Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess14164 J. Oleo Sci. 64, (2) 197-204 (2015)
Production of High Docosahexaenoic Acid by Schizochytrium sp. Using Low-cost Raw Materials from Food Industry Xiaojin Song＊ , Xiaonan Zang and Xuecheng Zhang＊ College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, China
Abstract: The low-cost substrates from food industry, including maize starch hydrolysate and soybean meal hydrolysate, were used to produce docosahexaenoic acid (DHA) by Schizochytrium limacinum OUC88. Glucose derived from maize starch hydrolysate was used as the carbon source and soybean meal hydrolysate as the nitrogen sources. In 10L bioreactor fermentation, by using the soybean meal hydrolysate as the main nitrogen source, the biomass of Schizochytrium limacinum OUC88 reached 85.27 g L–1, and the yields of DHA was 20.7g L–1. As a comparison, when yeast extract was used as the main nitrogen source, the yields of biomass and DHA were 68.93 g L–1 and 13.3 g L–1, respectively. From the results of this study, these hydrolysates can provide all the nutrients required for high-density cultivation of S. limacinum OUC88 and DHA production, that will improve the economical and competitive efficiency of commercial DHA production. Key words: docosahexaenoic acid (DHA), hydrolyzation, maize starch, Schizochytrium, soybean meal 1 Introduction Docosahexaenoic acid（DHA, 22:6 n-3）is widely recognized as an important nutritional component during invertebrate and vertebrate development1, 2）. It is an essential component of cell membranes in some human tissues, mainly in brain and retina3）. DHA plays a key role in improving neural and retinal development in infants. It was also reported that DHA could lower the incidence of certain cardiovascular diseases4−6）. Moreover, it has also been confirmed that marine fish larvae require n-3 highly unsaturated fatty acids, such as DHA for their development and survival7, 8）. Oceanic fish oil products are the typical dietary sources of DHA9）. However, due to emerging concerns over the sustainability of marine resources and of the levels of environmental contaminants present in fish, major efforts have been made to identify or create alternative sources of DHA10, 11）. Two genera of microorganisms, Crypthecodinium cohnii and Schizochytrium, have been developed as alternative commercial sources of DHA enriched oils12）. In developing a large-scale fermentation process for DHA, it is necessary to utilize inexpensive medium components to lower the cost. At present, most studies have used glucose and yeast extract as carbon and nitrogen
sources13）. However, these are expensive and may make the product less economically competitive. There have been a number of efforts to use cheaper sources of carbon and nitrogen for DHA production. Sweet sorghum juice14）, coconut water 15）, Shochu Distillery wastewater 16） and crude glycerol17） have been used as C-sources, ammonia18） and spent yeast autolysis19） have been used as N-sources. But all these trials were difficult to expand for industrial fermentation, because of the lack of material supply and low biomass yield. Maize starch and soybean meal are abundant farm products in China. If these low-cost materials could be used as C-source and N-source, the cost of the DHA product will be reduced sharply. The objective of this study is to investigate the feasibility of using maize starch hydrolysate as a carbon source and soybean meal hydrolysate as a nitrogen source for cell growth and DHA production by Schizochytrium limacinum OUC88.
2 Materials and methods 2.1 Microorganism and medium Schizochytrium limacinum OUC88 is kept by College
＊
Correspondence to: Xiaojin Song, College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, China; Xuecheng Zhang, College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, China; E-mail: [email protected], [email protected] Accepted September 25, 2014 (received for review July 25, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
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X. Song, X. Zang and X. Zhang
Table 1 Amino acid compositions of soybean meal hydrolysate and yeast extract medium. Composition
Fig. 1 The protocol of soybean meal hydrolyzation. of Marine Life Sciences, Ocean University of China20）. M1 medium, which contained glucose（ 30g L −1）and yeast extract（10g L −1）in 50％ artificial seawater（ASW）, was used as the basal medium21）. Seed culture was grown in a 500 ml flask containing 100 ml medium at 25℃ with shaking at 200 rpm for 3 d of cultivation without light. In the fermentation culture, the initial medium contains glucose 70 g/L, soybean meal hydrolysate 30 g/L, ASW 15g/ L, thiamine 50 mg/L, biotin 1 mg/L and cyanocobalamin 10mg/L（The initial medium was optimized by using single factor test, and the results were shown in Fig. S1）. With 10％ （v/v）inoculum size, Schizochytrium cells were grown at 25℃, pH 6.5. 2.2 Preparation of maize starch hydrolysate The starch hydrolysate was prepared by enzymatic hydrolysis of 1 kg maize starch in 3 L water. Heat-resistant amylase 1.5 mL（20,000U mL−1, Genencor Bioengineering, Wuxi, China）was added for gelatinization at 105℃ for 15 min, then the temperature was lowered to 95℃ for liquefaction for 2 h. The temperature was further lowered to 60℃ and 1.5 g glucoamylase（Genencor Bioengineering; 50,000U g−1）was added for saccharification for 24 h. The yield was 35–40％ dextrose equivalents in the filtrate, and the glucose yield was 0.98kg of glucose per kg of starch. 2.3 Preparation of soybean meal hydrolysate The soybean meal（XiangChi CO.LTD., Binzhou, China） was put into a 5000 L porcelain enamel reactor with water at a ratio 1:7（w/v）, and hydrochloric acid was added to regulate pH to 1.3 to hydrolyze the soybean meal. The hy-
Content (%,w/v) SMH
YE
Met
0.13±0.03
0.08±0.02
Asp
0.22±0.02
0.18±0.02
Ala
0.34±0.01
0.24±0.04
Glu
0.20±0.03
0.26±0.02
Ser
0.15±0.04
0.12±0.02
Lys
0.12±0.03
0.08±0.01
Leu
0.16±0.01
0.18±0.03
Pro
0.10±0.02
0.04±0.01
Gly
0.11±0.02
0.16±0.03
Arg
0.06±0.01
0.06±0.01
Phe
0.11±0.01
0.08±0.02
Ile
0.09±0.01
0.06±0.02
Thr
0.07±0.01
0.08±0.03
His
0.06±0.02
0.02±0.01
Tyr
0.07±0.02
0.04±0.01
Val
0.19±0.02
0.2 ±0.05
Total amino nitrogen
2.18±0.09
1.88±0.09
