Bioresource Technology xxx (2015) xxx–xxx

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Bioresource Technology

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Synergistic effect of fermentable and non-fermentable carbon sourcesenhances TAG accumulation in oleaginous yeast Rhodosporidiumkratochvilovae HIMPA1

http://dx.doi.org/10.1016/j.biortech.2015.02.0620960-8524/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +91 1332 285530 (office), +91 1332 285110 (Resi.),mobile: +91 9760214585; fax: +91 1332 273560.

E-mail address: parulaggarwalpruthi@gmail.com (P.A. Pruthi).

Please cite this article in press as: Patel, A., et al. Synergistic effect of fermentable and non-fermentable carbon sources enhances TAG accumulaoleaginous yeast Rhodosporidium kratochvilovae HIMPA1. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.062

Alok Patel, Vikas Pruthi, Rajesh P. Singh, Parul A. Pruthi ⇑Molecular Microbiology Laboratory, Biotechnology Department, Indian Institute of Technology Roorkee (IIT R), Roorkee, Uttarakhand 247667, India

h i g h l i g h t s

� Synergistic effect of fermenting andnon-fermenting carbon.� Maximum total lipid yield 9.26 g/l

observed in mix carbon sources.� Maximum TAG accumulation in lipid

droplets (5.35 ± 0.76 lm) in mixcarbon sources.� Enhanced (C14:0-C24:0) fatty acids in

mix carbon sources revealed by FAMEprofile.� Simultaneous utilization of mix

carbon sources by Rhodosporidiumkratochvilovae.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 December 2014Received in revised form 12 February 2015Accepted 13 February 2015Available online xxxx

Keywords:OleaginousYeastFermentingNonfermentingTAG

a b s t r a c t

Novel strategy for enhancing TAG accumulation by simultaneous utilization of fermentable and non-fermentable carbon sources as substrate for cultivation of oleaginous yeast Rhodosporidium kratochvilovaeHIMPA1 were undertaken in this investigation. The yeast strain showed direct correlation between thesize of lipid bodies, visualized by BODIPY stain (493–515 nm) and TAG accumulation when examinedon individual fermenting and non-fermenting carbon sources and their mixtures. Maximum TAGaccumulation (lm) in glucose (2.38 ± 0.52), fructose (4.03 ± 0.38), sucrose (4.24 ± 0.45), glycerol(4.35 ± 0.54), xylulose (3.94 ± 0.12), and arabinose (2.98 ± 0.43) were observed. Synergistic effect of theabove carbon sources (fermentable and non-fermentable) in equimolar concentration revealed maximumlipid droplet size of 5.35 ± 0.76 lm and cell size of 6.89 ± 0.97 lm. Total lipid content observed in mixedcarbon sources was 9.26 g/l compared to glucose (6.2 g/l). FAME profile revealed enhanced longer chain(C14:0-C24:0) fatty acids in mix carbon sources.

� 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Oleaginous yeasts have triggered significant attention as idealoil producing cell factories that can accumulate neutral lipids inthe form of triglycerides in their lipid bodies. These yeasts have

emerged as a promising feedstock since they can be easily cultivat-ed and can scale up for large scale production of biodiesel for oleo-chemical industries (Koutinas et al., 2014). The oleagenic propertyof these yeasts is due to the presence of additional enzymes and al-ternative metabolic pathway utilizing various cost effective carbonsubstrates (Ageitos et al., 2011; Papanikolaou and Aggelis, 2011;Yu et al., 2014). They are broadly classified into fermenting andnon-fermenting yeasts (Gancedo and Gancedo, 1971). Fermentingyeasts are able to thrive well on glucose, under aerobic conditions

