2014 [27]. The empirical formulas were derived for calculation of biodiesel properties [26,28]. The analysis of triacylglycerol (TAG) conversion into fatty acid methyl ester (FAME) was carried out by TLC using 15% ethyl acetate in hexane as developing solvent [35]. 2.8 Statistical analysis The data values are means ± standard deviation of three independent recorded values. One- way analysis of variance (ANOVA) using Microsoft Office Excel 2013 (Microsoft, USA) with p<0.05 was used for data acceptance. 3. Results and discussion 3.1 Phenol degradation by oleaginous yeast R. kratochvilovae HIMPA1 Initially, optimization studies were carried out to determine minimal initial cell density (0.063, 0.126 and 0.189 at OD600nm) of R. kratochvilovae HIMPA1 which showed dose-dependent behavior to degrade different phenol concentration from 0.5 g/l to 1.5 g/l with an interval of 0.25 g/l. Time course experiments of cell growth in term of cell density and phenol degradation is presented in Fig. 1. Data suggested that the cell growth was severely inhibited when > 0.5g/l phenol concentration was provided to R. kratochvilovae HIMPA1 at an initial cell density of 0.063 at OD600nm (Fig. 1a). Maximum phenol degradation (62.2%) was observed after 96 h of incubation when 0.5 g/l phenol was provided to yeast cells at cell density of 0.063 at OD600nm (Fig. 1b). When the phenol concentration was raised from 0.5 g/l to 0.75 g/l at similar cell density (0.063 at OD600nm) no growth was observed (Fig. 1a and b). Interestingly, when these cells were supplemented with glucose (70 g/l), enhanced growth was observed (Fig. 1b). On doubling the cell density (0.126 at OD600nm), complete degradation of phenol was observed after 108 h of incubation (Fig. 1c), but the supplementation of glucose (70 g/l) to the phenol containing medium extended

the phenol degradation period to 144 h (Fig. 1d). At high phenol concentration (1 g/l), complete phenol degradation was observed after 36 h of incubation when the cell density was raised to 0.189 at OD600nm. On further increase in phenol concentration to 1.25 g/l, the cells were not able to tolerate this high concentration of phenol (supplementary data Fig. 1). Interestingly, this could be due to the toxic effect of phenol on cell viability at these concentrations (1.25 g/l and 1.50 g/l). It was observed that the period for phenol degradation increases with respect to increment in phenol concentration in the medium (Fig. 1e). Moreover, the yeast cells showed higher cell density after glucose supplementation in phenol containing medium. Data infers that the time required for complete phenol degradation was reduced at higher cell density of 0.189 at OD600nm. Wang et al., suggested that the extent of cell density and the time required for phenol degradation varied as a function of the initial phenol concentration in the medium [36]. Phenol degradation was found to be a function of strain, time of incubation and initial phenol concentration along with simultaneous utilization of glucose. Several microorganisms can degrade phenol but their maximum degradable concentration (200-2000 mg/l) varies from species to species. Varma and Gaikwad in their study on biodegradation of phenol by C. tropicalis strain NCIM 3556, showed its ability to degrade phenol (200 mg/l) completely after 48 h of incubation [37]. Liu et al. has found maximum 2500 mg/l phenol tolerant yeast T. montevideense strain PHE1 [10]. 3.2 Proposed pathway for phenol degradation in oleaginous yeast linked to lipid synthesis

Both ortho- and meta-cleavage pathways were analyzed for the phenol degradation in R. kratochvilovae HIMPA1. For ortho-cleavage pathway, the product formation was analyzed with the help of catechol 1, 2-dioxygenase enzyme that gives absorbance at 260 nm, while 2-HMSA formed in the meta-pathway with catechol 2, 3-dioxygenase that gives absorbance at 375 nm [19]. Results of absorbance at 375 nm showed a linear increment in OD with respect to time and

