Lipid production from sugar beet molasses under non-aseptic culture conditions using the oleaginous yeast Rhodotorula glutinis TR29

Renewable Energy 99 (2016) 198e204

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Lipid production from sugar beet molasses under non-aseptic culture conditions using the oleaginous yeast Rhodotorula glutinis TR29 Mesut Taskin a, *, Serkan Ortucu b, Mehmet Nuri Aydogan c, Nazli Pinar Arslan c a

Department of Molecular Biology and Genetics, Science Faculty, Ataturk University, 25240 Erzurum, Turkey Department of Molecular Biology and Genetics, Science Faculty, Erzurum Technical University, Erzurum, Turkey c Department of Biology, Science Faculty, Ataturk University, 25240 Erzurum, Turkey b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 August 2015 Received in revised form 31 May 2016 Accepted 29 June 2016

In this study the lipid production potential of the isolated yeast Rhodotorula glutinis TR29 in molasses medium under non-aseptic culture conditions was investigated. Different molasses concentrations and initial pH values were tested to make R. glutinis TR29 cells more dominant population in the medium, thereby preventing undesired microbial contaminants. Contamination could be prevented by selecting the high molasses concentration (20%) and low initial pH (5.0). When these parameters were kept constant, the optimum temperature, additional nitrogen source concentration and incubation time for the lipid production were found to be 25
C, 4 g/L and 168 h, respectively. Under these culture conditions, the cell mass and lipid concentration were determined as 16.2 and 10.5 g/L, respectively. The lipid content was determined as 64.8%. The main cellular fatty acids of the yeast were oleic (63.5%), politic acid (15.4%), stearic acid (9.1%) and palmiteloic acid (7.2%). The yeast lipids seems to be a promising feedstock for biodiesel production due to a high content of C16 and C18 fatty acids. To our best knowledge, this is the first work on lipid production by yeasts in non-sterile molasses medium. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Rhodotorula glutinis Oleaginous Lipids Non-sterile conditions Molasses Optimization

1. Introduction In the recent years, biodiesel has received increasing attention because of the environmental pollution and energy crisis worldwide. Biodiesel can be used in the existing engines, produces less harmful gas emissions such as sulfur oxide and reduces net carbon dioxide emissions by 78% on a life-cycle basis when compared to conventional diesel fuel [1]. Biodiesel can be produced by transesterification of triacylglycerols from various renewable lipid resources [2,3]. If plant oil is used for biodiesel production, the cost of source has account to 70e85% of the whole production cost. Microorganisms have often been considered for the production of oils and fats as an alternative to agricultural and animal sources [4]. Some microorganisms can accumulate lipids to a level corresponding to more than 20% of their biomass, and they therefore are described as oleaginous. Oleaginous yeasts are generally found in genera such as Candida, Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon, and Yarrowia [4e8]. On average, these yeasts accumulate lipids to levels corresponding to 40% of their

* Corresponding author. E-mail address: [email protected] (M. Taskin). http://dx.doi.org/10.1016/j.renene.2016.06.060 0960-1481/© 2016 Elsevier Ltd. All rights reserved.

biomass. Besides, under conditions of nutrient limitation, they may accumulate lipids to levels exceeding 70% of their biomass [5]. Furthermore, oleaginous yeasts have the other advantages such as fast growth rate, high oil content and the resemblance of their triacylglycerol fraction to plant oil [4]. On the other hand, they can utilize waste agricultural materials as well as some industrial byproducts [9]. High carbon:nitrogen (C:N) ratio in the medium increases in lipid accumulation capacity of oleaginous microorganisms including yeasts [10,11]. Therefore, selection of appropriate substrate may be a good approach for enhancement of lipogenesis in oleaginous microorganisms. For example, Wu et al. [12] have reported that organic materials such as Jerusalem artichoke tuber juice, sewage sludge and monosodium glutamate wastewater are not favorable for microbial lipid production due to their high nitrogen content. In contrast, molasses seems a good lipid production substrate for oleaginous yeasts due to its high C:N ratio. Molasses is a co-product of sugar production from sugar beet or sugar cane. Sugar beet molasses contain 23e26% water, 47e48% sugar, 9e14% minerals (Mg, Mn, Al, Fe and Zn) and 8e12% nitrogenous compounds (aminoacids, proteins, etc.) [13e15]. So far, studies have reported that molasses can be used as a lipid

M. Taskin et al. / Renewable Energy 99 (2016) 198e204

production substrate for some microorganisms [2,16]. However, to our best knowledge, there is no work on usage of molasses as substrate for lipid production under non-sterile culture conditions. Furthermore, there are the limited studies on microbial lipid production under non-aseptic culture conditions [8,11,17,18]. Nonsterile culture technique can be effectively used in industrial scale, since it can reduce energy and time consumption as well as workload. Hence, the purpose of the present study was to produce lipids from isolated yeasts Rhodotorula glutinis TR29 under non-sterile culture conditions by using sugar beet molasses as substrate. 2. Materials and methods

