Microbial lipid production by cold-adapted ... - Wiley Online Library

Modeling and Analysis

Microbial lipid production by cold-adapted oleaginous yeast Yarrowia lipolytica B9 in non-sterile whey medium Mesut Taskin, Amir Saghafian, Mehmet Nuri Aydogan, Nazli Pinar Arslan, Ataturk University, Erzurum, Turkey Received September 6, 2014; revised March 2, 2015; accepted March 17, 2015 View online April 17, 2015 at Wiley Online Library (wileyonlinelibrary.com); DOI: 10.1002/bbb.1560; Biofuels, Bioprod. Bioref. 9:595–605 (2015) Abstract: Deproteinized whey was used as a substrate for the production of lipids by cold-adapted yeast Yarrowia lipolytica B9 under non-sterile culture conditions. Undesired microbial contamination in non-sterile whey medium could be prevented when appropriate culture parameters (inoculum size of 3 mL/100 mL, initial pH of 5.5 and incubation temperature of 15°C) were selected. In contrast to additional nitrogen (ammonium sulfate) and phosphorus (potassium dihydrogen phosphate) sources, additional carbon source (lactose) increased lipid accumulation. Under optimized culture conditions, biomass and lipid concentrations of the yeast were found as 7.4 g/L and 4.29 g/L, respectively. Lipid content was determined as 58% of total cell biomass. Fatty acids of the yeast were oleic acid (18:1), cis-10-heptadecenoic acid (C17:1), palmitoleic acid (16:1) and palmitic acid (16:0). The yeast was found to contain no polyunsaturated fatty acids. The content of C16 and C18 fatty acids was found to be 91.98% of total lipids. Monounsaturated fatty acids accounted for 80.54% of total lipids. Due to rich monounsaturated fatty acid composition, biomass of Y. lipolytica B9 may be used as feedstock for biodiesel production, especially operating in winter conditions. This is the first report on the use of cheese whey as a lipid production substrate for cold-adapted microorganisms including Y. lipolytica yeast. Besides, lipid production potential of Y. lipolytica under non-sterile culture conditions was investigated for the first time in this study. © 2015 Society of Chemical Industry and John Wiley & Sons, Ltd Keywords: Yarrowia lipolytica B9; cold-adapted yeast; biodiesel; lipids; non-sterile conditions; whey

Introduction iodiesel production using microbial lipids, which are named as single cell oils (SCOs), has attracted great attention around the world. SCOs are considered a very important feedstock for biodiesel production,

B

since their fatty acid composition is similar to those of common plants that are currently used for biodiesel production.1,2 Oleaginous micro-organisms convert excessive carbon sources into storage lipids under suitable culture conditions. In order to reduce the cost of lipid production from these micro-organisms, low-cost raw materials such

Correspondence to: Dr Mesut Taskin, Department of Molecular Biology and Genetics, Faculty of Science, Ataturk University, 25240 Erzurum, Turkey. E-mail: [email protected]

© 2015 Society of Chemical Industry and John Wiley & Sons, Ltd

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as raw glycerol, industrial sugars, energy crops, and lignocellulosic residues have been used as substrates.2 In general, yeasts and moulds can accumulate much more lipids than bacteria and microalgae.1 Oleaginous yeasts are typically found, but not exclusively, in genera such as Candida, Cryptococcus, Rhodotorula, Rhizopus, Trichosporon, Lipomyces, and Yarrowia. On average, these yeasts accumulate lipids to the levels corresponding to 40% of their biomass. However, under conditions of nutrient limitation, they may accumulate lipids to levels exceeding 70% of their biomass.3 Lipid accumulation in oleaginous yeasts and moulds has been demonstrated to occur when a nutrient (e.g. the nitrogen or the phosphorus source) in the medium becomes limited and the carbon source is present in excess. In other words, high carbon/nitrogen (C/N) or high carbon/phosphorus (C/P) ratio in the culture medium increases lipid accumulation in oleaginous species. Especially, nitrogen limitation is the most effective factor for inducing lipogenesis. Under nitrogen-limited conditions, the growth rate slows down and the synthesis of proteins and nucleic acids tends to cease. In non-oleaginous species, excessive carbon remains unutilized or is converted into storage polysaccharides, while, in oleaginous species, it is preferentially channeled toward lipid synthesis.4,5 Another important factor affecting lipid accumulation in oleaginous yeasts is temperature. Cold-adapted yeasts (obligate psychrophilic or psychrotolerant yeasts) are characterized as micro-organisms having both high lipid content and rich unsaturated fatty acids composition. Conventionally, obligate psychrophiles have a maximum temperature for growth < 20°C, and a minimum temperature of 0°C or lower. On the contrary, facultative psychrophiles (psychrotolerant) can tolerate the temperatures > 20°C and are capable of growing at 0°C. In other words, they can be thought of as mesophiles that evolved to tolerate cold. Therefore, this ability of cold-adapted yeasts to grow at low temperatures may be correlated with an increasing proportion of unsaturated fatty acids in the lipid phase of the cell membrane, which makes it more fluid, and a protein conformation functional at low temperature. Namely, changes in lipid metabolism of cold-adapted yeasts constitute the major adaptation of metabolic functions occurring during growth at low temperatures.5–10 Composition of lipids is also very important for the quality of biodiesel. Saturated fatty acids (such as C16:0 and C18:0) and monounsaturated fatty acids (such as C16:1 and C18:1) are generally preferred for biodiesel