tion in

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are non-oleaginous in nature such as Saccharomyces cerevisiae,Torulopsis salmanticensis and Hansenula anomala, while oleaginousyeast on the other hand, lack the ability to utilize glucose in the ab-sence of oxygen and are referred as non-fermenting yeasts such asRhodotorula glutinis, Rhodotorula mucilaginosa, Rhodotorula gracilis,Rhodotorula minuta, Sporobolomyces pararoseus and Debaryomyceshansenii. The difference in the two categories is based on cataboliterepression observed in the presence of glucose for utilization ofalternate sugar carbon sources (galactose, sucrose, maltose,melibiose) as well as non-sugar carbons such as ethanol, lactate,glycerol, acetate and oleate (Gancedo, 1998; Weinhandl et al.,2014). Most of microorganisms have their own carbon cataboliterepression (CCR) mechanism, which generally metabolize sugarssequentially (first glucose and then xylose) since glucose repressesother sugar utilization and also hinder to utilize mixed sugars ob-tained from hydrolysis of lignocellulose biomass (Stülke andHillen, 1999; Kim et al., 2010). Recent progress in understandingthe transcriptional regulation of genes involved in the use ofnon-fermentable carbon sources showed to get expressed 9–12times more than fermentable carbon sources utilized by oleagi-nous yeasts (Turcotte et al., 2010). These results in massive repro-gramming of gene expression including genes involved ingluconeogenesis, the glyoxylate cycle, and the tricarboxylic acidcycle (Gancedo, 1998; Weinhandl et al., 2014). Role of Snif1 kinasefor activation of gene responsible for utilization of non-fermentable carbon sources indicates networking of severalunexplained mechanism of sugar utilization. Classical diauxic shiftis usually observed with a shift from a fermentative to a non-fermentative mode of growth by yeast. Researcher have alsoshown differences in enzyme activities and indicated that isoci-trate lyase (ICL1), a key promoter enzyme of the TCA and glyoxy-late cycle enables the cell to grow on non-fermentable carbonsources is repressed by glucose and strongly induced by ethanolor acetate (Schöler and Schüller, 1994; Schüller, 2003). Similarly,a number of genes require for glycerol uptake (Stl1, GUT1, GUT2)are also up regulated when the cells were grown in the presenceof glycerol or ethanol while these genes are repressed in the pres-ence of glucose (Pavlik et al., 1993). Similarly, studies have alsoindicated that the non-fermenting oleaginous yeasts lack severalenzymes like PFK, F1,6DP activity, and metabolize glucose throughhexose monophosphate pathway but they possess specific enzymeATP: citrate lyase (EC 2.3.3.8) which generates acetyl-CoA, the keysubstrate for fatty acid biosynthesis, in the cytoplasm from citrate.Non oleaginous organisms lack this enzyme (Breuer and Harms,2006). Recent explorations of screening new oleaginous yeaststrains which are capable of simultaneous utilization of both C5and C6 sugars and can overcome the problem of glucose repressionled to the finding of red yeasts Rhodosporidium toruloides and R.glutinis. They can accumulate over 50% of its dry biomass as lipidthrough simultaneous utilization of mix sugars obtained from lig-nocellulosic hydrolysates (Yen et al., 2014). Rhodosporidium spp.have shown to produce the highest amount of lipids using a varietyof carbon sources, such as molasses, glycerol, and complex carbo-hydrates like rice and wheat hydrolysates, starch, inulin, laevoglu-cose, wheat straw, waste oils, animal fats, Jerusalem artichokehydrolysates, hydrolysate of cassava starch, wastewater andmixtures of glucose, xylose and glycerol (Zhao et al., 2008; Zhanet al., 2013; Yu et al., 2014; Gen et al., 2014). Although co-fermen-tation of mixed sugars has been intensively investigated forethanol production but there is vast lacuna in the studies to utilizemixed sugars for microbial lipid production from oleaginous mi-croorganisms (Yu et al., 2014). Few reports on mix sugar utilizationshowed that growth on dextrose can be enhanced through thesimultaneous addition of glycerol in R. glutinis (Easterling et al.,2009). Similarly, simultaneous utilization was also observed whenR toruloides was grown in combination of glycerol, dextrose and

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xylose (Wiebe et al., 2012). But oleaginous yeasts Lipomyces star-keyi and Trichosporon fermentans utilizes various carbon sourcesand their mixture in a sequential manner (Tapia et al., 2012; Sha,2013; Abghari and Chen, 2014). During this investigation, the abil-ity of novel oleaginous yeast Rhodosporidium kratochvilovaeHIMPA1 (Acc. No. KF772881) to grow on the wide variety of sugarsources (fermentable and non-fermentable sugars and their mix-tures) for obtaining maximum TAG accumulation was done. Thevariation in lipid body size in R. kratochvilovae HIMPA1 cultivatedon different sugar sources were monitored by BODIPY staining andimaged using new live in vivo fluorescence microscopy technique(Patel et al., 2014). The amount of dry cell weight (g/l), total lipidyield (g/l) and lipid content (%) produced were estimated and itsFAME profile was determined using GC–MS analysis. Based onthe above facts, a correlation of enhanced TAG accumulation withincrease in lipid body size was elucidated in the cells grown on amixture of fermentable and non-fermentable sugar sources togeth-er as compared to glucose alone.

2. Methods

2.1. Materials

Analytical grade solvents viz., n-hexane, chloroform, and metha-nol were purchased from Merck Ltd., Mumbai, India. Sugars(glucose, glycerol, sucrose, fructose, xylulose, arabinose) and otherchemicals (NaCl, KCl, anhydrous sodium sulfate, 14% methanolicBF3) were procured from HiMedia laboratories, Mumbai, India.FAME standard (AOCS low erucic rape seed oil O7756-1AMP) forGC–MS analysis and standard for TLC (Triolein) were obtained fromSigma Aldrich (St. Louis, USA). BODIPY 493/515 (4,4-difluro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indacene) was purchased fromInvitrogen (Life Technology, USA) for fluorescence staining.

2.2. Yeast strain and culture conditions

R. kratochvilovae HIMPA1 (Genbank Acc. No. KF772881) wascultivated at 30 �C on a rotary shaker at 200 rpm for 48 h usingYEPD broth as reported earlier (Patel et al., 2014). To obtain theseed culture, cells were harvested by centrifugation, washed twicewith sterilized distilled water and resuspended in 0.9% sterilizedsaline to attain cell density of 6.5–7.8 � 108 cells/ml. Batch cultiva-tions of R. kratochvilovae HIMPA1 for TAG accumulation wereperformed in 250 ml Erlenmeyer flasks where all fermenting (glu-cose, fructose, and sucrose), non-fermenting (glycerol, xylulose,and arabinose) carbon sources and mixture of both of them werestandardized in moles of carbon equivalents relating to 70 g/l glu-cose (388 mM), mixed with salt solution (g/l): KH2PO4, 1; MgSO4,0.5; (NH4)2SO4, 1; CaCl2, 0.1; trace elements (mg/l): boric acid,0.5; CuSO4, 0.04; KI, 0.1; FeCl3, 0.2; MnSO4, 0.4; NaMO3, 0.2;ZnSO4, 0.4; vitamin solution (mg/l): D-Biotin, 0.002; calcium pan-tothenate, 0.4; folic acid, 0.002; inositol, 2; niacin, 0.4; PABA, 0.2;pyridoxine HCl, 0.4; riboflavin, 0.2; thiamine, 0.4. All experimentswere done in triplicate.

