Synergistic effect of fermentable and non-fermentable carbon sources enhances TAG accumulation in oleaginous yeast Rhodosporidium kratochvilovae HIMPA1

Accepted Manuscript Synergistic effect of fermentable and non-fermentable carbon sources enhances TAG accumulation in oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 Alok Patel, Vikas Pruthi, Rajesh P. Singh, Parul A. Pruthi PII: DOI: Reference:

S0960-8524(15)00241-2 http://dx.doi.org/10.1016/j.biortech.2015.02.062 BITE 14635

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

1 December 2014 12 February 2015 13 February 2015

Please cite this article as: Patel, A., Pruthi, V., Singh, R.P., Pruthi, P.A., Synergistic effect of fermentable and nonfermentable carbon sources enhances TAG accumulation in oleaginous yeast Rhodosporidium kratochvilovae HIMPA1, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.02.062

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Synergistic effect of fermentable and non-fermentable carbon sources enhances

TAG

accumulation

in

oleaginous

yeast

Rhodosporidium

kratochvilovae HIMPA1

Alok Patel, Vikas Pruthi, Rajesh P Singh, Parul A Pruthi* Molecular Microbiology Laboratory, Biotechnology Department, 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]

Abstract Novel strategy for enhancing TAG accumulation by simultaneous utilization of fermentable and non-fermentable carbon sources as substrate for cultivation of oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 were undertaken in this investigation. The yeast strain showed direct correlation between the size of lipid bodies, visualized by BODIPY stain (493-515 nm) and TAG accumulation when examined on individual fermenting and non-fermenting carbon sources and their mixtures. Maximum TAG accumulation (µm) 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 the above carbon sources (fermentable and non-fermentable) in equimolar concentration revealed maximum lipid droplet size of 5.35 ± 0.76 µm and cell size of 6.89 ± 0.97 µm. Total lipid content observed in mixed carbon 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. Keywords: Oleaginous; Yeast; fermenting; nonfermenting; TAG; Biodiesel

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1

Introduction

2

Oleaginous yeasts have triggered significant attention as ideal oil producing cell factories that

3

can accumulate neutral lipids in the form of triglycerides in their lipid bodies. These yeasts have

4

emerged as a promising feedstock since they can be easily cultivated and can scale up for large

5

scale production of biodiesel for oleo-chemical industries (Koutinas et al., 2014). The oleagenic

6

property of these yeasts is due to the presence of additional enzymes and alternative metabolic

7

pathway utilizing various cost effective carbon substrates (Ageitos et al., 2011; Papanikolaou and

8

Aggelis, 2011; Yu et al., 2014). They are broadly classified into fermenting and non-fermenting

9

yeasts (Gancedo and Gancedo, 1971). Fermenting yeasts are able to thrive well on glucose,

10

under aerobic conditions are non-oleaginous in nature such as Saccharomyces cerevisiae,

11

Torulopsis salmanticensis and Hansenula anomala, while oleaginous yeast on the other hand,

12

lack the ability to utilize glucose in the absence of oxygen and are referred as non-fermenting

13

yeasts such as Rhodotorula glutinis, R. mucilaginosa, R. gracilis, R. minuta, Sporobolomyces

14

pararoseus and Debaryomyces hansenii. The difference in the two categories is based on

15

catabolite repression observed in the presence of glucose for utilization of alternate sugar carbon

16

sources (galactose, sucrose, maltose, melobiose) as well as non-sugar carbons such as ethanol,

17

lactate, glycerol, acetate and oleate (Gancedo, 1998; Weinhandl et al., 2014). Most of

18

microorganisms have their own carbon catabolite repression (CCR) mechanism, which generally

19

metabolize sugars sequentially (first glucose and then xylose) since glucose represses other sugar

20

utilization and also hinder to utilize mixed sugars obtained from hydrolysis of lignocellulose

21

biomass (Kim et al., 2010). Recent progress in understanding the transcriptional regulation of

22

genes involved in the use of non-fermentable carbon sources showed to get expressed 9-12 times

23

more than fermentable carbon sources utilized by oleaginous yeasts (Turcotte et al., 2010). These

1

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24

results in massive reprogramming of gene expression including genes involved in

25

gluconeogenesis, the glyoxylate cycle, and the tricarboxylic acid cycle (Gancedo, 1998;

26

Weinhandl et al., 2014). Role of Snif1 kinase for activation of gene responsible for utilization of

27

non-fermentable carbon sources indicates networking of several unexplained mechanism of

28

sugar utilization. Classical diauxic shift is usually observed with a shift from a fermentative to a

29

non-fermentative mode of growth by yeast. Researcher have also shown differences in enzyme

30

activities and indicated that isocitrate lyase (ICL1), a key promoter enzyme of the TCA and

31

glyoxylate cycle enables the cell to grow on non-fermentable carbon sources is repressed by

32

glucose and strongly induced by ethanol or acetate (Sch¨oler and Sch¨uller, 1994; Sch¨uller,

33

2003). Similarly, a number of genes require for glycerol uptake (Stl1, GUT1, GUT2) are also up

34

regulated when the cells were grown in the presence of glycerol or ethanol while these genes are

35

repressed in the presence of glucose (Pavlik et al., 1993). Similarly, studies have also indicated

36

that the non-fermenting oleaginous yeasts lack several enzymes like PFK, F1,6DP activity, and

37

metabolize glucose through hexose monophosphate pathway but they possess specific enzyme

38

ATP: citrate lyase (EC 2.3.3.8) which generates acetyl-CoA, the key substrate for fatty acid

39

biosynthesis, in the cytoplasm from citrate. Non oleaginous organisms lack this enzyme (Breuer

40

and Harms, 2006). Recent explorations of screening new oleaginous yeast strains which are

41

capable of simultaneous utilization of both C5 and C6 sugars and can overcome the problem of

42

glucose repression led to the finding of red yeasts Rhodosporidium toruloides and R. glutinis.

43

They can accumulate over 50% of its dry biomass as lipid through simultaneous utilization of

44

mix sugars obtained from lignocellulosic hydrolysates (Yen et al., 2014). Rhodosporidium spp.

45

have shown to produce the highest amount of lipids using a variety of carbon sources, such as

46

molasses, glycerol, and complex carbohydrates like rice and wheat hydrolysates, starch, inulin,

2

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47

laevoglucose, wheat straw, waste oils, animal fats, Jerusalem artichoke hydrolysates, hydrolysate

48

of cassava starch, wastewater and mixtures of glucose, xylose and glycerol (Zhao et al., 2008;

49

Zhan et al., 2013; Yu et al., 2014; Gen et al., 2014). Although co-fermentation of mixed sugars

50

has been intensively investigated for ethanol production but there is vast lacuna in the studies to

51

utilize mixed sugars for microbial lipid production from oleaginous microorganisms (Yu et al.,

52

2014). Few reports on mix sugar utilization showed that growth on dextrose can be enhanced

53

through the simultaneous addition of glycerol in R. glutinis (Easterling et al., 2009). Similarly,

54

simultaneous utilization was also observed when R toruloides was grown in combination of

55

glycerol, dextrose and xylose (Wiebe et al., 2012). But oleaginous yeasts L. starkeyi and T.

