Molasses was used as substrate for lipid production under non-sterile conditions.
Low pH and high molasses concentration could prevent unwanted contaminants.
The present process can decrease energy and time consumption as well as workload.
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.
- Rhodotorula glutinis;
- Non-sterile conditions;
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 . Biodiesel can be produced by transesterification of triacylglycerols from various renewable lipid resources  and . If plant oil is used for biodiesel production, the cost of source has account to 70–85% 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 .
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 , , ,  and . On average, these yeasts accumulate lipids to levels corresponding to 40% of their biomass. Besides, under conditions of nutrient limitation, they may accumulate lipids to levels exceeding 70% of their biomass . 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 . On the other hand, they can utilize waste agricultural materials as well as some industrial by-products .
High carbon:nitrogen (C:N) ratio in the medium increases in lipid accumulation capacity of oleaginous microorganisms including yeasts  and . Therefore, selection of appropriate substrate may be a good approach for enhancement of lipogenesis in oleaginous microorganisms. For example, Wu et al.  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 23–26% water, 47–48% sugar, 9–14% minerals (Mg, Mn, Al, Fe and Zn) and 8–12% nitrogenous compounds (aminoacids, proteins, etc.) ,  and . So far, studies have reported that molasses can be used as a lipid production substrate for some microorganisms  and . 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 , ,  and . Non-sterile 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
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 10−3 with sterile saline water. Afterwards, 0.1 mL of 10−3 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.
2.3. 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 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) . Amplification reactions were carried out in a 50-μl 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 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  and the sequence was compared to all known sequences in the Genbank by use of BLASTN 2.2.26+ program  and deposited with the GenBank database under the accession number KX017570.1. This isolate was identified as Rhodotorula glutinis.