199

for 1 min, with a final extension step of 72
C for 10 min. PCR products were analyzed in 1% (w/v) agarose gels by horizontal gel electrophoresis. DNAs were visualized by UV excitation after staining with ethidium bromide. The products were purified following the protocols of the PureLink® PCR Purification Kit (Life Technologies, Carlsbad, CA, USA). After purification, ITS rDNA gene was sequenced in both directions with ITS 1 and ITS 4 primers at Medsantek Co., Ltd., Turkey. Sequences chromatograms were assembled into one complete sequence using Bioedit Sequence Alignment Editor version 7.2.5 [20] and the sequence was compared to all known sequences in the Genbank by use of BLASTN 2.2.26þ program [21] and deposited with the GenBank database under the accession number KX017570.1. This isolate was identified as Rhodotorula glutinis.

2.1. Isolation of lipids-producing yeasts The first stage of the present study was focused on isolating the yeasts, which were capable of growing at high molasses concentrations and low pH as well as utilizing the molasses sucrose as carbon source. In this context, the soil contaminated with the effluent of the sugar fabric of Erzurum (Turkey) was used as the isolation source for the selection of the most potent yeast strains. For this purpose, approximately 1 g of the contaminated soil was suspended in 10 mL sterile water. The obtained mixture was vortexed for 1 min and serially diluted up to 103 with sterile saline water. Afterwards, 0.1 mL of 103 dilution sample was spread on 90-mm diameter petri dish containing 15 mL of sterilized molasses agar medium. This medium composed of molasses (12%), 20 g/L agar and mineral salts. The mineral salts were (g/L) (NH4)2 SO4 (ammonium sulfate) 3.0, KH2 PO4 1.5, MgSO4 1.0, CaCl2 0.3 and FeSO4 0.03. pH of the medium was adjusted to 4.0. All the petri dishes were incubated at 30
C for 72 h after inoculation. Yeast colonies developing on agar medium were picked up, sub-cultured and purified. 2.2. Screening of lipids-producing yeasts In the second step, the isolates were screened for their abilities to produce lipids in molasses broth medium (screening medium). This medium had the same composition with that of the molasses agar medium except for agar. Seed culture of each isolate was prepared in standard malt extract broth (MEB) medium at 30
C in a shaking incubator (ZHWY-200B, Zhicheng Analytical Co, Shanghai, China) at 200 rpm. After 48 h, cell final concentrations of seed culture were adjusted to106/mL cells using sterile saline water (0.9%). Then 1 mL of seed culture was used for the inoculation of the molasses broth medium in 250 mL flask. The flasks were placed in a shaking incubator and then incubated at 30
C and 200 rpm. At the end of a 96 h growth period, the cultures were analyzed for cell mass and lipid concentrations as well as lipid content. The isolate (TR-29) having the maximum lipid concentration was selected and then used for the subsequent experiments. The screening experiments were performed in sterile media under sterile culture conditions.

2.4. Optimization of culture conditions for lipid production under non-aseptic culture conditions In order to design non-aseptic culture conditions during the lipid production by the yeast strain TR-29, the molasses medium and apparatuses were not sterilized. The medium was prepared in a beaker and then transferred into the non-sterile flasks (the medium was prepared using tap water). Flasks were not sterilized and directly inoculated with the seed culture of the yeast strain. During cultivation the flasks were not covered with cotton plugs and therefore media and flasks were open to the environment. During the optimization experiments (molasses concentration, temperature, pH and additional nitrogen source), the degree of bacterial contamination in the culture medium was analyzed using only microscope. To do this, 1 mL sample taken from the culture broth was spread on a glass slide and then examined by using an Olympus BX51 microscope [11]. But, the bacterial contamination of culture broth during the optimization of incubation time was also analyzed according to the colony enumeration procedure on trypticase soy agar (TSA) medium and molasses agar (MA) medium. Cycloheximide was added in media at a final concentration of 4 mg/mL in order to detect unwanted bacteria by suppressing yeast growth. For the enumeration procedure, 1 mL sample taken from the culture broth was diluted with sterile-saline water, and 0.1 mL of the dilution sample was then spread on TSA and MA media. In case of low bacterial contamination, the culture broth was spread on media without pre-dilution. Degree of bacterial contamination on both media was evaluated as colony forming units per milliliter of molasses medium (CFUs/mL). The preliminary experiments were performed to determine the most suitable molasses concentration (4e28%) and initial pH (pH 3e7). Afterwards, different culture temperatures (10e40
C), additional nitrogen source (ammonium sulfate) concentrations (1e6 g/L) and incubation times (with 24-h intervals up to 192 h) were studied, respectively. In the case of screening experiments, one milliliter of seed culture was used for the inoculation of nonsterile molasses medium (100 mL) in a 250 mL flask, and the flasks were incubated in a shaking incubator at 200 rpm. 2.5. Analytical methods