56

fermentans utilizes various carbon sources and their mixture in a sequential manner (Tapia et al.,

57

2012; Sha, 2013; Abghari and Chen 2014). During this investigation, the ability of novel

58

oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 (Acc. No. KF772881) to grow on the

59

wide variety of sugar sources (fermentable and non-fermentable sugars and their mixtures) for

60

obtaining maximum TAG accumulation was done. The variation in lipid body size in R.

61

kratochvilovae HIMPA1 cultivated on different sugar sources were monitored by BODIPY

62

staining and imaged using new live in- vivo fluorescence microscopy technique (Patel et al.,

63

2014). The amount of dry cell weight (g/l), total lipid yield (g/l) and lipid content (%) produced

64

were estimated and its FAME profile was determined using GC-MS analysis. Based on the

65

above facts, a correlation of enhanced TAG accumulation with increase in lipid body size was

66

elucidated in the cells grown on a mixture of fermentable and non-fermentable sugar sources

67

together as compared to glucose alone.

68

2. Methods

69

2.1. Materials 3

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70

Analytical grade solvents viz., n-hexane, chloroform, and methanol were purchased from Merck

71

Ltd., Mumbai, India. Sugars (glucose, glycerol, sucroce, fructose, xylulose, arabinose) and other

72

chemicals (NaCl, KCl, anhydrous sodium sulphate, 14% methanolic BF3) were percured from

73

HiMedia laboratories, Mumbai, India. FAME standard (AOCS low erucic rape seed oil O7756-

74

1AMP) for GC–MS analysis and standard for TLC (Triolein) were obtained from Sigma Aldrich

75

(St. Louis, USA). BODIPY 493/515 (4,4-difluro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-

76

indacene) was purchased from Invitrogen (Life Technology, USA) for fluroscence staining.

77

2.2. Yeast strain and culture conditions

78

R. kratochvilovae HIMPA1 (Genebank Acc. No. KF772881) was cultivated at 30 ºC on a rotary

79

shaker at 200 rpm for 48 h using YEPD broth as reported earlier (Patel et al., 2014). To obtain

80

the seed culture, cells were harvested by centrifugation, washed twice with sterilized distilled

81

water and resuspended in 0.9% sterilized saline to attain cell density of 6.5-7.8 × 108 cells/ml.

82

Batch cultivations of R. kratochvilovae HIMPA1 for TAG accumulation were performed in 250

83

ml Erlenmeyer flasks where all fermenting (glucose, fructose, and sucrose), non-fermenting

84

(glycerol, xylulose, and arabinose) carbon sources and mixture of both of them were

85

standardized in moles of carbon equivalents relating to 70 g /l glucose (388 mM), mixed with

86

salt solution (g/l) :- KH2PO4, 1; MgSO4, 0.5; (NH4)2SO4, 1; CaCl2, 0.1; trace elements (mg/l) :-

87

boric acid, 0.5; CuSO4, 0.04; KI, 0.1; FeCl3, 0.2; MnSO4, 0.4; NaMO3, 0.2; ZnSO4, 0.4; vitamin

88

solution (mg/l) :- D-Biotin, 0.002; calcium pantothenate, 0.4; folic acid, 0.002; inositol, 2; niacin,

89

0.4; PABA, 0.2; pyridoxine HCl, 0.4; riboflavin, 0.2; thiamine, 0.4. All experiments were done

90

in triplicate.

91

2.3. Schematic procedure for fluorescence microscopy analysis

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92

For fluorescence microscopy BODIPY stock solution was prepared by adding 0.1 mg/ml with

93

DMSO (Govender et al., 2012). Initially, 10 µl of the culture broth of R. kratochvilovae grown

94

at different time intervals was pelleted and washed with 0.9% saline water. After washing, the

95

cells were suspended in 10 µl of 0.9% saline to obtain appropriate cell concentration (1 × 106

96

cells/ml). 2 µl (2 × 10-3 µg /µl) of BODIPY stock solution was then added to it and mixed well.

97

After five minutes of incubation in dark the cell suspension was washed with 0.9% saline water.

98

Fluorescence images were viewed on a digital inverted fluorescence microscope equipped with

99

EVOS GFP light cube (EVOS- FL, Advance Microscopy Group, AMG, USA) (Patel et al.,

100

2014).

101

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

102

The size of cells and intracellular LDs were analyzed by ImageJ 1.48a software. Images obtained

103

after fluorescence microscopy containing the fluorescence channel were converted to a Z-

104

projection in ImageJ (Wong and Franz, 2013). The obtained image was a color image (RGB); it

105

was necessary to split multi-color images into single channels and converted single channel color

106

images to grayscale before proceeding. The image created as binary images for threshold to

107

highlight all of the structures that is required to measure. Watershed programme is used for

108

separating two nearby lipid droplets by 1 pixel thick line. Using straight tool in ImageJ two lines

109

were manually drawn which bisect the cell and each fluorescent LD followed by adding the

110

region of interest ROI manager. Each line represents the diameter of the cell and LD. The length

111

and width of LDs were measured for droplets that show a spherical geometry.100 cells were

112

counted to calculate the average size of cells and intracellular LDs, which was presented as mean

113

± S.D.

114

2.5. Dry cell weight determination 5

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115

The yeast cells growth were observed by measuring cell optical density (OD) at 600 nm. Cell

116

biomass, expressed as dry cell weight (DCW) was harvested by centrifuging it at 3000 rpm for 5

117

min and its weight was determined gravimetrically after drying the cells in an oven at 105 ˚C for

118

12 h (Patel et al., 2014).

119

2.6. Lipid extraction and gravimetric determination

120

The total cellular lipid was extracted using modified protocol of Bligh and Dyer (1959). Firstly,

121

dried samples were mixed with distilled water and mixed vigorously for 15 seconds. Cell

122

suspension was subjected to sonication at 40 Hz for 5 min and after that 10 ml of chloroform:

123

methanol (2:1; v/v) was added to the suspension and stirred for 30 min. The extract was filtered

124

with sintered glass funnel. 5 ml of 0.034% MgCl2 was added to the extract and centrifuged at

125

3,000 rpm for 5 minutes. Upper aqueous layer was aspirated and washed the organic phase with

126

1ml of 2N KCl/methanol (4:1; v/v). After washing suspension was centrifuged again at 3000 rpm

127

for 5 minutes and washed repeatedly with 5 ml of upper phase (chloroform/methanol/water;

128

3:48:47; v/v) until the phase boundary becomes clear. The bottom chloroform layer was

129

transferred to a new screw cap test tube, and the lipid yield was determined gravimetrically. The

130

lipid yield is the amount of lipid extracted from the cells per litre of the fermentation medium

131

(g/l) while the lipid content Y (%), was calculated using the following equation: Y = WL/DCW

132

× 100. Where WL is the weight of the total lipid obtained gravimetrically and DCW is the weight

133

of the cell biomass.

