Article pubs.acs.org/EF
Biodiesel Potential of Oleaginous Yeast Biomass by NMR Spectroscopic Techniques A. S. Sarpal,* Paulo R. M. Silva, Juliana L. Martins, Julio J. Amaral, Marianne M. Monnerat, Valnei S. Cunha, Romeu J. Daroda, and Wanderley de Souza Instituto Nacional de Metrologia, Qualidade e Tecnologia−INMETRO, Avenida Nossa Senhora das Graças 50, Xerém, Duque de Caxias, RJ Brazil ABSTRACT: Analytical strategies based on NMR (1H and 13C), IR (infrared), and GC (gas chromatography) techniques have been developed for the molecular level characterization of Soxhlet and ultrasonic solvent extracts of yeast biomass samples generated on a lab scale by different yeast, feed, and diverse culture conditions, with an objective to explore biodiesel potential. The extraction efficiency of each solvent (cyclohexane, chloroform, methanol) toward extraction of neutral lipids (total glycerides (TG), free fatty acids (FFA), and polar lipids have been determined and compared with regards to the nature of fatty acid components extracted in each solvent fractions. The fatty acid composition of yeast extracts has been found to be similar to vegetable oils, mostly rich in C16:0, 18:0, and C18:N (N = 1−3) fatty acids as indicated by the combined NMR, GC, and GCMS analyses. The analytical protocol developed has established that 1H NMR techniques can be used directly and rapidly without any sample treatment and prior separation to determine total neutral lipid content (TG, FFA), nature of fatty acids/ester, polyunsaturated fatty esters (PUFE), iodine value, etc. NMR results of nature of unsaturated fatty acids/esters (C18:N, N = 1−3) have been validated by GC and GC-MS analyses. The results have shown the presence of C18:1 and C18:2 as the predominant unsaturated fatty acid components besides common saturated fatty acids. The content and composition of biomass has been found to be specific to types of yeast and feed used for cultivation. The NMR methods offer great potential for rapid screening of yeast for generation of yeast biomass with desired lipid content, quality, and biodiesel potential and value added PUFE, keeping in view of the cost economics of overall generation cost of the biomass.
■
INTRODUCTION
The NMR techniques are extensively used for the compositional analyses and quality control of oils and biodiesel from different sources.19,20,22,24−30 The NMR spectroscopic techniques have been applied to study the effect of culture condition such as feed, median etc. on the efficiency of production of neutral lipids,15,20 metabolic engineering of yeast strains for the production of eicosapentaenoic acid (EPA)13 and 31P NMR techniques to study the content and the degree of polymerization of inorganic linear polyphosphates during the growth of yeast cells under conditions of P extremes in the cultivation medium.14 The 1H NMR spectroscopy has been applied for rapid monitoring of neutral lipid extraction efficiency of ionic and polar solvents in yeast and microalgae biomass21 and estimation of neutral lipids in yeast biomass.22 In the present work, the analytical protocol comprising NMR, IR, and GC techniques have been used for the compositional characterization of yeast biomass generated in the lab from different yeast and feeds with an objective to explore biodiesel potential. The extraction efficiency of extraction techniques, Soxhlet and ultrasonic, have been compared with regards to extraction of neutral lipids as well as time of extraction.
Yeast oil lipids are produced by fermentative or nonfermentative ways by heterotrophic microorganism such as yeast utilizing feedstocks of biomass, sugars, glycerol, xylose, etc.1−15 The intracellular lipid content (TAG, DAG, MAG) accumulating yeasts are Rhodosporidium toruloides, Apiotrichum curvatum, Lipomyces starkeyi, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon fermentans, Yarrowia lipolytica, and Saccharomyces cerevisiae.2,7,8 These oleaginous organisms have been well studied for engineering their metabolism by synthetic biologist to enhance the content and quality of glyceride lipids along with specific types of structured TAG.1,7 The oleaginous yeasts produce lipid containing long carbon chain 12 to 18 saturated and unsaturated fatty acids such as lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic, etc., similar to that found in common vegetable oils.1,3,7,9 Besides neutral lipids, yeast contains glycolipids, phospholipids, and polar substances similar to those present in algae. The content and oil composition is specific to different yeast and can be enhanced or composition modified by optimization of medium, feed, and culturing conditions.2,10−14 The content and quality of potential neutral lipids and their fatty acid profile present in oleaginous biomass including yeast are determined by solvent extraction-gravimetric method, Nile Red staining microscopy, and application of chromatographic and spectroscopic techniques.16−24 The biodiesel potential (BP) of oleaginous biomass is determined by its total content and quality of neutral lipids (TAG, FFA).16 © XXXX American Chemical Society
Received: December 20, 2013 Revised: May 14, 2014
A
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Table 1. Neutral Lipid Distribution (% w/w) in Yeast Extracts by 1H NMR Techniquesa sample Y44GUMX Y44SUMX Y80GUMX Y80SUMX Y80SSXMX Y44SSXMX Y80XUCH Y80XUCL Y80XUME Y80XUMX
fat tri + di + mono (% w/w) 59.3 36.1 75.9 61.6 65.0 48.8 70.2 71.5 70.2 67.5
(6.5) (1.2) (11.0) (5.4) (13.3) (4.5) (10.7) (2.9) (4.3) (3.8)
FFA (% w/w) 20.4 28.6 8.0 22.4 20.4 18.2 9.0 12.1 12.9 20.0
(2.2) (0.9) (1.2) (1.9) (4.2) (1.7) (1.4) (0.5) (0.8) (1.1)
polars (% w/w) 20.3 35.3 16.1 16.0 14.1 33.0 20.8 15.4 16.9 12.5
(2.2) (1.1) (2.3) (1.4) (3.0) (3.1) (3.1) (0.7) (1.0) (0.8)
total extract in biomass (% w/w) 10.9 3.2 14.5 8.7 20.5 9.3 15.2 4.1 6.2 5.7
(10.7)* (4.6)* (22.5)* (13.3)* (181)* (67.8)* (14.9)* (4.1)* (6.1)* (4.7)*
neutral lipids fat + FFA (% w/w) 8.7 2.1 12.2 7.3 17.5 6.2 12.1 3.4 5.1 4.9
a
