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Optimization of lipid production by Mortierella isabellina using glycerol, a by-product of biodiesel production as a carbon source.

In recent years, much attention has been paid to the exploration and development of alternative oil sources from oleaginous microorganisms such as yeasts, fungi, bacteria, and microalgae (1). Oleaginous microorganisms are able to accumulate more than 20% of their dry biomass as oil (single cell oil-SCO) (2,3), which is viewed as a possible alternative for commercial oil producers as food and energy resources due to their fatty acid compositions being similar to that of common plant oils (4,5,6,7,8). Among the group of oleaginous microorganisms, fungi (molds) have attracted considerable attention because they are able to be cultivated on a wide range of substrates, particularly waste such as glycerol, as well as lignocellulosic biomass (rice hull and corn stover) hydrolysates (9,10), have short life cycles, and display rapid growth rates, as well as being unaffected by space or climatic variations and allowing production to be easily scaled up in the fermentation process to produce more lipid biomass (8). The oleaginous filamentous fungus Mortierella isabellina has been reported as one of the potential oleaginous fungal species due to its ability to accumulate large amounts of lipids by up to 80% of their cellular dry weight (11). It also has a high tolerance for cultivation on different residual materials as substrates (9,10) and short generation times with minimal nutrient requirements (11).

Biodiesel is one of the most promising renewable energy sources receiving significant attention worldwide. The rapid increase in demand for renewable fuels has also substantially increased biodiesel production (12). In Thailand, oil palm (Elaeis guineensis) is used as the major feedstock for biodiesel production. From the production of biodiesel via a transesterification reaction, approximately 10% by weight of glycerol is created as a major by-product, most of which is disposed of as a low-value product because refining glycerol is not feasible, especially for medium and small biodiesel plants. Therefore, several methods have been investigated for utilizing this abundant waste. The use of crude or partially-purified glycerol as a carbon source for cultivating oleaginous microorganisms is promising for the utilization of such abundant glycerol waste. It could also assist in reducing the cost of microbial oil production (13).

In this work, three strains of M. isabellina oleaginous fungus were screened for their ability to utilize glycerol, a by-product partially purified from palm oil-biodiesel production, as a carbon source for biomass and lipid production. The optimization of lipid production for the most promising oleaginous strain was conducted using response surface methodology (RSM).

MATERIALS AND METHODS

Glycerol sample

The glycerol sample (93.97% purity), a by-product of palm oil-biodiesel production, was obtained from Patum Vegetable Oil Co., Ltd., Thailand.

Microorganisms and culture conditions

Three fungal strains of M. isabellina, NBRC 7874, NBRC 7884, and NBRC 105998 were obtained from the Department of Biotechnology, National Institute of Technology and Evaluation (Chiba, Japan) and preserved on potato dextrose agar (PDA, Difco, USA). The growth medium was comprised of (per 1 L distilled water) a glycerol sample from biodiesel production 30 g, yeast extract 1 g, K[H.sub.2]P[O.sub.4] 7 g, [Na.sub.2]HP[O.sub.4] 2 g, MgS[O.sub.4] .7[H.sub.2]O 1.5 g, Fe[Cl.sub.3].6[H.sub.2]O 0.08 g, ZnS[O.sub.4].7[H.sub.2]O 0.001 g, MnS[O.sub.4].5[H.sub.2]O 0.0001 g, Ca[Cl.sub.2].2[H.sub.2]O 0.1 g, CuS[O.sub.4].5[H.sub.2]O 0.0001 g, Ca[(N[O.sub.3]).sub.3].[H.sub.2]O 0.0001 g, [(N[H.sub.4]).sub.2]S[O.sub.4] 0.5 g. All experiments were performed in 250 ml Erlenmeyer flasks containing 50 ml of growth medium and sterilised in an autoclave at 121 [degrees]C for 15 min. Five 7 mm fungal discs (9.35x[10.sup.6] spores) from a 7-day-old culture of each fungal strain was added to each flask. All cultures were incubated in a rotary shaker at 180 rpm and 30 [+ or -] 1 [degrees]C.

