Screening
One hundred and seven yeast strains previously isolated from boza were tested in YPD medium using commercial glycerol or crude glycerol as carbon source. It was determined that crude glycerol, provided by a private waste oil collection company, showed the following properties: pH 6.9 and purity 70.775 %. Ninety-two strains which could be growing both of the medium or only crude glycerol medium were stained with Sudan black B technique and observed under microscope for the presence of fat globules within the cell. Sixty yeast strains were found positive for showing fat globules within the cell (Fig. 1) and they were selected as potential lipid biomass producers (Table 1). It has been demonstrated that Sudan black B staining technique is a rapid method of screening lipid production. In their work Kitcha and Cheirsilp [16] were determined rapidly whether 889 yeast strains had lipid production ability by Sudan black B technique and they have revealed that 23 isolates are potential lipid producers.
Carbon source
Table 1
Growth on commercial/crude glycerol and results of staining
Carbon source
|
Number of yeast strains
|
|
Growth
|
Sudan black B staining
|
|
|
+
|
-
|
|
Crude glycerol
|
1
|
1
|
|
|
Crude glycerol and commercial glycerol
|
91
|
59
|
32
|
|
Commercial glycerol
|
6
|
no staining
|
no staining
|
|
In the first screening, we performed lipid screening on both crude glycerol and commercial glycerol medium. In the experiments, two separate glycerol’s were used to see the differences in metabolizing the crude and commercial glycerol of microorganisms. In general, 107 isolates grew on both glycerol media. Only 6 of them did not grow in crude glycerol and 9 of them did not grow in both (Table 1). Similar to our work previously shown that crude glycerol is a useful waste substrate for lipid production by Yarrowia lipolytica [22, 23] and some other yeasts [5].
Results of the secondary screening, summarized in Fig. 2, show the final OD600 values of the 60 yeasts in a medium containing crude glycerol as carbon source (40 g/l). Between the 60 yeast strains tested, 25 grew well and reached final OD600 of nearly 2 Units or up. They were selected for calculation of growth parameters and lipid yield. Good performance was exhibited by isolate 4 that reached final OD600 of 2.74 on the other hand isolate 37 grew most poorly (Fig. 2). Many researches have been done on the microbial conversion of crude glycerol. However, impurities in crude glycerol often negatively affected the success of these processes [24]. Since we are working with a crude glycerol containing higher impurities (purity 70.775 %) than various publications (14, 22, 23; 25), the selection was made among the isolates by looking at their ability to metabolize crude glycerol in the screening step. Also, in microbial lipid production studies, ~ 20–100 C/N ratio has been used because it is known that high C/N ratio promotes lipid production in some yeast species [14, 22, 23, 26]. On the other hand, as we mentioned above, the crude glycerol we used has high impurities for this reason a second stress factor affecting growth due to low nitrogen ratio was not used.
Calculation Of Lipid Yield Parameters And Biomass
Among 25 isolates, four of them (1, 4, 6 and 18) gave high lipid yield with lipid content higher 55% when grown in broth medium containing crude glycerol for 72h. Table 2 shows biomass and lipid yield parameters of all strains. The lowest amount of lipid yield obtained from 25 yeast isolates selected by first-screening (25, 55%) was indicative of the accuracy of the pre-screening. It was determined that isolate 1 gave highest lipid content and highest lipid yield up to 64.94% and 7.14 g/L, respectively (11 mg/mL of biomass). High lipid accumulation and also high lipid coefficient in crude glycerol media, isolate 1 was selected for the further studies.