Values are expressed as mean ± s .d . (n=3) SMH: soybean meal hydrolysate; YE : yeast extract drolysis reaction was kept for 20 min at 110℃. After cooling off, the pH was adjusted to 6.0 with 40％ NaOH, and the liquid was separated as the soybean meal hydrolysate. The process of soybean meal hydrolyzed project was shown in Fig. 1. The amino acid concentration after hydrolyzation was shown in Table 1. 2.4 Optimization of hydrolyzation conditions An orthogonal experiment was designed to optimize the hydrolyzation conditions of the soybean meal, including time（min）, temperature（℃）, and hydrochloric acid con（Table 2） . centration（mol L−1） 2.5 Comparison of the different nitrogen sources The fed-batch fermentation experiments for comparison of two nitrogen sources, soybean meal hydrolysate（SMH） and yeast extract（YE）, with the concentration glucose 70 g/L, N-source 30 g/L, ASW 15g/L, thiamine 50 mg/L, biotin 1 mg/L and cyanocobalamin 10mg/L, were carried out to check their efficiency by comparing the biomass, fatty acid composition, and amino acid composition of S. limacinum OUC88. Fermentation experiments were performed in 10 L bioreactors equipped with controls for pH, temperature, agitation and dissolved oxygen concentration（DO）. The tem-
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Production of High Docosahexaenoic Acid by Schizochytrium sp. Using Low-cost Raw Materials from Food Industry
Table 2 Optimization of hydrolyzed conditions by an orthogonal experiment. Run#
Hydrolyzed time (min)
Temperature (℃)
Hydrochloric acid density (mol L–1)
Biomass (g L–1)
DHA yield (g L–1)
1
15
100
0.2
70.04±1.43
18.6±0.13
2
15
110
0.6
62.72±1.59
15.7±0.32
3
15
120
1.0
50.44±0.72
11.5±0.28
4
20
100
0.6
88.52±1.21
20.6±0.31
5
20
110
1.0
72.28±2.32
15.1±0.15
6
20
120
0.2
58.92±1.48
20.6±0.17
7
25
100
1.0
76.12±1.32
10.3±0.26
8
25
110
0.2
50.64±0.55
12.2±0.42
9
25
120
0.6
50.08±1.36
14.6±0.37
Values are expressed as mean ± s .d . (n=3) In the fermentation culture, the initial medium contains glucose 70 g/L, soybean meal hydrolysate 30 g/L, ASW 15 g/L, thiamine 50 mg/L, biotin 1 mg/L and cyanocobalamin 10 mg/L. With 10% (v/v) inoculum size, 25℃, pH 6.5. perature was maintained at 25℃. The agitation speed automatically varied from 300 to 800 rpm at a fixed airflow rate of 1.2 VVM（air volume/culture volume/min）to maintain the DO at 20％ air saturation. The pH was maintained at 6.5 by adding 2 mol L −1 NaOH or 1 mol L −1 H 2SO 4 aqueous solutions automatically. To control foam formation, 1 mL of antifoam was added at the beginning of the run. To explore the reason for the different efficiency of the two nitrogen sources, an experiment of deficiency of single-amino acid was designed. 16 kinds of amino acids were examined. In each experiment one kind of amino acid was deleted in medium and the concentrations of other amino acids were 0.5 g L−1. Except N-source, other compositions were the same as the medium described above. 2.6 15000L Fermentation A 15000L fermentation system was setup at Zhenghai Ecological Science & Technology CO.LTD（Binzhou, China）. A seed was cultivated in a 100L fermentor for 2 days, and then the seed was shifted to a 1000L fermentor for another 2 days. Finally, the culture was switched to a 15000L fermentor to get the best biomass and DHA content for 4 days. The control of various parameters was performed by a control unit. During the fermentation, the bioreactor was operated at 25℃, and the aeration rate was 0.8 L min−1L−1（VVM）. The pH of the medium was automatically controlled at 6.5 by adding NaOH （40％） . The three-step fed-batch fermentation strategy was carried out with the initial starch hydrolysate 7％ （dextrose equivalents） , and the initial soybean meal hydrolysate 3％. From 12 h, the starch hydrolysate and the soybean meal hydrolysate concentration were controlled at 4％ （dextrose equivalents）and 0.5％ respectively by controlling the
feeding rate until 60 h. Then the N-feeding was stopped and the sugar concentration was controlled at 2％ for 20 h. Finally, the feeding was stopped for lipid and DHA accumulation for last 16 h. 2.7 Biomass determination The biomass of S. limacinum OUC88 is expressed in terms of the dry cell weight（DCW）. Cell suspensions 30 mL were centrifuged at 7,000 g and 4℃ for 10 min. Cell pellets were washed twice with 0.2 M phosphate buffer solution. Cell pellets were then freeze-dried to constant weight at −50℃ for approximately 60 h. 2.8 Analysis of glucose and nitrogen concentrations Glucose was analyzed by a biosensor equipped with a glucose oxidase electrode（SBA-40C, Institute of Biology, Shandong Academy of Sciences, China）. Nitrogen concentration in the broth was determined by alkaline potassium persulfate digestion and UV spectrophotometry according to the China Standard GB 11894-89 method22）. 2.9 Lipid and amino acid analysis The dried cells were suspended in 5 mL 0.4 M methanolic KOH for 1 h at 60℃, and then the lipids were esterified reagent at 60℃. After for 1 h in 5 mL BF3-methanol（14％） extraction with 10 mL n-hexane followed by evaporation, the fatty acid methyl esters（FAMEs）were dissolved in 1 mL n-hexane and analyzed using an Agilent 6890 GC （Agilent Technologies, Lexington, United States）equipped with an FID and a DB-23 capillary column（30 m×0.25 mm）. Nitrogen was used as carrier gas. Initial column temperature was kept at 50℃ for 1 min. Then the temperature was raised to 175℃ at 25℃ min−1, subsequently raised to 230℃ at 4℃ min−1, and maintained for 5 min. The FID de199
J. Oleo Sci. 64, (2) 197-204 (2015)
X. Song, X. Zang and X. Zhang
tector temperature was set at 280℃20）. FAMEs were identified by chromatographic comparison with authentic standards（Sigma Chemical Co., USA）. The quantity of DHA was estimated from the peak areas on the chromatogram using nonadecanoic acid （19:0） as an internal standard. An amino acid analyzer（L-8900; Hitachi, Tokyo, Japan） was used to measure the levels of free total amino acids （AAs）and those of 16 individual AAs: alanine（ Ala）, arginine（Arg）, aspartic acid（Asp）, glutamic acid（Glu）, glycine （Gly） , histidine （His） , isoleucine （Ile） , leucine （Leu） , lysine （Lys） , methionine （Met） , phenylalanine （Phe） , serine （Ser）, threonine（Thr）, tryptophan（Trp）, tyrosine（Tyr）, and valine （Val）. The quantitative ninhydrin assay was employed. All analyses were performed in duplicate.