thereafter, reaching a steady value which strongly supports the meta-cleavage activity for phenol degradation (Fig. 2). However, irregular absorbance was recorded at 260 nm which confirmed the absence of ortho-cleavage pathway. The meta-degradation pathway also depends on initial phenol concentration viz. 0.5, 0.75, 1 g/l present in the medium along with supplementation of glucose (70 g/l) measured by UV–vis spectrophotometer (Supplementary data Fig. 1). The data revealed from enzymatic assay confirms that in this oleaginous yeast, phenol is degraded via meta cleavage pathway. The proposed pathway for phenol degradation and lipid accumulation in oleaginous yeast is represented in Fig. 3. As observed, in oleaginous yeast R. kratochvilovae HIMPA1, phenol is converted into catechol by phenol hydroxylase (PH) followed by extradiol division of aromatic ring to form 2-hydroxymuconic acid-6-semialdehyde (HMSA) catalyzed by catechol 2, 3dioxygenase. HMSA gets further converted into 2-oxopent-4-dienoate (OE) by two different routes. The first route is 4-oxalocrotonate route which is catalyzed by HMSA dehydrogenase (HMSA-DH), 4-oxalocrotonate tautomerase (4OT) and 4-oxalocrotonate decarboxylase (4OD), respectively. While, by the second hydrolytic route it is converted directly into OE via HMSA hydrolase (HMSAH) in a single step. OE is further hydroxylated into 4-hydroxy-2-oxovalerate (HOV) by OE hydratase (OEH) and HOV is finally converted into pyruvate and acetaldehyde by HOV aldolase (HOVA) [3,17]. Thus the meta-cleavage pathway of phenol degradation provides pyruvate and acetaldehyde as end products [32]. Among them pyruvate is utilized by Krebs cycle without any modification while acetaldehyde dehydrogenase (AcDH) catalyzed the conversion of acetaldehyde to acetyl-CoA. In respiring cells of non-oleaginous yeast, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, while in fermenting cells pyruvate is directed towards the by-product ethanol. Once pyruvate enters the mitochondrion it can be converted to acetyl-CoA by the PDH complex,

however, this mitochondrial acetyl-CoA pool is not available to cytoplasmic biosynthesis pathways as the transport mechanism for shuttling acetyl-CoA from the mitochondrion to the cytoplasm does not exist in S. cerevisiae, but oleaginous yeasts shuttle acetyl-CoA through the intermediate citrate. Acetyl-CoA gets transported into cytosol from mitochondria through malatecitrate shuttle [38]. As citrate can be transported across the mitochondrial membrane, this metabolite basically becomes a carrier of acetyl-CoA from the mitochondria to the cytosol. An important fact is that oleaginous yeasts have ATP-citrate lyase (ACL) which is a ubiquitous enzyme located in the cytosol that uses citrate and converts it to acetyl-CoA and oxaloacetate. ACL is found in many different eukaryotic species, including fungi, plants and animals [39]. But it is absent in non-oleaginous yeasts such as S. cerevisiae suggesting that this enzyme ACL plays a crucial role in supplying the precursor acetyl-CoA necessary for lipid biosynthesis during the lipid accumulation phase in these organisms [25,40,41]. This is typified by oleaginous yeast species, which are defined by their ability to accumulate high levels of cytoplasmic acetyl-CoA derived triacylglycerides in nitrogen-limiting conditions [41,42]. The pyruvate obtained (from phenol degradation through the meta-pathway and fatty acid synthesis pathway) diffuse from cytosol to mitochondria due to permeability of inner mitochondrial membrane and get converted into oxaloacetate by pyruvate carboxylase and the TCA cycle is completed [40]. In this way, the final degradation products of phenol pathway buildup acetyl-CoA accumulation in the cytoplasm [25]. Acetyl-CoA is the precursor for de novo TAG (Triacylglycerols) biosynthesis pathway (Kennedy pathway) in oleaginous yeasts as further confirmed in Section 3.4. Degradation of phenol is found to be strain-specific phenomenon and their metabolic pathway for phenol degradation depends on different parameters of initial phenol concentrations [43]. Previous studies revealed that both ortho and meta routes can be active for the same microorganism depending on

the substrate [19,21,22]. Phenol biodegradation by other yeast strains of the genera Trichosporon cutaneum, Rhodotorula rubura and Acinetobacter calcoacetium, Aureobasidium follows orthocleavage pathway [7,44,45]. The cascades of these pathways can be induced for these phenolmetabolizing enzymes in T. cutaneum which occurs at low concentration (0.01-0.10 mM). Fully induced cells showed 50-400 times higher activities of phenol hydroxylase, catechol 1,2oxygenase, cis-cis-muconate cyclase and delactonizing enzyme, maleolyl acetate reductase than in non-induced T. cutaneum cells [45]. Another phenol-utilizing yeast, T. cutaneum POB 14 utilized phenol in preference to glucose in a medium containing both phenol (200 mg/l) and glucose (0.15%) as carbon sources has shown a partially constitutive activity of catechol 1,2oxygenase [44].