2.3. Schematic procedure for fluorescence microscopy analysis

For fluorescence microscopy BODIPY stock solution was pre-pared by adding 0.1 mg/ml with DMSO (Govender et al., 2012).Initially, 10 ll of the culture broth of R. kratochvilovae grown atdifferent time intervals was pelleted and washed with 0.9% salinewater. After washing, the cells were suspended in 10 ll of 0.9%saline to obtain appropriate cell concentration (1 � 106 cells/ml).2 ll (2 � 10�3 lg/ll) of BODIPY stock solution was then added toit and mixed well. After five minutes of incubation in dark the cell

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A. Patel et al. / Bioresource Technology xxx (2015) xxx–xxx 3

suspension was washed with 0.9% saline water. Fluorescence im-ages were viewed on a digital inverted fluorescence microscopeequipped with EVOS GFP light cube (EVOS-FL, AdvanceMicroscopy Group, AMG, USA) (Patel et al., 2014).

2.4. Estimation of cell size and LDs size by ImageJ 1.48a software

The size of cells and intracellular LDs were analyzed by ImageJ1.48a software. Images obtained after fluorescence microscopycontaining the fluorescence channel were converted to a Z-projec-tion in ImageJ (Wong and Franz, 2013). The obtained image was acolor image (RGB); it was necessary to split multi-color images intosingle channels and converted single channel color images tograyscale before proceeding. The image created as binary imagesfor threshold to highlight all of the structures that is required tomeasure. Watershed programme is used for separating two nearbylipid droplets by 1 pixel thick line. Using straight tool in ImageJ twolines were manually drawn which bisect the cell and each fluores-cent LD followed by adding the region of interest ROI manager.Each line represents the diameter of the cell and LD. The lengthand width of LDs were measured for droplets that show a sphericalgeometry.100 cells were counted to calculate the average size ofcells and intracellular LDs, which was presented as mean ± SD.

2.5. Dry cell weight determination

The yeast cells growth were observed by measuring cell opticaldensity (OD) at 600 nm. Cell biomass, expressed as dry cell weight(DCW) was harvested by centrifuging it at 3000 rpm for 5 min andits weight was determined gravimetrically after drying the cells inan oven at 105 �C for 12 h (Patel et al., 2014).

2.6. Lipid extraction and gravimetric determination

The total cellular lipid was extracted using modified protocol ofBligh and Dyer (1959). Firstly, dried samples were mixed with dis-tilled water and mixed vigorously for 15 s. Cell suspension wassubjected to sonication at 40 Hz for 5 min and after that 10 ml ofchloroform: methanol (2:1; v/v) was added to the suspensionand stirred for 30 min. The extract was filtered with sintered glassfunnel. 5 ml of 0.034% MgCl2 was added to the extract and cen-trifuged at 3000 rpm for 5 min. Upper aqueous layer was aspiratedand washed the organic phase with 1 ml of 2 N KCl/methanol (4:1;v/v). After washing suspension was centrifuged again at 3000 rpmfor 5 min and washed repeatedly with 5 ml of upper phase (chloro-form/methanol/water; 3:48:47; v/v) until the phase boundary be-comes clear. The bottom chloroform layer was transferred to a newscrew cap test tube, and the lipid yield was determined gravimet-rically. The lipid yield is the amount of lipid extracted from thecells per liter of the fermentation medium (g/l) while the lipid con-tent Y (%), was calculated using the following equation: Y = WL/DCW � 100. Where WL is the weight of the total lipid obtainedgravimetrically and DCW is the weight of the cell biomass.

2.7. Transesterification of fatty acids by methanolic BF3 anddetermination of FAMEs composition by GC–MS

Transesterification of extracted yeast lipid samples was doneusing the method of Morrison and Smith (1964). Briefly, fatty acidsamples dried with anhydrous sodium sulfate were evaporated todryness under nitrogen in a Teflon coated screw cap tube. Boronfluoride–methanol reagent was added under nitrogen, in the pro-portions 1 ml reagent per 4–16 mg of lipid. The samples were thenheated at 80 �C for 20 min in a boiling water bath. The esters wereextracted by adding 2 volumes of n-hexane and then 1 volume ofwater followed by centrifugation at 3500 g until both layers were

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clearly visible. The products of transesterification were analyzedby GC–MS (Agilent, Santa Clara, CA, USA). The column used wasa capillary column (DB-5MS) with dimensions 30 m � 0.25 mmID and 0.25 lm film thickness. The sample was injected in splitlessinjection mode (1 ll at 250 �C) using helium as a carrier gas (1 ml/min). The column temperature was initially set at 50 �C (1.5 min)where, after the temperature was ramped to 180 �C (25 �C/min)for 1 min, followed by a further increased to 220 �C (10 �C/min),it was held for 1 min. Finally, the temperature was increased to250 �C (15 �C/min) and held for 3 min. The mass transfer line andion source were set at 250 and 200 �C, respectively. The FAMEswere detected with electron ionization (70 eV) in scan mode(50–600 m/z).

2.8. Determination of sugar

The amount of residual sugars in the samples were determinedby HPLC equipped with an Aminex HPX-87H column (da Silvaet al., 2011). The amount of sugar consumption (%) were deter-mined as C = St0 � St/St0 � 100, where C is the amount of sugar con-sumption, St0 is the amount of initial sugar (g/l) added and St is theresidual sugar left at each sampling time.