Table 2
Charaterization of lipid production of yeasts and statistical data
Isolate number
|
Biomass (g/L)
|
Lipid yield (g/L)
|
Lipid %
|
Glycerol utilized (g/L)
|
Lipid coefficient (g lipid/g glycerol)
|
1
|
11 ± 0.55
|
7.14 ± 0.07
|
64.94 ± 0.65
|
17.49 ± 0.35
|
0.409 ± 0.004
|
2
|
9 ± 0.36
|
3.89 ± 0.12
|
43.17 ± 1.30
|
12.17 ± 0.49
|
0.319 ± 0.010
|
3
|
11 ± 0.44
|
5.49 ± 0.22
|
49.87 ± 1.99
|
27.75 ± 0.83
|
0.197 ± 0.008
|
4
|
9 ± 0.63
|
5.54 ± 0.22
|
61.59 ± 2.46
|
27.86 ± 1.39
|
0.199 ± 0.009
|
5
|
9.5 ± 1.14
|
4.34 ± 0.30
|
45.71 ± 3.20
|
8.48 ± 0.09
|
0.512 ± 0.033
|
6
|
11.5 ± 0.69
|
6.34 ± 0.06
|
55.16 ± 0.55
|
26.24 ± 0.79
|
0.242 ± 0.002
|
7
|
10.5 ± 0.53
|
4.40 ± 0.13
|
41.9 ± 1.26
|
27.82 ± 1.39
|
0.158 ± 0.005
|
8
|
15.5 ± 0.47
|
4.97 ± 0.20
|
32.07 ± 1.28
|
26.36 ± 0.53
|
0.189 ± 0.007
|
9
|
13 ± 0.65
|
5.26 ± 0.16
|
40.44 ± 1.21
|
20.16 ± 0.81
|
0.261 ± 0.008
|
10
|
11 ± 0.99
|
5.72 ± 0.11
|
51.95 ± 1.04
|
27.81 ± 0.56
|
0.206 ± 0.004
|
11
|
12 ± 0.48
|
5.89 ± 0.12
|
49.05 ± 0.98
|
27.85 ± 1.11
|
0.211 ± 0.005
|
12
|
8.5 ± 0.85
|
2.17 ± 0.07
|
25.55 ± 0.77
|
5.37 ± 0.06
|
0.404 ± 0.012
|
16
|
12.5 ± 0.37
|
5.54 ± 0.17
|
44.34 ± 1.33
|
27.6 ± 0.83
|
0.201 ± 0.006
|
18
|
10 ± 0.7
|
5.77 ± 0.12
|
57.71 ± 1.15
|
27.87 ± 1.39
|
0.207 ± 0.004
|
26
|
11.5 ± 0.35
|
5.77 ± 0.23
|
50.19 ± 2.00
|
27.87 ± 0.56
|
0.207 ± 0.008
|
30
|
11.5 ± 0.23
|
5.31 ± 0.32
|
46.21 ± 2.77
|
27.42 ± 1.10
|
0.194 ± 0.011
|
41
|
9.5 ± 0.86
|
5.09 ± 0.05
|
53.53 ± 0.54
|
27.87 ± 1.39
|
0.183 ± 0.002
|
63
|
11.5 ± 0.46
|
5.54 ± 0.11
|
48.19 ± 0.95
|
27.88 ± 0.83
|
0.199 ± 0.004
|
68 − 1
|
12.5 ± 0.38
|
6.17 ± 0.09
|
49.37 ± 0.75
|
27.61 ± 0.28
|
0.224 ± 0.003
|
68 − 2
|
11.5 ± 0.92
|
5.83 ± 0.06
|
50.68 ± 0.51
|
27.71 ± 0.28
|
0.210 ± 0.002
|
82
|
12.5 ± 0.25
|
5.54 ± 0.17
|
44.34 ± 1.33
|
27.55 ± 1.38
|
0.201 ± 0.006
|
84
|
11 ± 0.33
|
4.97 ± 0.20
|
45.19 ± 1.81
|
21.57 ± 0.64
|
0.231 ± 0.009
|
95 − 1
|
11 ± 0.66
|
4.74 ± 0.14
|
43.12 ± 1.29
|
27.87 ± 1.11
|
0.170 ± 0.005
|
95 − 2
|
10 ± 0.8
|
4.51 ± 0.09
|
45.14 ± 0.90
|
27.88 ± 0.56
|
0.162 ± 0.003
|
110
|
10 ± 0.6
|
5.49 ± 0.06
|
54.86 ± 0.55
|
27.83 ± 0.45
|
0.197 ± 0.002
|
After 72 hours of incubation, the analysis of residual crude glycerol is performed for calculation of lipid coefficient. Yeast isolates were not utilized glycerol completely. It was thought that the accumulated lipids in the yeast cells were not used for lipid-free biomass synthesis because yeasts do not use their stored lipids before the run out of extracellular carbon supply [27, 28, 29].