3.3 Effect of various phenol concentration on dry biomass (g/l), total lipid (g/l) and lipid content (%; wt/wt) of R. kratochvilovae HIMPA1 The dry biomass (g/l), total lipid yield (g/l) and lipid content (%; wt/wt) during the yeast growth on different concentration of phenol and on glucose synthetic medium (GSM) is represented in Fig. 4. When minimal media supplemented with 0.5 g/l phenol was used for the cultivating yeast, the dry biomass, total lipid yield and lipid content were 4.16 ± 0.29 g/l, 1.14 ± 0.31 g/l and 27.4 ± 0.34%, respectively, on increasing the concentration of phenol to 0.75 g/l, the dry biomass and total lipid were reported to be decreased as 3.81 g/l and 1.09 g/l respectively but the lipid content (%) with respect to dry biomass was enhanced. However, maximum lipid content (29.47%) was recorded when the concentration of phenol was raised to 1 g/l. The increased lipid content with higher concentration of phenol could be due to increased toxicity. After supplementation of glucose at similar concentration of phenol, the amounts of total lipid along with their dry biomass were increased. Interestingly, the dry biomass decreased from 10.98 g/l to 6.13 g/l with increased

amount of phenol from 0.5 g/l to 1 g/l, respectively in the presence of glucose. While the lipid contents were observed in opposite manner as highest lipid content (64.92%) was reported with 1 g/l phenol in the presence of glucose. 3.4 Determination of morphological variation in the Lipid droplets and TAG accumulation ability of R. kratochvilovae HIMPA1 in the presence of phenol The patterns of morphological variations and fluorescence images for LD formation in R. kratochvilovae HIMPA1 grown under different concentrations of phenol are shown in Fig. 5. The results obtained show a direct correlation between LDs size with the TAG accumulating ability. Fluorescence images inferred that the cells grown in phenol containing medium were small and slightly elongated, whereas cell size and lipid accumulation increased in those cultures where phenol was supplemented with glucose (Fig. 5). Maximum cell size (7.34± 0.67 µm) and LD size (6.12 ± 0.78 µm) were obtained in 1 g/l phenol supplemented with glucose as compared to cell size (4.53 ± 0.31µm) and LD size (2.38 ± 0.52 µm) of GSM grown cells (Fig. 5). Earlier researcher has shown that under nitrogen and phosphorus limiting conditions these yeast species have a general tendency to enlarge their cell size rather than their cell number [46]. Previous studies done on transcriptomics and proteomics analysis of several yeast species have shown that oleaginous yeasts behaves differently from non-oleaginous yeasts to utilize substrates and lipid accumulation in their cellular compartment [47]. Results concluded that phenol acts as a nutrient stress under which cell and lipid droplet size increases with increasing concentration of phenol. 3.5 Effect of phenol on lipid profile of R. kratochvilovae HIMPA1 Oleaginous yeasts usually accumulate lipids in the form of LDs in its cellular compartment and the main component of LD are usually triacylglycerols (TAG). The contents of fatty acids are

totally dependent on providing culture medium and culturing conditions [48]. It has been earlier reported that the yeast species R. kratochvilovae HIMPA1 can grow on various substrates such as hemp seed aqueous extract and synthesize 62.5% of saturated fatty acids (SFA), 37.5% of monounsaturated fatty acids along with an unusual fatty acid C27:0 [27]. While in another study this oleaginous yeast synthesized SFA (71.8%) and MUFA (17.34%) as major fatty acids when grown in non-edible lignocellulosic biomass of Cassia fistula L. fruit pulp [30]. In this study, an increased amount of MUFA was observed when R. kratochvilovae HIMPA1 grown in the presence of phenol as compared to the GSM grown cells. The percentage increase in MUFA was directly proportional to the amount of phenol in the medium as 51.87%, 58.33% and 62.98% MUFA were synthesized with 0.5, 0.75 and 1 g/l of phenol (Fig. 6). While R. kratochvilovae HIMPA1 grown in glucose synthetic medium (GSM) synthesize 60.35% SFA; 23.15 %, MUFA; and 2.23 %, PUFA respectively (Fig. 6). It was observed that R. kratochvilovae HIMPA1 grown in the presence of phenol exhibited enhanced MUFA content. This could be due to the conversion of stearic acid (C18:0) into oleic acid (C18:1) by Δ-9-desaturase which added double-bond through an oxidative process known as desaturation that requires oxygen, NADH, NADPH, and the substrate. This process prevents the accumulation of reactive oxygen species under stress condition [49]. It has been previously described by various researcher that the amount of MUFA increases in unsaturated fatty acid mutants (UfaMs) of A. curvature by mutagenic treatment [50] or by using sterculia oil as Δ-9-desaturase inhibitor [51] that block the conversion of stearic (C18:0) to oleic acid (C18:1). 3.6 Estimation of TAG conversion into fatty acid methyl esters (biodiesel) by TLC The conversion of total extracted lipid into FAME was demonstrated by TLC (Fig. 7). Data showed that 51% and 75% TAG were converted into FAME after 2 h and 4 h of the transesterification reaction, respectively. The conversion of TAG into FAME depends on various factors such as