2.9. TLC analysis for neutral lipid determination

Triacylglycerol (TAG) analysis in the extracted total lipid wascarried out by TLC using 0.25-mm-thick silica gel G-60 F254 plates(Merck, India) and chromatograms were developed in hexane: di-ethyl ether: acetic acid (85:15:1, v/v/v) with triolein as standard.For quantification of SEs and TAGs, plates were dipped intomethanolic MnCl2 solution (0.63 g MnCl2�4H2O, 60 ml water,60 ml methanol and 4 ml concentrated sulfuric acid), dried andheated at 120 �C for 15 min (Fei et al., 2009). Conversion of TAG in-to FAMEs was detected by double development of TLC. The platewas firstly developed to 2.5 cm from the origin with hexane:tert-butyl methyl ether: acetic acid (50:50:0.5, v/v/v), and afterair dried, redeveloped to 8 cm from the origin with hexane: tert-butyl methyl ether: acetic acid (97:3:0.5, v/v/v) as a developingagent. The samples were visualized by spraying sulfuric acid 50%(w/w) and then heating at 135 �C (Patel et al., 2014).

3. Results and discussion

3.1. The effect of fermentable, non-fermentable and mixture of bothfermentable and non-fermentable carbon sources on the growth andlipid production of R. kratochvilovae HIMPA1

R. kratochvilovae HIMPA1 showed a wide range of carbon sourceutilization when grown on fermentable glucose (Glu), fructose(Fru) and sucrose (Suc), non-fermentable glycerol (Gly), arabinose(Arb) and xylulose (Xyl) as well as on mixture of both carbonsources. All individual sources of carbon for each batch ex-periments were provided in moles of carbon equivalents relatingto 70 g/l glucose (388 mM) while mixture of both fermentableand non-fermentable carbon sources were provided in equal pro-portion of 64.75 mM to make it equivalent to 388 mM. The resultsof all batch experiments for cell dry weight (CDW) in g/l, total lipidyield (g/l) and lipid content (%) were presented in Fig. 1. Thegrowth of R. kratochvilovae HIMPA1 on individual carbon sourcesresulted in the highest CDW of 14.15 ± 0.78 g/l on glycerol whileit was 13.26 ± 0.98, 13.22 ± 0.54, 13.38 ± 0.56 g/l for Glu, Fru andSuc, respectively, which were higher than pentose sugars Xyl(6.9 ± 0.43 g/l) and Arb (7.64 ± 0.76 g/l) used. The CDW results ob-tained on mixture of Fermentable (Glu, Fru and Suc) and nonfer-mentable carbon sources were (Gly, Arb and Xyl) 13.35 ± 0.92 g/l

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and 13.58 ± 0.71 g/l, respectively, while the mixture of all ferment-ing and non-fermenting carbon sources together had highest CDWof 15.56 ± 0.54 g/l comparable to the mixture of Glu + Gly having14.75 ± 0.78 g/l. The total lipid yields measured gravimetricallyshowed maximum value of 9.26 ± 0.54 g/l on mixture of both fer-mentable (Glu + Fru + Suc) and non-fermentable (Gly + Xyl + Arb)carbon sources together. While on individual carbon the total lipidyields were 8.0 ± 0.65 > 7.72 ± 0.87 > 7.5 ± 0.43 > 6.2 ± 0.58 >4.0 ± 0.98 > 3.84 ± 0.32 g/l respectively in the following orderGly > Suc > Fru > Glu > Arb > Xyl. Data showed that the total lipidyields (7.99 ± 0.79 g/l) on non-fermentable (Gly + Xyl + Arb)sources were more than fermentable carbon sources(Glu + Fru + Suc) 7.14 ± 0.71 g/l, while it was higher in the mixtureof both Glu and Gly (8.45 ± 0.49 g/l). The lipid content(59.51 ± 0.59%) obtained from the mixture of fermenting andnon-fermenting carbon sources together utilized by R. kratochvilo-vae HIMPA1 clearly indicates the synergistic effect in the lipid ac-cumulating property. Similarly, mixture containing Gly + Xyl + Arbrecorded high lipid content (58.83 ± 0.75%) while on Gly + Glu lipidcontent was 57.28 ± 0.35%, respectively. The lipid contentof R. kratochvilovae HIMPA1 observed for individual carbon sourceswere 57.69 ± 0.71% > 56.73 ± 0.34% > 56.6 ± 0.69% > 55.65 ± 0.38% >52.35 ± 0.88% > 46.76 ± 0.61% in the following order Suc > Fru >Gly > Xyl > Arb > Glu, respectively. The sugar mixtures of non-fermentable carbon sources (Gly + Xyl + Arb) together have lipidcontent of 58.83 ± 0.75%. These values are 10–12% higher than thatof glucose as sole carbon source (46.76 ± 0.61%), which enhanceslipid accumulation in above carbon source (Gly + Xyl + Arb) to1.38-fold when compared with glucose. Interestingly, the mixturecontaining both fermentable and non-fermentable carbon sourcestogether also had 12–13% higher lipid content which is 1.2 timeshigher than glucose grown cells (Fig. 1). The maximum cell dryweight (15.56 ± 0.54 g/l) and lipid yield (9.26 ± 0.67 g/l) were ob-served when combinations of fermentable and non-fermentablecarbon sources were used. The present investigation suggestedthat when fermentable carbon sources were mixed with non-fer-mentable carbon sources, it leads to increase in both cell dryweight and total lipid yield of oleaginous yeast R. kratochvilovaeHIMPA1 which support the co-utilization of sugars simultaneously.The combination of both types of mixtures would probably triggermultiple pathways for sugar uptake that enhances maximum lipidaccumulation as has been supported by earlier reports (Turcotteet al., 2010). Earlier, reports on production of biodiesel from simul-taneous utilization of mixtures of glycerol in combination withglucose or xylose as a growth substrate to produce TAG correlates

Fig. 1. Graph showing dry cell weight (g/l), total lipid yield (g/l) and lipid content (%) of Rfermentable (Gly + Xyl + Arb), and mixture of both fermentable and non-fermentable ca