Lipid extraction and fatty acid determination

The lipid content of each fungal biomass was extracted to determine lipid accumulation efficiency at 4, 8, 12 and 16 days according to Folch et al.(1957) (14) with a slight modification. The extracted lipids were measured using a calibrated balance. Lipid content per 100 g of cell dry weight was calculated. Lipid transesterification and fatty acid extraction were carried out following the procedures described by Riengsilchai et al. (2013) (15). The FAMEs were analysed using a gas chromatograph (GC) fitted with a flame ionization detector (FID) (Shimadzu, Japan). A fused capillary column BPX70 (0.32 mm i.d. x 30 m, 0.25 [micro]m film thickness; SGE, Australia) was used. GC-FID was performed using the following conditions: carrier gas He; flow rate 1.5 ml/min; injection temperature 250 [degrees]C; oven temperature programmed from 210 [degrees]C (9 min hold) to 240 [degrees]C at 20 [degrees]C/min (6 min hold); detector temperature 280 [degrees]C. Individual fatty acids were identified by comparing them to the retention time of authentic fatty acid standards obtained from Sigma Co., USA.

Optimization of cultivation conditions for the best oil-producing strain obtained by response surface methodology (RSM)

Response surface methodology (RSM) was performed using a 5-level 2-factor central composite design (CCD). There were 13 experiments involving the two variables investigated for glycerol concentration [15.86-44.14 (g/L)] and inoculum size [2-8 fungal discs (1.87x[10.sup.6] spores/disc)]. The factors and their respective levels are summarized in Table 1.

RESULTS AND DISCUSSION

Screening of potential M. isabellina strains for lipid accumulation

Three M. isabellina strains were cultured in a growth medium (using glycerol at a concentration of 30 g/L) and incubated at 30 [degrees]C on a shaker set at 180 rpm. The fungal biomass of each strain was isolated and lipid accumulation in the fungal mass was extracted at 4, 8, 12 and 16 days. The data for fungal mass (weight) and lipid content accumulation for each fungal strain at different periods of time are given in Table 2.

The results showed that M. isabellina NBRC 105998 was the best lipid-accumulating strain among the three strains tested, with a total biomass of 0.71 g/L and a lipid content of 35.02% after 12 days.

Optimization of cultivation conditions for the best oil-producing strain and determination of fatty acid profiles in lipid samples

The optimum cultivation condition for the best oil-producing strain obtained (M. isabellina NBRC 105998) was evaluated using response surface methodology (RSM). A 5-level 2-factor central composite design (CCD) was employed for the optimization of culture conditions, as shown in Table 3. A second-order polynomial equation was used to fit the predicted model to the experimental data. The development of design matrices and analysis of variance (ANOVA) for the RSM model was conducted.

The effect of the combination of glycerol concentration and inoculum size on the lipid accumulation of M. isabellina NBRC 105998 is shown in Table 4. Based on these data, the equation that follows is expressed as in Equation 1, which predicts lipid accumulation in the linear regression model.

Y = 9.791[X.sub.1] - 2.729[X.sub.2] - 0.592[X.sub.1][X.sub.2] - 3.883[X.sub.1.sup.2] - 0.077[X.sub.2.sup.2] + 34.156 (1)

where

Y represents Lipid content (%)

[X.sub.1] represents Glycerol concentration (g/L)

[X.sub.2] represents Inoculum size (fungal discs)

The optimum cultivation condition for M. isabellina NBRC 105998 obtained in this study (Fig. 1) was identified when the media were set as follows: glycerol concentration in the medium (44.14 g/L), inoculum size (2 fungal discs; 3.74x[10.sup.6] spores) and incubation time at 30 [degrees]C for 12 days. This optimum condition provided 45.21% of the lipid content. The lipid content produced was analyzed further by GC-FID for its fatty acid compositions. The GC chromatogram and relative percentage of fatty acid compositions in the lipid sample are shown in Fig. 2 and Table 5, respectively.