3 Results and Discussion 3.1 Optimization of hydrolyzation conditions The results of optimization experiment for hydrolyzation conditions were shown in Table 2. The best soybean meal hydrolyzation condition was 0.2 mol L−1 HCl, 110℃ and 20 min. The influences of the three experiment factors to the biomass from big to small were hydrolyztion time, hydrochloric acid concentration and temperature. Under the best conditions, the final biomass of S. limacinum OUC88 was 78.37 g L−1, the DHA yield is 17.5 g L−1. During the hydrolyzation, along with the concentration of amino acid in the hydrolysate approached the max value, its increment became very slow, and the yield of byproduct increased rapidly. In the hydrolyzation process, many carbohydrates in soybean meal became pentoses and hexoses and then were dehydrated into furfural and carboxymethyl furfural, and finally they were changed to levulinic acid and formic acid which can polymerize with protein hydrolysate23, 24）. Moreover, the hydrolyzation process also born sulfur compounds, such as H2S, methyl mercaptan, and dimethyl sulfide. Therefore, the hydrolyzation time should be optimized to maximize the amino acid concentration and minimize the subsequence reactions. In our experiments, we found the best hydrolyzation time was 20 min. The hydrochloric acid dosage relates the production cost and hydrolyzation efficiency. Insufficiency of the hydrochloric acid dosage will result in bad heat transmission and make the product difficult to separation. Otherwise, the excessive hydrochloric acid dosage will increase the pair reactions and influence the hydrolyzation efficiency. We found that the highest amino acid content was obtained at 0.2 mol L−1 hydrochloric acid concentration, when the proportion between solid and liquid was 1:7. 3.2 Comparison of the different nitrogen sources In a 10L ferment experiment, the biomass, lipid produc-
Fig. 2 The comparison fed-batch fermentation experiment of two nitrogen sources (soybean meal hydrolysate and yeast extract). A, the growth cruve of two nitrogen sources. (SMH, filled square; YE, filled triangle); B, the lipid and DHA accumulation with two nitrogen sources (lipid in SMH, filled square; DHA in SMH, open square; lipid in YE, filled triangle, DHA in YE, open triangle). tion and the DHA yields of S. limacinum OUC88 were 85.27 g L−1, 44.68 g L−1 and 20.7 g L−1, respectively, using the soybean meal hydrolysates as the nitrogen source. When yeast extracts was used as the nitrogen source, these were 68.93 g L−1, 34.95 g L−1 and 13.3 g L−1 respectively（Fig. 2）. Fatty acid compositions of Schizochytrium limacinum OUC88 culture with different nitrogen sources were shown in Table 3. The OUC88 strain grown with the soybean meal hydrolysates as nitrogen source had higher polyunsaturated fatty acid content（58.88％ of total fatty acids）and DHA content（46.33％ of total fatty acids）, comparing to the values（ 46.74％ and 38.06％, respectively）when it was grown with yeast extracts as nitrogen source. Amino acids play different roles in the process of growth and DHA accumulation of Schizochytrium limacinum
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Table 3 Fatty acids compositions of S. limanium OUC88 cultivated with different nitrogen sources. Fatty acid
Content (%, total fatty acids) SMH
YE
C12:0
0.15±0.04
0.22±0.07
C14:0
3.74±0.43
8.71±0.24
C15:0
0.38±0.07
2.01±0.19
C16:0
33.94±0.43
38.54±1.47
C17:0
0.62±0.09
0.74±0.11
C18:0
1.11±0.36
1.65±0.12
C18:2 n-6
0.42±0.07
0.26±0.05
C18:3 n-3
0.44±0.06
0.58±0.07
C18:3 n-6
0.27±0.04
0.23±0.11
C20:0
0.16±0.02
0.50±0.07
C20:3 n-6
0.15±0.05
0.35±0.12
C20:4 n-6
0.32±0.06
0.43±0.09
C22:0
0.15±0.01
0.42±0.03
C20:5 n-3
0.46±0.16
0.52±0.14
C22:5 n-6
10.49±0.34
7.31±0.25
C22:6 n-3
46.33±0.51
38.06±0.41
Saturated
39.25
51.79
Unsaturated
58.88
47.74
Values are expressed as mean ± s .d . (n=3) SMH: soybean meal hydrolysate; YE : yeast extract
Fig. 3 The effect of single-deficiency of amino acid. OUC88. From Fig. 3, compared with the control group （no amino acid deficiency） , amino acids, such as Met, Ala, Glu, Lys, Pro et al., had positive effect on the growth of S. limacinum OUC88 and the DHA accumulation. The groups whose medium deleted amino acid Gly, Arg and Thr, had more biomass, announced these amino acids had negative effect on the growth of this strain. Other kinds of amino acids had no impact on the growth of S. limacinum