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with the data obtained. Easterling et al. (2009) demonstrated thatthe oleaginous yeast R. glutinis cultured on medium containingdextrose, xylose, glycerol, dextrose and xylose, xylose and glycerol,or dextrose and glycerol accumulated 16%, 12%, 25%, 10%, 21%, and34% TAG on a dry cell weight basis, respectively. They further sup-ports simultaneous utilization of Glu and Gly enhances TAG accu-mulation and glycerol grown R. glutinis accumulates more lipidunder these experimental conditions than dextrose grown and xy-lose grown cultures. They also observed that when R. glutinis wasgrown on dextrose plus glycerol, the monounsaturated fatty acidscontent in total fatty acids were increased when compared to cellsgrown on dextrose or glycerol individually. But the addition ofglycerol as a carbon source along with dextrose or xylose resultedin a decrease in the amount of saturated fatty acids. Studies revealthat the capability of simultaneous sugar utilization greatly varieswith different strains and also depends on culture conditions. Gonget al. (2012) demonstrated that L. starkeyi simultaneously utilizedcellobiose and xylose at their mass ratios of 2:1 and 1:1. Yu et al.(2014) demonstrated that the cell biomass and lipid productionon the different sugar mixtures of glucose/xylose, glucose/cel-lobiose and cellobiose/xylose were similar in Cryptococcus curvatus.They found an effective approach for alleviating glucose repressionfor microbial lipid production by C. curvatus through xylose/cel-lobiose co-utilization. According to Sha (2013), the oleaginousyeast Yarrowia lipolytica produced more biomass and lipid whengrown on the mixture of glucose and xylose. In the case of non-oleaginous yeast S. cerevisiae preferentially utilize glucose as solecarbon source but in the absence of this fermentable carbon sourceit has been shifted to utilize other variety of non-fermentable sugarsource (glycerol) and suppressed the transcriptional expression ofgenes responsible for utilization of these carbon sources but inoleaginous yeast both the pathways of sugar utilization were si-multaneously activated (Turcotte et al., 2010). Since most microbespossess carbon catabolite repression (CCR), mixed sugars derivedfrom the lignocellulose are consumed sequentially, reducing theefficacy of the overall process (Stülke and Hillen, 1999). Contraryto this R. kratochvilovae HIMPA1 exhibit the simultaneous utiliza-tion of mixed sugars and combination of fermentable and non-fermentable sugar mixtures have showed enhanced total lipidproduction as compare to individual sugars utilization (Fig. 1). Anearlier report supports this foundation based on presence of twoindependent promoter elements present in the triosephosphatedehydrogenase 3 gene (TDH3) which is a glycolytic enzyme geneand is abundantly transcribed in S. cerevisiae. The promoter regionof the TDH3 gene is known to exhibit high transcription activity

hodosporidium kratochvilovae HIMPA1 grown on fermentable (Glu + Fru + Suc), non-rbon sources together (Glu + Fru + Suc + Gly + Xyl + Arb).

able and non-fermentable carbon sources enhances TAG accumulation in2015), http://dx.doi.org/10.1016/j.biortech.2015.02.062

A. Patel et al. / Bioresource Technology xxx (2015) xxx–xxx 5

regardless of the fermentability of the carbon source and has beenwidely utilized to synthesize heterologous gene products in S. cere-visiae (Kuroda et al., 1994) They identified three distinct promoterelements within the TDH3 promoter that are controlled by thefermentability of carbon source One is fermentable carbonsource-dependent (UAS), and another is nonfermentable carbonsource-dependent (UAS2) and a fermentable carbon source-depen-dent upstream repression sequence (URS). They showed that thefermentable carbon source dependence of both UASl and the URSis regulated by a ‘‘switching protein,’’ the GPEB. Whether oleagi-nous yeasts have similar promoters and regulated mechanism asof S cerevisiae is still need to be deciphered. Although, more under-standing of molecular mechanism responsible for this enhance-ment would be possible by studying the transcriptionalregulation of genes involved.

3.2. Using Image J to quantify the cell size and lipid droplets (LDs) sizein R. kratochvilovae HIMPA1 grown on different carbon sources

The accumulation of triglycerides occurs in lipid bodies of thecells grown on various carbon sources can be stained specificallywith BODIPY 493/515 nm. This lipophilic bright green fluorescentdye can bind to neutral lipids (TAGs) stored in lipid bodies of R. kra-tochvilovae HIMPA1 (Supplementary data Fig 1). The results of cellsize and lipid droplets were statistically verified and measured usingImage J software (Wong and Franz, 2013). Data presented in Table 1show LDs size (lm) and cell size (lm) of R. kratochvilovae HIMPA1grown on individual fermentable (Glu, Fru, Suc), non-fermentable(Gly, Xyl, Arb) carbon sources and their mixtures (Glu + Gly),(Glu + Fru + Suc), (Gly + Xyl + Arb) and (Glu + Fru + Suc + Gly +Xyl + Arb). A direct correlation of lipid body size with the TAG accu-mulating ability of R. kratochvilovae HIMPA1 was observed as hasbeen reported earlier (Patel et al., 2014). The result demonstratedthe maximum lipid body size (4.35 ± 0.54 lm) on glycerol growncells, which is comparable to lipid body size of fructose(4.03 ± 0.38 lm) and sucrose (4.24 ± 0.45 lm) grown cells thus indi-cating thereby that the maximum TAG accumulation occurs in glyc-erol and lowest in glucose. The cell size (lm) of R. kratochvilovaeHIMPA1 were observed in the following order Suc (6.67 ± 0.34) >Fru (6.32 ± 0.19) > Gly (6.01 ± 0.76) > Xyl (5.67 ± 0.15) > Glu (5.53 ±0.31) > Arb (4.34 ± 0.89). When the mixture of fermenting andnon-fermenting sugar sources (Glu + Gly) were taken together, in-crease in LDs size (4.59 ± 0.56 lm) and cell size (6.53 ± 0.09 lm)was observed. Maximum LDs size (5.35 ± 0.36 lm) and cell size(6.89 ± 0.97 lm) were recorded when the mixture of fermentableand non-fermentable carbon sources (Glu + Fru + Suc + Gly +Xyl + Arb) was provided. Again the synergistic effect in TAG