For indicating further availability of using strains selected in this study for single cell oil production from crude glycerol, lipid production results of this study were compared with other oleaginous microorganisms using crude glycerol as the sole carbon source. The biomass, lipid content and lipid yield of Yarrowia lipolytica ATCC 20460 after fermentation were 11.6 g/L, 31% and 3.6 g/L, respectively [30]. The biomass, lipid content and lipid yield of Cryptococcus curvatus ATCC 20509 after fermentation were 29.2 g/L, 26.0% and 7.7 g/L, respectively [31]. The biomass, lipid content and lipid yield of Kodamaea ohmeri BY4-523 and Trichosporanoides spathulata JU4-57 after fermentation were 10.3 g/L, 53.0%, 5.5 g/L and 17.0 g/L, 43%, 7.4 g/L, respectively [32]. The biomass, lipid content, lipid yield and lipid coefficient of Rhodotorula glutinis TISTR 5159 were 8.17 g/L, 53%, 4.3 g/L, and 7%, respectively [33]. The biomass, lipid content, lipid yield and lipid coefficient of Mortierella isabellina were 6.2 g/L, 53%, 3.3 g/L and 12% respectively [34]. The biomass, lipid content, lipid yield, and lipid coefficient of Trichosporon fermentans and Trichosporon cutaneum after 8-day fermentation were 16.0 g/L, 32.4%, 5.2 g/L, 16.5%, and 17.4 g/L, 32.2%, 5.6 g/L, 17.0%, respectively [35]. Maximum lipid content of lipid-engineered Yarrowia lipolytica strain JMY4086 after continuous culture with molasses and crude glycerol under different oxygenation conditions was 31% [23]. Biomass and lipid yield parameters of selected 25 isolates in this study by two step screening were moderate or higher than strains above, exhibiting that strains in current study were encouraging strains for single cell oil production using crude glycerol as the sole carbon source.
Fatty Acid Compositions
Lipid samples produced by fermentation on crude glycerol were analysed by GC/MS. Percentages of total fatty acids were shown in Table 3. The fatty acid contents of fatty yeasts are generally known to consist of long chain fatty acids such as C16 and C18 [36]. Lipids of selected 25 oleaginous fungi were composed mainly of long-chain fatty acids with 16 and 18 carbon atoms. Main fatty acids of microbial lipids are an important indicator that whether it can be used for biodiesel production. For the quality of biodiesel and optimum fuel properties Fatty acid composition is quite important. Increasing the chain length of fatty acids increases the cetane number, combustion temperature, melting point and viscosity of clean oil compounds of biodiesel. Furthermore, the high amount of oleic acid in fatty acid content has a positive effect on biodiesel properties [36]. Presence of C16:0, C18:0, C18:1 and C18:2 fatty acids were detected by GC-MS. FAME analysis of yeast strains are relevant from a biodiesel standpoint and also to discriminate closely related strains. The total unsaturated fatty acids, oleic acid and linoleic acid similar to that of plant oils were > 50 % of microbial oils except isolate 7. Knothe [37] was reported that the best fatty acid composition for biodiesel production is palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). This indicates that lipids from oleaginous yeasts selected in this study have potential use as a feedstock for second generation biodiesel production. These fatty acids were reported by Kumar et al. for lipid production from municipal waste in the glycerol medium, and they also concluded that these fatty acids are suitable for biodiesel production [25].