2.3. Identification of the best lipid producing yeast strain The identification of the strain TR-29 was performed by sequencing a fragment of genome. The primers used for the polymerase chain reaction (PCR) were ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) [19]. Amplification reactions were carried out in a 50-ml reaction volume under the following PCR cycling conditions: one cycle of denaturation at 94
C for 2 min, followed by 30 cycles of denaturation at 95
C for 45 s, annealing at 55
C for 1 min, and elongation at 72
C

Total carbon (C) and nitrogen (N) contents of the molasses medium as well as the molasses were determined using a Leco CHNS-932 apparatus. Total sugar content of molasses was analyzed by phenolesulfuric acid method [22]. pH of the molasses medium as well as raw molasses was assayed using Ohaus Starter 3100 pH meter (Greifensee, Switzerland). Yeast cell mass was determined by cell dry weight. To do this, wet yeast cells obtained by centrifugation (at 5000 rpm for 5 min) was washed twice with 5 mL of distilled water and then dried at

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80
C to constant weight. For the determination of lipid concentration, the extraction of total lipids was performed with chloroformemethanol (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 chloroformemethanol mixture were added on dried cells in this tube. Following this, the tube was centrifuged for 5 min and the supernatant was transferred into another tube. Chloroformmethanol extraction was applied 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 cell mass was expressed as lipid concentration (g/L). The lipid content was determined as fallows: Lipid content (%) ¼ [Lipid concentration (g/L)/Cell mass (g/ L)]  100. For determination of fatty acid composition, the dried lipid residue was re-dissolved in 500 ml BF3-methanol 10% (FLUKA, 15716) and incubated in a sealed vial in a 95
C heater for 20 min. FAMEs were extracted with 300 ml n-hexane after the addition of 300 ml saturated NaCl in water. Then, analysis of fatty acids composition of lipids was performed by GC-MS using an Agilent Technologies 7890A-5975C GC-MS system equipped with a HP-88 capillary column (60 m  0.25 mm  0.20 mm). Fatty acid methyl esters (FAMEs) mix C8eC24 (SUPELCO, USA) was used as a standard. The FAME peaks were identified and quantitated by comparing the unknown responses to those generated from known standards.

2.6. 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.

3. Results and discussion 3.1. Isolation and screening of lipids-producing yeast strains The first step of the present study was focused on isolating yeast strains, capable of tolerating low pHs and high concentrations of molasses as well as utilizing molasses sucrose as carbon source. For this purpose, soil samples taken from molasses-contaminated soils were used as an isolation source. The isolation of yeast strains was performed at pH 4.0 on the sterilized agar medium containing high amounts (12%) of molasses. At the end of isolation experiments, a total of 35 yeast strains having these properties could be isolated. The screening experiments demonstrated that twenty-five of 35 isolates showed low growth and/or lipid accumulation potential. Therefore it was considered that these isolates were not suitable for purpose of the present study (data not shown). The rest 10 isolates possessed relatively higher cell mass and lipid concentrations (Table 1). It was also determined that 10 isolates had lipid content above 20%. It has been widely documented that microorganisms accumulating lipids above 20% are defined as oleaginous [5]. Hence we assumed that 10 isolates might be oleaginous. From the screening experiments, it was also seen that although the maximum lipid content (37.9%) could be reached using the strain TR15, too low cell mass (7.7 g/L) and lipid (2.9 g/L) concentration were attained for this isolate when compared to the other 9 isolates. Whereas, the maximum lipid concentration (5.5 g/L) could be obtained for the strain TR29. Furthermore, the second highest lipid content (35.0%) and cell mass (15.7 g/L) was obtained for the strain TR29. This strain with the orange-red color was identified as Rhodotorula glutinis and then used for the subsequent experiments.

Table 1 Screening of lipids-producing yeast isolates. IC

CM (g/L)

LC1

TR-7 TR-13 TR-15 TR-16 TR-21 TR-23 TR-24 TR-29 TR-30 TR-33

8.1 ± 0.06h 9.3 ± 0.09g 7.7 ± 0.12i 10.3 ± 0.13e 10.4 ± 0.09e 9.9 ± 0.06f 11.7 ± 0.10d 15.7 ± 0.14b 14.4 ± 0.06c 16.8 ± 0.17a

2.4 2.7 2.9 3.5 2.9 2.5 3.9 5.5 4.7 4.9

LC2 ± ± ± ± ± ± ± ± ± ±

0.03g 0.08ef 0.05e 0.15d 0.06e 0.06fg 0.09c 0.08a 0.12b 0.05b

24.5 29.1 37.9 34.3 28.5 25.6 33.5 35.0 32.6 29.2

± ± ± ± ± ± ± ± ± ±

0.14i 0.12f 0.23a 0.12c 0.14g 0.10h 0.12d 0.16b 0.20e 0.12f

All values are mean ± standard error of six determinations (n ¼ 6). Same alphabet letters in the same column are not significantly different at p  0.05. The experiments were performed under sterile culture conditions. Culture conditions: molasses concentration ¼ 12%, additional nitrogen source (ammonium sulfate) concentration ¼ 3 g/L, initial pH 4.0, temperature ¼ 30
C, shaking speed ¼ 200 rpm and incubation time ¼ 96 h. IC, isolate code; MC, molasses concentration; CM, cell mass; LC1, lipid concentration; LC2, lipid content and CD, contamination degree.