The value in the parentheses corresponds to actual content in the biomass; fat = more than 90% triglycerides, feed G = glycerin, S = sabouraud, X = xylose, U = ultrasonic, FFA = free fatty acids, CH = cyclohexane, CL = chloroform, ME = methanol, MX = mix solvent (CHCl3/MeOH, 1:2), SX = Soxhlet, (*) = content in mg. with mix solvent system (CHCl3/MeOH, 1:2) marked Y80XUMX. The extraction yield in each case is given in Table 1. 2.2.2. Soxhlet Extraction. The Soxhlet extraction of the samples Y80S (881 mg) and Y44S (729 mg) were carried out in chloroform and methanol mixture (1:2) using cellulosic membranes and time of extraction of 510 min. The solvent was removed, dried and extracts were weighed, marked as Y44SSX (Sabouraud) and Y80SSX (Sabouraud) and kept in the refrigerator (Table 1). The Soxhlet extraction of other samples could not be carried as the quantities of samples provided were not sufficient. In order to prevent auto-oxidation of neutral and polar lipids, particularly PUFA constituents, the solvent from the extracts was evaporated by slow heating on a water bath. The addition of acetone expedited the removal of traces of water and solvents. All the extracts were kept in the refrigerator at −4 °C before being subjected to instrumental analyses and hydrolyses. The auto-oxidation of lipids on account of presence of PUFE/A (C18:3) was monitored by measurement of integral intensities of unsaturated region 5.0−5.5 ppm at frequent interval of times. The variation in the intensities as shown in parentheses for samples Y80XUCH (4.92 to 4.95%), Y80SSXMX (4.57 to 4.61%) and Y44SUMX (3.32 to 3.29%) was found to be between 0.59 and 0.87%. The variation is within the precision of measurement of integral values. This indicates practically no auto-oxidation. Similarly, no variation in the integral intensities of allylic protons in the chemical shift region of 2.7−3.0 ppm has been observed with time. This indicates practically no auto-oxidation. Allylic CH2 protons of unsaturated fatty acids, particularly PUFE, are susceptible to oxidation when kept at room temperature.31 In the present case, lack of auto-oxidation has been ascribed to two reasons. First, the solvent extracts were kept all the time in the refrigerator at −4° before recording of 1H NMR spectra. Second, all the spectra of the extracts were recorded with in 4 days after the extractions of lipids. Samples are found to contain only 1 to 2% of the C18:3 and no trace of EPA and DHA, which are easily prone to oxidation. 2.3. Hydrolyses of Yeast Extracts for FAME Conversion. The ultrasonic and Soxhlet extracts were converted into fatty acid methyl ester (FAME) by adopting the usual methanol/KOH hydrolysis procedure. The hydrolysis procedure was optimized for right quantity of MeOH/KOH and BF3/MeOH to be taken for complete conversion into FAME. The time was found to be optimum in the range of 60 min. Depending upon the quantity of the extracts available, 15 to 35 mg of yeast extracts were taken in a 10 mL flask. Freshly prepared 2 to 3 mL of 0.5N MeOH/KOH solution (2.8 g of KOH in 100 mL of MeOH) was added in a flask. The resulting solution was refluxed on a water bath for 60 min at 70−75 °C. The solution was cooled and added ∼0.5 to 1 mL of BF3/MeOH and again refluxed for 25 min. The flask was cooled and added about 1 mL of saturated NaCl solution. The solution was transferred into the separating funnel and added 2 mL of water. The resulting solution was shaken with 5 mL of hexane. Water washings were given to the hexane layer to remove KOH or NaCl. The hexane layer was allowed to settle for 45 min in order to
2. EXPERIMENTAL SECTION 2.1. Materials and Methods. Yeasts were isolated from the digestive tract of the conch Achatina f ulica, and the samples were cultured by using the yeast marked Y80 and Y44 in the laboratories. These yeasts were selected to culture oleaginous yeast biomass with potential to produce biodiesel meeting B100 specification. The feeds glycerin (G) and Sabouraud (S) (2% glucose/dextrose) were used for culturing yeast samples. Typical composition of S in g/L is peptone from meat = 5.0 g, peptone from casein = 5.0 g, D+ glucose = 20.0 g. Peptone is an enzymatic hydrolysate of animal tissues used as culture media ingredient in variety of media. Samples of yeast biomass generated utilizing these feeds are mentioned in Table 1. 2.2. Extraction and Analytical Strategy. The extraction of neutral lipids (TAG, FFA, hydrocarbons) were carried out by ultrasonic and Soxhlet extraction methods as per the single step extraction procedure. The ultrasonic procedure using various solvents was optimized and developed for achieving the maximum extraction efficiency. Before extractions all the samples were dried in oven at 60 °C and powdered in a mortar. The extracts were analyzed by NMR, IR, and GC techniques. 2.2.1. Ultrasonic Extraction by Single Step Solvent Method. Samples grown using different yeast, feed, culture media and conditions as were extracted in a mixture of chloroform and methanol (1:2 v/v) in an ultrasonic bath by single step extraction method. The biomass samples of 97−152 mg Y44G (Yeast Y44 and feed glycerin) and Y80G (Yeast Y80 and feed glycerin) were extracted with 10 mL of mixture of solvents (chloroform/methanol 1:2) for 30 min. The solution was decanted on a filter paper/ and the residue in the tube was extracted a second time with same quantity of the mix solvent for another 30 min. The solvent in extracts in both the cases was evaporated on a water bath to remove solvent completely. The extracts so obtained were treated with 5 mL of acetone and evaporated to dryness on a water bath to remove traces of water. Similarly, residues in the tubes were transferred in the small beakers (10 mL) and evaporated to dryness using acetone for removal of traces of water. The extracts and the residues were weighed, labeled as Y44GU and Y80GU, and kept in the refrigerator at −4 °C. Similarly, the extracts Y44SU (Sabouraud) and Y80SU (Sabouraud) were obtained following the same procedure. Each extract and residue were dried, weighed, marked properly, and kept in the refrigerator for compositional analyses by various analytical techniques. The residues in each extraction step were dried, labeled, and kept in the refrigerator at −4 °C to prevent auto-oxidation of polyunsaturated fatty acids (PUFA). The amount of dried extracts obtained from ultrasonic extraction of yeast samples is given in Table 1. It has been observed that 90% of the lipids were extracted in the first extraction. The extracts of yeast biomass Y80X from feed xylose were obtained by ultrasonic single step solvent extraction method using solvent cyclohexane (CH), chloroform (CL), and methanol (ME) and marked accordingly as mentioned in Table 1. The biomass was also extracted B
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Table 2. Integral Intensities (% II) of Functional Groups at Different Relaxation Times (RD)a sample
NIST RD 5s
NIST RD 10 s
NIST RD 15 s
Soya B RD 5s
Soya B RD 10 s
Soya B RD 15 s
7.68 7.93
8.37 8.54
8.44 8.59
7.64
8.02
8.10
8.18
8.46
8.28
2.63 8.89
2.50 9.34
2.48 9.20
CHCH(II) 5.0−5.5 ppm OCH3(II) 3.66 ppm OCH2(II) 4.05−4.37 ppm CH3(II) 0.6−1.0 ppm
COA RD 5s
9.17
COA RD 10 s
COA RD 15 s
5.46
5.55
9.53
a
s = seconds, Soya B = blend of soya oil and commercial oleic acid, COA = commercial oleic acid containing C18:N (N = 0−3), II = % integral intensities, NIST = NIST2772 CRM.