Table 1Lipid droplets size (lm) and cell size (lm) of R. kratochvilovae HIMPA1 cells measured(Glu + Fru + Suc), (B) non-fermentable (Gly + Xyl + Arb) and (C) mixture of both fermentab

Carbon sources

(A) Fermentable carbon sourcesGlucoseFructoseSucrose

(B) Non-fermentable carbon sourcesGlycerolXyluloseArabinose

(C) Mixture of fermentable and non-fermentable carbon sourcesGlucose + GlycerolGlucose + Fructose + SucroseGlycerol + Arabinose + XyluloseGlycerol + Arabinose + Xylulose + Glucose + Fructose + Sucrose

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accumulation as revealed by increase in LD size on the mixture offermentable and non-fermentable carbon sources (Glu + Fru +Suc + Gly + Xyl + Arb) together confirms previous data obtained fortotal lipid yield in Section 3.1. The mixture containing non-fermentable carbon sources (Gly + Xyl + Arb) had bigger LD size of4.98 ± 0.43 lm than fermentable carbon sources (Glu + Fru + Sur)depicting thereby, better TAG accumulating ability in non-ferment-ing carbon sources than fermenting sources. Data obtained confirmsthe enhancement in maximum TAG accumulation that can be ob-tained from this new strategy of using the combination of ferment-ing and non-fermenting sugars. Since TAG stained with BODIPYexclusively depicts accumulated neutral lipid in the lipid body, themaximum LD size obtained on various sugar and their mixturesconfirms preferential utility of that sugar substrate displaying max-imum accumulation. The strategy of using combination of ferment-ing and non-fermenting sugars sources simultaneously in thisoleaginous yeast is reported for the first time and elucidate en-hanced lipid yield specific TAG accumulation in R. kratochvilovaeHIMPA1. The mechanism of enhanced lipid accumulation onnon-fermentable carbon sources is not yet clearly defined, but dere-pression of several alternate sugar utilizing promoters which get in-duced in the presence of their respective sugar sources could be oneof the probable reason. Galactose, maltose, sucrose, and some otherfermentable carbon sources, as well as oleate, glycerol, acetate orethanol, as non-fermentable carbon sources, can be considered as al-ternative inducers for regulated gene expression, since the genesthat are involved in the particular metabolism are repressed, as longas the preferred carbon source glucose is available (Weinhandl et al.,2014). The shift from a fermentable carbon source, glucose, to a non-fermentable carbon source, glycerol, resulted in a marked reductionin overall translation of mRNAs (Gancedo, 1998). Glycerol is a majorinducer of many promoters (ICL1 and FBP1); prior to actively inducetranscription activation by another inducer, such as ethanol, metha-nol or acetate. The promoter of ICL1, which encodes for isocitratelyase, a key enzyme of the TCA and glyoxylate cycle, enables the cellto grow on non-fermentable carbon sources. It is repressed by glu-cose, derepressed by depletion of glucose and strongly induced byethanol or acetate. The promoter region of FBP1, encoding fruc-tose-1,6-bisphosphatase, is another promoter which is repressedby sugars like glucose (Gancedo, 1998). This also shows a Mig1 bind-ing site in the upstream sequence from �200 to �184 and carries aCat8 and Sip4 recognition site (UAS2) for activation of transcriptionwhen non-fermentable carbon sources (ethanol, acetate, glycerol)are available (Schüller, 2003). S. cerevisiae glycerol kinase (GUT1)is another example of a gene whose expression is mainly inducedby glycerol, but also by ethanol, lactate, acetate or oleate.Complete depletion of glucose is necessary to derepress the

using ImageJ software when grown on different carbon sources; (A) fermentablele and non-fermentable carbon sources (Glu + Fru + Suc + Gly + Xyl + Arb).

Cell size (lm) Lipid droplets size (lm)

5.53 ± 0.31 2.38 ± 0.526.32 ± 0.19 4.03 ± 0.386.67 ± 0.34 4.24 ± 0.45

6.01 ± 0.76 4.35 ± 0.545.67 ± 0.15 3.94 ± 0.124.34 ± 0.39 2.98 ± 0.43

6.53 ± 0.09 4.59 ± 0.566.78 ± 0.23 4.87 ± 0.546.63 ± 0.53 4.98 ± 0.436.89 ± 0.27 5.35 ± 0.36

able and non-fermentable carbon sources enhances TAG accumulation in2015), http://dx.doi.org/10.1016/j.biortech.2015.02.062

Fig. 2. Graph showing sugar consumption of R. kratochvilovae HIMPA1 grown at different time intervals; on Panel I. Fermentable carbon sources (Glu + Fru + Suc), Panel II.Non-fermentable carbon sources (Gly + Xyl + Arb) and Panel III. Mixture of fermentable and non-fermentable carbon sources together (Glu + Fru + Suc + Gly + Xyl + Arb).

6 A. Patel et al. / Bioresource Technology xxx (2015) xxx–xxx

promoter. However, the mechanism of TAG accumulation in the LDsbased on different substrates utilization still needs to be explored.