3.2. Design of non-sterile culture technique and the optimization of some culture conditions for lipid production In non-sterile culture process the production medium and equipments can be used without sterilization. Furthermore this process can be performed under open fermentation conditions. Therefore, non-sterile culture process is considered to reduce energy and time consumption as well as workload [11]. This process is based on a strategy, which makes the test microorganism more dominant population in growth medium but eliminates or restricts undesired microorganisms. For this purpose, the environmental and nutritional factors are designed according to the growth requirements of the test microorganism [11]. For instance, some studies have demonstrated that fungal lipid production can be performed under non-sterile culture conditions. Santamauro [17] informed that lipid production from the yeast Metschnikowia pulcherrima could be accomplished under non-aseptic culture conditions by selecting a combination of low temperature and restricted nutrient availability during the cultivation. Moustogianni et al. [18] showed that oleaginous fungi under non-aseptic conditions could produce lipids on selective medium containing essential oils and/or antibiotics. Taskin et al. [11] demonstrated that an appropriate combination of low temperature and acidic pH as well as high inoculum size could prevent undesired contaminations in nonsterile whey medium during the lipid production by cold-adapted yeast Yarrowia lipolyttica. Ling et al. [8] revealed that when a mixed culture of oleaginous yeast (Rhodosporidium toruloides) and microalga (Chlorella pyrenoidosa) was used, lipid production could be achieved under non-sterile conditions. Ling et al. [23] demonstrated that the selection of high initial cell density for the yeast R. toruloides could make the lipid production possible even in real non-sterile wastewater medium. Yen et al. [24] demonstrated that a low pH level of 4.0 could suppress the growth of undesired contaminants under non-sterile conditions. The preliminary optimization experiments of the present study were performed to determine the optimal molasses concentration for lipid production under non-sterile culture conditions. The experiments demonstrated that cell mass and lipid concentration as well as lipid content were significantly increased by the molasses concentrations from 4 to 12%. When higher molasses concentrations (16 and 20%) were tested, cell mass decreased but lipid concentration and lipid content increased. Molasses concentrations above 20% significantly decreased both lipid concentration and lipid content in the yeast. When pH of the molasses medium was kept at 4.0, little undesired contamination took place at the

M. Taskin et al. / Renewable Energy 99 (2016) 198e204

molasses concentrations of 4e8% but the contamination became invisible at the molasses concentrations above 8%. In brief, the molasses concentration of 20% not only prevented undesired bacterial contaminations but also resulted in the maximum lipid concentration and lipid content (Table 2). Prevention of undesired bacterial contaminations at the molasses concentration above 8% could be attributed to the presence of some bactericidal or bacteriostatic compounds inside the molasses. In other words, the total content of toxic compounds increased in the medium at higher molasses concentrations. Eventually, these toxic compounds exerted more inhibitory effect on bacterial growth. This assumption should not be too surprising, since a previously published study demonstrated that molasses contains some phenolic compounds possessing antibacterial effect [25]. In contrast to the contaminant bacteria, the test yeast could grow in the molasses medium because it had a high tolerance against phenolic compounds. The yeast might have gained this high tolerance on the soil that was constantly contaminated with the effluent containing molasses. When the molasses concentration was kept at 20%, there was a little contamination at pH 5.5 and above. Whereas undesired bacterial contaminations could be completely prevented at the pH values of 5.0. These results are in good agreement with earlier reports demonstrating that low initial pH can help the prevention of undesired microbial contaminants in microbial culture media [11,26]. As illustrated in Table 3, the cell growth and lipid accumulation potential of the yeast increased up to pH 5.5 but both of them decreased at pH levels above 5.5. Besides, no significant difference was observed in total lipid concentration of the yeast at pH 5.0 and 5.5. Although slightly higher lipid concentration (7.8 g/L) was reached at pH 5.5, the subsequent experiments were carried out at pH 5.0 where the bacterial contamination became invisible and the second highest lipid concentration (7.6 g/L) was obtained. These results suggested that a combination of low initial pH (5.0) and high molasses concentration (20%) not only prevented undesired bacterial contaminations in the medium but also resulted in favorable lipid accumulation in the yeast. The experiments for the temperature optimization exhibited that although a temperature of 25
C resulted in slightly more lipid concentration and cell mass, no significant difference was observed in cell growth and lipid accumulation potential at 20e35
C (Table 4). These results indicate that the yeast may be cultivated for lipid production in a wide temperature range without any cooling or heating systems. Therefore, this process may make energy saving possible in non-sterile cultures. Although the temperatures of 10 and 15
C resulted in higher lipid contents in the yeast, significant