Fig. 4 The fed-batch cruves of 15000 L fermentation system.(Biomass, filled circle; Lipid accumulation, filled square; DHA yield, filled triangle; Glucose concentration, open square; N concentration, open triangle). OUC88. From the results of this study, compared with yeast extract, the soybean meal hydrolysates were more appropriate nitrogen sources for the growth and the DHA production of S. limacinum OUC88, because the soybean meal hydrolysates contained more positive amino acids and were more consistent with the amino acid composition of S. limacinum OUC88. This consistency can reduce the consumption of energy and reducing power such as ATP and NADPH which play key roles in lipid and DHA accumulation25, 26）. Therefore, more energy and reducing power could be utilized in the lipid synthesis pathway to produce lipids and DHA. 3.3 Results of the 15000 L fermentation system After the 15000 L fermentation, the biomass of S. limacinum OUC88 was 81.84 g L−1, and the yields of total lipid and DHA were 43.13 g L−1 and 19.2 g L−1（Fig. 4）. In contrast to only about 20 percent crude protein in cell, over 50 percent of the whole cell components were fatty acids. The amino acid compositions and the fatty acid compositions of S. limacinum OUC88 were displayed in Table 4. The main fatty acids in cell were palmitic acid（C16:0） and docosahexaenoic acid（DHA, C22:6 n-3）which were 34.30％ and 44.54％ of the total fatty acids, respectively. The fermentation substrate is not the only determinant for industrialization of DHA production, but this factor is undoubtedly the base of DHA production and directly influences the possibility and potential of its industrialization27）. This declines the use of expensive high quality substrates such as glucose and yeast extract to cultivate DHArich microalgae. To reduce the cost, many efforts focused on using low-cost materials as media for DHA production （Table 5）. Maize starch and soybean meal are very cheap 201
J. Oleo Sci. 64, (2) 197-204 (2015)
X. Song, X. Zang and X. Zhang
Table 4 Amino acid and Fatty acids composition of S. limanium produced by 15000 L ferment. Amino acid
Content (%,dry weight)
Fatty acid
Content (%,total fatty acid)
Asp
1.14±0.12
C12:0
0.18±0.04
Thr
0.44±0.09
C14:0
3.45±0.43
Ser
0.78±0.11
C15:0
1.02±0.32
Glu
0.97±0.15
C16:0
34.30±0.51
Gly
0.70±0.07
C17:0
0.45±0.09
Ala
1.03±0.04
C18:0
1.63±0.36
Cys
0.22±0.02
C18:1 n-6
0.24±0.07
Val
1.85±0.17
C18:3 n-3
0.49±0.06
Ile
0.46±0.06
C18:3 n-6
0.37±0.12
Leu
0.77±0.09
C20:0
0.34±0.05
Tyr
0.35±0.08
C20:3 n-6
0.25±0.10
Phe
0.54±0.04
C20:4 n-6
0.47±0.06
Lys
0.77±0.10
C22:0
0.18±0.03
His
0.39±0.05
C20:5 n-3
0.56±0.16
Arg
0.63±0.02
C22:5 n-6
9.81±0.34
Pro
0.77±0.03
C22:6 n-3
44.54±0.51
Total
11.81
Saturated
41.55
Unsaturated
56.73
Values are expressed as mean ± s .d . (n=3) the biomass was cultivated with a medium based on starch and soybean hydrolysates.
Table 5 T he biomass and DHA yield by the strains Schizochytrium and Aurantiochytrium using low-cost feedstock. Biomass （g/L）
DHA yield （g/L）
Strain
Feedstock
Schizochytrium limacinum SR21
Sweet sorghum juice
2.5
13
Schizochytrium mangrovei Sk-02
coconut water
28
6
14
Schizochytrium sp. KH105
Shochu Distillery Wastewater
30
3.4
15
Schizochytrium limacinum SR21
crude glycerol
11.78
1.74
24
Schizochytrium sp. G13/2S
ammonia
63.3
6
16
Aurantiochytrium sp. KRS101
Spent yeast
31.8
4.15
17
Schizochytrium limacinum OUC88
maize starch hydrolysate and soybean meal hydrolysate
81.84
in China（the price are shown in Table 6） with a production approximately 26 million tons of maize starch and 32 million tons of soybean meal in 2009 （Agricultural Statistics of China crop year 2009- 2010）. From the results of this study, these hydrolysates can provide all the nutrients required for high-density cultivation of S. limacinum OUC88 and DHA production, thus utilization of these low-cost substrates will improve the economical and competitive efficiency of commercial DHA production.
9.4
19.2
Ref.
This study
4 Conclusion This study showed that maize starch and soybean meal, raw materials from food industry, can serve as low-cost substrates for cultivation of Schizochytrium limacinum OUC88 for producing DHA. These low-cost substrates provided sufficient nutrients for high-density cultivation of S. limacinum OUC88, and produced high amount of DHA. Thus, the use of maize starch hydrolysate and soybean meal hydrolysate will significantly reduce the cost of DHA
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J. Oleo Sci. 64, (2) 197-204 (2015)
Production of High Docosahexaenoic Acid by Schizochytrium sp. Using Low-cost Raw Materials from Food Industry
Table 6 Potential price and fermentation cost of different fermentation substrates. Exp. C-source dosage (W/W)a a
N-source dosage (W/W)
c
Price of C-source ($/ton)
b
Cost of C-source ($/ton powder) Price of N-source ($/ton)c
b
Cost of N-source ($/ton powder)
b
Cost of other auxiliary material ($/ton powder)
Cost of total fermentation process ($/ton powder)
b
Tradition craft
Our experiment
2.5
2.3
0.42
0.67
555-667
445-524
1387.5-1667.5
1023.5-1205.2
6200-10000
480-720
2604-4200
321.6-482.4
619.1-952.4
619.1-952.4
4610.6-6819.9
1964.2-2640
a
: The dosage for producing per gramme powder of S.limacinum OUC88. b : The cost for producing per ton powder of S.limacinum OUC88. c : The data were according to information published on www.alibaba.com. production.