134

2.7. Transesterification of fatty acids by methanolic BF3 and determination of FAMEs

135

composition by GC–MS

136

Transesterification of extracted yeast lipid samples was done using the method of Morrison and

137

Smith (1964). Briefly, fatty acid samples dried with anhydrous sodium sulphate were evaporated

6

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138

to dryness under nitrogen in a Teflon coated screw cap tube. Boron fluoride–methanol reagent

139

was added under nitrogen, in the proportions 1 ml reagent per 4–16 mg of lipid. The samples

140

were then heated at 80 ˚C for 20 min in a boiling water bath. The esters were extracted by adding

141

2 volumes of n-hexane and then 1 volume of water followed by centrifugation at 3500g until

142

both layers were clearly visible. The products of transesterification were analyzed by GC-MS

143

(Agilent, Santa Clara, CA, USA). The column used was a capillary column (DB- 5MS) with

144

dimensions 30 m × 0.25mm ID and 0.25 µm film thickness. The sample was injected in splitless

145

injection mode (1 µl at 250 °C) using helium as a carrier gas (1 ml/min). The column

146

temperature was initially set at 50 °C (1.5 min) where, after the temperature was ramped to 180

147

°C (25 °C/min) for 1 min, followed by a further increased to 220 °C (10 °C/min), it was held for

148

1 min. Finally, the temperature was increased to 250 ° C (15 °C/min) and held for 3 min. The

149

mass transfer line and ion source were set at 250 °C and 200 °C, respectively. The FAMEs were

150

detected with electron ionization (70 eV) in scan mode (50–600m/z).

151

2.8. Determination of sugar

152

The amount of residual sugars in the samples were determined by HPLC equipped with an

153

Aminex HPX-87H column (Silva et al., 2011). The amount of sugar consumption (%) were

154

determined as C = St0 – St / St0 × 100, where C is the amount of sugar consumption, St0 is the

155

amount of initial sugar (g/l) added and St is the residual sugar left at each sampling time.

156

2.9. TLC analysis for neutral lipid determination

157

Triacylglycerol (TAG) analysis in the extracted total lipid was carried out by TLC using 0.25-

158

mm-thick silica gel G-60 F254 plates (Merck, India) and chromatograms were developed in

159

hexane: diethyl ether: acetic acid (85:15:1, v/v/v) with triolein as standard. For quantification of

160

SEs and TAGs, plates were dipped into methanolic MnCl2 solution (0.63 g MnCl2 · 4H2O, 60 ml 7

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161

water, 60 ml methanol and 4 ml concentrated sulfuric acid), dried and heated at 120 °C for 15

162

min (Fei et al., 2009). Conversion of TAG into FAMEs was detected by double development of

163

TLC. The plate was firstly developed to 2.5 cm from the origin with hexane: tert-butyl methyl

164

ether: acetic acid (50:50:0.5, v/v/v), and after air dried, redeveloped to 8 cm from the origin with

165

hexane: tert-butyl methyl ether: acetic acid (97:3:0.5, v/v/v) as a developing agent. The samples

166

were visualized by spraying sulphuric acid 50% (w/w) and then heating at 135 °C (Patel et al.,

167

2014).

168

3. Results and Discussion

169

3.1. The effect of fermentable, non-fermentable and mixture of both fermentable and non-

170

fermentable carbon sources

171

HIMPA1

172

R. kratochvilovae HIMPA1 showed a wide range of carbon source utilization when grown on

173

fermentable glucose (Glu), fructose (Fru) and sucrose (Suc), non-fermentable glycerol (Gly),

174

arabinose (Arb) and xylulose (Xyl) as well as on mixture of both carbon sources. All individual

175

sources of carbon for each batch experiments were provided in moles of carbon equivalents

176

relating to 70 g /l glucose (388 mM) while mixture of both fermentable and non-fermentable

177

carbon sources were provided in equal proportion of 64.75 mM to make it equivalent to 388mM.

178

The results of all batch experiments for cell dry weight (CDW) in g/l, total lipid yield (g/l) and

179

lipid content (%) were presented in Figure 1. The growth of R. kratochvilovae HIMPA1 on

180

individual carbon sources resulted in the highest CDW of 14.15 ± 0.78 g/l on glycerol while it

181

was 13.26 ± 0.98 g/l, 13.22 ± 0.54g/l, 13.38 ± 0.56 g/l for Glu, Fru and Suc respectively, which

182

were higher than pentose sugars Xyl (6.9 ± 0.43 g/l) and Arb (7.64 ± 0.76 g/l) used. The CDW

183

results obtained on mixture of Fermentable (Glu, Fru and Suc) and nonfermentable carbon

on the growth and lipid production of R. kratochvilovae

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184

sources were (Gly, Arb and Xyl) 13.35 ± 0.92 g/l and 13.58 ± 0.71 g/l, respectively, while the

185

mixture of all fermenting and non-fermenting carbon sources together had highest CDW of 15.56

186

± 0.54 g/l comparable to the mixture of Glu + Gly having 14.75 ± 0.78 g/l. The total lipid yields

187

measured gravimetrically showed maximum value of 9.26 ± 0.54 g/l on mixture of both

188

fermentable (Glu + Fru + Suc) and non-fermentable (Gly + Xyl + Arb) carbon sources together.

189

While on individual carbon the total lipid yields were 8.0 ± 0.65 > 7.72 ± 0.87 > 7.5 ± 0.43 > 6.2

190

± 0.58 > 4.0 ± 0.98 > 3.84 ± 0.32 g/l respectively in the following order Gly > Suc > Fru > Glu >

191

Arb > Xyl. Data showed that the total lipid yields (7.99 ± 0.79 g/l) on non-fermentable (Gly +

192

Xyl + Arb) sources were more than fermentable carbon sources (Glu + Fru + Suc) 7.14 ± 0.71

193

g/l, while it was higher in the mixture of both Glu and Gly (8.45 ± 0.49 g/l). The lipid content

194

(59.51 ± 0.59%) obtained from the mixture of fermenting and non-fermenting carbon sources

195

together utilized by R. kratochvilovae HIMPA1 clearly indicates the synergistic effect in the lipid

196

accumulating property. Similarly, mixture containing Gly + Xyl + Arb recorded high lipid

197

content (58.83 ± 0.75%) while on Gly + Glu lipid content was 57.28 ± 0.35% respectively. The

198

lipid content of R. kratochvilovae HIMPA1 observed for individual carbon sources were 57.69 ±

199

0.71 > 56.73 ± 0.34 > 56.6 ± 0.69 > 55.65 ± 0.38 > 52.35 ± 0.88 > 46.76 ± 0.61 % in the

200

following order Suc > Fru > Gly > Xyl > Arb > Glu, respectively. The sugar mixtures of non-

201

fermentable carbon sources (Gly + Xyl + Arb) together have lipid content of 58.83 ± 0.75%.