Table 2 Effect of molasses concentration on lipid accumulation potential of R. glutinis TR29. MC (%)

CM (g/L)

LC1

4 8 12 16 20 24 28

4.9 ± 0.09f 11.3 ± 0.17d 15.7 ± 0.12a 14.4 ± 0.09b 12.3 ± 0.11c 11.1 ± 0.06d 8.7 ± 0.12e

1.4 3.7 5.5 5.9 6.2 5.4 3.9

LC2 ± ± ± ± ± ± ±

0.14e 0.12d 0.13bc 0.16ab 0.12a 0.17c 0.09d

29.8 32.7 35.0 41.2 50.4 48.9 45.3

CD ± ± ± ± ± ± ±

0.23g 0.20f 0.46e 0.29d 0.64a 0.69b 0.46c

þ þ     

All values are mean ± standard error of six determinations (n ¼ 6). Same alphabet letters in the same column are not significantly different at p  0.05. The experiments were performed under non-sterile culture conditions. Culture conditions: additional nitrogen source (ammonium sulfate) concentration ¼ 3 g/L, initial pH 4.0, temperature ¼ 30
C, shaking speed ¼ 200 rpm and incubation time ¼ 96 h. MC, molasses concentration; CM, cell mass; LC1, lipid concentration; LC2, lipid content and CD, contamination degree. () ¼ invisible contamination, (þ) ¼ low (little) contamination and (þþ) ¼ moderate contamination.

201

Table 3 Effect of initial pH on lipid accumulation potential of R. glutinis TR29. pH

CM (g/L)

LC1

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

5.1 ± 0.09h 8.9 ± 0.12g 12.3 ± 0.40e 13.4 ± 0.29cd 14.5 ± 0.14ab 14.9 ± 0.40a 13.8 ± 0.23bc 12.7 ± 0.20de 10.1 ± 0.60f

1.7 3.9 6.2 6.9 7.6 7.8 7.0 5.9 4.4

LC2 ± ± ± ± ± ± ± ± ±

0.03f 0.09e 0.12c 0.11b 0.14a 0.23a 0.18b 0.12c 0.06d

33.3 43.8 50.4 51.5 52.6 52.3 50.7 46.4 43.5

CD ± ± ± ± ± ± ± ± ±

0.17e 0.75d 0.23b 0.29ab 0.61a 0.55ab 0.17ab 0.81c 0.81d

     þ þ þ þ

All values are mean ± standard error of six determinations (n ¼ 6). Same alphabet letters in the same column are not significantly different at p  0.05. The experiments were performed under non-sterile culture conditions. Culture conditions: molasses concentration ¼ 20% (94.4 g/L total sugar), additional nitrogen source (ammonium sulfate) concentration ¼ 3 g/L, temperature ¼ 30
C shaking speed ¼ 200 rpm and incubation time ¼ 96 h. CM, cell mass; LC1, lipid concentration; LC2, lipid content and CD, contamination degree. () ¼ invisible contamination, (þ) ¼ low (little) contamination and (þþ) ¼ moderate contamination.

Table 4 Effect of temperature on lipid accumulation potential of R. glutinis TR29. T (
C)

CM (g/L)

LC1

10 15 20 25 30 35 40

8.1 ± 0.13c 11.9 ± 0.20b 14.4 ± 0.23a 14.8 ± 0.29a 14.5 ± 0.23a 14.9 ± 0.12a 11.1 ± 0.52b

4.5 6.5 7.7 7.9 7.6 7.8 4.3

LC2 ± ± ± ± ± ± ±

0.14c 0.18b 0.14a 0.15a 0.14a 0.20a 0.29c

55.7 55.6 53.9 53.3 52.6 52.3 38.7

CD ± ± ± ± ± ± ±

0.40a 0.61ab 0.52bc 0.17bc 0.31c 0.33c 0.92d

      

All values are mean ± standard error of six determinations (n ¼ 6). Same alphabet letters in the same column are not significantly different at p  0.05. The experiments were performed under non-sterile culture conditions. Culture conditions: molasses concentration ¼ 20% (94.4 g/L total sugar), additional nitrogen source (ammonium sulfate) concentration ¼ 3 g/L, initial pH ¼ 5.0, shaking speed ¼ 200 rpm and incubation time ¼ 96 h. T, temperature; CM, cell mass; LC1, lipid concentration; LC2, lipid content and CD, contamination degree. () ¼ invisible contamination, (þ) ¼ low (little) contamination, (þþ) ¼ moderate contamination and (þþþ) ¼ high contamination.