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Modeling and Analysis: Biodiesel feedstock production from Y. lipolytica B9

production. Even so, C16:0 and C18:0 saturated fatty acids, which are solid at room temperature, can be a disadvantage in the production of biodiesel, operating in winter conditions. Whereas, monounsaturated fatty acids, which are in liquid form at room temperature, have better flow properties, and are therefore accepted as more advantageous for biodiesel production operating in winter conditions.11 Therefore, cold-adapted (psychrophilic or psychrotolerant) yeasts having high content of monounsaturated fatty acids may be used as more suitable feedstock for biodiesel production operating in winter conditions. However, there is only one report on the production of lipids by cold-adapted oleaginous yeasts, with the aim to develop a novel potential alternative feedstock for biodiesel industry.5 Cheese whey is a green-yellowish liquid resulting from the precipitation and removal of milk casein in cheese making processes. Although several possibilities of cheese whey utilization have been explored, a major portion of the world cheese whey production is discarded as effluent. Its disposal as waste causes serious pollution problems for the surrounding environment. It results in a decrease in crop yield by influencing the physical and chemical structure of soil. It also threatens aquatic life by depleting the dissolved oxygen, when released into water bodies. Typically, cheese whey is composed of 92–95% w/w water and 5–8% w/w dry matter. The main components of cheese whey, in addition to water, are lactose (44–52 g/L), whey proteins (6–10 g/L) and minerals. It also contains vitamins, organic acids and lipids.12–14 Up to now, several reports have showed that cheese whey can be utilized as a growth substrate in the production of valuable microbial products such as carotenoid, polysaccharides, ethanol, lactic acid, and hydrogen.15–19 Recently, some studies also reported that whey could be utilized as a substrate in the production of lipids by mesophilic micro-organisms.20,21 However, there is no report on the use of cheese whey as substrate for the production of lipids by cold-adapted microorganisms including the yeast Y. lipolytica. On the other hand, there are only two reports that are newly published on microbial lipid production under non-sterile culture conditions.22,23 However, we think that further studies on microbial lipid production under non-sterile culture are required. Besides, there is no report on the lipid production by the yeast Y. lipolytica under nonsterile conditions. Therefore, this study was performed to investigate the usability of deproteinized cheese whey as substrate for microbial lipid production by a cold-adapted strain of Yarrowia lipolytica strain under non-sterile culture conditions.

© 2015 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. 9:595–605 (2015); DOI: 10.1002/bbb


Materials and methods Preparation of deproteinized whey Cheese whey was obtained from Dairy Milk Processing System (Department of Food Engineering, Ataturk University, Erzurum). It had a pH of 4.7. Protein precipitation was induced by heating the whey at 90°C for 5–25 min. Precipitated proteins were removed by filtration and centrifugation (5000 rpm for 5 min).15 In the present study, deproteinized whey possessing highest total sugar/total protein ratio was chosen for the subsequent experiments. The total sugar contents of non-treated and deproteinized whey were determined by phenol–sulfuric acid method using glucose as a standard.24 Crude protein contents were determined according to AOAC methods.25 Nitrogen content was measured using a micro-Kjeldahl apparatus (Labconco Corporation, Kansas City, MO, USA), and crude protein was estimated by multiplying the nitrogen content by 6.25.

Isolation and screening of lipidsproducing psychrotolerant yeasts Yeast strains were isolated from different soil samples obtained from different localities of Erzurum, Turkey. In brief, 1 g of soil sample was suspended in 10 mL sterilesaline water. The obtained suspension was diluted up to 10−3 with sterile-saline water. The suspension of 0.1 ml was plated on a petri dish containing 20 mL of deproteinized whey agar (DWA) medium. This medium was prepared by dissolving 20 g agar in 1 L deproteinized whey, and its pH was adjusted to 6.0. The inoculated petri dishes were incubated at 4°C for 15 days. At the end of this incubation period, faster-growing and larger-size colonies were picked up, subcultured and purified. In this way, a total of 55 yeast strains could be isolated. These strains were then screened for the determination of the best lipid-producing yeast. Screening experiments were performed in 250 mL flasks containing 100 ml of deproteinized whey broth (DWB) medium (pH 6.0). This medium containing only deproteinized whey was used in the screening experiments without sterilization. The yeast starter cultures were prepared in malt extract broth medium (MEB) at 15°C for 48 h. At the end of a 48-incubation period, absorbance of yeast starter cultures was adjusted to 2.0 (at 600 nm) using sterile saline-water. Then, 3 mL of yeast starter culture was used for the inoculation of DWB medium. In the case of preparation of yeast starter cultures, inoculated-DWB media were incubated at 15°C for 48 h at 150 rpm in a shaking incubator (ZHWY-200B, Zhicheng Analytical Co., Shanghai, China). After 48 h, the cultures

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were centrifuged at 5,000 rpm for 5 min. The biomass and lipid concentrations of obtained cells were determined. Strain B9 was found to be the best lipid-producing isolate. This strain was identified as Yarrowia lipolytica and it was selected for the subsequent experiments.