reaction temperature, reaction time, catalyst to lipid ratio, methanol to lipid ratio and the presence of moisture. The current improved method applied for lipid and FAME separation results in terms of regular patterns that allow quantitative analysis and devoid of heavy dark background as previously reported in the staining procedure based on KMnO4 [35,52]. 10% ethyl acetate in hexane was used as developing solvent which gives darker spots of separated compounds [30]. 3.7 Estimation of biodiesel properties The quality of biodiesel is totally influenced by the fatty acids composition of feedstocks, production process, refining process and post production parameters besides this, biodiesel properties are also affected by compositional variations including fatty acids type, chain length and number, position and isomers of double bonds [53–55]. Studies done on the biodiesel properties of R. kratochvilovae HIMPA1 grown on various phenol concentrations are listed in Table 1. The biodiesel obtained from GSM grown cells have higher oxidative stability (55.474 h) as compared to the cells grown in the different concentration of phenol. High saturated fatty acids (SFA) contents present in biodiesel impart great self-life by preventing it from autoxidation while the presence of high MUFA contents causes autoxidation and predict the better utility of fuel at low-temperature condition only. Yeasts cells grown in 1 g/l phenol showed lower CFPP (-14.102 °C), while GSM grown cells exhibited high CFPP (27.943 °C). Kinematic viscosity is another important fuel property of biodiesel that increases with increasing chain length of fatty acid or saturation of fatty acid, however, the viscosity of unsaturated fatty acid depends on number and nature of double bonds but less affected by position [56]. The data for kinematic viscosity show that all biodiesel types listed in Table 1 fall within a narrow range of 3.5–5 mm2/s. Cetane number (CN) is yet another important factor which affects the various parameters of engine performance such as noise, emissions of CO and stability [57]. The biodiesel having high CN provides a quick

start to engines and better cold start behavior along with complete combustion of fuel that leads to reduced emission of smoke and particulate matters [54,58]. Yeast cells grown in GSM showed highest CN value (71.251) while cells grown in 1 g/l of phenol showed low CN value (53.092) as listed in Table 1. Thus, high saturation in fatty acid profile supports the CN, kinematic viscosity and oxidative stability while unsaturation in fatty acid profile supports the cold flow behavior, density and high heating value of biodiesel. The fatty acids profile and biodiesel physical properties abide by ASTM D6751 and EN 14214 biodiesel standards, signifying its applicability in diesel engines. Conclusion The proposed pathway of Phenol degradation follows meta cleavage in the oleaginous yeast R. kratochvilovae HIMPA1 which was found to enhance TAG accumulation in the lipid droplets of its cellular compartment when fed on the high concentration of phenol (1 g/l). Meta pathway of the phenol degradation is established by enzyme assays buildup pyruvate and acetaldehyde concentration in the cytosol that further flux towards acetyl-CoA accumulation. ATP-citrate lyase plays a crucial role in acetyl-CoA generation during the lipid accumulation phase in this organism. Acetyl-CoA feeds the de novo TAG biosynthesis pathway in the cytoplasm of oleaginous yeasts which was monitored by TAG accumulation through fluorescence microscopic study of BODIPY505-5015 stained live cells revealing larger LDs size (6.12 ± 0.78 µm) when R. kratochvilovae HIMPA1 cells were grown in the presence of phenol (1 g/l). The accumulated TAG was transesterified to produce Biodiesel. Acknowledgements Authors are grateful for financial funding from the DBT, Govt. of India, (Grant No.: DBT-608BIO) and SRF to AKP from UGC, India (Grant No.: 6405-35-044).