Table 3. Fatty Acid Profile of Yeast Extracts (Area %) by GC-FIDa sample/carbon number
Y44SSX MX
Y80SSX MX
Y44SU MX
Y80GU MX
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 (N-3) UI SF
1.0 16.3 10.5 4.8 44.8 9.8 1.9 10.9 22.1
0.4 17.3 1.2 2.5 54.8 19.0
0.8 22.5 2.5 8.6 42.5 9.4 2.3 11.4 31.9
0.5 20.4 0.8 19.3 35.5 19.8 1.0 2.7 40.2
4.8 20.2
Y80SU MX 26.6 7.5 49.3 16.2 nd 34.1
Y80XU MX
rapeseed
1.0 32.3 2.4 (C17) 5.0 50.5 6.0
0.1 4.3
2.8 38.3
2.2 65.8 16.9 8.1 2.6 6.6
jatropha
kartuma 6.9
13.4 0.7 6.9 42.3 36.6 0.1 20.3
4.7 30.7 57.7
11.6
a
S = Sabouraud, G = glycerin, X = xylose, SX = Soxhlet, UI = unidentified, U = ultrasonic solvent, MX = mix solvent (CHCl3/MeOH), SF = saturated fatty acids, nd = not detected. allow formation of clearly transparent layer at the top. The superannuated layer was separated and hexane evaporated on a water bath. Small quantity of acetone was added during evaporation to remove traces of water. The final dried FAME samples were analyzed by GC for fatty acid composition and by NMR and IR for monitoring of FAME conversion.25 The conversion efficiency has been found to be around 60−70% depending upon the amount of polar lipids present in the extracts. A small amount of the higher fatty acids such as C18:3 or C18:2 was found to be lost during hydrolysis, as indicated by the comparative NMR analyses of the extracts and their corresponding FAME. 2.4. Instrumental Analyses. 2.4.1. NMR Recordings. All the 1H NMR spectra were recorded on a Bruker 600 MHz NMR Spectrometer equipped with dual probe (1H/13C). The solutions were prepared by dissolving approximately 5 to 10 mg of the yeast extracts, FAMEs, vegetable oils, and biodiesels in 0.7 mL of CDCl3 containing internal standard TMS. Instrument parameters such as relaxation delay (RD) and receiver gain (RG) were optimized and 90° PW calibrated in order to sufficiently relax the nuclei to get the quantitative spectra. In order to study the effect of relaxation times on the quantitative results, the NMR recordings were carried out at different interscan delay of 8 s (RD = 5.0 s), 13 s (RD =10 s), and 18 s (RD = 15 s) on standard NIST2772 and soya oil. The results shown in Table 2 did not reflect any variation in the integral intensities of unsaturated and glycerides protons at inter scan delay of 13 and 18 s. Thus, RD of 10 s and acquisition time (AT) of 3 s were used for all the recordings. The S/N ratio of CHCH (5.0−5.5 ppm) and OCH3 (3.66 ppm) signals in the NMR spectrum of soya oil (5 μL in 0.7 mL) was measured at 310 and 582, respectively. This meets the minimum requirement of S/N ratio of 250 for carrying out the quantitative analyses. In order to achieve the higher S/N ratio of yeast extracts for better integral measurement accuracy, number of scans (NS) = 16 was given for each recording of yeast extracts and oils for concentration of 5−10 mg/0.7 mL of CHCl3. The higher number of scans enabled to measure accurately the integral intensities of signals of low intensities, such as C18:2 (2.77 ppm), C18:3 (2.81 ppm), and 0.98 ppm for CH3 of N-3 PUFA). It was required to use NS = 32 for samples of FAMEs of yeast extracts in order to get higher S/N of signals as the quantity of sample generated was less than 5 mg in certain cases. The following parameters were used for each recording:
Acquisition time = 3.0 s. Relaxation delay (D1) = 10 s. P1 (90° PW) = 11.01 micro second. NS = 16 or 32. Chemical shift range = 0−12 ppm. All the spectra were integrated thrice after proper phase and baseline corrections and average percentage integral intensities were taken for calculation of structural parameters. The 13C NMR spectra of yeast extracts were recorded on a Bruker 500 MHz NMR spectrometer equipped with BBO probe in CDCl3 solution of yeast extracts (15−20 mg/0.7 mL of CDCl3) containing internal standard TMS. Since the amount of samples available for 13C NMR recording was 10−20 mg, overnight recordings (∼10 000 scans) with a relaxation delay time of 3.0 s in CPD mode were carried out to achieve the reasonable S/N ratio. However, 20% weight by volume solutions of vegetable oil or biodiesel were used for 13C NMR recordings with maximum number of scans 1024 and delay time of 3.0 s on 600 MHz NMR instrument equipped with dual probe. 2.4.2. GC Analyses for Fatty Acid Composition. The fatty acid profile identification and composition was carried out on an Agilent gas chromatograph with FID (flame ionization detector). A capillary polar wax column, PEG coated (length 30 m, diameter 0.25 mm, film thickness 0.25 μm), and helium as carrier gas was used for the separation of unsaturated and saturated fatty acid esters by the method EN14103. The GC conditions, such as split ratio 100:1 for yeast FAMEs (3−4 mg/mL) and biodiesel (10 mg/mL), detector temperature 250 °C, the injector temperature 250 °C, the oven/ column temperature at 240 °C and gas flow, were used as mentioned in the method. The retention times of methyl esters (FAME) were confirmed by analyzing the NIST2772 certified reference material containing C14, C16, C18:0, C18:1, C18:2, and C18:3 FAMEs. The other fatty acids including DHA and EPA were compared with the blends prepared from individual standards of FAME supplied by Aldrich. The yeast FAME samples were analyzed with or without C19:0 standards in order to detect C17:0 FAME in the samples. The area percentages of each peak in the chromatogram were taken for determination of individual FAME composition (Table 3). The identification in some of the samples of yeast was also confirmed by GC-MS under the identical GC conditions. 2.4.4. FTIR Analyses. The FTIR-ATR recordings were done on a Nicolet 6700 FTIR instrument equipped with diamond ATR C
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(Madison w1-2002). The powdered solid and residues (3−4 mg) and liquid extracts of 2−3 mg in chloroform of yeast biomasses were properly spread on the plate so that diamond tip touches the mass equally and homogeneously. Chloroform was evaporated completely before recording. Blank correction and 32 or 64 numbers of scans were given for each recording. The spectral processing including area percentages measurements were done using OMNIC E.S. P2 software.