3.3. Time course of growth chart of R. kratochvilovae HIMPA1 onvarious carbon sources

The time course studies of R. kratochvilovae HIMPA1 grown onvarious carbon sources was measured by using optical density(O.D.). The prominent feature of this data suggested that the sugarmixtures used in this time course experiments does not show anydiauxic shifts on multiple substrates. Data indicated that simulta-neous utilization of mixed carbon sources and lack of cataboliterepression could be due to the indigenous property of thisCAT negative oleaginous yeast R. kratochvilovae HIMPA1(Supplementary Fig. 2). Since most of the organisms utilize prefer-ably glucose over other sugars and do not shift to other sugars untilglucose is consumed, however, simultaneously utilization ofglucose and other sugar sources by microorganisms are rarely ob-served (Gancedo, 1998; Stülke and Hillen, 1999). The microorgan-isms generally metabolize sugars sequentially (first glucose andthen other sugars) since glucose represses other sugar utilizationdepending on intrinsic metabolic pathways (Kim et al., 2010).The major challenges for oleochemical industry is to developnew strains which are capable of simultaneous utilization of bothC5 and C6 sugars and to overcome with the problem of glucose re-pression by modifying the genes involved in glucose signaling,regulatory pathways and expressing the genes which are responsi-ble for transporting secondary sugars and catabolic enzymes.Recently, cellodextrin transporter and an intracellular b-glucosi-dase (BGL) were introduced in host microorganism to accumulatelittle amount of glucose in the medium and to enhance the overallconsumption of cellodextrin and non-glucose sugars (Kim et al.,2012). In this regard, R. kratochvilovae HIMPA1 species is a noveloleaginous yeast that is capable of simultaneous sugar utilization.However, each species have dissimilar property to accumulatelipid and totally dependent on the nature of carbon source provid-ed. In R. toruloides CBS14, C5 carbohydrates were utilized less effi-ciently than glucose for lipid production while it was more efficientfor xylulose than arabinose among C5 carbohydrates. This yeastshowed higher lipid production when mixtures of carbohydrateswere provided in fed-batch cultivation (Wiebe et al., 2012).

Please cite this article in press as: Patel, A., et al. Synergistic effect of fermentoleaginous yeast Rhodosporidium kratochvilovae HIMPA1. Bioresour. Technol. (

3.4. Sugar consumption by R. kratochvilovae HIMPA1

Sugar consumption by R. kratochvilovae HIMPA1 was studied atdifferent time intervals for fermentable (Panel-I), non-fermentable(Panel-II) and mixture of both carbon sources (Panel-III) as shownin Fig. 2. Data revealed individual fermentable and non-fermentable carbon sources were completely consumed after192 h cultivation, but cultivation time was increased to 264 hwhen these carbon sources were given in combination (Fig. 2).Maximum utilization of carbon by R. kratochvilovae HIMPA1 after48 h of cultivation was observed in case of glycerol (43.15%) ascompared to glucose (20.94%). After 120 h of cultivation, the con-sumption of fructose, sucrose, and xylulose were 85.71%, 82.05%and 87.7%, respectively, which was higher than that of the glucose(63.42%) but interestingly the consumption of these carbon sourceswere lower than glucose after 48 h of cultivation. R. kratochvilovaeHIMPA1 showed similar pattern of sugars consumption when allsugars provided in combination (Glu + Fru + Suc + Gly + Xyl + Arb)and the results depicted that all sugar sources were consumedsimultaneously rather than sequentially. Similar observations werealso recorded by Yu et al., 2014 during their studies on co-utiliza-tion of glucose, xylose and cellobiose by the oleaginous yeast C.curvatus. Their results indicated that the consumption of bothxylose and cellobiose was repressed by glucose, while both couldbe simultaneously consumed at similar consumption rates andremained constant at about 0.6 g L�1 h�1. The lipid content of C.curvatus grown on glucose, xylose and cellobiose individually,reached around 40% (w/w) of dry cell weight with no significantdifferences on the sugar mixtures. Co-fermentation of cellobioseand xylose by the oleaginous yeast L. starkeyi, showed simultane-ous utilization of both carbon sources (Gong et al., 2012).

3.5. Fatty acid analysis

The lipid profiles and analysis of fatty acids composition of R.kratochvilovae HIMPA1 were investigated on fermentable, non-fermentable as well as on mixture of both carbon sources. The ana-lysis was done by GC–MS and results were presented in Table 2.When glucose was provided as carbon source to this yeast, the fat-ty acids profile contain mainly myristic acid (C14:0) 1.89 ± 0.11%,pentadecylic acid (C15:0) 0.37 ± 0.09%, palmitic acid (C16:0)3.72 ± 0.21%, oleic acid (C18:1) 15.31 ± 0.1%, linoleic acid (C18:2)

able and non-fermentable carbon sources enhances TAG accumulation in2015), http://dx.doi.org/10.1016/j.biortech.2015.02.062

Table 2Comparison of the total percentage of fatty acid methyl esters (FAMEs) produced by R. kratochvilovae HIMPA1 when grown on different fermentable (Glu + Fru + Suc), and non-f entable carbon sources (Gly + Xyl + Arb) and mixtureof both fermentable and non-fermentable carbon sources (Glu + Fru + Suc + Gly + Xyl + Arb).