Supporting Information This material is available free of charge via the Internet at http://dx.doi.org/jos.64.10.5650/jos.ess.14164
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EPA and ArA enrichment materials for marine aquaculture using single cell heterotrophs. Aquaculture 213, 347-362（2002）. 8）Song, X. J.; Zhang, X. C.; Guo, N.; Zhu, L. Y.; Kuang, C. H. Assessment of marine thraustochytrid Schizochytrium limacinum OUC88 for mariculture by enriched feeds. Fisheries Science 73, 565-573（2007）. 9）Graham, I. A.; Larson, T.; Napier, J. A. Rational metabolic engineering of transgenic plants for biosynthesis of omega-3 polyunsaturates. Current Opinion in Biotechnology 18, 142-147（2007）. 10）Nakahara, T.; Yokochi, T.; Higashihara, T.; Tanaka, S.; Yaguchi, T.; Honda, D. Production of docosahexaenoic and docosapentaenoic acids by Schizochytrium sp isolated from Yap islands. J. Am. Oil Chem. Soc. 73, 1421-1426（1996）. 11）Singh, A.; Wilson, S.; Ward, O. P. Docosahexaenoic acid （DHA）production by Thraustochytrium sp ATCC 20892. World J. Microb. Biotechnol. 12, 76-81 （1996）. 12）Ratledge, C. Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production. Biochimie 86, 807-815（2004）. 13）Honda, D.; Yokochi, T.; Nakahara, T.; Erata, M.; Higashihara, T. Schizochytrium limacinum sp. nov., a new thraustochytrid from a mangrove area in the west Pacific Ocean. Mycological Res. 102, 439-448（1998）. 14）Liang, Y.; Sarkany, N.; Cui, Y.; Yesuf, J.; Trushenski, J.; Blackburn, J. W. Use of sweet sorghum juice for lipid production by Schizochytrium limacinum SR21. Biores. Technol. 101, 3623-3627（2010）. 15）Unagul, P.; Assantachai, C.; Phadungruengluij, S.; Suphantharika, M.; Tanticharoen, M.; Verduyn, C. Coconut water as a medium additive for the production of docosahexaenoic acid（C22:6 n3）by Schizochytrium mangrovei Sk-02. Biores. Technol. 98, 281-287 （2007）. 203
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16）Yamasaki, T.; Aki, T.;Shinozaki, M.; Taguchi, M.; Kawamoto, S.; Ono, K. Utilization of Shochu distillery wastewater for production of polyunsaturated fatty acids and xanthophylls using thraustochytrid. J. Biosci. Bioeng. 102, 323-327 （2006） . 17）Ethier, S.; Woisard, K.; Vaughan, D.; Wen, Z. Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid. Biores. Technol. 102, 88-93 （2011）. 18）Ganuza, E.; Anderson, A. J.; Ratledge, C. High-celldensity cultivation of Schizochytrium sp. in an ammonium/pH-auxostat fed-batch system. Biotechnol. Lett. 30, 1559-1564 （2008） . 19）Ryu, B.-G.; Kim, K.; Kim, J.; Han, J.-I.; Yang, J.-W. Use of organic waste from the brewery industry for highdensity cultivation of the docosahexaenoic acid-rich microalga, Aurantiochytrium sp. KRS101. Biores. Technol. 129, 351-359 （2012） . 20）Zhu, L. Y. Effects of environmental conditions on the growth and fatty acid composition of Schizochytrium limacinum and cloning and molecular characterization of squalene synthase gene and upstream sequence. Ph.D Thesis. Ocean University of China. pp, 26-30 （2006） . 21）Song, X. J.; Zhang, X. C.; Kuang, C. H.; Zhu, L. Y.; Guo, N. Optimization of fermentation parameters for the biomass and DHA production of Schizochytrium limacinum OUC88 using response surface methodology.
Process Biochem. 42, 1391-1397（2007） . 22）Ren, L. J.; Ji, X. J.; Huang, H.; Qu, L.; Feng, Y.; Tong, Q. Q.; Ouyang, P. K. Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp. Appl. Microbiol. Biotechnol. 87, 1649-1656（2010）. 23）Assary, R. S.; Kim, T.; Low, J. J.; Greeley, J.; Curtiss, L. A. Glucose and fructose to platform chemicals: understanding the thermodynamic landscapes of acid-catalysed reactions using high-level ab initio methods. Physical Chem. Chemical Phys. 14, 16603-16611 （2012）. 24）Daorattanachai, P.; Khemthong, P.; Viriya-empikul, N.; Laosiripojana, N.; Faungnawakij, K. Conversion of fructose, glucose, and cellulose to 5-hydroxymethylfurfural by alkaline earth phosphate catalysts in hot compressed water. Carbohyd. Res. 363, 58-61（2013）. 25）Ratledge, C. Regulation of lipid accumulation in oleaginous micro-organisms. Biochem. Soc. Trans. 30, 1047-1050（2002）. 26）Zhang, Y.; Adams, I. P.; Ratledge, C. Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation. Microbiology 153, 2013-2025（2007）. 27）Huang, C.; Chen, X.-f.; Xiong, L.; Chen, X.-d.; Ma, L.-l.; Chen, Y. Single cell oil production from low-cost substrates: The possibility and potential of its industrialization. Biotechnol. Adv. 31, 129-139（2012）.
