Single Cell Oil from Oleaginous Yeast Grown on Sugarcane Bagasse

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Single Cell Oil from Oleaginous Yeast Grown on Sugarcane Bagasse-Derived Xylose: An Approach toward Novel Biolubricant for Low Friction and Wear Sheetal Bandhu, Mahesh B Khot, Tripti Sharma, Om P Sharma, Diptarka Dasgupta, Swati Mohapatra, Saugata Hazra, Om Prakash Khatri, and Debashish Ghosh ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02425 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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ACS Sustainable Chemistry & Engineering

Single Cell Oil from Oleaginous Yeast Grown on Sugarcane Bagasse-Derived Xylose: An Approach toward Novel Biolubricant for Low Friction and Wear Sheetal Bandhu1,2,‡, Mahesh B Khot1,‡, Tripti Sharma1,2, Om P Sharma3, Diptarka Dasgupta1,2, Swati Mohapatra4, Saugata Hazra4, Om P Khatri2,3, *, Debashish Ghosh1,2,*

1

Biotechnology Conversion Area, Bio Fuels Division, CSIR-Indian Institute of Petroleum,

Dehradun – 248 005, India 2

Academy of Scientific and Innovative Research, Chennai – 600 113, India

3

Chemical Science Division, CSIR-Indian Institute of Petroleum, Dehradun – 248 005, India

4

Department of Biotechnology, Indian Institute of Technology, Roorkee – 247667, India

‡These authors contributed equally. * Corresponding authors: [email protected]; [email protected]

Keywords: Single cell oil, yeast, fatty acids, biolubricant, friction

ACS Paragon Plus Environment

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ABSTRACT Yeast lipid as single cell oil (SCO) is evaluated as an alternative renewable source of vegetable oils for the biolubricant formulation. The Rhodotorula mucilaginosa IIPL32 yeast strain is cultivated on lignocellulosic pentosans derived from sugarcane bagasse to produce the SCO. The chemical composition and distribution of variable fatty acids in the yeast SCO are characterized by NMR, FTIR, and GC × GC analyses. The high viscosity index and a low pour point of yeast SCO owing to the favorable composition of saturated and unsaturated fatty acids promise its potential as renewable and environmentally-friendly lube base oil. The yeast SCO as lube base oil significantly reduced the coefficient of friction (72 %) and the wear (24 %) compared to conventional mineral lube base oil (SN 150). The fatty acids in the yeast SCO formed a good quality tribo-chemical thin film on the engineering surfaces, which not only reduced the friction but also protected the contact interfaces against the wear. This study demonstrates that yeast SCO being renewable, biodegradable and non-toxic; provides favorable physicochemical and tribophysical properties for good quality lubricant formulation and it can be a good alternative to the conventional mineral lube oil-based lubricants.


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INTRODUCTION Climate change, diminishing petroleum reserves, increasing oil demand and rising CO2 level in the environment are widespread challenges and cause adverse impacts on the ecosystem and health hazards to living-beings. In this context, bioeconomy-based energy and associated products from renewable sources are gaining large attention. The single cell oil (SCO) referring to lipids of oleaginous yeast has been studied as a source of unsaturated fatty acids for a diversified range of applications. Recently, triacylglycerol accumulating yeasts have emerged as a promising feedstock for the fuels and oleochemicals.1-4 The suitability of oleaginous yeasts as SCO source lies in their capability to produce biomass in conventional bioreactors; no competition with food production; the rapid growth rates with high biomass and lipid productivity; growth independent of space, facile approach to adopt light or climatic variations; ability to utilize lignocellulosic sugars; ease of scale-up and amenable to genetic manipulations etc.5 As a result, oleaginous yeast shows immense potential for producing SCO as an intermediate ‘‘building block’’ of fuels (biodiesel, biojet-fuel), soaps, plastics, paints, detergents, textiles, rubber, surfactants, lubricants, additives for the food and cosmetic industries, and other oleochemicals. The SCO can be used in its native form or converted to desired commodities and high-value specialty oleo-chemicals through a variety of chemical, physical, and biochemical methods.6 The SCO has been successfully commercialized for polyunsaturated fatty acid (PUFA) enriched specialty oils for use in the food and supplement industries.7 The production of yeastderived SCO for renewable oleo-chemicals is still in its preliminary stage and attracts immense interest for a diversified range of applications as an alternate to edible and non-edible oleochemical commodities.