fat and glycolipids employing ultrasonic extraction have been found to be in the following order (Table 4): fat/ester content = Y80GU > Y44GU = Y80SU > Y44SU
Table 4. Neutral Lipid Extraction Efficiency by FTIR of Yeast Biomasses by Soxhlet and Ultrasonic Extraction Methods Using Mixed solvent (CHCl3/MEOH, 1:2)a
3.0. RESULTS AND DISCUSSION 3.1. Monitoring of Solvent Extraction Process Efficiency of Biomass by FTIR. In order to monitor extraction efficiency of different solvents by ultrasonic and Soxhlet methods toward extraction of neutral lipids, the extracts, solids, and residues of yeast biomasses after extraction with different solvents were analyzed by FTIR using ATR accessory. The spectra are shown in the Figures 1−3. The
yeast
Y44GU
Y44SU
EF (%)
81.2
90.2
Y80GU Y80SU 72.0
59.6
Y80SSX
Y44SSX
Y80XU
81.1
>95
>95
a
EF = extraction efficiency, U = ultrasonic, SX = Soxhlet, G = glycerin, S = Sabouraud, X = xylose.
The comparative results show that much higher content of fats (neutral lipids) can be grown by using glycerin as feedstocks using yeast Y80 or Y44. The extractions and NMR results have substantiated the results of IR analyses. The extraction efficiency (EF) has been estimated by the following equation and given in Table 4: EF (%) = (area biomass of band 1740 − 44 cm−1 − area residue of band 1740 − 44 cm−1) /(area biomass of band 1740 − 44 cm−1) × 100
Comparing the results given in Table 4, it is understood that the ultrasonic extraction efficiency in respect of ester/fat content of yeast biomasses has been achieved in the range from 72 to 95%, as indicated by the decrease in the intensity of the ester band of residues at 1744 cm−1 compared to parent biomasses except in case of ultrasonic extraction residue Y80SUR of Y80S biomass, where the efficiency was found to be 59.6%. It is interesting to note that Soxhlet and ultrasonic extraction efficiencies in case of extracts Y44SSX and Y44SU of sample Y44S are almost similar, that is, >95 and 90.2%, respectively (Table 4). This is in spite of the much higher time of extraction of 600 min in case of Soxhlet compared to 30−60 min for ultrasonic. The amount of solvent (∼20 mL), sample (30−50 mg), and time requirements are significantly less for ultrasonic compared to Soxhlet (150 mL, 200−500 mg). Thus, ultrasonic extraction method developed in the present studies is a concept of green chemistry and cost economy. The extraction efficiency of single step single solvent extracts Y44XUCH, Y44XUCL, and Y44XUME of yeast biomass Y44X (feed xylose) using solvents cyclohexane (CH), chloroform (CL), and methanol (ME) were compared with mix solvent (MX) extract Y44XUMX (CHCl3/MeOH, 1:2). It is evident that MX solvent system extracts nearly the same amount (5.7% w/w) compared to chloroform (4.1% w/w) and methanol (6.2% w/ w). However, cyclohexane extracts much higher amount of components (15.2% w/w) (Table 1). This has been attributed to high preference of cyclohexane toward less polar components such as glycerides. The IR spectra of ultrasonic cyclohexane, chloroform, and methanol extracts of yeast biomass Y80X (feed xylose) marked Y80XUCH, Y80XUCL, and Y80XUME, respectively, and chloroform/methanol extract of biomass Y44S (feed Sabouraud) marked Y44SUMX, as shown in Figure 3, present both ester and acid bands at 1744.15 and 1710.25 cm−1, respectively. The appearance of weak intensity bands at 1645.25 cm−1 is indicative of the presence of small amount of amides of protein in the extracts besides bands of weak intensity due to
Figure 1. FTIR-ATR spectra of yeast biomass Y44S (feed Sabouraud) and residue (Y44SSXR) after Soxhlet extraction.
superimposed spectra of biomass of Y44S and residue of Soxhlet extraction Y44SSXR (feed Sabouraud), as given in Figure 1, show significant variation in the intensity of ester (1744 cm−1), amides (1637.45, 1545.50 cm−1), and glycolipids/ sugar (1045.20 cm−1, 1164 cm−1) bands indicating different amount of these components in the biomass and its residue. Similarly, the spectra of residues of Y80G and Y80S biomasses after ultrasonic and Soxhlet extractions (Y80GUR and Y80SSXR (feed S = Sabouraud, G = glycerin, Yeast Y80) clearly depict the extent of extraction of neutral lipids (Figure 2). The intensity of the ester bands at 1744 cm−1 due to TAG
Figure 2. FTIR-ATR spectra of yeast Y80G (feed glycerin) and Y80S (feed Sabouraud) and their residue after ultrasonic (Y80GUR) and Soxhlet extraction (Y80SXR). D
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compared to vegetable oils (2−7%). The yeast extracts have been found to contain C16:1 in the range 0.7−1.2%, except 10.5% in Y44SSXMX grown with Sabouraud (S). The C17:0 has been detected in sample cultivated with xylose (Y80XUMX). From the comparative FAME analyses, it is evident that cultivation of both saturated and unsaturated esters are dependent on the types of feed (dextrose or glycerin) and types of yeast. The results have shown the presence of C18:1 and C18:2 as the predominant unsaturated fatty acid components besides common saturated fatty acids. The nature is similar to those present in vegetable oil except their relative concentration. 3.3. 1H NMR Analyses of Yeast Biomass Extracts. The 1 H NMR spectral features of yeast biomass ultrasonic extracts by mixed solvent (CHCl3/MeOH, 1:2) are similar to those of vegetable oils such as jatropha, soya, and NIST2772 CRM, except variation in the relative concentration of various fatty acids constituting glycerides, as depicted in the Figures 4−7.