decreases in cell mass and lipid concentration were detected at these temperatures. Obtaining of higher lipid contents at the temperatures of 10 and 15
C compared to higher temperatures might be correlated with an adaption mechanism, which was developed by the yeast. Because it has been reported that changes in lipid metabolism of cold-adapted yeasts constitute the major adaptation of metabolic functions occurring during growth at low temperatures. Especially cold-adapted yeasts increase the content of unsaturated fatty acids making cell membranes more fluid, thereby growing at low temperatures. Accordingly, the adaptation mechanism of R. glutinis TR29 could be attributed to an increase in the content of total lipids and/or unsaturated fatty acids [10,11,27,28]. Considering the present results, the subsequent experiments were performed at the temperature of 25
C where the maximum lipid concentration was obtained and the bacterial growth could be prevented. pH of the molasses (100%) was determined as 8.7. The total sugar content of the molasses (100%) was found as 47.2 g. The total carbon and nitrogen contents of the molasses were 31.81% and 1.36%, respectively. Carbon:nitrogen (C:N) ratio of molasses was calculated as 23.39 (31.81:1.36). The production medium contained 20% molasses and the other mineral salts except for additional nitrogen source. Carbon and nitrogen contents of this medium were found to be 10.12% and 0.43%, respectively (C:N ratio was 23.53). The total sugar content of the production medium was determined as 94.4 g/L.

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As seen from Table 5, no significant cell growth and lipid accumulation occurred in this medium (additional nitrogen source-free medium). This result implied that nitrogenous compounds of molasses were not suitable to support the yeast growth or lipid synthesis. Consequently, we considered that an additional nitrogen source should be added to the medium. Ammonium sulfate was selected as an additional nitrogen source for this study. Because it is cheaper than organic nitrogen sources such as yeast extract and peptone. Furthermore it has been reported that Rhodotorula strains produce more lipids in the presence of ammonium sulfate compared to the other inorganic nitrogen sources [29,30]. Effect of ammonium sulfate as additional nitrogen source on lipid production was tested at the different concentrations from 0 to 6 g/L. C:N ratio was also analyzed after the additional nitrogen source was added into the molasses medium. There was a gradual decrease in C:N ratio when ammonium sulfate concentration was increased from 0 to 6 g/L. Whereas, a gradual increase in cell mass was observed at all the tested concentrations of ammonium sulfate. For example, the maximum cell mass for the yeast could be reached at the ammonium sulfate concentration of 6 g/L where the lowest C:N ratio (12.23) was detected. In contrast to the cell mass, the maximum lipid concentration (8.8 g/L) and lipid content (55.3%) were attained at the ammonium sulfate concentration of 4.0 g/L where C:N ratio of the production medium was 14.94. But, applications above 4.0 g/L rise to significant reductions in lipid concentration and lipid content. This inhibitory effect can be explained by the fact that excessive nitrogen source or low C:N ratio limits lipid accumulation in oleaginous microorganisms including Rhodotorula strains [5,8,10,12]. Therefore, the subsequent experiments were performed at the ammonium sulfate concentration of 4 g/L. As seen from Fig. 1, cell growth and sugar consumption in the medium became rapid within about the first 48 h (especially between 24 and 48 h) of fermentation but progressed slowly after 48 h. In contrast to cell growth, lipid accumulation was at low levels within the first 48 h. There was a little increase in lipid accumulation potential of the yeast between 48 and 72 h. Notably, the most increments in lipid accumulation were detected after 72 h. This situation might be ascribed to the increase in C:N ratio (the decrease in nitrogen source concentration). This is because nitrogen limitation is reported to be the most efficient factor for inducing lipogenesis [10]. The sugar content (94.4 g/L) of the medium was completely depleted after 168 h. The maximum yeast biomass (16.6 g/L) could be reached at the end of 120-h cultivation period, and further incubation periods did not cause an increase in biomass

Table 5 Effect of additional nitrogen source concentration on lipid accumulation potential of R. glutinis TR29. ACS (g/L)

C/N

CM (g/L)

LC1

0 1 2 3 4 5 6

23.53 20.61 18.32 16.45 14.94 13.64 12.23

4.1 ± 0.50e 9.0 ± 0.50d 12.7 ± 0.36c 14.8 ± 0.32bc 15.9 ± 0.12b 17.0 ± 0.23ab 17.6 ± 0.59a

1.9 4.3 6.5 7.9 8.8 7.1 5.2

LC2 ± ± ± ± ± ± ±

0.05g 0.05f 0.20d 0.16b 0.10a 0.22c 0.12e

46.3 48.1 51.5 53.3 55.3 41.7 30.1

CD ± ± ± ± ± ± ±

0.64e 0.64d 0.72c 0.16b 0.27a 0.40f 0.29g

      