202

These values are 10-12% higher than that of glucose as sole carbon source (46.76 ± 0.61%),

203

which enhances lipid accumulation in above carbon source (Gly + Xyl + Arb) to 1.38 fold when

204

compared with glucose. Interestingly, the mixture containing both fermentable and non-

205

fermentable carbon sources together also had 12-13% higher lipid content which is 1.2 times

206

higher than glucose grown cells (Figure 1). The maximum cell dry weight (15.56 ± 0.54 g/l) and

9

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207

lipid yield (9.26 ± 0.67 g/l) were observed when combinations of fermentable and non-

208

fermentable carbon sources were used. The present investigation suggested that when

209

fermentable carbon sources were mixed with non-fermentable carbon sources, it leads to increase

210

in both cell dry weight and total lipid yield of oleaginous yeast R. kratochvilovae HIMPA1

211

which support the co-utilization of sugars simultaneously. The combination of both types of

212

mixtures would probably trigger multiple pathways for sugar uptake that enhances maximum

213

lipid accumulation as has been supported by earlier reports (Turcotte et al., 2010). Earlier,

214

reports on production of biodiesel from simultaneous utilization of mixtures of glycerol in

215

combination with glucose or xylose as a growth substrate to produce TAG correlates with the

216

data obtained. Easterling et al., 2009 demonstrated that the oleaginous yeast R. glutinis cultured

217

on medium containing dextrose, xylose, glycerol, dextrose and xylose, xylose and glycerol, or

218

dextrose and glycerol accumulated 16, 12, 25, 10, 21, and 34% TAG on a dry cell weight basis,

219

respectively. They further supports simultaneous utilization of Glu and Gly enhances TAG

220

accumulation and glycerol grown R. glutinis accumulates more lipid under these experimental

221

conditions than dextrose grown and xylose grown cultures. They also observed that when R.

222

glutinis was grown on dextrose plus glycerol, the monounsaturated fatty acids content in total

223

fatty acids were increased when compared to cells grown on dextrose or glycerol individually.

224

But the addition of glycerol as a carbon source along with dextrose or xylose resulted in a

225

decrease in the amount of saturated fatty acids. Studies reveals that the capability of

226

simultaneous sugar utilization greatly varies with different strains and also depends on culture

227

conditions. Gong et al., 2012 demonstrated that Lipomyces starkeyi simultaneously utilized

228

cellobiose and xylose at their mass ratios of 2:1 and 1:1. Yu et al., 2014 demonstrated that the

229

cell biomass and lipid production on the different sugar mixtures of glucose/xylose,

10

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230

glucose/cellobiose and cellobiose/xylose were similar in Cryptococcus curvatus. They found an

231

effective approach for alleviating glucose repression for microbial lipid production by C.

232

curvatus through xylose/cellobiose co-utilization. According to Sha, 2013, the oleaginous yeast

233

Y. lipolytica produced more biomass and lipid when grown on the mixture of glucose and xylose.

234

In the case of non-oleaginous yeast Saccharomyces cerevisiae preferentially utilize glucose as

235

sole carbon source but in the absence of this fermentable carbon source it has been shifted to

236

utilize other variety of non- fermentable sugar source (glycerol) and suppressed the

237

transcriptional expression of genes responsible for utilization of these carbon sources but in

238

oleaginous yeast both the pathways of sugar utilization were simultaneously activated (Turcotte

239

et. al., 2010). Since most microbes possess carbon catabolite repression (CCR), mixed sugars

240

derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall

241

process. Contrary to this R. kratochvilovae HIMPA1 exhibit the simultaneous utilization of

242

mixed sugars and combination of fermentable and non-fermentable sugar mixtures have showed

243

enhanced total lipid production as compare to individual sugars utilization (figure 1). An earlier

244

report supports this foundation based on presence of two independent promoter elements present

245

in the triosephosphate dehydrogenase 3 gene (TDH3) which is a glycolytic enzyme gene and is

246

abundantly transcribed in Saccharomyces cerevisiae. The promoter region of the TDH3 gene is

247

known to exhibit high transcription activity regardless of the fermentability of the carbon source

248

and has been widely utilized to synthesize heterologous gene products in S. cerevisiae (Kurada et

249

al., 1994) They identified three distinct promoter elements within the TDH3 promoter that are

250

controlled by the fermentability of carbon source One is fermentable carbon source-dependent

251

(UAS), and another is nonfermentable carbon source-dependent (UAS2) and a fermentable

252

carbon source-dependent upstream repression sequence (URS). They showed that the

11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

253

fermentable carbon source dependence of both UASl and the URS is regulated by a “switching

254

protein,” the GPEB. Whether oleaginous yeast have similar promoters and regulated mechanism

255

as of S cerevisiae is still need to be deciphered. Although, more understanding of molecular

256

mechanism responsible for this enhancement would be possible by studying the transcriptional

257

regulation of genes involved.

258

3.2. Using Image J to quantify the cell size and lipid droplets (LDs) size in R. kratochvilovae

259

HIMPA1 grown on different carbon sources

260

The accumulation of triglycerides occurs in lipid bodies of the cells grown on various carbon

261

sources can be stained specifically with BODIPY 493/515nm. This lipophilic bright green

262

fluorescent dye can bind to neutral lipids (TAGs) stored in lipid bodies of R. kratochvilovae

263

HIMPA1 (supplementary data Fig 1). The results of cell size and lipid droplets were statistically

264

verified and measured using Image J software (Wong and Franz, 2013). Data presented in table 1

265

show LDs size (µm) and cell size (µm) of R. kratochvilovae HIMPA1 grown on individual

266

fermentable (Glu, Fru, Suc), non-fermentable (Gly, Xyl, Arb) carbon sources and their

267

mixtures(Glu + Gly), (Glu + Fru + Suc), (Gly + Xyl + Arb) and (Glu + Fru + Suc+ Gly + Xyl +

268

Arb). A direct correlation of lipid body size with the TAG accumulating ability of R.