Optimization of culture conditions for lipid production Optimization experiments for lipid production were performed in 250-mL flasks containing 100 mL of DWB medium (pH 6.0). Preliminary experiments were undertaken to determine the optimal concentration of inoculum size (1–6 mL/100 mL). Subsequently, initial pH (pH 3.5–6.5) and temperature (5–35°C) were optimized for lipid production, respectively. On the other hand, different concentrations of lactose (0–20 g/L), ammonium sulfate (0–2 g/L) and potassium dihydrogen phosphate (0–2 g/L) as additional carbon, nitrogen and phosphorus sources were also tested for maximum lipid production, respectively. Effect of incubation time on the lipid production was tested from 24 to 168 h. Final experiments were performed to determine the composition of fatty acids of the yeast. While non-sterile culture conditions were designed for the experiments, the medium components (lactose, deproteinized whey, ammonium sulfate and potassium dihydrogen phosphate) and apparatuses were not sterilized. After deproteinized whey was prepared, it was transferred into non-sterile flasks in a short time. The deproteinized whey medium in the flask was not sterilized and directly inoculated with the yeast seed culture. Even, the flasks were not covered with cotton plugs during the cultivation. Namely the media and growth flasks were open to environment. To determine the degree of possible contamination in the medium, 0.1 mL sample taken from the culture medium was spread on a glass slide and then examined using a Leica microscope.

Determination of the fatty acid composition as well as biomass and lipid contents Yeast biomass concentration was determined by cell dry weight. For this, wet yeast cells obtained by centrifugation was washed twice with 5 mL of distilled water and then dried at 80°C to constant weight. For the determination of lipid concentration, the extraction of total lipids was performed with chloroform–methanol (2:1, v/v) mixture. For this purpose, dried yeast cells were powdered and transferred into a tube of 50 mL. Then, 5 mL of chloroform– methanol mixture were added on dried cells in this tube. Following this, the tube was centrifuged for 5 min


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and the supernatant was transferred into another tube. Chloroform-methanol extraction was applied as four cycles to the yeast cells inside the tube. After extraction, cells were re-dried at 80°C to constant weight. The decrease in total biomass was expressed as lipid concentration (g/L). The lipid content was determined as follows: Lipid content (%) = [Lipid concentration (g/L) / biomass concentration (g/L)] x 100. Analysis of saturated, monounsaturated and polyunsaturated fatty acids of lipids was performed by GC-MS using an Agilent 7890A-5975C GCMS Network system. For this, extracted and dried fatty acids were methylated with BF3. After this, obtained fatty acid methyl esters (FAME) were analyzed with GC-MS. The mass spectrum of new peak was compared with that of standard for the identification of fatty acid (37 Comp. FAME Mix 10 mg/ml in CH2CL2; Supelco, Bellefonte, PA, USA).

Identification of the best lipid-producing yeast strain The identification of the yeast strain was performed by DNA sequence analysis. Yeast DNA was obtained using PureLink TM Genomic DNA Mini Kits (Invitrogen, Carlsbad, CA, USA), according to supplier’s specifications. Polymerase chain reactions (PCRs) were performed in a final volume of 25 μL, containing 2.5 μL of 10 X PCR buffer, 2.5 μL of 50 mM MgCl2, 0.5 μL of 10 mM dNTP, 1 μL of the primers 1TS 1 and ITS 2, 2.5 μL of DNA, 15.5 μL of ddH2O and 0.5 μL of Taq DNA Polymerase (Invitrogen). PCR conditions were as following: initial denaturation at 95°C for 2 min; followed by 40 cycles of 1 min at 94°C, annealing at 59°C for 1 min and extension at 72°C for 1 min; and a final extension at 72°C for 3 min. PCR products were purified and sequenced. Obtained sequences were compared to all known sequences in the Genbank by use of BLASTN 2.2.26. program,26 and deposited into GenBank with access number KF486913.

Statistical analysis Each experiment was repeated at least three times in two replicates. The analysis of variance was conducted using one-way ANOVA test using SPSS 13.0 for Microsoft Windows, and means were compared by Duncan test at the 0.05 level of confidence.

Results and discussions Preparation of deproteinized whey It is well known that the selection of a low-cost substrate as well as a hyper producer microbial strain in commercial

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production of microbial products is accepted as a major aspect. In this regard, it has been shown that cheap organic materials such as crude glycerol, sugarcane bagasse hydrolyzate, rice bran hydrolyzate, molasses, olive-mill wastewaters and food-processing wastes can be harnessed as alternative substrates for oleaginous yeast Y. lipolytica in the production of lipids-rich biomasses.27–31 However, usability of cheese whey as substrate for lipid production from Y. lipolytica was tested for the first time in the present study. Up to now, many studies have reported that carbon/ nitrogen (C/N) ratio significantly improves lipid accumulation in oleaginous microorganisms. Especially it has been reported that more lipid accumulation in oleaginous microorganisms occurs when nitrogen in the medium is limited but the carbon source is present in excess.3,32 Under the light of this knowledge, we interpreted that no-treated (normal) whey might be unsuitable for lipid production due to its high nitrogen content. To solve this problem, cheese whey was exposed to a temperature treatment of 90°C, and obtained denatured whey proteins were then removed by filtration and centrifugation. In this way, deproteinized whey having high total sugar/nitrogen content was prepared. In the present study, it was assumed that the deproteinized whey containing high total sugar and low protein content might have high C/N ratio. As seen from Table 1, denaturation of whey proteins drastically increased as treatment time increased, and the maximum protein denaturation was achieved for 25 min. In contrast to protein content, total sugar content of whey showed a small decrease depending on the treatment duration. Even so, the highest total sugar/total protein (14.16) ratio (namely the highest C/N ratio) for whey was achieved with a temperature treatment of 90°C for 15 min. Hence, the following experiments were carried out with this optimal whey. The