Six different fatty acids were identified in the lipid content produced from M. isabellina NBRC 105998 under optimum condition. Stearic acid (57.28%) was the dominant fatty acid found, followed by palmitic acid (19.93%), oleic acid (13.15%), and linoleic acid (5.80%). There were also small amounts of linoleic acid (2.77%) and myristic acid (1.07%). The entire fatty acid profile present in the produced lipid content showed that it possessed potential utilization for biodiesel production (10,16,17).

CONCLUSION

M. isabellina is an oleaginous fungal species with a high capability for utilization of glycerol, a by-product of palm oil-biodiesel production, as an alternative carbon source. In this study, the NBRC 105998 strain showed the most ideal lipid accumulation among the 3 strains tested, with a total biomass of 0.71 g/L and a lipid content of 35.02% after 12 days when using a concentration of glycerol in a medium at 30 g/L. Moreover, the lipid content of the best oil-producing strain (M. isabellina NBRC 105998) can be increased significantly by optimizing two of the principal factors in the culture, namely glycerol concentration and inoculum size using RSM. The optimum cultivation condition for M. isabellina NBRC 105998 identified this study included the glycerol concentration in the medium (44.14 g/L), the inoculum size (2 fungal discs; 3.74x[10.sup.6] spores) and incubation at 30 [degrees]C for 12 days. This optimum condition provided 45.21% of the lipid content. Further, six fatty acids were found in the lipid content produced from this condition, composed mostly of long chain saturated fatty acid (78.28%), which had suitable properties for use as an alternative feedstock for biodiesel production.

ACKNOWLEDGEMENTS

This research was financially supported by Kasetsart University Research and Development Institute (KURDI) and King Mongkut's University of Technology Thonburi Thailand.

REFERENCES

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(6.) Gatenby, C.M., Orcutt, D.M., Kreeger, D.A., Parker, B.C., Jones, V.A., Neves, R.J. Biochemical composition of three algal species proposed as food for captive freshwater mussels. J. Appl. Phycol., 2003; 15: 1-11.

(7.) Alvarez, H.M., Steinbuchel, A. Triacylglycerols in prokaryotic microorganisms. Appl. Microbiol. Biotechnol., 2002; 60: 367-376.

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(9.) F akas, S., Papanikolaou, S., Batsos, A., Galiotou-Panayotou, M., Mallouchos, A., Aggelis, G. Evaluating renewable carbon sources as substrates for single cell oil production by Cunninghamella echinulata and Mortierella isabellina. Biomass Bioenerg., 2009; 33: 573-580.

(10.) Ruan, Z., Zanotti, M., Wang, X., Ducey, C., Liu, Y. Evaluation of lipid accumulation from lignocellulosic sugars by Mortierella isabellina for biodiesel production. Bioresour. Technol., 2012; 110: 198-205.

(11.) Chatzifragkou, A., Fakas, S., Galiotou-Panayotou, M., Komaitis, M., Aggelis, G., Papanikolaou, S. Commercial sugars as substrates for lipid accumulation in Cunninghamella echinulata and Mortierella isabellina fungi. Eur. J. LipidSci. Technol., 2010; 112(9): 1048-1057.

(12.) Haas, M.J., McAloon, A.J., Yee, W.C., Foglia, T.A. A process model to estimate biodiesel production costs. Bioresour. Technol., 2006; 97: 671-678.

(13.) Easterling, E.R., French, W.T., Hernandez, R., Licha, M. The effect of glycerol as a sole and secondary substrate on the growth and fatty acid composition of Rhodotorula glutinis. Bioresour. Technol., 2009; 100: 356-361.

(14.) Folch, J., Lees, J.M., Slane-Stanley, J. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 1957; 226: 497-509.

(15.) Riengsilchai, A., Siramon, P., Punsuvon, V Screening the potential oleaginous yeast strains for lipid accumulation on glycerol, a by-product from biodiesel production. Adv. Mat. Res., 2013; 781-784: 2445-2451.

(16.) Gopinath, A., Puhan, S., Nagarajan, G. Theoretical modeling of iodine value and saponification value of biodiesel fuels from their fatty acid composition. Renew. Energy, 2009; 34: 1806-1811.