269

kratochvilovae HIMPA1 was observed as has been reported earlier (Patel et al., 2014). The result

270

demonstrated the maximum lipid body size (4.35 ± 0.54 µm) on glycerol grown cells, which is

271

comparable to lipid body size of fructose (4.03 ± 0.38 µm) and sucrose (4.24 ± 0.45 µm) grown

272

cells thus indicating thereby that the maximum TAG accumulation occurs in glycerol and lowest

273

in glucose. The cell size (µm) of R. kratochvilovae HIMPA1 were observed in the following

274

order Suc (6.67 ± 0.34) > Fru (6.32 ± 0.19) > Gly (6.01 ± 0.76) > Xyl (5.67 ± 0.15) > Glu (5.53 ±

275

0.31) > Arb (4.34 ± 0.89). When the mixture of fermenting and non-fermenting sugar sources 12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

276

(Glu + Gly) were taken together, increase in LDs size (4.59 ± 0.56 µm) and cell size (6.53 ± 0.09

277

µm) was observed. Maximum LDs size (5.35 ± 0.36 µm) and cell size (6.89 ± 0.97 µm) were

278

recorded when the mixture of fermentable and non-fermentable carbon sources (Glu + Fru + Suc

279

+ Gly + Xyl + Arb) was provided. Again the synergistic effect in TAG accumulation as revealed

280

by increase in LD size on the mixture of fermentable and non-fermentable carbon sources (Glu +

281

Fru + Suc + Gly + Xyl + Arb) together confirms previous data obtained for total lipid yield in

282

Section 3.1. The mixture containing non-fermentable carbon sources (Gly + Xyl + Arb) had

283

bigger LD size of 4.98 ± 0.43 µm than fermentable carbon sources (Glu + Fru + Sur) depicting

284

thereby, better TAG accumulating ability in non-fermenting carbon sources than fermenting

285

sources. Data obtained confirms the enhancement in maximum TAG accumulation that can be

286

obtained from this new strategy of using the combination of fermenting and non-fermenting

287

sugars. Since TAG stained with BODIPY exclusively depicts accumulated neutral lipid in the

288

lipid body, the maximum LD size obtained on various sugar and their mixtures confirms

289

preferential utility of that sugar substrate displaying maximum accumulation. The strategy of

290

using combination of fermenting and non-fermenting sugars sources simultaneously in this

291

oleaginous yeast is reported for the first time and elucidate enhanced lipid yield specific TAG

292

accumulation in R. kratochvilovae HIMPA1. The mechanism of enhanced lipid accumulation on

293

non-fermentable carbon sources is not yet clearly defined, but derepression of several alternate

294

sugar utilizing promoters which get induced in the presence of their respective sugar sources

295

could be one of the probable reason. Galactose, maltose, sucrose, and some other fermentable

296

carbon sources, as well as oleate, glycerol, acetate or ethanol, as non-fermentable carbon sources,

297

can be considered as alternative inducers for regulated gene expression, since the genes that are

298

involved in the particular metabolism are repressed, as long as the preferred carbon source

13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

299

glucose is available (Weinhandl et al., 2014). The shift from a fermentable carbon source,

300

glucose, to a non-fermentable carbon source, glycerol, resulted in a marked reduction in overall

301

translation of mRNAs (Gancedo, 1998). Glycerol is a major inducer of many promoters (ICL1

302

and FBP1); prior to actively induce transcription activation by another inducer, such as ethanol,

303

methanol or acetate. The promoter of ICL1, which encodes for isocitrate lyase, a key enzyme of

304

the TCA and glyoxylate cycle, enables the cell to grow on non-fermentable carbon sources. It is

305

repressed by glucose, derepressed by depletion of glucose and strongly induced by ethanol or

306

acetate. The promoter region of FBP1, encoding fructose-1,6-bisphosphatase, is another

307

promoter which is repressed by sugars like glucose (Gancedo, 1998). This also shows a Mig1

308

binding site in the upstream sequence from -200 to -184 and carries a Cat8 and Sip4 recognition

309

site (UAS2) for activation of transcription when non-fermentable carbon sources (ethanol,

310

acetate, glycerol) are available (Schüller, 2003). S. cerevisiae glycerol kinase (GUT1) is another

311

example of a gene whose expression is mainly induced by glycerol, but also by ethanol, lactate,

312

acetate or oleate. Complete depletion of glucose is necessary to derepress the promoter.

313

However, the mechanism of TAG accumulation in the LDs based on different substrates

314

utilization still needs to be explored

315

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

316

The time course studies of R. kratochvilovae HIMPA1 grown on various carbon sources was

317

measured by using optical density (O.D). The prominent feature of this data suggested that the

318

sugar mixtures used in this time course experiments does not show any diauxic shifts on multiple

319

substrates. Data indicated that simultaneous utilization of mixed carbon sources and lack of

320

catabolite repression could be due to the indigenous property of this CAT negative oleaginous

321

yeast R. kratochvilovae HIMPA1(Supplementary Fig. 2). Since most of the organisms utilize 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

322

preferably glucose over other sugars and do not shift to other sugars until glucose is consumed,

323

however, simultaneously utilization of glucose and other sugar sources by microorganisms are

324

rarely observed (Gancedo, 1998; Stulke and Hillen, 1999). The microorganisms generally

325

metabolize sugars sequentially (first glucose and then other sugars) since glucose represses other

326

sugar utilization depending on intrinsic metabolic pathways (Kim et al., 2010). The major

327

challenges for olechemical industry is to develop new strains which are capable of simultaneous

328

utilization of both C5 and C6 sugars and to overcome with the problem of glucose repression by

329

modifying the genes involved in glucose signaling, regulatory pathways and expressing the

330

genes which are responsible for transporting secondary sugars and catabolic enzymes. Recently,

331

cellodextrin transporter and an intracellular b-glucosidase (BGL) were introduced in host

332

microorganism to accumulate little amount of glucose in the medium and to enhance the overall

333

consumption of cellodextrin and non-glucose sugars (Kim et al., 2012). In this regard, R.

334

kratochvilovae HIMPA1 species is a novel oleaginous yeast that is capable of simultaneous sugar

335

utilization. However, each species have dissimilar property to accumulate lipid and totally

336

dependent on the nature of carbon source provided. In R. toruloides CBS14, C5 carbohydrates

337

were utilized less efficiently than glucose for lipid production while it was more efficient for

338

xylulose than arabinose among C5 carbohydrates. This yeast showed higher lipid production

339

when mixtures of carbohydrates were provided in fed-batch cultivation (Wiebe et al., 2012).