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Production of High Docosahexaenoic Acid by Schizochytrium sp. Using Low-cost Raw Materials from Food Industry Xiaojin Song＊ , Xiaonan Zang and Xuecheng Zhang＊ College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, China
Abstract: The low-cost substrates from food industry, including maize starch hydrolysate and soybean meal hydrolysate, were used to produce docosahexaenoic acid (DHA) by Schizochytrium limacinum OUC88. Glucose derived from maize starch hydrolysate was used as the carbon source and soybean meal hydrolysate as the nitrogen sources. In 10L bioreactor fermentation, by using the soybean meal hydrolysate as the main nitrogen source, the biomass of Schizochytrium limacinum OUC88 reached 85.27 g L–1, and the yields of DHA was 20.7g L–1. As a comparison, when yeast extract was used as the main nitrogen source, the yields of biomass and DHA were 68.93 g L–1 and 13.3 g L–1, respectively. From the results of this study, these hydrolysates can provide all the nutrients required for high-density cultivation of S. limacinum OUC88 and DHA production, that will improve the economical and competitive efficiency of commercial DHA production. Key words: docosahexaenoic acid (DHA), hydrolyzation, maize starch, Schizochytrium, soybean meal 1 Introduction Docosahexaenoic acid（DHA, 22:6 n-3）is widely recognized as an important nutritional component during invertebrate and vertebrate development1, 2）. It is an essential component of cell membranes in some human tissues, mainly in brain and retina3）. DHA plays a key role in improving neural and retinal development in infants. It was also reported that DHA could lower the incidence of certain cardiovascular diseases4−6）. Moreover, it has also been confirmed that marine fish larvae require n-3 highly unsaturated fatty acids, such as DHA for their development and survival7, 8）. Oceanic fish oil products are the typical dietary sources of DHA9）. However, due to emerging concerns over the sustainability of marine resources and of the levels of environmental contaminants present in fish, major efforts have been made to identify or create alternative sources of DHA10, 11）. Two genera of microorganisms, Crypthecodinium cohnii and Schizochytrium, have been developed as alternative commercial sources of DHA enriched oils12）. In developing a large-scale fermentation process for DHA, it is necessary to utilize inexpensive medium components to lower the cost. At present, most studies have used glucose and yeast extract as carbon and nitrogen
sources13）. However, these are expensive and may make the product less economically competitive. There have been a number of efforts to use cheaper sources of carbon and nitrogen for DHA production. Sweet sorghum juice14）, coconut water 15）, Shochu Distillery wastewater 16） and crude glycerol17） have been used as C-sources, ammonia18） and spent yeast autolysis19） have been used as N-sources. But all these trials were difficult to expand for industrial fermentation, because of the lack of material supply and low biomass yield. Maize starch and soybean meal are abundant farm products in China. If these low-cost materials could be used as C-source and N-source, the cost of the DHA product will be reduced sharply. The objective of this study is to investigate the feasibility of using maize starch hydrolysate as a carbon source and soybean meal hydrolysate as a nitrogen source for cell growth and DHA production by Schizochytrium limacinum OUC88.
2 Materials and methods 2.1 Microorganism and medium Schizochytrium limacinum OUC88 is kept by College
＊
Correspondence to: Xiaojin Song, College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, China; Xuecheng Zhang, College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, China; E-mail: [email protected], [email protected] Accepted September 25, 2014 (received for review July 25, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs 197
X. Song, X. Zang and X. Zhang
Table 1 Amino acid compositions of soybean meal hydrolysate and yeast extract medium. Composition
Fig. 1 The protocol of soybean meal hydrolyzation. of Marine Life Sciences, Ocean University of China20）. M1 medium, which contained glucose（ 30g L −1）and yeast extract（10g L −1）in 50％ artificial seawater（ASW）, was used as the basal medium21）. Seed culture was grown in a 500 ml flask containing 100 ml medium at 25℃ with shaking at 200 rpm for 3 d of cultivation without light. In the fermentation culture, the initial medium contains glucose 70 g/L, soybean meal hydrolysate 30 g/L, ASW 15g/ L, thiamine 50 mg/L, biotin 1 mg/L and cyanocobalamin 10mg/L（The initial medium was optimized by using single factor test, and the results were shown in Fig. S1）. With 10％ （v/v）inoculum size, Schizochytrium cells were grown at 25℃, pH 6.5. 2.2 Preparation of maize starch hydrolysate The starch hydrolysate was prepared by enzymatic hydrolysis of 1 kg maize starch in 3 L water. Heat-resistant amylase 1.5 mL（20,000U mL−1, Genencor Bioengineering, Wuxi, China）was added for gelatinization at 105℃ for 15 min, then the temperature was lowered to 95℃ for liquefaction for 2 h. The temperature was further lowered to 60℃ and 1.5 g glucoamylase（Genencor Bioengineering; 50,000U g−1）was added for saccharification for 24 h. The yield was 35–40％ dextrose equivalents in the filtrate, and the glucose yield was 0.98kg of glucose per kg of starch. 2.3 Preparation of soybean meal hydrolysate The soybean meal（XiangChi CO.LTD., Binzhou, China） was put into a 5000 L porcelain enamel reactor with water at a ratio 1:7（w/v）, and hydrochloric acid was added to regulate pH to 1.3 to hydrolyze the soybean meal. The hy-
Content (%,w/v) SMH
YE
Met
0.13±0.03
0.08±0.02
Asp
0.22±0.02
0.18±0.02
Ala
0.34±0.01
0.24±0.04
Glu
0.20±0.03
0.26±0.02
Ser
0.15±0.04
0.12±0.02
Lys
0.12±0.03
0.08±0.01
Leu
0.16±0.01
0.18±0.03
Pro
0.10±0.02
0.04±0.01
Gly
0.11±0.02
0.16±0.03
Arg
0.06±0.01
0.06±0.01
Phe
0.11±0.01
0.08±0.02
Ile
0.09±0.01
0.06±0.02
Thr
0.07±0.01
0.08±0.03
His
0.06±0.02
0.02±0.01
Tyr
0.07±0.02
0.04±0.01
Val
0.19±0.02
0.2 ±0.05
Total amino nitrogen
2.18±0.09
1.88±0.09