Table 1. Effect of temperature treatment on total sugar and protein contents of whey. TT (min) 0 (control)

TP (g/L)

TS (g/L) a

7.2 ± 0.13

b

TS/TP ratio

45.1 ± 2.1

a

6.26 ± 0.20d

a

6.53 ± 0.42d

5

6.9 ± 0.09

45.1 ± 1.7

10

4.7 ± 0.14c

44.8 ± 1.3a

9.53 ± 0.63c

15

3.1 ± 0.10d

43.9 ± 1.1a

14.16 ± 0.44a

20

2.9 ± 0.10

e

b

13.65 ± 0.35ab

25

2.7 ± 0.13e

35.4 ± 1.2c

13.11 ± 0.26b

39.6 ± 2.0

*All values are mean ± standard error of six determinations (n = 6). Same alphabet letters in the same column are not significantly different at p ≤ 0.05. TT, temperature treatment; TS, total sugar and TP, total protein. Control (0): non-temperature treated whey.



total sugar and protein contents of this optimal whey were determined as 43.9 and 3.1 g/L, respectively.

Isolation and screening of lipidsproducing psychrotolerant yeasts The isolation experiments were performed at 4°C on the medium containing only deproteinized whey and agar (DWA medium). The aim of this application was to isolate the cold-adapted yeast strains having potential to use whey lactose as main carbon source. A total of 55 cold-adapted yeast strains that appear capable of metabolizing whey lactose at low temperatures could be isolated from different soil samples. On the other hand, screening procedure of isolated new microorganisms is accepted as a very powerful tool in microbiological studies. This is because that the screening method used for the determination of the best isolate can reduce time and energy consumption as well as working load. The present screening experiments showed that 44 of a total of 55 yeast isolates possessed poor growth potential in deproteinized whey broth (DWB) medium at 15°C. Therefore, the screening results for only the rest 11 isolates were given in Table 2. Among 11 isolates, 6 isolates were found to have lipid content over 20%, and they therefore were assumed to be oleaginous yeast. The maximum biomass (4.8 g/L) and lipid (1.39 g/L) concentrations were attained for the isolate B9 among all isolates tested. Besides, the maximum lipid content (28%) was achieved for the same isolate. Taking these results into account, the subsequent experiments were performed with the B9 isolate. This isolate was identified as Yarrowia lipolytica Table 2. Screening of isolated yeasts. IC

BC (g/L)

LC1 (g/L)

B1

3.3 ± 0.08f

0.52 ± 0.09fg

16

e

19

f

15

B2

de

3.5 ± 0.06

d

LC2 (%)

0.66 ± 0.08

B3

3.7 ± 0.12

B4

4.3 ± 0.10c

0.86 ± 0.10cd

20

B5

g

3.1 ± 0.08

0.68 ± 0.06

e

22

B6

4.6 ± 0.14b

0.69 ± 0.05e

15

B7

4.3 ± 0.10

c

0.90 ± 0.11

c

21

B8

4.5 ± 0.15b

0.99 ± 0.08b

22

B9

4.8 ± 0.10

a

a

28

B10

3.5 ± 0.13

e

0.80 ± 0.06

d

23

B11

3.1 ± 0.10g

0.46 ± 0.02g

15

0.55 ± 0.08

1.34 ± 0.12

*All values are mean ± standard error of six determinations (n = 6). Same alphabet letters in the same column are not significantly different at p ≤ 0.05. IC, isolate code; BC, biomass concentration; LC1, lipid concentration and LC2, lipid content.