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Pornpun Siramon [1]*, Vittaya Punsuvon [2] and Aksorn Riengsilchai [2]

[1] King Mongkut's University of Technology Thonburi (Ratchaburi Campus), Ratchaburi, 70150, Thailand.

[2] Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

(Received: 03 January 2016; accepted: 06 March 2016)

* To whom all correspondence should be addressed. E-mail: siramon_p@hotmail.com

Caption: Fig. 1. Response surface and contour plot of the combined effects of glycerol concentration and inoculum size on lipid production by M. isabellina NBRC 105998

Caption: Fig. 2. GC chromatogram of fatty acid compositions in the lipid produced from M. isabellina NBRC 105998 when grown under optimum condition
Table 1. Experimental ranges of the two independent
variables used in RSM

Variables                           Levels

                         -1.414       -1     0    +1     +1.414
                       (-[alpha])                      (+[alpha])

[X.sub.1], Glycerol      15.86        20     30   40     44.14
concentration [g/L]

[X.sub.2], Inoculum        2          3      5    7        8
size [fungal
discs] *

* 1.87x[10.sup.6] spores/disc

Table 2. Cell mass and lipid content accumulation
during growth of M. isabellina

Strains       Times     Biomass[g    Lipid content
              [days]   dry wt/L] *   [% w/w] *,**

NBRC 7874       4         0.22           14.43
                8         0.45           20.86
                12        0.43           20.59
                16        0.45           20.70
NBRC 7884       4         0.44           15.25
                8         0.58           16.14
                12        0.63           24.61
                16        0.66           24.98
NBRC105998      4         0.49           18.68
                8         0.67           32.26
                12        0.71           35.02
                16        0.72           35.01

* values are means of three replications

** based on dry mass

Table 3. Experimental design and predicted values of
lipid production yield for CCD

               Code

Run    [X.sub.1]   [X.sub.2]        [X.sub.1]
no.                                 [Glycerol
                               concentration (g/L)]

1         -1          -1                20
2         +1          -1                40
3         -1          +1                20
4         +1          +1                40
5       -1.414         0              15.86
6        1.414         0              44.14
7          0         1.414              30
8          0         1.414              30
9          0           0                30
10         0           0                30
11         0           0                30
12         0           0                30
13         0           0                30

                             Y [Lipid content (%)]

Run        [X.sub.2]       Experimental   Predicted
no.     [Inoculum size        values       values
       (fungal discs) *]

1              3              22.84         22.54
2              3              45.86         43.31
3              7              17.01         18.27
4              7              37.66         36.67
5              5              13.49         12.55
6              5              38.00         40.24
7              2              36.11         37.86
8              8              30.60         30.14
9              5              35.62         34.16
10             5              35.03         34.16
11             5              34.42         34.16
12             5              33.10         34.16
13             5              32.61         34.16

* 1.87x [10.sup.6] spores/disc

Table 4. Analysis of variance (ANOVA) for the
quadratic model of the lipid production yield

Parameters            Coefficients   p-value

Constant                 34.156       0.000
[X.sub.1]                9.791        0.000
[X.sub.2]                -2.729       0.005
[X.sub.1][X.sub.2]       -0.592       0.549
[X.sub.1][X.sub.1]       -3.883       0.001
[X.sub.2][X.sub.2]       -0.077       0.917

R-squared ([R.sup.2]) = 0.956

Table 5. Relative percentage of fatty acid compositions
in the lipid content extracted from M. isabellina NBRC
105998 when grown under the optimum condition

Fatty acid              Composition (%)

Myristic acid (C14:0)          1.07
Palmitic acid (C16:0)          19.93
Stearic acid (C18:0)           57.28
Oleic acid (C18:1)             13.15
Linoleic acid (C18:2)          5.80
Linolenic acid (C18:3)         2.77
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Author:Siramon, Pornpun; Punsuvon, Vittaya; Riengsilchai, Aksorn
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Jun 1, 2016
Words:2754
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