Table 3
Percentages of total fatty acids from selected isolates
Isolate No
|
Myristic acid (%)
|
Pentadecanoic acid (%)
|
Palmitic acid (%)
|
palimitoleic acid (%)
|
margaric acid (%)
|
stearic acid (%)
|
oleic acid (%)
|
linoleic acid (%)
|
α-linolenic acid (%)
|
lignoseric acid (%)
|
Total unsaturated fatty acids (%)
|
Total saturated fatty acids (%)
|
|
C14:0
|
C15:0
|
C16:0
|
C16:1
|
C17:0
|
C18:0
|
C18:1
|
C18:2
|
C18:3
|
C24:0
|
|
|
1
|
0
|
0
|
11.95
|
0
|
0
|
36.82
|
17.51
|
33.72
|
0
|
0
|
51.23
|
48.77
|
2
|
0
|
0
|
13.04
|
0
|
0
|
9.86
|
34.78
|
42.32
|
0
|
0
|
77.10
|
22.90
|
3
|
0.07
|
0
|
10.20
|
0
|
0
|
3.33
|
37.82
|
48.26
|
0.23
|
0
|
86.30
|
13.60
|
4
|
0.25
|
0.58
|
19.73
|
0
|
0.68
|
7.18
|
61.89
|
9.69
|
0
|
0
|
71.58
|
28.42
|
5
|
0.34
|
0
|
15.41
|
0.73
|
0
|
5.71
|
46.16
|
29.27
|
0
|
1,71
|
76.16
|
23.17
|
6
|
0.21
|
0
|
20.96
|
0
|
0
|
7.05
|
58.67
|
11.15
|
0
|
0
|
69.83
|
28.23
|
7
|
1.03
|
0
|
55.86
|
0
|
0
|
20.76
|
14.84
|
5.00
|
0
|
0
|
14.84
|
82.65
|
8
|
0.43
|
0
|
18.54
|
0.91
|
0
|
12.28
|
52.86
|
13.76
|
0
|
0
|
67.54
|
31.25
|
9
|
0.23
|
0
|
9.76
|
0.99
|
0
|
5.98
|
36.71
|
44.40
|
0
|
1.83
|
82.09
|
17.80
|
10
|
0.46
|
0
|
23.48
|
0.66
|
0
|
11.52
|
46.29
|
15.45
|
0
|
0
|
62.40
|
35.46
|
11
|
0
|
0
|
33.91
|
0
|
0
|
13.77
|
13.88
|
38.44
|
0
|
0
|
52.32
|
47.68
|
12
|
0
|
0
|
11.85
|
0
|
0
|
5.97
|
31.46
|
50.72
|
0
|
0
|
82.18
|
17.82
|
16
|
0
|
0
|
12.38
|
0
|
0
|
5.02
|
47.81
|
34.56
|
0
|
0
|
82.37
|
17.39
|
18
|
0
|
0
|
17.09
|
0
|
0
|
6.17
|
53.75
|
21.77
|
0
|
0
|
75.51
|
23.27
|
26
|
0.15
|
0
|
10.33
|
0
|
0
|
3.48
|
34.04
|
51.71
|
0
|
0
|
85.75
|
13.95
|
30
|
0
|
0.19
|
8.67
|
1.53
|
0.45
|
4.10
|
29.67
|
55.39
|
0
|
0
|
86.58
|
13.42
|
41
|
0.25
|
0.30
|
12.96
|
0
|
0.40
|
5.52
|
40.65
|
39.33
|
0
|
0
|
79.97
|
19.43
|
63
|
0.51
|
0.28
|
25.13
|
0.93
|
0.40
|
9.69
|
48.90
|
11.67
|
0
|
0
|
61.50
|
36.01
|
68 − 2
|
0.18
|
0
|
13.34
|
0
|
0
|
5.17
|
38.35
|
41.63
|
0,60
|
0
|
80.58
|
18.69
|
68 − 1
|
0.20
|
0.21
|
17.96
|
0
|
0.16
|
7.53
|
52.48
|
19.88
|
0
|
0
|
72.36
|
26.05
|
82
|
0.27
|
0.21
|
19.63
|
0
|
0.21
|
8.41
|
58.54
|
10.69
|
0
|
0
|
69.23
|
28.74
|
84
|
0.55
|
0.41
|
21.04
|
1.12
|
0.46
|
10.61
|
52.75
|
11.07
|
0
|
0
|
64.94
|
33.07
|
95 − 1
|
0.42
|
0
|
20.53
|
0.61
|
0
|
6.25
|
33.42
|
38.12
|
0
|
0
|
72.14
|
27.20
|
95 − 2
|
0.37
|
0
|
18.34
|
0
|
0
|
5.24
|
23.65
|
52.02
|
00
|
0
|
75.67
|
23.95
|
110
|
0.23
|
0.42
|
14.65
|
0.24
|
0.57
|
5.90
|
51.36
|
25.95
|
0
|
0
|
77.55
|
21.77
|
The predominant fatty acid in the lipid from isolate 1 that gave highest lipid content and highest lipid yield on crude glycerol was stearic acid (36,82%) followed by linoleic acid (33.72 %), oleic acid (17.51 %), and palmitic acid (11.95 %). The results of fatty acid analysis after lipid production and optimization studies with Rhodotorula sp. [38], Psuedozyma sp. [1] were similar to ours. Areesirisuk et al. [1] were also remarked that these fatty acid profiles were the same as the vegetable oils used in biodiesel production and fatty acid profiles of some oleaginous yeasts, and also that the fatty acid composition used in biodiesel production affected biodiesel quality.