340

3.4. Sugar Consumption by R. kratochvilovae HIMPA1

341

Sugar consumption by R. kratochvilovae HIMPA1 was studied at different time intervals for

342

fermentable (Panel-I), non-fermentable (Panel-II) and mixture of both carbon sources (Panel-III)

343

as shown in Figure 2. Data revealed individual fermentable and non-fermentable carbon sources

344

were completely consumed after 192 h cultivation, but cultivation time was increased to 264 h 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

345

when these carbon sources were given in combination (Figure 2). Maximum utilization of

346

carbon by R. kratochvilovae HIMPA1 after 48 h of cultivation was observed in case of glycerol

347

(43.15%) as compared to glucose (20.94%). After 120 h of cultivation, the consumption of

348

fructose, sucrose, and xylulose were 85.71%, 82.05% and 87.7% respectively, which was higher

349

than that of the glucose (63.42%) but interestingly the consumption of these carbon sources were

350

lower than glucose after 48 h of cultivation. R. kratochvilovae HIMPA1 showed similar pattern

351

of sugars consumption when all sugars provided in combination (Glu + Fru + Suc + Gly + Xyl +

352

Arb) and the results depicted that all sugar sources were consumed simultaneously rather than

353

sequentially. Similar observation were also recorded by Yu et al., 2014 during their studies on

354

co-utilization of glucose, xylose and cellobiose by the oleaginous yeast Cryptococcus curvatus.

355

Their results indicated that the consumption of both xylose and cellobiose was repressed by

356

glucose, while both could be simultaneously consumed at similar consumption rates and

357

remained constant at about 0.6 g L-1h-1. The lipid content of C. curvatus grown on glucose,

358

xylose and cellobiose individually, reached around 40% (w/w) of dry cell weight with no

359

significant differences on the sugar mixtures. Co-fermentation of cellobiose and xylose by the

360

oleaginous yeast Lipomyces starkeyi, showed simultaneous utilization of both carbon sources

361

(Gong et al., 2014).

362

3.5. Fatty acid analysis

363

The lipid profiles and analysis of fatty acids composition of R. kratochvilovae HIMPA1 were

364

investigated on fermentable, non-fermentable as well as on mixture of both carbon sources. The

365

analysis was done by GC-MS and results were presented in Table 2. When glucose was provided

366

as carbon source to this yeast, the fatty acids profile contain mainly myristic acid (C14:0) 1.89 ±

367

0.11%, pentadecylic acid (C15:0) 0.37 ± 0.09%, palmitic acid (C16:0) 3.72 ± 0.21%, oleic acid 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

368

(C18:1) 15.31 ± 0.1%, linoleic acid (C18:2) 3.88 ± 0.32%, along with arachidic acid (C20:0) 0.36 ±

369

0.1%, behenic acid (C22:0) 1.78 ± 0.34%. Additional fatty acid, stearic acid (C18:0) were present in

370

sucrose (2.36 ± 0.46%) and fructose (13.24 ± 0.3%) containing fermentable carbon source as

371

medium respectively. However, palmitic acid (C16:0) was absent in both above mentioned

372

fermentable carbon sources. Mono-unsaturated fatty acids content (%) in this yeast was high in

373

glucose and fructose while in sucrose containing medium the content of poly-unsaturated fatty

374

acids were high. Galafassi et al., 2012 in their studies have shown that R. graminis produced

375

more saturated fatty acid when grown on glucose as compared to glycerol which supports these

376

observations. When combination of glucose and glycerol were given to oleaginous yeast R.

377

kratochvilovae HIMPA1, the fatty acid profile containing higher poly-unsaturated fatty acid

378

linolenic acid (C18:3) 24.97 ± 0.3% was obtained which showed the high percentages of linoleic

379

acid (C18:2) 45.34 ± 0.01%. Easterling et al., 2009 reported similar results, when R. glutinis was

380

grown on glycerol along with dextrose as carbon sources, the saturation of fatty acids was less

381

when dextrose was used alone and mixing glycerol with either of the other two carbon sources

382

(glucose or xylulose) serves to decrease the saturation of FAMEs produced by R. glutinis. The

383

results showed that combination of fermenting and non-fermenting sugars together enhances the

384

percentage of individual fatty acids varying from (C14:0, C16:0 , C18:1, C18:2, C18:3 , C20:0, C22:0,

385

C24:0) which were in lesser amounts in individual carbon sources.

386

Conclusion

387

Enhanced total lipid yield of (9.26 ± 0.67 g/l) was obtained when the novel oleaginous yeast R.

388

kratochvilovae HIMPA1 was cultivated on fermentable and non-fermentable carbon source

389

mixture showed to be 12% more than glucose alone. Synergistic effect in TAG accumulation

390

was observed in the LDs having max. size (5.35 ± 0.36 µm) when grown on mix sugars as

17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

391

compare to glucose (2.38 ± 0.52µm). Consumption of sugars lack diauxic shift depicting

392

simultaneous utilization of mix sugars. FAME profile revealed higher longer chain (C14:0-

393

C24:0) fatty acids. New strategy for enhancing TAG production in oleaginous yeast reported for

394

the first time.

395

Supplementary data

396

Figure 1. Fluorescence microscopic images of Rhodosporidium kratochvilovae HIMPA1 live

397

cells stained with BODIPY 493/515 nm showing TAG accumulated in LDs and cell size was

398

measured using ImageJ software when grown on (Panel: I) fermentable (Glu + Fru + Suc),

399

(Panel: II) non-fermentable (Gly + Xyl + Arb) and (Panel: III) mixture of both fermentable

400

and non-fermentable carbon sources (Glu + Fru + Suc+Gly + Xyl + Arb).

401

Figure 2. Optical density measurements (OD600nm) of R. kratochvilovae HIMPA1 while

402

grown on different fermentable (Glu + Fru + Suc), non-fermentable (Gly + Xyl + Arb) and

403

mixture of both fermentable and non-fermentable carbon sources (Glu + Fru + Suc+Gly +

404

Xyl + Arb).

405

Acknowledgement

406

Authors are thankful for financial support by the Department of Biotechnology, Govt. of India,

407

BioCare Programme, DBT Sanction No.: 102/IFD/SAN/3539/2011-2012 (Grant No. : DBT-608-

408

BIO) and JRF fellowship to Alok Kumar Patel from University Grant Commission, India (Grant

409

No. : 6405-35-044).

410

Legends to figures and table.

411

Figure 1. Graph showing dry cell weight (g/l), total lipid yield (g/l) and lipid content (%) of

412

Rhodosporidium kratochvilovae HIMPA1 grown on fermentable (Glu + Fru + Suc), non-

18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

413

fermentable (Gly + Xyl + Arb), and mixture of both fermentable and non-fermentable carbon

414

sources together (Glu + Fru + Suc+Gly + Xyl + Arb).

415

Figure 2. Graph showing sugar consumption of R. kratochvilovae HIMPA1 grown at different

416

time intervals; on Panel I. Fermentable carbon sources (Glu + Fru + Suc), Panel II. Non-

417

fermentable carbon sources (Gly + Xyl + Arb) and Panel III. Mixture of fermentable and non-

418

fermentable carbon sources together (Glu + Fru + Suc+Gly + Xyl + Arb).