Figure 3. FTIR-ATR spectra of ultrasonic cyclohexane, chloroform, and methanol extracts of yeast biomass Y80X (feed xylose) and chloroform/methanol extract of Y44S (Y44SUMX) (feed Sabouraud).
glycolipids/sugars. The weak intensity bands at 3007.56 cm−1 are indicative of unsaturation components in the extracts. The higher intensity of the acid band of Y44SUMX extract compared to all extracts of Y80X is indicative of the presence of higher amount of FFA in Y44SUMX. The NMR results of FFA content in algal extracts has also shown higher content in Y44SUMX (28.6% w/w) compared to Y80XUMX (9−12.9% w/w) (Table 1). Similarly, other extracts of algae yeast biomass samples were analyzed to monitor extraction of various components by different solvents. From these results, it can be concluded that FTIR-ATR technique can conveniently be used to monitor the extraction efficiency and nature of components extracted by different solvent systems. This is a new addition to the application of FTIR-ATR and information provided by using this developed methodology will be very useful for optimizing the culture condition, selection of an appropriate feed, medium, yeast, and time duration for modification and enhancement of neutral lipids content of yeast biomass. 3.2. Fatty Acid Profile of Solvent Extracts by GC/FID. The yeast extracts were found to be different in fatty acid profile, compared to algal extracts.1,15 However, results are matching with the fatty acid profile of most of vegetable oil with regards to the concentration of C18:3 (Table 3). The major difference is in the presence of C16:N (N = 1−4), C18:3, and higher carbon number acids such as C20:5 (EPA) and C22:6 (DHA) in quite high amount in the algal extracts. The content of C18:3 has been found to be less than 2% in all the yeast samples, commonly present in jatropha, kartuma, sunflower, etc. However, it is in the range 15−25% in algal extracts compared to yeast extracts and vegetable oils (1−8.1%) (Table 3). The oleic acid (C18:1) content in the extracts were found to be in the range 22.1−54.8%, which is commonly found in the usual vegetable oils. The C18:2 content is in the range 6.0− 19.8%, which is low compared to jatropha (36.6%), kartuma (57.7%), and other similar types of oils. The total saturated fatty esters content including C14:0. C16:0, and C18:0 have been found to be different in various yeast samples specific to types of yeast and feed. It is in the order of 22.1% in Y44SSXMX (Yeast 44, feed dextrose, S), 20.2% Y80SSXMX (Yeast 80, feed dextrose, S), 40.2% in Y80GUMX (Yeast 80, feed glycerin, G), and 38.3% in Y80XUMX (Yeast 80, feed xylose). This is quite high as compared to vegetable oils of rapeseed (6.6%), jatropha (20.3%), and kartuma (11.6%) (Table 3). The C18:0 is unusually very high (19.3%) in Y80G
Figure 4. 600 MHz 1H NMR spectra of mix solvent ultrasonic extracts Y80SUMX (feed Sabouraud) and Y80GUMX (feed G = glycerin). The spectra of jatropha oil and NIST2772 CRM are shown for comparison.
The spectra of extracts Y80GUMX (feed glycerin) and Y80SUMX (feed Sabouraud) of respective biomasses from yeast Y80 exhibit signals at 4.07 to 4.37 ppm (sn1,3; OCH2), 2.30 ppm (CH2CO), 5.25 ppm (sn2; OCH), 5.05−5.65 ppm (CHCH), 2.0 ppm (CH2CHCH), 1.26 and 1.30 ppm ((CH2)n), and 0.88 ppm (CH3) characteristic of triglycerides comprising of both saturated and unsaturated fatty acid chain (Figure 4). The appearance signal at 2.77 ppm due to CH2 protons of allylic CHCHCH2CHCH is indicative of the presence of linoleic (C18:2) chain in the triglycerides. The presence of very low intensities signals at 2.81 ppm (allylic CH2) and at 0.98 ppm due to terminal CH3 of N-3 fatty acids is indicative of the presence of small amount of C18:3 chain, which is also supported by the fatty acid profile by GC (Table 3). The broad and sharp signals of very weak intensities in the region of 3−4.0 ppm are due to glyco/ phospholipids. The presence of free fatty acids (FFA) are visible by triplet at 2.35 ppm, which is overlapped to some extent with the triplet of protons of ester group CH2CO at 2.30 ppm. The expanded spectra of regions of 2.1−4.50 ppm E
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Figure 7. 600 MHz 1H NMR spectra of mix solvent ultrasonic extract Y44SUMX (feed S = Sabouraud) and Soxhlet extract Y44SSXMX (feed S = Sabouraud).
Figure 5. 600 MHz 1H NMR spectra of cyclohexane extract (Y80XUCH), chloroform extract (Y80XUCL), methanol extract (Y80XUME), and mixed solvent extract (Y80XUMX) of yeast biomass Y80X (feed X = xylose) after ultrasonic extraction. The spectra of soya oil and biodiesel are shown for comparison.
Table 1. The spectra clearly show higher amount of FFA present in ultrasonic extract (28.6% w/w) compared to Soxhlet extraction (18.2% w/w). The polars ie glyco/phospho lipids are also present in the same amount (33−35.3% w/w) in both the extracts. The 1H NMR spectra of ultrasonic extracts of yeast Y80X (feed xylose) with mix solvents (MX) (Y80XUMX) as well as single solvents (CH, CL, ME) (Y80XUCH, Y80XUCL, Y80XUME) also exhibit spectral feature similar to extracts of yeast prepared from feed glycerin and Sabouraud and vegetable oils as discussed above except difference in the intensity of polar lipids in the region of 3−4 ppm (Figure 5). The spectra of soya oil, jatropha oil, NIST2772 CRM, and soya biodiesel are shown in the Figures 4−7 for comparison of spectral features with the spectral features of yeast extracts. All the spectra in Figure 5 indicate the absence of C18:3 as no signals at 2.81 ppm characteristic of C18:3 fatty acids/esters are observed in the spectra. The spectra show the presence of C18:2 components as evident from the appearance of signals at 2.77 ppm. The expanded part of the 2.1−4.4 ppm region of spectra of all extracts indicates the presence of FFA in different proportion as depicted in the region of 2.1−2.9 ppm (Figure 6). The CH2CO signals corresponding to FFA and ester are clearly distinguishable as marked in Figure 6. Since the signal at 3.66 ppm due to OCH3 protons of biodiesel are quite distinguishable from the signals of oil at 4.05−4.3 ppm due to OCH2 protons; therefore, both can be identified when present in a mixture. The appearance of weak intensities signals at 3.66−3.67 ppm in the spectra of extracts of biomass Y80X may be an indication of the presence of small amount of biodiesel. However, it needs to be confirmed as the signals of glycerol phosphate also appear in these regions. The results of single solvent extracts (CH, CL, ME) and mix solvent (CL-ME) (MX) given in the Tables 1 and 5 reveal not much differences in the content of neutral and polar lipids and iodine values except extraction of higher amount of FFA in MX solvent in spite of the difference in the polarity of solvents. This has been attributed to the presence of much higher amount of neutral lipids (64.7−85.1% w/w) compared to polar lipids. Thus, solvents have not shown any preference toward extraction of particular components.