Values are expressed as mean ± s .d . (n=3) SMH: soybean meal hydrolysate; YE : yeast extract drolysis reaction was kept for 20 min at 110℃. After cooling off, the pH was adjusted to 6.0 with 40％ NaOH, and the liquid was separated as the soybean meal hydrolysate. The process of soybean meal hydrolyzed project was shown in Fig. 1. The amino acid concentration after hydrolyzation was shown in Table 1. 2.4 Optimization of hydrolyzation conditions An orthogonal experiment was designed to optimize the hydrolyzation conditions of the soybean meal, including time（min）, temperature（℃）, and hydrochloric acid con（Table 2） . centration（mol L−1） 2.5 Comparison of the different nitrogen sources The fed-batch fermentation experiments for comparison of two nitrogen sources, soybean meal hydrolysate（SMH） and yeast extract（YE）, with the concentration glucose 70 g/L, N-source 30 g/L, ASW 15g/L, thiamine 50 mg/L, biotin 1 mg/L and cyanocobalamin 10mg/L, were carried out to check their efficiency by comparing the biomass, fatty acid composition, and amino acid composition of S. limacinum OUC88. Fermentation experiments were performed in 10 L bioreactors equipped with controls for pH, temperature, agitation and dissolved oxygen concentration（DO）. The tem-
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Table 2 Optimization of hydrolyzed conditions by an orthogonal experiment. Run#
Hydrolyzed time (min)
Temperature (℃)
Hydrochloric acid density (mol L–1)
Biomass (g L–1)
DHA yield (g L–1)
1
15
100
0.2
70.04±1.43
18.6±0.13
2
15
110
0.6
62.72±1.59
15.7±0.32
3
15
120
1.0
50.44±0.72
11.5±0.28
4
20
100
0.6
88.52±1.21
20.6±0.31
5
20
110
1.0
72.28±2.32
15.1±0.15
6
20
120
0.2
58.92±1.48
20.6±0.17
7
25
100
1.0
76.12±1.32
10.3±0.26
8
25
110
0.2
50.64±0.55
12.2±0.42
9
25
120
0.6
50.08±1.36
14.6±0.37
Values are expressed as mean ± s .d . (n=3) In the fermentation culture, the initial medium contains glucose 70 g/L, soybean meal hydrolysate 30 g/L, ASW 15 g/L, thiamine 50 mg/L, biotin 1 mg/L and cyanocobalamin 10 mg/L. With 10% (v/v) inoculum size, 25℃, pH 6.5. perature was maintained at 25℃. The agitation speed automatically varied from 300 to 800 rpm at a fixed airflow rate of 1.2 VVM（air volume/culture volume/min）to maintain the DO at 20％ air saturation. The pH was maintained at 6.5 by adding 2 mol L −1 NaOH or 1 mol L −1 H 2SO 4 aqueous solutions automatically. To control foam formation, 1 mL of antifoam was added at the beginning of the run. To explore the reason for the different efficiency of the two nitrogen sources, an experiment of deficiency of single-amino acid was designed. 16 kinds of amino acids were examined. In each experiment one kind of amino acid was deleted in medium and the concentrations of other amino acids were 0.5 g L−1. Except N-source, other compositions were the same as the medium described above. 2.6 15000L Fermentation A 15000L fermentation system was setup at Zhenghai Ecological Science & Technology CO.LTD（Binzhou, China）. A seed was cultivated in a 100L fermentor for 2 days, and then the seed was shifted to a 1000L fermentor for another 2 days. Finally, the culture was switched to a 15000L fermentor to get the best biomass and DHA content for 4 days. The control of various parameters was performed by a control unit. During the fermentation, the bioreactor was operated at 25℃, and the aeration rate was 0.8 L min−1L−1（VVM）. The pH of the medium was automatically controlled at 6.5 by adding NaOH （40％） . The three-step fed-batch fermentation strategy was carried out with the initial starch hydrolysate 7％ （dextrose equivalents） , and the initial soybean meal hydrolysate 3％. From 12 h, the starch hydrolysate and the soybean meal hydrolysate concentration were controlled at 4％ （dextrose equivalents）and 0.5％ respectively by controlling the
feeding rate until 60 h. Then the N-feeding was stopped and the sugar concentration was controlled at 2％ for 20 h. Finally, the feeding was stopped for lipid and DHA accumulation for last 16 h. 2.7 Biomass determination The biomass of S. limacinum OUC88 is expressed in terms of the dry cell weight（DCW）. Cell suspensions 30 mL were centrifuged at 7,000 g and 4℃ for 10 min. Cell pellets were washed twice with 0.2 M phosphate buffer solution. Cell pellets were then freeze-dried to constant weight at −50℃ for approximately 60 h. 2.8 Analysis of glucose and nitrogen concentrations Glucose was analyzed by a biosensor equipped with a glucose oxidase electrode（SBA-40C, Institute of Biology, Shandong Academy of Sciences, China）. Nitrogen concentration in the broth was determined by alkaline potassium persulfate digestion and UV spectrophotometry according to the China Standard GB 11894-89 method22）. 2.9 Lipid and amino acid analysis The dried cells were suspended in 5 mL 0.4 M methanolic KOH for 1 h at 60℃, and then the lipids were esterified reagent at 60℃. After for 1 h in 5 mL BF3-methanol（14％） extraction with 10 mL n-hexane followed by evaporation, the fatty acid methyl esters（FAMEs）were dissolved in 1 mL n-hexane and analyzed using an Agilent 6890 GC （Agilent Technologies, Lexington, United States）equipped with an FID and a DB-23 capillary column（30 m×0.25 mm）. Nitrogen was used as carrier gas. Initial column temperature was kept at 50℃ for 1 min. Then the temperature was raised to 175℃ at 25℃ min−1, subsequently raised to 230℃ at 4℃ min−1, and maintained for 5 min. The FID de199
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X. Song, X. Zang and X. Zhang
tector temperature was set at 280℃20）. FAMEs were identified by chromatographic comparison with authentic standards（Sigma Chemical Co., USA）. The quantity of DHA was estimated from the peak areas on the chromatogram using nonadecanoic acid （19:0） as an internal standard. An amino acid analyzer（L-8900; Hitachi, Tokyo, Japan） was used to measure the levels of free total amino acids （AAs）and those of 16 individual AAs: alanine（ Ala）, arginine（Arg）, aspartic acid（Asp）, glutamic acid（Glu）, glycine （Gly） , histidine （His） , isoleucine （Ile） , leucine （Leu） , lysine （Lys） , methionine （Met） , phenylalanine （Phe） , serine （Ser）, threonine（Thr）, tryptophan（Trp）, tyrosine（Tyr）, and valine （Val）. The quantitative ninhydrin assay was employed. All analyses were performed in duplicate.