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Pretreatment of vegetal biomass is a pre-requisite for its utilization as a fermentable sugar source for yeast mediated bioprocess. The pentose sugars (e.g., xylose) making up the hemicellulosic fraction are not readily metabolized by the majority of the yeasts or so with weak efficiency. Consequently, the xylose sugars act as a barrier for efficient conversion of total biomass to lipids by oleaginous yeasts making it crucial factor to achieve economically favorable process of converting lignocellulosics to fuels and chemicals.8,9 The production of yeast SCO from hemicellulosic hydrolysates was not explored significantly compared to lipid production from sugars in the synthetic media. Moreover, none of the studies on oleaginous yeasts have reported maximum yield of 0.2 g lipid per gram sugar, which is an optimized conversion parameter, needed for the process feasibility.10,11

Sugarcane bagasse represents a major agro-residue in tropical countries and is being used for steam and heat generation in sugar and alcohol industries. The remaining stockpiled bagasse is of low economic value and constitutes an environmental hazard to sugar mills and surrounding areas. It would be economically and environmentally beneficial to utilize bagasse as a renewable substrate for cultivation of oleaginous yeasts from a biorefinery perspective.12 Moreover, bagasse represents a suitable fermentation substrate for lipid accumulation due to its high C/N ratio and low ash content. It has been utilized as substrate for biodiesel production from both oleaginous yeasts and algae.13-16

The capability of yeast to produce lipid as a starting material for biodiesel has been successfully demonstrated at pilot-scale.17,18 However, the potential of yeast lipid for lubricant applications has not been explored, although it carries variable fatty acids, which are considered as good


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friction-modifiers. The fatty acids and their esters exhibit inherent lubricious properties to form a tribo-chemical thin film on the engineering surfaces and offer low shearing while preventing direct contact between the interacting surfaces under the tribological stress.19,20 The exposure of conventional mineral lube base oils to the environment and their entry into the aquatic bodies lead to damage of the ecosystem and health hazards to living beings. As a result, bio-based lubricant formulations such as environmentally acceptable lubricants (EALs, biolubricants) are gaining considerable attention, which is associated with their biodegradable and non-toxic nature. In general, lubricants contain 70-90% base oil and remaining 10-30% variable additives depending on targeted lubricant applications. The vegetable oils make up the most common category of lube base oil of EALs besides the synthetic esters and polyalkylene glycols. However, vegetable oils possess several drawbacks for lubricant applications namely high cost, poor low-temperature fluidity and thermo-oxidative instability, which compromise their performance, particularly at low and high temperatures.21,22 These shortcomings can be addressed with the use of the yeast-derived SCO. The major fatty acids of most of the yeast lipids are oleic and palmitic acids3, which makes yeast SCO as attractive substitutes to vegetable lube base oils for EAL formulation. Moreover, SCO offers similar environmental benefits on account of their high biodegradability and non-toxic nature.

In this study, lipid accumulating yeast Rhodotorula mucilaginosa IIPL32 was used for the production of SCO using pentose sugar stream derived from acid-pretreated sugarcane bagasse. The chemical analysis of SCO was carried out to probe the fatty acids composition and distribution. The physicochemical and tribological properties of yeast SCO such as viscosity, pour point, friction and wear were examined by ASTM standard protocols to explore its potential


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as lube base oil. Furthermore, lubrication properties of well-established mineral and synthetic lube base oils were analyzed for the comparison. The tribo-characteristics of yeast SCO as a lubricant were evaluated using steel tribo-pairs under rolling contact geometry. This study revealed that SCO produced from yeast could be a good alternative to conventional and vegetable lube base oils, where the friction, wear, and environment are of prime importance.

EXPERIMENTAL SECTION Materials: Yeast strain Rhodotorula mucilaginosa IIPL32 (MTCC 25056) was used throughout the study. The stock culture was maintained in a medium containing 10.0 g/L yeast extract, 20.0 g/L peptone, 20.0 g/L dextrose and 20.0 g/L agar-agar. Sugarcane bagasse procured locally from Doiwala Sugar Mill, Dehradun, India was used as a lignocellulosic feedstock for culturing the yeast after mechanical and hydrothermal treatments. Briefly, size of sugarcane bagasse was reduced to 3-5 mm with a crusher and digested in a reactor with dilute acid (0.25 %, w/w) and steam with the solid-liquid loading of 1:8, for 90 min at 140 °C under autogenous pressure (~5 bar). In post digestion process, the xylose-rich liquid fraction was separated from a screw feeder unit and clarified by over-liming to adjust the pH (~4.5) before it was used as a carbon source for yeast biomass production.23-25