Figure 6. 600 MHz 1H NMR part expanded spectra (TG, FFA, C18:N (N = 2−3)) cyclohexane extract (CH), chloroform extract (CL), methanol extract (ME) and mix solvent extract (MX) of different yeast biomass samples after ultrasonic (U) and Soxhlet (SX) extraction generated from feed G (glycerin), X (xylose), and S (Sabouraud). The spectra of soya oil is shown for comparison.
depicting both ester and acid signals of extracts are given in Figure 6. The CH2CO signal at 2.30 ppm may also contain a little contribution of CH2CNO of amide group of proteins. The presence of amides has been confirmed by IR analyses (section 3.1). The 1H NMR spectra of the extracts Y44SUMX and Y44SSXMX obtained from yeast biomass Y44S by ultrasonic and Soxhlet extraction do not show difference in the unsaturated fatty acid profile of C18:N (N = 1−3), as shown in the Figures 6 and 7. The comparison is evident from the identical spectral features and intensities of signals due to C18:2 and C18:3 fatty acid components as marked in Figure 7. These observations are in line with the composition of C18:N (N = 1−3) determined by GC (Table 3). However, there is a marked difference in the amount of total extract generated by ultrasonic (3.2%) and Soxhlet (9.3%) extraction methods as given in F
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Table 5. Content of Various Parameters (% w/w) of Different Yeast Extracts by 1H NMRa extract
5.75−5.0 ppm of CHCH Iv
4.37−4.10 ppm of OCH2 TG
TG*
TG**
2.33−2.38 ppm of CH2CO FFA
neutral lipids TG + FFA
polars
Y44GUMX Y44SUMX Y80GUMX Y80SUMX Y80SSXMX Y44SSXMX Y80CGLMX Y80XUCH Y80XUCL Y80XUME Y80XUMX
77.0 48.9 58.6 70.6 67.4 68.2 66.1 72.6 68.6 72.9 76.2
59.3 36.1 75.9 61.6 65.0 48.8 42.1 70.2 71.5 70.2 67.5
60.6 36.9 77.6 62.9 66.4 49.9 43.0 71.7 73.1 71.7 69.0
60.0 36.6 76.9 62.3 66.6 48.1 42.6 71.1 72.4 71.1 68.4
20.4 28.6 8.0 22.4 20.4 18.2 24.5 9.0 12.1 12.9 20.0
79.7 64.7 83.9 84.0 85.4 67.0 93.9* 79.2 83.6 83.1 87.5
20.3 35.3 16.1 16.0 14.6 33.0 6.1 20.8 15.4 16.9 12.5
a
Iv = iodine value g/100 g, TG = triglycerides, FFA = free fatty acids, polars = glycolipids/phospholids/amides, U = ultrasonic, SX = Soxhlet, CH = cyclohexane, CL = chloroform, ME = methanol, S = Sabouraud, G = feed glycerin, X = xylose, MX = mix solvent (CHCl3/MEOH, 1:2), * Contain 27.3% biodiesel, CGL = commercial glycerin, TG* = by factor Ktg = 26.58, TG** = by factor Ktg = 26.33, TG = by factor Ktg = 26.0.
The 1H NMR spectra of all yeast extracts obtained either by single or mix solvent using Soxhlet and ultrasonic extraction methods have not indicated signals due to mono- and diglycerides, as evident from the chemical shift region 3.60− 4.35 ppm, generally assigned to OCH2/CH2OH/CHOH protons of mono and diglycerides. This region may also contain these groups contributed by glycolipids. The vertical expansion of this region show only very weak signal intensities, perhaps as a result of phosphates or mono- and diglycerides. Certainly, their content cannot be more than 10% of the total glycerides. In order to compare quantitatively the distribution of C18:1, C18:2, and C18:3 fatty acids in the glycerides by NMR and GC, the percentage integral intensities (II) of chemical shift regions corresponding to these acids have been measured and compare with the fatty acid profile determined by GC (Tables 2 and 3). The integral intensities of signals at 2.77 and 2.81 ppm (allylic CH2) corresponding to C18:2 and C18:3 in the 1 H NMR spectra of different solvent extracts show a direct relationship with fatty acid profile determined by GC. Similarly, the II of regions 2.04−1.95 ppm and 2.1−2.04 ppm (CH2 CHCH) corresponding to C18:1 and C18:2 show a relationship with the GC profile. Thus, it is concluded that NMR II can be used to find out the presence and relative content of C18:N (N = 1−3) in the solvent extracts of yeast biomass. It is possible to determine quantitative distribution of C18:N (N = 1−3) and saturated fatty acids from the 1H NMR spectra of vegetable oils.21 However, in the present study, this methodology cannot used for this type of analyses as the yeast extracts are composed of both FFA and TAG. Thus, IIs have been used for comparison with the GC results. 3.4. Quantitative Analyses of Neutral Lipids. The basis of calculation is comparing NMR results of yeast and vegetable oils with the fatty acid profile determined by GC analyses. The fatty acid profile of yeast extracts is composed of C14, C16:0, and C18:0 (22.1−40.2%) and C18:N (N = 1−3) (76.9−59.8%) besides unsaturated C16:1 acids (Table 3). Following approaches based on eq 1 have been adopted to determine the total content of glycerides (TG): TG = K tgItg(4.05−4.38 ppm)
Approach 1. The average carbon chain of the fatty acid part of the glycerides is taken as C18:1, as shown by GC results of fatty acid profile (Table 3). In order to estimate TG, theoretical/experimental proportionality constant, which is 100 divided by percentage of the protons of functional group OCH2 (sn1, sn3) in the 1H NMR spectra of reference C18:1 triglycerides or its theoretical values have been calculated. The percentage of OCH2 protons is 3.84 (4/104) and accordingly Ktg is 26 (104/3.84) as calculated from eq 1. Results of TG in different solvent extracts determined by this factor using eq 1 is given in Table 1 and 5. Approach 2. In order to consider the contribution of all types of fatty acids, three blends of 10 different vegetable oils, marked as mixoil1, mixoil2, and mixoil3, were prepared containing different proportions of soya, kartuma, babassu, cotton, jatropha, rapeseed, sunflower, canola, palm kernel, and turnip. The NMR spectra were recorded and Ktg measured by the application of eq 1. The value of the constant, Ktg were found to be 26.91, 26.24, and 26.66 for mixoil1, mixoil2, and mixoil3, respectively. The average value of Ktg 26.58 has been used for determination ester content (TG*). Results are given in Table 5. Approach 3. Blends of oils and free fatty acids The results in Table 1 and 5 showed the presence of both free fatty acids (FFA) and TG. Therefore, in order to study the effect of FFA on the quantization of ester content, the blends of FFA of soya oil and mixoil1 in different proportion of each were prepared. The glycerides (TG**) content in the yeast extracts were determined from the calibration graph, TG vs Itg, generated from the NMR spectra of these blends (Figure 8). The results of TG** obtained by using the value of Ktg = 26.33, are given in Table 5. The results obtained by adopting the above-discussed approaches are quite close to each other. Thus, any of the methodologies can be used to determine TG content in yeast extracts. 3.4.1. Analyses of Polyunsaturated Fatty Acid/Ester and Iodine Value. The polyunsaturated fatty acids/esters (PUFA/ E) content and iodine values (Iv) have been estimated based on equations described in our earlier published work (see refs 26 and 29 for eqs 2 and 3, respectively). The following equations are used for the estimations:
(1)
where Itg is the integral intensity of the region 4.05−4.38 ppm and Ktg is the proportionality constant.