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according to sequence analysis of conserved sequences in 5.8S rDNA and 28S rDNA. Up to now, several investigators have reported that Y. lipolytica strains does not possess capacity to assimilate lactose as carbon source.33,34 Therefore, one of the most interesting findings of this work was that the domestic strain (B9) of Y. lipolytica B9 was capable of assimilating the whey-lactose as carbon source. This finding should not be considered as a conflict, since it is well known that the ability to utilize the same carbon source is a highly variable phenotype among even different strains of the same microbial strain. For example, it has been reported that more than 90% of wild-type strains of Klebsiella spp. but less than 50% of Escherichia coli and less than 10% of Salmonella strains can assimilate sucrose as a sole carbon source.35,36 Similarly, it was reported that the E. coli wild-type isolate EC3132 in contrast to E. coli K-12 was capable of utilizing sucrose.37 Kitpreechavanich et al.38 reported that there was a difference in sucrose assimilation potential of two fungal isolates that were identified as Rhizopus oryzae according to DNA sequence analysis. Kreger-van Rij39 reported that Trichosporon fermentas was a lactose-negative yeast; however, Zhu et al.32 demonstrated that a strain (CICC 1368) of this yeast species could use lactose as carbon source. Although Rhodotorula glutinis was reported to be lactose negative yeast by Kurtzman et al., 34 whey lactose could be used as a carbon source by an isolated strain of this yeast species in a published study.40 Kurtzman et al.34 has informed that Y. lipolytica is not able to assimilate sucrose; however, molasses sucrose has been used as carbon source for Y. lipolytica (Candida lipolytica) in a study of Karatay and Dönmez.27 Maldonade et al.41 indicated that the L108 and L135 strains of Rhodotorula mucilaginosa yeast could not assimilate xylitol but the L12 and L135 strains assimilated this carbon source. Furthermore, it has been reported that Kluyveromyces marxianus is a lactose variable species.42 Furthermore, it has been reported that assimilation tests of carbon sources alone are unreliable as a universal means of yeast identification, because of numerous new species, variability of strains and increasing coincidence of assimilation profi les.43 Stratford44 has informed that 50% of spoilage yeasts are incorrectly identified by using carbon assimilation tests. Spencer et al. 43 have used DNA sequence analysis and carbon assimilation tests in order to identify several yeast isolates, and they have noted that 67% of the yeast isolates are misidentified by carbon assimilation tests. Therefore, it has been documented that in order to obtain an accurate identification, it is necessary to use molecular techniques based on the detection of conserved sequences in 18S rDNA, 5.8S rDNA and 28S


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rDNA.43,45 Hence, we considered that the isolate B9 identified as Y. lipolytica according to DNA sequence analysis might be a novel strain of Y. lipolytica yeast.


Table 3. Effect of inoculum size, pH, and temperature on cell growth and lipid accumulation. BC (g/L)

Effect of inoculum size, pH and temperature on lipid and biomass production Production of microbial substances is generally performed in sterile culture conditions. Nonetheless, investigators have declared that if appropriate values of culture parameters such as pH and temperature are chosen, production of microbial substances can be also performed in nonsterile media. For example, thermophilic microorganisms are chosen to prevent the contamination of their mesophilic counterparts when lactic acid production is carried out under non-sterile conditions.46 Similarly, it has been reported that when fermentation is performed at low temperatures, most undesired mesophilic contaminants may be prevented or limited.47 Furthermore, it has been reported that ethanol production can be performed at low pH of 4.5 under non-sterile culture conditions by using an acid-tolerant Zymomonas mobilis.48 Newly published two studies22,23 have also showed that microbial lipid production can be performed under non-sterile culture conditions thanks to the selection of favourable culture parameters. Even so, the lipid production potential of the yeast Y. lipolytica under non-sterile culture conditions has not been investigated, yet. Therefore, we decided to perform the production of lipids by Y. lipolytica in non-sterile whey medium. In this context, a combination of high inoculum size as well as low temperature and pH was selected to make Y. lipolytica B9 cells more dominant population in the medium, thereby preventing undesired microbial contaminants. Experiments showed that when inoculum sizes of 1 and 2 mL/100 mL were chosen, little bacterial contamination was observed in DWB medium at pH 6.0 and 15°C. Conversely, no contamination was detected in DWB medium when the experiments were conducted at the inoculum sizes above and 3 mL/100 mL at the pH 6.0 and 15°C. It is also clear from Table 3 that the maximum lipid content (28%) as well as the maximum biomass (4.38 g/L) and lipid (1.34 g/L) concentrations were obtained with an inoculum size of 3 mL/100 mL. Higher inoculum sizes significantly decreased the cell growth and lipid accumulation. This situation can be explained by the fact that Y. lipolytica cells prefer whey lactose as energy source instead of more lipid accumulation due to the nutrient competition in high cell density. As for the effect of initial pH on cell growth and lipid accumulation, it was seen that initial

600

Inoculum size (mL)

Initial pH

Temperature (°C)

d

LC1 (g/L) 0.89 ± 0.7

LC2 (%)

c

1

3.2 ± 0.13

2

4.2 ± 0.09c

1.17 ± 0.15ab

28

3

4.8 ± 0.12

b

a

28

4

5.1 ± 0.10a

1.27 ± 0.10ab

25

5

5.2 ± 0.14

a

b

22

c

1.34 ± 0.12

1.14 ± 0.09

c

28

6

4.3 ± 0.12

3.5

PG

ND

ND

4

2.7 ± 0.12e

0.40 ± 0.04e

15

4.5

4.7 ± 0.14c

0.89 ± 0.05d

19

b

c

21

0.86 ± 0.11

20

5

5.2 ± 0.10

5.5

7.0 ± 0.18a

2.73 ± 0.13a

39

6.0

c

1.34 ± 0.10

b

28

0.81 ± 0.08

d

19

4.8 ± 0.10

d

1.09 ± 0.10

6.5

4.3 ± 0.12

5

2.9 ± 0.10e

1.24 ± 0.11d

43

c

c

41

10

4.8 ± 0.14

1.97 ± 0.10

15

7.0 ± 0.25a

2.73 ± 0.13a

39

20

6.7 ± 0.18

b

b

36

25

4.2 ± 0.15

d

e

21

30

1.6 ± 0.10f

0.28 ± 0.06f

18

35

NG

ND

ND

2.41 ± 0.10

0.88 ± 0.09

*All values are mean ± standard error of six determinations (n = 6). Same alphabet letters in the same column are not significantly different at p ≤ 0.05. BC, biomass concentration; LC1, lipid concentration; LC2, lipid content; ND, not determined; PG, poor growth and NG, no growth.