Yeast Culturing and SCO Recovery: R. mucilaginosa IIPL32 was cultivated in a 15.0 L in-situ sterilizable tank fermenter (Andel BioSac, India) with a working volume of 12.0 L. The fermenter was operated with automated monitoring and control of pH, temperature and dissolved oxygen (DO) through supervisory control and data acquisition (SCADA) system. A two-stage yeast culture was performed; wherein the yeast strain was initially allowed to grow in a growth


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medium to produce biomass while maintaining low C/N ratio. In the second phase, nitrogen limitation was applied to trigger intracellular lipid accumulation by feeding bagasse-derived xylose (high C/N ratio). The yeast culture was initiated by inoculating 10 % seed culture (v/v) into the growth medium composed of bagasse hydrolysate (initial xylose concentration 20 g/L) supplemented with 1.98 g/L (NH4)2SO4, 1.0 g/L yeast extract, 1.26 g/L KH2PO4, 0.748 g/L Na2HPO4, 0.7 g/L MgSO4, 0.05 g/L CaCl2, 0.005 g/L MnSO4, 0.005 g/L H3BO3, 0.005 g/L CoNO3 and 0.005 g/L NH4MoO4 in water. The dry cell weight and lipid yield were monitored, together with the xylose and nitrogen concentration, to ensure the nutrient availability during fermentation. The xylose and other sugars (glucose, arabinose) were analyzed before and during yeast cultivation by liquid chromatography (UFLC, Shimadzu, Japan) having PL Hiplex-H acid 8 µm column (PL Polymer laboratory, UK) using a refractive index detector. The column was eluted with a mobile phase of 1 mM sulfuric acid at a flow rate of 0.7 mL/min. Total nitrogen in fermentation broth was analyzed using TN 3000 Total Nitrogen Analyzer (Thermo Fisher, USA) as per ASTM D 4629 standard protocol. The fermentation temperature and pH were maintained as 32 °C and 4.5, respectively, throughout the fermenter operation including cell biomass generation and lipid maturation. The microaerobic conditions were maintained during lipid maturation phase with aeration fixed at 0.15 vvm (volume of air/volume of the media) and the DO was maintained at 7-10 % saturation. The culture was interrupted when xylose concentration dropped to 0.5 g/L. The fermentation broth was allowed for settling of cells. The harvested yeast biomass was washed twice with distilled water followed by drying at 60 °C. The dried biomass was pulverized in a grinder. Total SCO was extracted in Soxhlet apparatus with chloroform/methanol (2:1, v/v). The total lipid extract was further treated with n-hexane to remove polar lipids if any from neutral lipids. The lipid fraction soluble in hexane was washed


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with distilled water, passed through anhydrous sodium sulfate and then the solvent was removed using a rotary evaporator. The collected SCO was estimated gravimetrically.

Chemical and Physico-chemical Analysis of SCO: The yeast-derived SCO was characterized by FTIR and 1H NMR techniques. The FTIR spectrum of yeast oil was collected using ThermoNicolet 8700 Research spectrometer under transmission mode at a resolution of 4 cm-1. A uniform thin film of SCO was prepared on KBr pallet to obtain the FTIR spectrum in the range of 4000 – 400 cm-1. The 1H NMR spectrum of SCO was recorded on a Bruker Advance III 500 MHz spectrometer. The CDCl3 was used as a deuterated solvent to prepare the NMR samples containing TMS as an internal reference for 1H spectra. The fatty acids distribution in SCO was probed by multi-dimensional gas chromatographic analysis (GC × GC) using Agilent 7890B Gas Chromatograph system fitted with a thermal modulator assembly, FID and three different capillary columns (1st dimension non-polar column: 30 m × 250 µm × 0.25 µm; 2nd dimension mid-polar column: 10 m × 320 µm × 0.25 µm and bleed column: 4.7 m × 100 µm × 0.25 µm) connected serially with thermal modulator. The oven temperature was programmed from 40 °C (7 min hold up) to 270 °C (20 min hold up) with different ramping rates. Helium was used as a carrier gas under constant flow (0.8 mL/min) mode. All samples were analyzed in split less mode (100:1) at an injection temperature of 250 °C.

The kinematic viscosity of SCO was measured using a Stabinger ViscometerTM (Model: SVMTM 3000, Anton Paar, UK) as per the ASTM D7042 standard protocol. The changes in viscosity of SCO as a function of temperature was compared with conventional mineral lube base oil (SN 150 supplied by Bharat Petroleum Corporation Limited, India) and synthetic lube base oil


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pentaerythritol tetraoleate (polyol, procured from Mohini Organic Pvt. Ltd. India). The Viscosity Index of SCO, SN 150 and polyol were calculated according to ASTM D2270 method using their kinematic viscosities measured at 40 and 100 °C. The pour points of all samples were evaluated by following an ASTM D97 standard test method using the pour point bath (Stanhopeseta, UK). The tubes of required specification charged with lube sample were placed in a pour point bath, and temperature of the bath is gradually decreased until the lube sample is completely frozen.