Iv = 14.75Iun(5−5.65) G
(2)
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range of 83.9−87.45%, but large variation in the fatty acid profile, FFA content (8−22.4%) and iodine values (58.6−76.2 g I2/100g) have been obtained. From the above results, it can be concluded that nature of neutral lipids with regards to fatty acid profile and iodine values are specific to the types of yeast and feed used for culturing biomass. Although, Soxhlet extraction method has extracted higher amount of total extracts due to higher temperature and time of extraction compared to ultrasonic extraction, the neutral lipid content including fatty acid profile and iodine value has not shown any trend and preference toward polarity of solvent.
Figure 8. Plot of TG (% Total Glycerides) vs Itg (% integral intensity of region 4.05−4.38 ppm in the 1H NMR spectra of blends of mixoil1 and soya FFA).
PUFE (3& > 3) = 10.2Ipf (0.98 ppm)
4.0. 13C NMR ANALYSES OF YEAST EXTRACTS The 13C NMR spectral analyses of yeast extracts have been carried out to confirm the findings of 1H NMR spectral analyses with regard to distribution of unsaturated fatty acid components; particularly direct observation of C18:N=1−3 chain in the glycerides. The spectra of extract Y80XUMX obtained by ultrasonic using mix solvent (CHCl3-MeOH) and mixoil1 (blend of 10 different vegetable oils) are given in Figure 9. Since it was not possible to spare sufficient quantity of
(3)
where Iun and Ipf are the integral intensities of functional groups of unsaturation (CHCH) and PUFE (CH3) in their respective chemical shift regions. Results obtained by these equations are mentioned in the Tables 1 and 5. 3.5. Comparison of Component Profile of Soxhlet and Ultrasonic Extracts. The 1H NMR spectral features of Soxhlet and ultrasonic extracts of biomass from feed Sabouraud (S) and yeast Y80 and Y44 using mix solvents were compared to find out differences in the components extracted and to determine structural parameters including fatty acid component profile. The ultrasonic (Y80SUMX) and Soxhlet (Y80SSXMX) extracts of biomass from yeast Y80 shows similar spectral features with regard to neutral lipids and nature of fatty acid profile except in the variation in their relative composition. However, there are significant differences found in the content and composition of components of neutral lipids (TAG, FFA), polar lipids, and iodine values in the Soxhlet extract Y44SSXMX and ultrasonic extract Y44SUMX from biomass generated from yeast Y44. The FFAs have been extracted in quite high amount (28.6%) in ultrasonic extract compared to 18.2% in Soxhlet extract. The neutral lipids are extracted in higher amount (85.4%) in Soxhlet extract compared to 64.7% in ultrasonic extract. The iodine value of Soxhlet extract (68.2 g I2/100 g) is higher than the iodine value of ultrasonic extract (48.9 g I2/100 g). This has been attributed to the difference in the composition of saturated (22.1%) and unsaturated fatty acid/ester (31.9%) extracted in both the extracts as shown by GC analyses (Tables 3 and 5). The NMR intensity data given in Table 6 support these results. The 1H NMR spectral analyses of ultrasonic mix solvent extracts of yeast biomasses generated with yeast Y80 and feeds glycerin (Y80GUMX), Sabouraud (Y80SUMX), and xylose (Y80XUMX) show interesting results regarding neutral lipid contents, fatty acids profile and iodine values (Iv) (Tables 3 and 5). The total neutral lipid content is nearly the same in the
Figure 9. 150 MHz 13C NMR spectra of ultrasonic yeast extract Yeast Y80XMX (feed xylose) biodiesel. MX = mix solvent. The spectra of mix oil (blend of different 10 vegetable oils) is shown for comparison.
samples for quantitative analyses, the 13C NMR spectral analyses results with relaxation delay of 3.0 s were used for identification and qualitative analyses of types of fatty acids constituting TAG. The spectral features are similar to those of mix oils with respect to similar chemical nature of triglycerides composed of acyl group and fatty acids chain commonly observed in the spectra of vegetable oils. The spectrum of a yeast extract indicates signals due to OCH2 (sn1, sn3 carbons)
Table 6. Percentage Integral Intensities (II) of Functional Groups of C18:N (N = 2−3)a sample/chemical shift (ppm)
Y44GU MX
Y44SU MX
Y80GU MX
Y80SU MX
Y80SSX MX
Y44SSX MX
Y80XU CH
Y80XU CL
Y80XU ME
2.1−2.04 C18:2 2.04−1.95 C18:1 2.77 C18:2 2.81 C18:3
2.9 5.7 1.0 0.5
2.0 5.2 0.7 0.4
2.5 4.1 1.3 0.2
2.3 5.6 1.2 nd
1.8 5.7 1.3 nd
1.4 6.5 0.5 0.1
4.0 3.8 1.9 nd
3.3 3.2 1.6 nd
3.6 3.5 1.7 nd
a
S = Sabouraud, G = glycerin, X = xylose, SX= Soxhlet, U = ultrasonic, G = glycerin, MX = mix solvent, CH = cyclohexane, CL = CHCl3, ME = methanol, nd = not detectable. H
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Figure 10. 150 MHz 13C NMR spectra of ultrasonic yeast extract Y80CGLYUMX (feed glycerin).