pH significantly affected both cell growth and lipid accumulation in the yeast, and the maximum biomass (7.0 g/L) and lipid (2.73 g/L) concentrations were reached at pH 5.5 (Table 3). Similarly, the maximum lipid content (39%) was obtained at the same optimal pH. This result was consistent with the fact that medium pH had a significant effect on the cell growth and lipid accumulation potential of oleaginous microorganisms.49,50 As seen from Table 3, cold-adapted Y. lipolytica B9 was able to grow between 5 and 30°C. On the other hand, no cell growth for the yeast was detected at a temperature of 35°C. Based on these results, it was concluded that coldadapted Y. lipolytica B9 had a psychrotolerant character. This is because that obligate psychrophiles are not able to grow at temperatures above 20°C, whereas facultative psychrophiles (psychrotolerant) can tolerate temperatures above 20°C. 8 No bacterial contamination was observed at 10 and 15°C, whereas significant bacterial contamination



occurred at the temperatures (especially at 25 and 30°C) above 15°C. The experiments also showed that the maximum biomass and lipid concentrations were reached at a temperature of 15°C. On the other hand, it was seen that lipid accumulation was also significantly affected by the changes in temperature. This result should not be so surprising, because of the fact that an increase or a decrease in temperature significantly affects lipid accumulation in oleaginous microorganisms. 32,49 Lipid accumulation significantly increased as temperature gradually decreased from 30 to 5°C. For example, the maximum lipid content (43%) was obtained at 5°C. Even so, a temperature of 15°C providing the maximum lipid concentration was selected for the subsequent experiments instead of the temperatures of 5 and 10°C.

Effect of additional carbon and nitrogen sources on biomass and lipid production Experiments exhibited that the maximum lipid concentrations (3.16 g/L) was attained in DWB medium containing 15 g/L lactose as additional carbon source, whereas the maximum biomass concentration (7.8 g/L) was reached when DWB medium was supplemented with 10 g/L lactose (Table 4). At the lactose concentration of Table 4. Effect of additional carbon, nitrogen, and phosphorus sources on cell growth and lipid accumulation. g/L Additional carbon source (lactose)

Additional nitrogen source (ammonium sulphate)

Additional phosphorus source (potassium dihydrogen phosphate)

0

BC (g/L) 7.0 ± 0.09

c

b

LC1 (g/L)

LC2 (%) c

39

bc

40

2.73 ± 0.12

5

7.2 ± 0.10

2.88 ± 0.08

10

7.8 ± 0.19a

3.03 ± 0.10ab

42

15

b

7.2 ± 0.17

a

3.16 ± 0.13

44

20

6.0 ± 0.13d

2.76 ± 0.09c

46

0

bc

a

44

b

7.2 ± 0.10

ab

3.16 ± 0.09

0.5

7.3 ± 0.10

2.99 ± 0.10

41

1

7.4 ± 0.15a

2.73 ± 0.12c

37

c

d

32

1.5

7.1 ± 0.10

2.27 ± 0.10

2

6.6 ± 0.16d

1.71 ± 0.26e

26

0

7.2 ± 0.14

c

a

3.16 ± 0.10

44

0.5

7.3 ± 0.10bc

3.06 ± 0.14ab

42

a

b

39

c

1

7.5 ± 0.10

ab

2.92 ± 0.12

1.5

7.4 ± 0.12

2.44 ± 0.14

33

2

6.9 ± 0.09d

1.79 ± 0.10d

26

*All values are mean ± standard error of six determinations (n = 6). Same alphabet letters in the same column are not significantly different at p ≤ 0.05. BC, biomass concentration; LC1, lipid concentration and LC2, lipid content.

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15 g/L, lipid content was found to be 44% of the biomass. Based on the results obtained, the following experiments were performed in DWB medium containing 15 g/L as an additional carbon source. As for the effect of additional nitrogen source on lipid and biomass production, it was seen that all concentrations of (NH4)2SO4 (ammonium sulfate) as additional nitrogen source significantly decreased lipid production. The maximum lipid concentration (3.16 g/L) and lipid content (%44) was reached in the control medium (non-ammonium sulfate containing DWB medium) containing 15 g/L lactose. These results could be attributed to C/N ratio of DWB medium as previously reported.21 Namely the present data confirmed that the excess of the carbon source had a large positive effect on lipid production by Y. lipolytica B9. Experiments showed that when KH2PO4 (potassium dihydrogen phosphate) was used as an additional phosphorus source, all of its concentrations significantly decreased the lipid accumulation in Y. lipolytica B9 (Table 4). In contrast to lipid production, more biomass was attained when DWB medium was supplemented with KH2PO4. For instance, the maximum yeast biomass concentration was reached in DWB medium containing 1 g/L KH2PO4. These results elucidated that whey was a more favourable substrate in lipid production without additional phosphorus source. It is well known that cheese whey is rich in phosphorus 51 and excessive phosphorus significantly decreases lipid accumulation in oleaginous yeasts.4 Therefore, it should not be so surprising that additional phosphorus source decreases the lipid accumulation in the oleaginous yeast Y. lipolytica B9. Based on these results, the following experiments were carried out in DWB medium containing only 15 g/L additional lactose without additional nitrogen and phosphorus sources.