Tribo-Evaluation of SCO: The tribological properties of SCO oil were examined using a fourball tribo-tester (Ducom, India) under rolling contact geometry of steel balls. The steel balls of 12.7 mm diameter (φ), material: AISI 52100, surface finish: grade 25 EP and hardness of 64-66 Rc were used for the tribo-tests. M/s. Ducom India supplied the steel balls. Each steel ball was sonicated in the hexane before tribo-test. In a typical tribo-test, one steel ball was rotated over three stationary balls establishing the rotating point contact and resultant coefficient of friction and wear parameters were probed. Each test was run for one hour at a rotational speed of 1200 rpm under the load of 392 N. The temperature of sample pot having SCO as lubricant sample was maintained at 75 °C throughout the test. The WSD of three stationary balls of each test were measured by optical microscopy and reported values are an average of six test-balls of two repetitive experiments. The SN 150 mineral oil and polyol were used as reference lube base oils for a comparative study. The morphological features of worn surfaces of steel balls after the tribo-tests were examined by field emission scanning electron microscopy (FESEM, FEI Quanta 200F). The roughness profiles of worn scars were measured by scanning the surface of worn areas of steel balls using the Atomic Force Microscope (NT-MDT INTEGRA scanning probe


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microscope). The 3D profile images of worn surface area are shown for the roughness illustration. The RMS roughness of each sample was calculated based on four AFM measurements on each worn area. The elemental distribution and mapping on the worn area of steel balls were recorded using the energy-dispersive X-ray spectroscopy (EDX) coupled with FESEM.

RESULTS AND DISCUSSION

Figure 1: Growth profile of Rhodotorula mucilaginosa IIPL32 yeast on xylose stream derived from acid hydrolyzed sugarcane bagasse. It shows changes in cell biomass (), total lipid (), residual sugar () and total nitrogen (▲) as a function of cultivation time.


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Table 1: Biomass and lipid production from oleaginous yeasts on hemicellulosic sugarcane bagasse hydrolysate Yeast sp.

Cell Biomass

Lipid concentration

Lipid content

Lipid productivity

Reference

g/L Yarrowia lipolytica 11.42 Po1g

g/L 6.68

%, w/w 58.49

g/L/h 0.04

13

Lipomyces starkeyi 9.3 DSM 70296

2.5

26.9

0.03

14

Rhodosporidium 7.6 toruloides CCT 0783

1.2

16

0.016

15

Rhodotorula 15.35 mucilaginosa IIPL32

3.75

24.43

0.07

Present study

Production of SCO from Yeast Cells Cultivated on Bagasse Derived Xylose: The oleaginous yeasts were cultivated on sugar-based growth media and the lipid accumulation was increased after depletion of nitrogen at carbon excess conditions. Therefore, SCO productivity is highly dependent on the ratio of available carbon and nitrogen (C/N ratio). In fact inducing lipid accumulation in oleaginous yeast requires a nitrogen starvation mechanism.26 The process of lipid accumulation was induced at the molar ratio of C/N > 20.27 In the present study, a simple nitrogen starvation strategy was employed by cultivating R. mucilaginosa IIPL32 strain in two stages. The first phase involved the yeast growth at balanced C/N ratio of 20 and generated cell mass (8.94 g/L) with less storage lipid and negligible lipid yield (0.02 g/g). In the subsequent step, concentrated xylose from pretreated sugarcane bagasse was fed to the cultivation medium resulting in an increase of C/N ratio up to 40 triggering the lipid accumulation. The SCO yield and titer estimated to be 0.17 g/g (of xylose) and 3.75 g/L of medium respectively (Fig.1). The corresponding lipid content of the yeast cells varied from 24.43 – 28.12 % (w/w) on dry weight basis during 38 to 56 h of the fermentation process. The low amounts of other sugars namely