Fatty acids (%) Fermentable carbon sources Non-fermentable carbon sources Mixture of fermentable and non-fermentablecarbon sources together

Glucose Fructose Sucrose Glu + Fru + Su Glycerol Xylulose Arabinose Gly + Arb + l Glu + Gly Glu + Fru + Suc + Gly + Arb + Xyl

Saturated Caprylic acid (C8:0) ND ND ND ND ND ND ND ND ND 0.18 ± 0.01Pelargonic acid (C9:0) ND ND ND ND ND ND ND ND ND 0.19 ± 0.02Myristic acid (C14:0) 1.89 ± 0.11 0.58 ± 0.05 0.62 ± 0.12 1.93 ± 0.34 0.57 ± 0.09 0.33 ± 0.10 1.12 ± 0.18 2.47 ± 0.16 ND 14.45 ± 0.00Pentadecylic acid (C15:0) 0.37 ± 0.09 6.41 ± 0.36 ND 3.76 ± 0.45 ND ND ND ND ND 1.68 ± 0.21Palmitic acid (C16:0) 3.72 ± 0.21 ND ND 3.13 ± 0.65 7.70 ± 0.56 7.32 ± 0.21 6.00 ± 0.34 5.34 ± 0.29 2.73 ± 0.26 8.12 ± 0.12Stearic acid (C18:0) ND 2.36 ± 0.46 12.22 ± 0.37 8.89 ± 0.76 ND ND 0.77 ± 0.43 ND ND NDArachidic acid (C20:0) 0.36. ± 0.10 2.85 ± 0.65 24.61 ± 0.87 25.65 ± 0.61 ND 3.41 ± 0.43 2.09 ± 0.34 1.67 ± 0.18 7.26 ± 0.19 15.98 ± 0.48Behenic acid (C22:0) 1.78 ± 0.34 ND ND 2.34 ± 0.53 3.53 ± 0.60 ND ND ND ND 4.84 ± 1.20Lignoceric acid (C24:0) ND ND ND ND ND ND ND ND ND 2.29 ± 0.43

Mono-unsaturated Oleic acid (C18:1) 15.31 ± 0.12 26.24 ± 0.78 0.78 ± 0.17 18.58 ± 0.59 23.83 ± 0.58 34.05 ± 0.15 26.17 ± 0.32 32.43 ± 0.31 2.37 ± 0.65 28.12 ± 0.32

Poly-unsaturated Linoleic acid (C18:2) 3.88 ± 0.32 15.31 ± 0.23 37.15 ± 0.87 22.44 ± 0.31 10.28 ± 1.10 4.24 ± 0.12 7.62 ± 0.10 12.36 ± 0.50 45.34 ± 0.01 3.09 ± 0.41Linolenic acid (C18:3) ND ND ND ND 4.19 ± 0.94 ND ND 4.98 ± 0.21 24.97 ± 0.30 0.71 ± 0.01

ND = Non detectable.

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3.88 ± 0.32%, along with arachidic acid (C20:0) 0.36 ± 0.1%, behenicacid (C22:0) 1.78 ± 0.34%. Additional fatty acid, stearic acid (C18:0)were present in sucrose (2.36 ± 0.46%) and fructose (13.24 ± 0.3%)containing fermentable carbon source as medium respectively.However, palmitic acid (C16:0) was absent in both above mentionedfermentable carbon sources. Mono-unsaturated fatty acids content(%) in this yeast was high in glucose and fructose while in sucrosecontaining medium the content of poly-unsaturated fatty acidswere high. Galafassi et al. (2012) in their studies have shown thatR. graminis produced more saturated fatty acid when grown on glu-cose as compared to glycerol which supports these observations.When combination of glucose and glycerol were given to oleagi-nous yeast R. kratochvilovae HIMPA1, the fatty acid profile contain-ing higher poly-unsaturated fatty acid linolenic acid (C18:3)24.97 ± 0.3% was obtained which showed the high percentages oflinoleic acid (C18:2) 45.34 ± 0.01%. Easterling et al. (2009) reportedsimilar results, when R. glutinis was grown on glycerol along withdextrose as carbon sources, the saturation of fatty acids was lesswhen dextrose was used alone and mixing glycerol with either ofthe other two carbon sources (glucose or xylulose) serves to de-crease the saturation of FAMEs produced by R. glutinis. Earlier stud-ies by Tuller et al., (1999) revealed that the ratio of unsaturated tosaturated fatty acids is enhanced in S. cerevisiae cells grown onnon-fermentable carbon sources such as ethanol or lactate as com-pared to glucose and also results in an increase of mitochondrialcardiolipin. The present data showed that combination of ferment-ing and non-fermenting sugars together enhances the percentageof long chain fatty acids varying from (C14:0, C16:0, C18:1, C18:2,C18:3, C20:0, C22:0, C24:0) which were in lesser amounts in individualcarbon sources.

4. Conclusion

Enhanced total lipid yield of (9.26 ± 0.67 g/l) was obtainedwhen the novel oleaginous yeast R. kratochvilovae HIMPA1 wascultivated on fermentable and non-fermentable carbon sourcemixture showed to be 12% more than glucose alone. Synergisticeffect in TAG accumulation was observed in the LDs having max.size (5.35 ± 0.36 lm) when grown on mix sugars as compare toglucose (2.38 ± 0.52 lm). Consumption of sugars lack diauxic shiftdepicting simultaneous utilization of mix sugars. FAME profilerevealed higher longer chain (C14:0-C24:0) fatty acids. New strategyfor enhancing TAG production in oleaginous yeast reported for thefirst time.

Acknowledgements

Authors are thankful for financial support by the Department ofBiotechnology, Govt. of India, BioCare Programme, DBT SanctionNo. 102/IFD/SAN/3539/2011-2012 (Grant No. DBT-608-BIO) andJRF fellowship to Alok Kumar Patel from University GrantCommission, India (Grant No. 6405-35-044).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2015.02.062.

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