Effect of incubation time on biomass and lipid production Experiments showed that the maximum increases in the cell biomass and lipid accumulation occurred in 24–48 h. A small increase in the cell biomass was observed in 48–72 h. The cell biomass reached to the maximum (7.4 g/L) within 72 h and no further cell growth was observed after 72 h. In contrast to cell growth, lipid accumulation showed a significant increase after 48 h and reached to maximum (4.29 g/L) within 120 h. Similarly, the maximum lipid content (58%) was obtained within 120 h. On the other hand, a high amount (11.2 g/L) of carbon source (lactose) was detected in the medium when the cell growth completely ceased after 72 h. Content of total sugar (58.9 g/L) dropped to a very low


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utilize storage lipids as energy sources in the absence of sugars such as glucose and lactose. Another reason of this decrease can be attributed to the conversion of stored lipids into citric acid, as reported by Makri et al. 54 These results are in good agreement with those reported by investigators, who show that incubation time strongly affects the cell growth and lipid accumulation in different oleaginous microorganisms including the yeast Y. lipolytica.5,49,54,55

Determination of fatty acids composition of the yeast Figure 1. Effect of incubation time on sugar consumption, cell growth, and lipid accumulation in Y. lipolytica B9.

level (0.2 g/L) after 120 h. Time profiles obtained for lipid accumulation, cell growth and sugar consumption in the present study were similar to those reported by the previous studies.5,28 Although cell growth completely stopped after 72 h, the continuance of lipid accumulation after the 72nd h might be due to the complete depletion or decreasing of nitrogen source in the whey medium. This is because it has been reported that after the stationary phase of yeast growth, the restriction or depletion of nitrogen source in the medium does not prevent lipid accumulation potential of yeasts. In direct contradiction, the remaining excessive carbon source in the medium after the depletion of nitrogen source is channelled toward lipid synthesis or organic acid synthesis (citric acid and/or isocitric acid) in oleaginous yeast Y. lipolytica. 4,5,52,53 The time profile of the yeast cultivation also demonstrated that no increase in lipid concentration and lipid content was observed between 120 and 144 h (Fig. 1). A reduction in the biomass concentration was observed after 144 h. Even, a small decrease was observed in lipid concentration and lipid content after 144 h. This decrease may be explained by the fact that microorganisms

So far, researchers have showed that the composition of fatty acids in oleaginous micro-organisms is depended on the cultivation parameters such as the microbial growth stage, temperature, the initial substrate concentration and carbon/nitrogen ratio.5,50,54 However, we think that if the lipids of the yeasts will be used as biodiesel feedstock, a large amount of lipids should be preferably produced. Therefore, we decided to analyze the fatty acid composition of the lipids that were produced only under optimized culture conditions. Even so, the effect of temperature on fatty acid composition was studied in detail since the yeast had a cold-adapted character. Experiments showed that the same fatty acids were obtained at all the temperatures tested. They were oleic acid (18:1), cis-10-heptadecenoic acid (C17:1), palmitoleic acid (16:1) and palmitic acid (16:0). Namely, temperature did not change the kind of fatty acids (Table 5). The fatty acid composition of Y. lipolytica B9 was similar to those of other Y. lipolytica strains reported by previous studies. For example, Wang et al. 56 informed that fatty acids of Y. lipolytica W29 were oleic acid, palmitic acid, palmitoleic acid and linoleic acid. Katre et al. 11 reported that Y. lipolytica NCIM 3589 cells were very rich in oleic acid and palmitic acid. On the other hand, present experiments showed that total content of

Table 5. Fatty acids composition of Y. lipolytica B9. Fatty acid content (%) Fatty acids Palmitic acid (C16:0) Palmitoleic acid (C16:1)

Fatty acid kind

10°C

15°C c

20°C b

25.53 ± 0.51a 14.46 ± 0.30c

Saturated

16.87 ± 0.63

19.46 ± 0.40

Mono-unsaturated

18.72 ± 0.26a

17.76 ± 0.22b

a

8.02 ± 0.10

b

cis-10-heptadecenoic acid (C17:1)

Mono-unsaturated

8.36 ± 0.14

Oleic acid (C18:1)

Mono-unsaturated

56.05 ± 0.20a

54.76 ± 0.25b

a

a

7.91 ± 0.11b 52.1 ± 0.10c

Total content of C16 and C18 fatty acids

91.64 ± 0.38

91.98 ± 0.17

92.09 ± 0.12a

Total content of mono-unsaturated fatty acids

83.13 ± 0.21a

80.54 ± 0.32b

74.47 ± 0.18c

Total content of saturated fatty acids

16.87 ± 0.25c

19.46 ± 0.23b

25.53 ± 0.26a

*All values are mean ± standard error of six determinations (n = 6). Same alphabet letters with in the same line are not significantly different at p ≤ 0.05.