Table 7. Chemical Shift (ppm) Assigned for Identification for Algae and Yeast Oil chemical shift
TG
FFA
BD
C18:2
C18:3
C22:6
PUFA
CHCH Iv
4.05−4.38
2.34−2.35(2.33−2.38)
3.66
2.77
2.81
2.40
0.98
5.0−5.7
Table 8. 13C NMR Chemical Shift (ppm) Assignments for Vegetable and Yeasta group
jatropha
mix oil
fish oil
yeast1
yeast2
OCH2, OCH CH3 C18:0 C18:1 C18:2 C18:3 CHCH C22:6 CO
68.93, 62.06
68.95, 62.09
68.95, 62.06
68.92, 62.16
69.07, 61.99
14.13
14.16
14.19
14.18
14.09
14.11 14.32 132.5−127.0
14.15(s)
14.14
127.96, 128.13−14, 129.75, 129.78, 130.085, 130.098, 127.05 132.09 173.34, 173.38
127.95, 128.11, 129.77, 130.07, 130.27 172.6
130.5−127.5 173.20, 173.26
C22:6 C22:5 a
126.95, 131.90, 126.89, 131.82
172.87, 173.29, 173.32 172.28, 172.64
173.15 173.52
(s) superscript = shoulder.
signals were confirmed by spectral analyses of a blend of FFA, biodiesel, and oil. Similarly, the spectra of few of the yeast extracts available with reasonable quantities show signals of chemical nature similar to those observed for Y80XUMX and mix oil. The 13C NMR spectra of yeast extracts show low intensities signals due to methyl carbons of phospholipids in the chemical shift regions of 51−55 ppm, which has also been observed in their respective 1H NMR spectra between 3.2 and 3.67 ppm. The higher carbon number fatty acid esters such as C20:5 and C22:6 have been found to be absent as no characteristics signals were observed in the 1H and 13C NMR spectra of yeast extracts. The unsaturated region of the spectrum between 125 and 132 ppm reveals information regarding the presence of C18:N=1−3 fatty acids/ester components. The presence of C18:3 is clearly shown by the appearance of weak intensities
(62.16 ppm) and OCH (sn2 carbon) (68.92 ppm) of acyl groups, CHCH (120−132 ppm), ester carbonyl CO (172.99−173.38 ppm), acid carbonyl CO (179.63 ppm), terminal methyl (14.15, 14.19 ppm) of unsaturated and saturated fatty acid chain of glycerides, β-carbon (22.64− 24.93 ppm), γ-carbon (31.59−31.99 ppm), and intense signals due to long alkyl fatty chain part (28.5−30 ppm). The signals at 14.15 ppm and 14.19 have been assigned to C18:2, and combine of C18:1 and saturated chain of C18:0, C16:0, and C14:0 components, respectively. These components are also confirmed by appearance of intense signals at 34.12 and 34.26 ppm (CH2CO) due to C18:2 and other fatty acid chain, respectively. The features are similar to mix oil except the appearance of low intensity signal at 14.30 ppm (CH3) and 34.69 ppm (CH2CO) due to C18:3 fatty acid chain for mix oils, as shown in the same Figure 9. The identities of these I
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signals at 127.15 and 131.99 ppm in the spectrum of mix oil in contrast to spectrum of yeast oil not showing any signals in this region confirming the absence of C18:3 components in yeast extracts. The other prominent signals due to C18:1 and C18:2 are observed at 127.96−128.14 ppm (C18:2) and 129.76− 130.09 ppm (C18:1, C18:2) in the spectra of both mix oil and yeast extract. The glycerin produced by transesterification of vegetable oils in the laboratory was used for cultivation of yeast biomass (Y80CGLU) by utilization of yeast Y80. The analyses of the glycerin by NMR have shown the presence of oil, biodiesel, and glycerin as the major products besides impurities of unknown nature. The analyses of ultrasonic mix solvent extracts Y80CGLYUMX by 1H and 13C NMR indicate the presence of triglycerides, biodiesel (51.34 ppm), and FFA (179.52 ppm) as the main products (Table 5, Figure 10). The characteristics chemical shift regions assigned to various carbons corresponding to groups/components of yeast oil and mix oil along with jatropha oil are compiled in Tables 7 and 8.
Figure 11. Plot of FFA (% free fatty acids) vs Iffa (% integral intensity of region 2.33−2.37 ppm in the 1H NMR spectra of blends of mixoil1 and soya FFA).
5.0. FREE FATTY ACID (FFA) ANALYSES IN THE EXTRACTS 1 The H NMR analyses of the extracts have clearly shown the presence of free fatty acid from the appearances of signals at 2.34−2.35 ppm as shown in the expanded part of spectra of the yeast extracts (Figure 6). The assignments of signals at 2.34− 2.35 ppm due to FFA were confirmed by internal addition of mixture of saturated and unsaturated fatty acids of soya oil as well as standards of unsaturated fatty acids in the yeast extracts. It has been observed that chemical shift of CH2CO signal of FFA are independent of the nature and carbon chain length of fatty acids, saturated or unsaturated. Signals due to saturated (C14−C18:0) and unsaturated fatty acids (C18:N, N = 1−3) are observed at 2.34−2.35 ppm, partly overlapped with ester signals at 2.30−2.31 ppm. The presence of free fatty acids was also confirmed by the 13C NMR spectral analyses of the few extracts, which has clearly indicated a signal at 179−180 ppm (Figure 10). The FTIR analyses of the solvent extracts have also shown bands at 1707−1710 cm−1 due to acid carbonyl group besides the ester bands at 1738−42 cm−1. In the earlier published work on the estimation of FFA in vegetable oils, the estimation of FFA has been carried out from the combined integral intensity of chemical shift region of 2.20−2.40 ppm due to CH2CO protons of FFA and ester.28 Since, yeast oils are composed of both neutral and polar lipids, this method cannot be applied for the estimation of FFA. The CH2CO signals at 2.4−2.1 ppm also contain contribution from fatty acid part of glyco/phospho-lipids and amides. In order to estimate FFA, the same blends of fatty acids of soya oil in mix oil in different proportions, as described for the determination of TG content, were analyzed by 1H NMR. The FFA content has been found to directly proportional to the integral intensities (Iffa) of the chemical shift region of 2.33− 2.37 ppm due to signals of CH2CO of FFA as shown in the graphs of Figures 11 and 12. The proportionality constant Kffa has been calculated to be 17.05. FFA = K ffaIffa
Figure 12. Part 1H NMR spectra of blends BL20%FFA and BL40% FFA (20% and 40% soya FFA blended in mixoil1) and yeast extracts in MX (mix solvent, CHCl3/MeOH).
(1) The content and composition of biomass has been found to be specific to types of yeast and feed used for cultivation. (2) The extraction efficiency of each solvent (cyclohexane, chloroform, methanol, and mixed solvents) toward extraction of neutral lipid (glycerides TAG, DAG, and MAG and free fatty acids (FFA)) and polar lipids have been determined in the range of 59.6−95% using Soxhlet and ultrasonic extraction procedures. (3) The triacyl glycerides (TAG) are composed of more than 90% of the triglycerides as apparent from their integral intensity values in the region of 4.05−4.38 ppm due to glycerides. (4) The fatty acids/esters are primarily composed of both saturated and unsaturated fatty acids (C14:0, C16:0, C18:0, C18:1, C18:2, C18:3). The C18:1 and C18:2 fatty acid components are present in much higher amount compared to C18:3 in the extracts obtained from all types of feeds from yeast Y80 and Y44. The amount of C18:3 components are produced in small amount (1− 2.3%) by yeast Y44 compared to yeast Y80 (
