SE184:/S1/M1

Sample Set Information

ID	TSE1342
Title	Wax ester and lipophilic compound profiling of Euglena gracilis by gas chromatography-mass spectrometry: toward understanding of wax ester fermentation under hypoxia
Description	Lipids are being increasingly used as biodiesel feedstock, and several saturated wax esters from Euglena gracilis are candidates for outdoor bulk production. Wax ester fermentation in Euglena is strongly increased by hypoxia, but key events underlying the metabolic shift toward wax ester biosynthesis are poorly understood. Profiling of wax esters and other lipophilic compounds is the first step for research toward the clarification of wax ester fermentation molecular mechanisms, and thus, a simple and comprehensive platform for their profiling is required. In this study, we established a profiling method for wax esters and lipophilic compounds in Euglena using gas chromatography-mass spectrometry (GC–MS). Using this method, we compared accumulation profiles of wax esters and lipophilic compounds between a wild-type Euglena Z strain and a bleached SM-ZK strain. Both the wild-type and the bleached strains contained C14:0 fatty acid-C14:0 fatty alcohol as a dominant wax ester. Wax ester fermentation initiated 4 h after the cessation of oxygen supply by halting the culture agitation resulting in linear increase and proportional changes of wax ester amounts during 24 h. However, complete anoxia by nitrogen gas aeration inhibited wax ester production and the addition of bicarbonates reversed the inhibition, suggesting that there is a need for an additional carbon source for wax ester fermentation under anoxia. Our simple method enables the investigation of metabolic responses leading to wax ester fermentation in Euglena.
Authors	Furuhashi, T., Ogawa, T., Nakai, R., Nakazawa, M., Okazawa, A., Padermschoke, A., Nishio, K., Hirai, M.Y., Arita, M. and Ohta, D.
Reference	Metabolomics, February 2015, Volume 11, Issue 1, pp 175–183
Comment

http://link.springer.com/article/10.1007%2Fs11306-014-0687-1

Sample Information

ID	S1
Title	Euglena gracilis
Organism - Scientific Name	Euglena gracilis
Organism - ID	NCBI taxonomy 3039
Compound - ID
Compound - Source
Preparation	In this study, Euglena gracilis wild-type Z strain and a streptomycin-bleached mutant SM-ZK with less chloroplasts (Mccalla, 1963; Oda et al., 1982) were used. They were maintained in 150 mL of Koren-Hutner medium (pH 5.0) in Sakaguchi flasks at 27 °C under continuous light on a shaker (120 rpm) for 2 days (early-log phase) or 4 days (late-log phase) (Koren and Hutner 1967). Wax ester fermentation was induced either by halting the culture agitation, which stops aeration and induces hypoxia, or by aerating N2 gas into culture medium for 10 min to remove oxygen and other gases, including carbon dioxide, completely. For anoxic conditions induced by N2 gas aeration, different NaHCO3 concentration (1 and 10 mM) were added to investigate the effect of a carbon source on wax fermentation. Cells were collected 0, 4, and 24 h after the initiation of anaerobiosis and were immediately immersed in liquid nitrogen followed by lyophilization.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M1
Title	GC-MS
Method Details ID	MS1
Sample Amount	1 μL
Comment

Analytical Method Details Information

ID	MS1
Title	GC-MS
Instrument	GC Agilent 6890N gas chromatograph / MS Waters GTC premier
Instrument Type
Ionization	EI
Ion Mode	positive
Description	Lipophilic compound extraction The method for the extraction of lipophilic compounds was modified from Furuhashi and Weckwerth (2013). Approximately 5 mg of lyophilized Euglena sample was homogenized with quartz sands (Wako, Osaka, Japan) (approximately 300 mg) in 1 mL of MCA solvent (methanol/chloroform/2 % acetic acid, 5:2:1, v/v/v) using a Mixer Mill MM400 (Retsch, Haan, Germany) for 90 s at 30 Hz. For absolute quantification, 60 µg C20:0 FAME (Larodan, Sweden) was added into 1 mL MCA solvent before homogenizing. The sample was centrifuged at 21,000×g for 3 min. The supernatant was divided into two fractions, retaining 200 µL for wax ester analysis and the remaining 800 µL for total lipophilic compound analysis. Phase separation was conducted by adding 500 µL of water and 400 µL of chloroform to the supernatant. The sample was centrifuged at 21,000×g for 3 min, and an upper polar phase was removed. The remaining inter-phase and the bottom apolar phase were desiccated using a centrifugal evaporator (Thermo Fischer, Waltham, MA, USA). Derivatization for wax ester profiling For wax esters and other free form lipophilic compound profiling, the dried apolar (with inter-phase) pellet was incubated with 20 µL methoxamine-HCl (Sigma-Aldrich, St. Louis, MO, USA) solution (40 mg/mL pyridine) for 90 min at 30 °C. N-methyl-N-(TMS)trifuoroacetamide (80 µL; MSTFA: Sigma-Aldrich) for silylation was added and incubated for 30 min at 37 °C, and 1 µL of the sample was injected into the GC–MS. Methyl esterification and derivatization for total lipophilic compound profiling Alkaline methyl esterification converted only the bounded form of fatty acids (e.g., glycerides and wax esters) into FAMEs. The resulting glycerol and fatty alcohols (FALs) with free fatty acids (FFAs) and steroids contained in the original sample were silylated during the subsequent MSTFA treatment (Furuhashi and Weckwerth, 2013). For the lipophilic compound analysis, the dried apolar (with inter-phase) pellet was incubated with 500 µL of 0.5 M sodium methoxide in methanol for 90 min at 60 °C. The sample was then cooled to room temperature and 1 mL of 1 % acetic acid was added to stop the reaction. Chloroform (400 µL) was added and phase separation was conducted by centrifugation at 21,000×g for 3 min. An upper polar phase was removed, 1 mL of ultrapure water was added and vortexed. The sample was then centrifuged at 21,000×g for 3 min and an upper polar phase was removed again, leaving an inter-phase and a bottom apolar phase that were desiccated using a centrifugal evaporator (Thermo Fischer). The dried pellet was incubated with 20 µL of methoxamine solution (40 mg/mL pyridine) for 90 min at 30 °C. MSTFA (80 µL) was added and incubated for 30 min at 37 °C. The sample (1 µL) was injected into the GC–MS. Retention time was compared to that of authentic alkane standard compounds according to a previous study (Furuhashi and Weckwerth, 2013). Measurements were performed in a batch. GC–MS conditions GC–MS measurements were carried out on a gas chromatography-time of flight-mass spectrometer (GC–TOF-MS) (Auto sampler, JASCO PAL GC-xt; GC, Agilent 6890N; MS, Waters GTC premier) instrument (JASCO, Tokyo, Japan; Agilent Technologies, Santa Clara, CA; Waters, Milford, MA, USA). The injector temperature was 230 °C using a cold trap splitless mode. The flow rate of carrier He gas was 1 ml/min. GC separation was performed on an HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm) (Agilent Technologies, Santa Clara, CA). After a 1-min isothermal period at 70 °C, the temperature was programmed to 76 °C at a heating rate of 1 °C min−1, then to 350 °C at a heating rate of 6 °C min−1 and maintained for 1 min. The temperatures of the GC–MS transfer line and the source of the mass spectrometer were 340 and 250 °C, respectively. The mass spectrometer was operated in an electron-impact mode (EI) at 70 eV in a scan range of m/z 40–600.
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