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C16 and C18 fatty acids was not significantly affected by the changes in temperature. For example, total content of C16 and C18 fatty acids was determined as 91.64, 91.98 and 92.09% of total lipids at 10, 15 and 20°C, respectively. This is an expected result, since oleaginous yeasts contain mainly C16 and C18 fatty acids. 3,5,55 Unlike the kind of fatty acids, the content of each fatty acid was affected by culture temperature. Contents of unsaturated fatty acids (oleic acid, cis-10-heptadecenoic acid and palmitoleic acid) increased as temperature decreased from 20 to 10°C. For example, oleic acid content was 52.1% at 20°C; however, it assayed as 54.76% and 56.05% at 15 and 10°C, respectively. Total content of unsaturated fatty acids was found to be 83.13%, 80.54%, and 74.47% of total lipids at 10, 15 and 20°C, respectively. The experiments also elucidated that the yeast did not possess polyunsaturated fatty acids at the end of a 120-h cultivation period, and unsaturated fatty acid content is composed of only monounsaturated fatty acids (Table 5). Unsaturated fatty acid content of Y. lipolytica B9 was found to be higher than those reported for several oleaginous yeast strains in previous studies. For example, while total monounsaturated fatty acid content of Y. lipolytica B9 was 80.54% at 15°C, Katre et al. 11 reported that total saturated fatty acid (mono and polyunsaturated) contents of Y. lipolytica NCIM 3589, Y. lipolytica NCIM 3590 and Y. lipolytica NCIM 3229 were only 65.2, 35.9 and 29.4%, respectively. In the same study, Katre et al. 11 informed that monosaturated fatty acid content of Y. lipolytica NCIM 3450 was only 12.8% and Y. lipolytica NCIM 3472 did not contain monounsaturated fatty acids. Karatay and Dönmez 27 informed that unsaturated fatty acid contents of Candida lipolytica, C. tropicalis and Rhodotorula mucilaginosa yeasts were 36.2%, 7.3% and 33.7%, respectively. It has been reported that methyl esters from monounsaturated fatty acids such as palmitoleic and oleic acids are liquid at room temperature and have better flow properties in cold weather. Therefore, biodiesel from monounsaturated fatty acids-rich feedstock has high potential to operate in winter conditions. Conversely, a high total polyunsaturated content results in increased viscosity, which is undesirable for biodiesel. 11,57,58 Therefore, we gather that monounsaturated fatty acids-rich biomass of Y. lipolytica B9 can serve as a good feedstock for the production of biodiesel, efficiently operating in winter conditions.

Conclusions The present study demonstrated that deproteinized whey with high C/N ratio could be prepared by using heat

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treatment. This whey could be effectively used as a growth substrate for lipid production from a lactose-positive and cold-adapted strain of yeast Y. lipolytica under non-sterile culture conditions. Undesired microbial contaminations could be prevented by the selection of appropriate inoculum size, pH and temperature. When ambient temperature of environments such as laboratories and production plants become in the range of 10–20°C, production media do not require any cooling and heating since the yeast can grow in this temperature range. This process can not only make energy saving possible but also reduce time consumption and workload. Therefore, this process may also be applied for the production of various microbial products in industrial scale. Especially countries having cold climate may use this process without temperature control. Content of monounsaturated fatty acids as well as C16 and C18 fatty acids was found to be very high. No polyunsaturated fatty acids were detected in the yeast. Considering these results, we assume that lipid-rich biomass of Y. lipolytica B9 may be a good feedstock for biodiesel production, especially operating in winter conditions. On the other hand, cheese whey can be utilized as a cheap growth substrate in the production of microbial lipids.

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Mesut Taskin Mesut Taskin was born in Trabzon, Turkey. He graduated with a BSc in Biological Education from Ataturk University in 2005. He has a Ph.D. in Applied Microbiology and Biotechnology from Ataturk University. He is currently an Associate Professor of Molecular Biology and Genetics at Ataturk University. His main works focus on cell immobilization, strain development and microbial genetics as well as the microbial production of medically and/or industrially important substances such as recombinant proteins, biofuels and secondary metabolites.

Amir Saghafian Amir Saghafian holds a Bachelor’s degree in Department of Biology from Shahid Chamran University, Ahwaz, Iran. He holds a Master’s degree in Applied Microbiology and Biotechnology from Ataturk University, Erzurum, Turkey.

Mehmet Nuri Aydogan Mehmet Nuri Aydogan was born in Erzurum, Turkey. He is an Assistant Professor of Biology Department at Ataturk University. His MSc and PhD studies were focused on microbial biotechnology. His research domains include biofertilizers and microbial enzymes as well as the isolation, screening and identification techniques of industrially important microorganisms.

Nazli Pinar Arslan Nazli Pinar Arslan was born in Samsun, Turkey. She is currently a PhD student in the Department of Biology at Ataturk University. She holds a Master’s degrees in Applied Microbiology from the same university. Her recent works focus on cell immobilization, strain development and microbial natural products.


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