All values are mean ± standard error of six determinations (n ¼ 6). Same alphabet letters in the same column are not significantly different at p  0.05. The experiments were performed under non-sterile culture conditions. Culture conditions: molasses concentration ¼ 20% (94.4 g/L total sugar), initial pH ¼ 5.0, temperature ¼ 25
C shaking speed ¼ 200 rpm and incubation time ¼ 96 h. ACS, ammonium sulfate concentration; C/N, carbon:nitrogen ratio; CM, cell mass; LC1, lipid concentration; LC2, lipid content and CD, contamination degree. () ¼ invisible contamination, (þ) ¼ low (little) contamination and (þþ) ¼ moderate contamination.

concentration. Conversely, lipid accumulation continued between 120 and 168 h. Namely, it was seen that the prolonged incubation time increased lipogenesis in the oleaginous R. glutinis TR29 in case of the other oleaginous microorganisms [2,11,31]. Therefore, the maximum lipid concentration (10.5 g/L) and lipid content (64.8%) could be attained after 168 h (Fig. 1). From these results, it can be concluded that since the nitrogen source was exhausted or became limited within 120 h, no increase in cell mass was detected after 120 h. Conversely, the lipid accumulation in the yeast continued after 120 h since there was still carbon source in the medium. This result is accepted as a general property of oleaginous species including yeasts. In oleaginous species, excessive carbon is channeled toward lipid synthesis, in contrast, in non-oleaginous species, it remains unutilized or is converted into storage polysaccharides. Therefore, lipid content of oleaginous yeasts can reach to the levels exceeding 70% of cell mass [5,10]. Karatay and Donmez [2] reported that the maximum lipid contents of the yeasts Candida lipolytica, Candida tropicalis and Rhodotorula mucilaginosa were 59.9, 46.8 and 69.5%, respectively. Zhao et al. [3] found that lipid content of R. mucilaginosa reached to 48.8% in the medium containing inulin hydrolysate. Economou et al. [6] reported that when rice hull hydrolysate was used as substrate for lipid production by the oleaginous fungus Mortierella isabellina, the maximum lipid accumulation into fungal biomass was 64.3%. Amaretti et al. [9] demonstrated that oleaginous psychrophilic yeast Rhodotorula glacialis DBVPG 4785 could accumulate lipids up to 68% (lipids/ biomass). Taskin et al. [10] determined that the maximum lipid content of the yeast Yarrowia lipolytica B9 was 58%. Li et al. [25] indicated that when R. mucilaginosa TJY15a was cultivated in the medium containing cassava starch hydrolysate, it could accumulate 52.9% (w/w) of lipids during the fed-batch cultivation. Zhang et al. [32] found that the oleaginous yeast R. glutinis (ATCC 15125) was capable of producing up to 40% lipid on lignocellulosic hydrolysates. In a previous work [15], the maximum lipid content of R. glutinis was reported as 39% of dry cell biomass. Cescut et al. [33] showed that R. glutinis could accumulate 64% lipid. Finally, it was seen that lipid content (64.8%) of the present yeast was comparable to or even higher than those of many fungal strains, which were used in previous studies. Also, we consider that lipid accumulation production potential of the strain may be enhanced, if the other culture parameters such as shaking speed, aeration and additional carbon source are optimized. Furthermore, molasses and crude glycerol may be tested as co-substrate for lipid production by this yeast strain. However, further studies are required to prove these assumptions. On the other hand, there was a reduction in lipid concentration and lipid content between 168 and 172 h. This decrease could be attributed to the utilization of storage lipids as energy source after the depletion of fermentable sugars in the medium, as reported in the previous studies [11,34]. During the initial optimization experiments (molasses concentration, temperature, pH and additional nitrogen source), unwanted bacterial contaminants were monitored using only microscope. But, the presence of these contaminants during the final optimization experiments (incubation time) was also analyzed according to the colony enumeration procedure on trypticase soy agar (TSA) medium and molasses agar (MA) medium as described in the materials and methods section. During the experiments, pH of the enumeration media was adjusted to 5.0 and the incubation temperature was selected as 25
C. The microscope-based analyses of the culture broth exhibited that there was no bacterial contamination under the optimized culture conditions (molasses concentration of 20%, initial pH of 5.0, temperature of 25
C and ammonium sulfate concentration of 4 g/L) for 192 h. Therefore, the culture broth was directly spread on both enumeration media without pre-dilution.

M. Taskin et al. / Renewable Energy 99 (2016) 198e204

203

Fig. 1. Effect of incubation time on sugar consumption, cell growth and lipid accumulation potential of Rhodotorula glutinis TR-29 under non-aseptic culture conditions. Non-sterile culture conditions: molasses concentration ¼ 20%, additional nitrogen source (ammonium sulfate) concentration ¼ 4 g/L, initial pH ¼ 5.0, temperature ¼ 25
C and shaking speed ¼ 200 rpm.