Identification Of Oleaginous Yeasts
The 25 oleaginous yeast isolates that selected by screening in the glycerol medium were identified by the molecular diagnosis methods based on sequencing of the ITS region of the rDNA and D1/D2 regions of 26 S of the rDNA. Results showed high sequence similarity (98–99%) with strains in the GenBank. The GenBank accession numbers of the ITS and D1/D2 regions of strains sequenced in this study are given in Table 4. 25 isolates were identified as 13 different strains (Table 4). Isolate 7 and 26 could not be identified, they might be new strains. Two of strains were reported for the first time as lipid- producing yeast, because no information on lipid production of Pichia cactophila and Clavispora lusitaniae was found in the literature. The results of this study have been shown that there were plentiful new microorganism resources which produce lipids by using glycerol as sole carbon source.
Table 4
Identification results of 25 oleaginous yeasts.
İsolate no.
|
Sequenced region
|
GenBank accession number
|
Species Name
|
1
|
ITS
|
MW493237
|
Pichia cactophila
|
D1/D2
|
MW487301
|
2
|
ITS
|
MW493238
|
Pichia fermentans
|
D1/D2
|
MW487302
|
3
|
ITS
|
MW493239
|
Rhodotorula mucilaginosa
|
D1/D2
|
MW487303
|
4
|
ITS
|
MW493240
|
Pichia fermentans
|
D1/D2
|
MW487304
|
5
|
ITS
|
MW493241
|
Pichia fermentans
|
D1/D2
|
MW487305
|
6
|
ITS
|
MW493242
|
Rhodotorula mucilaginosa
|
D1/D2
|
MW487306
|
7
|
ITS
|
not identified
|
D1/D2
|
8
|
ITS
|
MW493243
|
Clavispora lusitaniae
|
D1/D2
|
MW487307
|
9
|
ITS
|
MW493244
|
Saccharomyces cerevisiae
|
D1/D2
|
MW487308
|
10
|
ITS
|
MW493245
|
Wickerhamomyces anomalus
|
D1/D2
|
MW487309
|
11
|
ITS
|
MW493246
|
Rhodotorula mucilaginosa
|
D1/D2
|
MW487310
|
12
|
ITS
|
MW493247
|
Rhodotorula mucilaginosa
|
D1/D2
|
MW487311
|
16
|
ITS
|
MW493248
|
Candida glabrata
|
D1/D2
|
MW487312
|
18
|
ITS
|
MW493249
|
Pichia fermentans
|
D1/D2
|
MW487313
|
26
|
ITS
|
not identified
|
D1/D2
|
30
|
ITS
|
MW493250
|
Yarrowia lipolytica
|
D1/D2
|
MW487314
|
41
|
ITS
|
MW493251
|
Candida inconspicua
|
D1/D2
|
MW487315
|
63
|
ITS
|
MW493252
|
Wickerhamomyces anomalus
|
D1/D2
|
MW487316
|
68 − 1
|
ITS
|
MW493253
|
Pichia fermentans
|
D1/D2
|
MW487317
|
68 − 2
|
ITS
|
MW493254
|
Pichia fermentans
|
D1/D2
|
MW487318
|
82
|
ITS
|
MW493255
|
Pichia fermentans
|
D1/D2
|
MW487319
|
84
|
ITS
|
MW493256
|
Rhodotorula mucilaginosa
|
D1/D2
|
MW487320
|
95 − 1
|
ITS
|
MW493257
|
Candida albicans
|
D1/D2
|
MW487321
|
95 − 2
|
ITS
|
MW493258
|
Pichia anomala
|
D1/D2
|
MW487322
|
110
|
ITS
|
MW493259
|
Candida inconspicua
|
D1/D2
|
MW487323
|