419

Table 1. Lipid droplets size (µm) and cell size (µm) of R. kratochvilovae HIMPA1 cells

420

measured using ImageJ software when grown on different carbon sources; (A) fermentable (Glu

421

+ Fru + Suc), (B) non-fermentable (Gly + Xyl + Arb) and (C) mixture of both fermentable and

422

non-fermentable carbon sources(Glu + Fru + Suc+Gly + Xyl + Arb).

423

Table 2. Comparison of the total percentage of fatty acid methyl esters (FAMEs) produced by

424

R. kratochvilovae HIMPA1 when grown on different fermentable (Glu + Fru + Suc), and non-

425

fermentable carbon sources (Gly + Xyl + Arb) and mixture of both fermentable and non-

426

fermentable carbon sources(Glu + Fru + Suc+Gly + Xyl + Arb).

427 428

References;

429

1. Abghari, A., Chen, S., 2014. Yarrowia lipolytica as an Oleaginous Cell Factory Platform

430

for Production of Fatty Acid-Based Biofuel and Bioproducts. Front. Energy Res. 2, 1–21.

431

doi:10.3389/fenrg.2014.00021

432

2. Ageitos, J.M., Vallejo, J.A., Veiga-Crespo, P., Villa, T.G., 2011. Oily yeasts as

433

oleaginous cell factories. Appl. Microbiol. Biotechnol. 90, 1219–27. doi:10.1007/s00253-

434

011-3200-z

19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

435 436 437 438

3. Bligh, E. G., Dyer, W. J., 1959. A rapid method for total lipid extraction and purification. Can. J. Biochem Phys. 37, 911–917. 4. Breuer, U., Harms, H., 2006. Debaryomyces hansenii—an extremophilic yeast with biotechnological potential. Yeast 415–437. doi:10.1002/yea.1374

439

5. Easterling, E.R., French, W.T., Hernandez, R., Licha, M., 2009. The effect of glycerol as

440

a sole and secondary substrate on the growth and fatty acid composition of Rhodotorula

441

glutinis. Bioresour. Technol. 100, 356–61. doi:10.1016/j.biortech.2008.05.030

442

6. Fei, W., Wang, H., Fu, X., Bielby, C., Yang, H., 2009. Conditions of endoplasmic

443

reticulum stress stimulate lipid droplet formation in Saccharomyces cerevisiae. Biochem.

444

J. 424, 61–7.

445 446

7. Gancedo, J., 1998. Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev. 62, 334–361

447

8. Gancedo, J., Gancedo, C., 1971. Fructose-1, 6-diphosphatase, phosphofructokinase and

448

glucose-6-phosphate dehydrogenase from fermenting and non fermenting yeasts. Arch.

449

Mikrobiol. 138, 132–138.

450

9. Gen, Q., Wang, Q., Chi, Z.-M., 2014. Direct conversion of cassava starch into single cell

451

oil by co-cultures of the oleaginous yeast Rhodosporidium toruloides and immobilized

452

amylases-producing yeast Saccharomycopsis fibuligera. Renew. Energy 62, 522–526.

453

doi:10.1016/j.renene.2013.08.016

454

10. Gong, Z., Wang, Q., Shen, H., Hu, C., Jin, G., Zhao, Z.K., 2012. Co-fermentation of

455

cellobiose and xylose by Lipomyces starkeyi for lipid production. Bioresour. Technol.

456

117, 20–4. doi:10.1016/j.biortech.2012.04.063

20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

457

11. Govender, T., Ramanna, L., Rawat, I., Bux, F., 2012. BODIPY staining, an alternative to

458

the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae.

459

Bioresour. Technol. 114, 507–11.

460 461

12. Guo, Y., Cordes, K.R., Farese, R. V, Walther, T.C., 2009. Lipid droplets at a glance. J. Cell Sci. 122, 749–52. doi:10.1242/jcs.037630

462

13. Huang, C., Chen, X., Xiong, L., Chen, X., Ma, L., Chen, Y., 2013. Single cell oil

463

production from low-cost substrates: the possibility and potential of its industrialization.

464

Biotechnol. Adv. 31, 129–39. doi:10.1016/j.biotechadv.2012.08.010

465 466

14. Ichihara, K., Fukubayashi, Y., 1996. Preparation of fatty acid methyl esters for gas-liquid chromatography. J. Lipid Res. 51, 635–40.

467

15. Kim, J.-H., Block, D.E., Mills, D. a, 2010. Simultaneous consumption of pentose and

468

hexose sugars: an optimal microbial phenotype for efficient fermentation of

469

lignocellulosic biomass. Appl. Microbiol. Biotechnol. 88, 1077–85. doi:10.1007/s00253-

470

010-2839-1

471

16. Kim, S.R., Ha, S.-J., Wei, N., Oh, E.J., Jin, Y.-S., 2012. Simultaneous co-fermentation of

472

mixed sugars: a promising strategy for producing cellulosic ethanol. Trends Biotechnol.

473

30, 274–82. doi:10.1016/j.tibtech.2012.01.005

474

17. Koutinas, A. a., Chatzifragkou, A., Kopsahelis, N., Papanikolaou, S., Kookos, I.K., 2014.

475

Design and techno-economic evaluation of microbial oil production as a renewable

476

resource

477

doi:10.1016/j.fuel.2013.08.045

for

biodiesel

and

oleochemical

production.

Fuel

116,

566–577.

21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

478

18. Kuroda, S., Otaka, S., Fujisawa, Y., 1994. Fermentable and nonfermentable carbon

479

sources sustain constitutive levels of expression of yeast triosephosphate dehydrogenase

480

3 gene from distinct promoter. J. Biol. Chem, 269, 6153-6162.

481

19. Morrison, W.R., Smith, L.M., 1964. Preparation of Fatty Acid Methyl Esters and

482

Dimethylacetals From Lipids With Boron Fluoride--Methanol. J. Lipid Res. 5, 600–8.

483

20. Papanikolaou, S., Aggelis, G., 2011. Lipids of oleaginous yeasts. Part II: Technology and

484

potential

applications.

485

doi:10.1002/ejlt.201100015

Eur.

J.

Lipid

Sci.

Technol.

113,

1052–1073.

486

21. Patel, A., Pravez, M., Deeba, F., Pruthi, V., Singh, R.P., Pruthi, P. A., 2014. Boosting

487

accumulation of neutral lipids in Rhodosporidium kratochvilovae HIMPA1 grown on

488

hemp (Cannabis sativa Linn) seed aqueous extract as feedstock for biodiesel production.

489

Bioresour. Technol. 165, 214–22. doi:10.1016/j.biortech.2014.03.142

490 491

22. Pavlik P1, Simon M, Schuster T, Ruis H., 1993. The glycerol kinase (GUT1) gene of Saccharomyces cerevisiae: cloning and characterization. Curr. Genet. 24(1-2):21-5.