No bacterial colony was observed on MA medium after 24, 48 and 72 h. In contrast to MA medium, the contaminants could form colonies on TSA medium. However, their numbers were too low. For example, numbers of bacterial colonies on TSA were determined as only 150, 185 and 205 CFU/mL after 24, 48 and 72 h, respectively. These results implied that even if undesired bacteria penetrated inside the molasses broth medium (pH 5.0) under open fermentation conditions, they could not show a good growth performance due to the bacteriostatic effect of some molasses compounds. Eventually, the density of contaminant bacteria became restricted in the molasses broth medium. Therefore, bacterial growth could not be visualized in this broth medium using a microscope. Furthermore, they could not develop colony when transferred on MA medium (pH 5.0) containing the compounds with bacteriostatic effect. Nevertheless, the damaged or inactive individuals of the bacterial contaminants in the molasses broth medium became active, repair possible cell damage and developed colonies when transferred on antibacterial (bacteriostatic) compounds-free TSA medium. So far, investigators have demonstrated that molasses can be used as substrate for lipid production under sterile culture conditions [2,16,35]. However, there is no work on microbial lipid production under non-sterile culture conditions using molasses as substrate. Furthermore, the ability of molasses to suppress the interference of unwanted microorganisms during a non-sterile fermentation process was demonstrated for the first time in this study. Yen et al. [24] informed that although low pH level of 4.0 prevented the unwanted contaminants during the lipid production by R. glutinis, it caused too low lipid accumulation (21%) in glycerol

medium. Harkins et al. [36] informed that although low temperature and pH allowed the yeast R. glutinis to compete with undesired bacterial contaminants, these conditions were detrimental to the lipid accumulation in the yeast. Furthermore, lowering the culture temperature increases energy consumption, and therefore it is not economical. Whereas, the present study showed that use of molasses as substrate not only provided high lipid content (64.8%) but also suppressed the undesired microbial contaminants. Furthermore, there was no requirement to increase or decrease the culture temperature in order to prevent bacterial contaminants. Therefore, we consider that the present non-sterile culture process is simple and economical. The experiments also demonstrated that a pH drop occurred during the cell cultivation, especially between 24 and 96. After 168 h, the final pH of the medium was determined as 3.2. This decrease was probably due to excretion of some organic acids. The similar results for Rhodotorula strains were also reported in some studies. For example, Calvente et al. [37] informed that the yeasts R. glutinis and Rhodotorula rubra produced rhodotorulic acid in the medium containing ammonium sulfate, thereby decreasing the culture pH. Cho et al. [38] demonstrated that when the extracellular polysaccharide production by R. glutinis was performed, the final pH of culture dropped below 2.0 (initial pH was 4.0) in the presence of ammonium salts (ammonium sulfate, ammonium chloride, ammonium nitrate). They presumed that the utilization of ammonium ions as nitrogen source produced an expulsion of protons from the cells, causing the medium to become acidic. Based on the present results, we presumed that unwanted bacterial contaminants could not proliferate in the molasses medium owing to the

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continuous decrease in pH as well as the presence of antibacterial compounds. 3.3. Determination of fatty acids composition of the yeast The final experiments were undertaken to determine the composition of fatty acids in the yeast. The main cellular fatty acids were oleic (C18:1) (63.5%), palmitic acid (C16:0) (15.4%), stearic acid (C18:0) (9.1%) and palmiteloic acid (C16:1) (7.2%). Other fatty acids produced in lower amounts were myristic (C14:0) (3.0%) and linoleic (C18:2) (1.8%). This fatty acid composition was similar to those of some R. glutinis strains, which were reported in the previous studies [32,39]. The analyses also revealed that the fatty acids of the yeast strain mainly composed of C16 and C18 series (total 95.2%), especially C18:1. Hence, we consider that the yeast biomass can be used as a potential biodiesel feedstock. This is because it is known that C16 and C18 fatty acids are more suitable for biodiesel production [40].

[14]

[15]

[16]

[17]

[18]

[19] [20]

[21]

4. Conclusions

[22]

The present study revealed that the soil contaminated by sugar fabric effluent was a good isolation source of yeasts, capable of utilizing the molasses sucrose as carbon source as well as tolerating high molasses concentrations. The yeast R. glutinis TR29 was found to be the most effective lipid producer. The study also demonstrated that when the appropriate culture conditions (high molasses concentration and low initial pH) were selected, lipid production by R. glutinis TR29 could be performed in molasses medium under non-aseptic conditions. The proposed non-aseptic culture process is simple and cheap. Therefore, application of this process in industrial scale can decrease energy and time consumption as well as workload. Besides, lipids of the yeasts may be evaluated as a biodiesel feedstock due to rich content of C16 and C18 fatty acids.

[23]

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