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glucose (1.54 g/L) and arabinose (0.019 g/L) were detected in the bagasse hydrolysate. The glucose level was further monitored during yeast fermentation, and the available amount was found to be consumed entirely within 24 h. The compositions of sugarcane bagasse hydrolysate reported in the literature vary according to procedures used by different researchers, and the variation was mainly due to variabe types of hydrolysis conditions.13 The acid and steam pretreatment procedures used in the present study have been successfully demonstrated for sugarcane bagasse to generate xylose-rich stream with a minimal concentration of other sugars.23-25 The increment in lipid yield exhibited by R. mucilaginosa IIPL32 from 0.02 to 0.17 g on xylose was significant considering the 0.22 g/g maximum practical yield of synthetic medium cultures.28 Table 1 shows the comparative evaluation of R. mucilaginosa IIPL32 with other oleaginous yeasts cultivated on xylose-rich (hemicellulosic) hydrolysate of bagasse. It is clear that the lipid productivity in the present study is higher than other yeasts while the lipid titer and content are intermediate. The conversion of xylose from lignocellulosic feedstock to SCO is necessary for sustainable fuels and chemicals production. Yarrowia lipolytica, a model oleaginous yeast for bio-oil production is unable to grow on xylose as the sole carbon source naturally.29 As xylose catabolism is not fastidious in this organism, pathway engineering is essential to improve the xylose catabolic phenotype in Y. lipolytica.30 In the present study, lipid yield facilitated by R. mucilaginosa IIPL32 on bagasse derived xylose demonstrates significantly enhanced oil production from the pentose fraction of abundantly available sugarcane bagasse waste in the tropical countries. The present study brings out operating strategies that can be used to achieve high cell density and lipid production in the low cultivation time.


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Chemical and physicochemical features of SCO: Figure S1 (Electronic Supplementary Information) shows 1H NMR spectrum of SCO extracted from yeast R. mucilaginosa IIPL32. The 1H NMR spectrum exhibited chemical shifts at 4.14 - 4.289, 2.30, 5.15 – 5.376, 2.0, 1.26 & 1.3 and 0.88 ppm; attributed to OCH2, CH2C═O, CH═CH, CH2CH═CH, (CH2)n, and CH3 features of glycerides comprising of both saturated and unsaturated fatty acid chains. The presence of linoleic (C18:2) chain in the glycerides of SCO was deduced from a chemical shift at 2.77 ppm corresponding to CH2 protons of allylic (—CH═CHCH2—CH═CH—) functionality. The low-intensity chemical shifts at 2.81 and 0.99 ppm, corresponding to allylic CH2 and terminal CH3 of N-3 fatty acids revealed the presence of a small fraction of C18:3 chains. The presence of free fatty acids in the SCO was confirmed by a triplet chemical shift at 2.35 ppm, which was partially overlapped with a triplet of protons of ester group CH2C═O at 2.30 ppm. 100

80

% Transmittance

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


60

40

20

0 3500

3000

2500

2000

1500

1000

-1

Wavenumber, cm

Figure 2: FTIR spectrum of SCO extracted from yeast R. mucilaginosa IIPL32


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Figure 2 shows FTIR spectrum of yeast SCO. The strong and broad vibrational modes at 2926 and 2855 cm-1 are assigned to methylene asymmetric and symmetric stretches of long alkyl chains of fatty acids in the SCO. The vibrational peaks at 3006, 1662 and 930 cm-1, ascribed to =C-H stretch, -C=C- stretch, and =C-H bending modes, respectively, confirmed the presence of unsaturated fatty acid chains in the SCO. A strong band at 1743 cm-1 is attributed to stretching mode of C=O and revealed the presence of ester group in the SCO. Furthermore, a C=O stretching band with lower intensity at 1711 cm-1 confirmed the presence of free fatty acids. The appearance of C-O stretches at 1160, and 1108 cm-1 are attributed to the presence of ester linkage in the SCO. The strong vibrational modes at 1462 and 1375 cm-1 are assigned to C-H bending modes of fatty acids in the SCO. These vibrations confirmed the presence of fatty acid esters and free fatty acids in the SCO. The distribution of variable fatty acids in the SCO was deduced from GC × GC analysis, and it exhibited the abundance of C16 and C18 fatty acids. The monounsaturated cis-∆9-octadecenoic (oleic, 18:1) acid was noted as a major fatty acid (50.48 %), followed by polyunsaturated cis,cis-∆9,12-Octadecadienoic (linoleic, 18:2; 29.8%) and linolenic (18:3, 3.1%) fatty acid. Among saturated fatty acids, hexadecanoic acid (16:0) was noted to be a major component (13.2 %) followed by octadecanoic acid (18:0, stearic; 3.3 %).

Table 2: Physico-chemical characteristics of lube base oils Physico-chemical Characteristics Lube Sample

Kinematic Viscosity, mm2.s-1 (ASTM D 7042)

Viscosity Index

Pour Point, °C

(ASTM D2270)

(ASTM D97)

At 40 °C

At 100 °C

SN 150

30.27

5.42

114.4

-21

Polyol

63.46

11.86

185.9

Single Cell Oil from Oleaginous Yeast Grown on Sugarcane Bagasse

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