492

23. Sch¨oler A. and Sch¨uller H. J., 1994. A carbon source-responsive promoter element

493

necessary for activation of the isocitrate lyase gene ICL1 is common to genes of the

494

gluconeogenic pathway in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 14, 3613–

495

3622.

496

24. Schüller, H.-J., 2003. Transcriptional control of nonfermentative metabolism in the yeast

497

Saccharomyces cerevisiae. Curr. Genet. 43, 139–60. doi:10.1007/s00294-003-0381-8

498

25. Sha, Q., 2013. A comparative study on four oleaginous yeasts on their lipid accumulating

499

capacity. Master’s Degree, Swedish University of Agricultural Sciences, Uppsala.

22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

500

26. Tapia, E. V, Anschau, A., Coradini, A.L. V, Franco, T.T., Deckmann, A.C., 2012.

501

Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random

502

mutagenesis coupled to cerulenin screening. AMB Express. 64, 1–8.

503

27. Tuller, G., Nemec, T., Hrastnik, C., Daum, G., 1999. Lipid composition of subcellular

504

membranes of an FY1679‐derived haploid yeast wild‐type strain grown on different

505

carbon sources. Yeast 1564, 1555–1564.

506

28. Turcotte, B., Liang, X.B., Robert, F., Soontorngun, N., 2010. Transcriptional regulation

507

of nonfermentable carbon utilization in budding yeast. FEMS Yeast Res. 10, 2–13.

508

doi:10.1111/j.1567-1364.2009.00555.x

509

29. Wiebe, M.G., Koivuranta, K., Penttilä, M., Ruohonen, L., 2012. Lipid production in

510

batch and fed-batch cultures of Rhodosporidium toruloides from 5 and 6 carbon

511

carbohydrates. BMC Biotechnol. 12, 26. doi:10.1186/1472-6750-12-26

512 513

30. Weinhandl, K., Winkler, M., Glieder, A., Camattari, A., 2014. Carbon source dependent promoters in yeasts. Microb. Cell Fact. 13, 5. doi:10.1186/1475-2859-13-5

514

31. Wong, D.M., Franz, A.K., 2013. A comparison of lipid storage in Phaeodactylum

515

tricornutum and Tetraselmis suecica using laser scanning confocal microscopy. J.

516

Microbiol. Methods 95, 122–8. doi:10.1016/j.mimet.2013.07.026

517

32. Yen, H.-W., Chen, P.-W., Chen, L.-J., 2014. The synergistic effects for the co-cultivation

518

of oleaginous yeast-Rhodotorula glutinis and microalgae-Scenedesmus obliquus on the

519

biomass

520

doi:10.1016/j.biortech.2014.09.113

and

total

lipids

accumulation.

Bioresour.

Technol.

23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

521

33. Yu, X., Zheng, Y., Xiong, X., Chen, S., 2014. Co-utilization of glucose, xylose and

522

cellobiose by the oleaginous yeast Cryptococcus curvatus. Biomass and Bioenergy 1–10.

523

doi:10.1016/j.biombioe.2014.09.023

524

34. Zhan, J., Lin, H., Shen, Q., Zhou, Q., Zhao, Y., 2013. Potential utilization of waste

525

sweetpotato vines hydrolysate as a new source for single cell oils production by

526

Trichosporon

527

doi:10.1016/j.biortech.2012.08.068

fermentans.

Bioresour.

Technol.

135,

622–9.

528

35. Zhao, X., Kong, X., Hua, Y., Feng, B., Zhao, Z. (Kent), 2008. Medium optimization for

529

lipid production through co-fermentation of glucose and xylose by the oleaginous yeast

530

Lipomyces

531

doi:10.1002/ejlt.200700224

starkeyi.

Eur.

J.

Lipid

Sci.

Technol.

110,

405–412.

24

Table 1.

Carbon sources (A) Fermentable carbon sources Glucose Fructose Sucrose (B) Non-fermentable carbon sources Glycerol Xylulose Arabinose (C) Mixture of fermentable and nonfermentable carbon sources Glucose + Glycerol Glucose + Fructose + Sucrose Glycerol + Arabinose + Xylulose Glycerol + Arabinose + Xylulose + Glucose + Fructose + Sucrose

Cell size (µm)

Lipid droplets size (µm)

5.53 ± 0.31 6.32 ± 0.19 6.67 ± 0.34

2.38 ± 0.52 4.03 ± 0.38 4.24 ± 0.45

6.01 ± 0.76 5.67 ± 0.15 4.34 ± 0.39

4.35 ± 0.54 3.94 ± 0.12 2.98 ± 0.43

6.53 ± 0.09 6.78 ± 0.23 6.63 ± 0.53 6.89 ± 0.27

4.59 ± 0.56 4.87 ± 0.54 4.98 ± 0.43 5.35 ± 0.36

Table 2. Fermentable carbon sources

Caprylic acid (C8:0) Pelargonic acid (C9:0) Myristic acid (C14:0) Pentadecylic acid (C15:0) Palmitic acid (C16:0) Stearic acid (C18:0) Arachidic acid (C20:0) Behenic acid (C22:0) Lignoceric acid (C24:0) Oleic acid (C18:1)

ND

ND

ND

ND

ND

ND

ND

ND

Mixture of fermentable and nonfermentable carbon sources together Glu + Gly Glu+Fru+ Suc+Gly+Arb+ Xyl ND 0.18±0.01

ND

ND

ND

ND

ND

ND

ND

ND

ND

0.19±0.02

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±0.00

0.37±0.09

6.41±0.36

ND

3.76±0.45

ND

ND

ND

ND

ND

1.68±0.21

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±0.12

ND

2.36±0.46

12.22±0.37

8.89±0.76

ND

ND

0.77±0.43

ND

ND

ND

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±0.48

1.78±0.34

ND

ND

2.34±0.53

3.53±0.60

ND

ND

ND

ND

4.84±1.20

ND

ND

ND

ND

ND

ND

ND

ND

ND

2.29±0.43

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

Linoleic acid (C18:2) Linolenic acid (C18:3)

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±0.41

ND

ND

ND

ND

4.19±0.94

ND

ND

4.98±0.21

24.97±0.30

0.71±0.01

Fatty acids (%)

Saturated

Monounsaturated Polyunsaturated

ND = Non detectable

Glucose

Fructose

Sucrose

Non-fermentable carbon sources

Glu+Fru+ Suc

Glycerol

Xylulose

Arabinose

Gly+Arb+ Xyl

Figure 1.

Figure 2.

Synergistic effect of fermenting and non-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 µm) in mix carbon sources Enhanced (C14:0 - C24:0) fatty acids in mix carbon sources revealed by FAME profile Simultaneous utilization of mix carbon sources by Rhodosporidium kratochvilovae