References;1; Table 1. Biodiesel properties of transesterified products from R. kratochvilovae HIMPA1. Biodiesel Properties

Culture medium A, GSM

B, Phenol (0.5 g/l)

C, Phenol (0.75 g/l)

D, Phenol (1 g/l)

G, Phenol (0.5 g/l) + Glu (70g/l)

H, Phenol (0.75 g/l) + Glu (70g/l)

I, Phenol (1 g/l) + Glu (70g/l)

60.35

12.79

13.85

8.6

12.69

11.78

13.71

23.15

51.87

58.33

62.98

66.99

67.15

68.47

2.23

24.77

18.36

22.86

9.8

8.96

4.12

27.61

101.41

95.05

108.7

86.59

85.07

76.71

178.694

179.958

182.333

189.173

179.891

176.663

173.616

24.857

91.511

85.71

98.042

77.992

76.614

69.034

Cetane number

71.251

56.039

56.95

53.092

59.093

59.957

62.205

Long Chain Saturated Factor Cold Filter Plugging Point Cloud Point

14.139

1.335

1.504

0.756

1.088

1.211

1.685

27.943

-12.283

-11.752

-14.102

-13.059

-12.672

-11.183

13.308

0.715

1.525

-1.015

0.731

0.904

1.294

Pour Point

7.625

-6.045

-5.165

-7.923

-6.028

-5.839

-5.417

Allylic Position Equivalent Bis-Allylic Position Equivalent Oxidation Stability Higher Heating Value Kinematic Viscosity Density

27.61

101.41

95.05

108.7

86.59

85.07

76.71

2.23

24.77

18.36

22.86

9.8

9.14

4.12

55.474

7.351

9.014

7.749

14.624

15.752

31.214

33.816

35.33

35.78

37.333

35.386

34.758

34.139

3.337

3.331

3.422

3.591

3.43

3.357

3.319

0.744

0.785

0.793

0.829

0.783

0.769

0.755

Saturated Fatty Acid Mono Unsaturated Fatty Acid Poly Unsaturated Fatty Acid Degree of Unsaturation Saponification Value Iodine Value

Legends to Figures Fig. 1. The growth profile of R. kratochvilovae HIMPA1 when grown on various phenol concentration (0.5 g/l to 1.5 g/l with an interval of 0.25 g/l) at various initial cell densities (Fig.

1.a) 0.063 at OD600nm (1.c) 0.126 at OD600nm (1.e) 0.189 at OD600nm And their corresponding residual phenol concentration in the medium was represented in Fig. 1.b, 1.d, and 1.f, respectively. Fig. 2. Analysis of meta and ortho pathway for phenol degradation in oleaginous yeast by enzymatic assay. Fig. 3. Proposed pathway for phenol degradation in oleaginous yeast that leads to enhanced fatty acids synthesis. Fig. 4. Graph showing the dry biomass (g/l), total lipid (g/l) and lipid content (%; wt/wt) of R. kratochvilovae HIMPA1. Fig. 5. Fluorescence Images of lipid droplets formation in R. kratochvilovae HIMPA1were taken when cells achieved its stationary phase using fluorescence microscopy of live cells stained with BODIPY505/515nm. All scale bars represent 10 μM. Fig. 6. The total percentage of fatty acid methyl esters (FAME) produced by R. kratochvilovae HIMPA1 while grown on different substrates A: GSM, B: phenol (0.5 g/l), C: phenol (0.75 g/l), D: phenol (1 g/l), G: phenol (0.5 g/l) + Glu (70g/l), H: phenol (0.5 g/l) + Glu (70 g/l), I: phenol (1 g/l) + Glu (70 g/l). Fig. 7. Chromatogram showing separation of MAG, DAG, TAG, FFA and FAME on TLC-plate. In lane 1: standard of FAME (palmitic acid methyl ester) and TAG (Triolein); lane 2 and 3 the transesterification reaction mixture from different time intervals (2 h and 4 h) were spotted. Optical densities of the spots plotted as the functions of the corresponding area of FAME and TAG (for details see section 2.9).

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Figr-1Figure 1.

Figr-2Figure 2.

Figr-3Figure 3.

Figr-4Figure 4.

Figr-5Figure. 5

Figr-6Figure 6.

Figr-7Figure 7.