3 Results and Discussion 3.1 Optimization of hydrolyzation conditions The results of optimization experiment for hydrolyzation conditions were shown in Table 2. The best soybean meal hydrolyzation condition was 0.2 mol L−1 HCl, 110℃ and 20 min. The influences of the three experiment factors to the biomass from big to small were hydrolyztion time, hydrochloric acid concentration and temperature. Under the best conditions, the final biomass of S. limacinum OUC88 was 78.37 g L−1, the DHA yield is 17.5 g L−1. During the hydrolyzation, along with the concentration of amino acid in the hydrolysate approached the max value, its increment became very slow, and the yield of byproduct increased rapidly. In the hydrolyzation process, many carbohydrates in soybean meal became pentoses and hexoses and then were dehydrated into furfural and carboxymethyl furfural, and finally they were changed to levulinic acid and formic acid which can polymerize with protein hydrolysate23, 24）. Moreover, the hydrolyzation process also born sulfur compounds, such as H2S, methyl mercaptan, and dimethyl sulfide. Therefore, the hydrolyzation time should be optimized to maximize the amino acid concentration and minimize the subsequence reactions. In our experiments, we found the best hydrolyzation time was 20 min. The hydrochloric acid dosage relates the production cost and hydrolyzation efficiency. Insufficiency of the hydrochloric acid dosage will result in bad heat transmission and make the product difficult to separation. Otherwise, the excessive hydrochloric acid dosage will increase the pair reactions and influence the hydrolyzation efficiency. We found that the highest amino acid content was obtained at 0.2 mol L−1 hydrochloric acid concentration, when the proportion between solid and liquid was 1:7. 3.2 Comparison of the different nitrogen sources In a 10L ferment experiment, the biomass, lipid produc-
Fig. 2 The comparison fed-batch fermentation experiment of two nitrogen sources (soybean meal hydrolysate and yeast extract). A, the growth cruve of two nitrogen sources. (SMH, filled square; YE, filled triangle); B, the lipid and DHA accumulation with two nitrogen sources (lipid in SMH, filled square; DHA in SMH, open square; lipid in YE, filled triangle, DHA in YE, open triangle). tion and the DHA yields of S. limacinum OUC88 were 85.27 g L−1, 44.68 g L−1 and 20.7 g L−1, respectively, using the soybean meal hydrolysates as the nitrogen source. When yeast extracts was used as the nitrogen source, these were 68.93 g L−1, 34.95 g L−1 and 13.3 g L−1 respectively（Fig. 2）. Fatty acid compositions of Schizochytrium limacinum OUC88 culture with different nitrogen sources were shown in Table 3. The OUC88 strain grown with the soybean meal hydrolysates as nitrogen source had higher polyunsaturated fatty acid content（58.88％ of total fatty acids）and DHA content（46.33％ of total fatty acids）, comparing to the values（ 46.74％ and 38.06％, respectively）when it was grown with yeast extracts as nitrogen source. Amino acids play different roles in the process of growth and DHA accumulation of Schizochytrium limacinum
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Table 3 Fatty acids compositions of S. limanium OUC88 cultivated with different nitrogen sources. Fatty acid
Content (%, total fatty acids) SMH
YE
C12:0
0.15±0.04
0.22±0.07
C14:0
3.74±0.43
8.71±0.24
C15:0
0.38±0.07
2.01±0.19
C16:0
33.94±0.43
38.54±1.47
C17:0
0.62±0.09
0.74±0.11
C18:0
1.11±0.36
1.65±0.12
C18:2 n-6
0.42±0.07
0.26±0.05
C18:3 n-3
0.44±0.06
0.58±0.07
C18:3 n-6
0.27±0.04
0.23±0.11
C20:0
0.16±0.02
0.50±0.07
C20:3 n-6
0.15±0.05
0.35±0.12
C20:4 n-6
0.32±0.06
0.43±0.09
C22:0
0.15±0.01
0.42±0.03
C20:5 n-3
0.46±0.16
0.52±0.14
C22:5 n-6
10.49±0.34
7.31±0.25
C22:6 n-3
46.33±0.51
38.06±0.41
Saturated
39.25
51.79
Unsaturated
58.88
47.74
Values are expressed as mean ± s .d . (n=3) SMH: soybean meal hydrolysate; YE : yeast extract
Fig. 3 The effect of single-deficiency of amino acid. OUC88. From Fig. 3, compared with the control group （no amino acid deficiency） , amino acids, such as Met, Ala, Glu, Lys, Pro et al., had positive effect on the growth of S. limacinum OUC88 and the DHA accumulation. The groups whose medium deleted amino acid Gly, Arg and Thr, had more biomass, announced these amino acids had negative effect on the growth of this strain. Other kinds of amino acids had no impact on the growth of S. limacinum
Fig. 4 The fed-batch cruves of 15000 L fermentation system.(Biomass, filled circle; Lipid accumulation, filled square; DHA yield, filled triangle; Glucose concentration, open square; N concentration, open triangle). OUC88. From the results of this study, compared with yeast extract, the soybean meal hydrolysates were more appropriate nitrogen sources for the growth and the DHA production of S. limacinum OUC88, because the soybean meal hydrolysates contained more positive amino acids and were more consistent with the amino acid composition of S. limacinum OUC88. This consistency can reduce the consumption of energy and reducing power such as ATP and NADPH which play key roles in lipid and DHA accumulation25, 26）. Therefore, more energy and reducing power could be utilized in the lipid synthesis pathway to produce lipids and DHA. 3.3 Results of the 15000 L fermentation system After the 15000 L fermentation, the biomass of S. limacinum OUC88 was 81.84 g L−1, and the yields of total lipid and DHA were 43.13 g L−1 and 19.2 g L−1（Fig. 4）. In contrast to only about 20 percent crude protein in cell, over 50 percent of the whole cell components were fatty acids. The amino acid compositions and the fatty acid compositions of S. limacinum OUC88 were displayed in Table 4. The main fatty acids in cell were palmitic acid（C16:0） and docosahexaenoic acid（DHA, C22:6 n-3）which were 34.30％ and 44.54％ of the total fatty acids, respectively. The fermentation substrate is not the only determinant for industrialization of DHA production, but this factor is undoubtedly the base of DHA production and directly influences the possibility and potential of its industrialization27）. This declines the use of expensive high quality substrates such as glucose and yeast extract to cultivate DHArich microalgae. To reduce the cost, many efforts focused on using low-cost materials as media for DHA production （Table 5）. Maize starch and soybean meal are very cheap 201
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Table 4 Amino acid and Fatty acids composition of S. limanium produced by 15000 L ferment. Amino acid
Content (%,dry weight)
Fatty acid
Content (%,total fatty acid)
Asp
1.14±0.12
C12:0
0.18±0.04
Thr
0.44±0.09
C14:0
3.45±0.43
Ser
0.78±0.11
C15:0
1.02±0.32
Glu
0.97±0.15
C16:0
34.30±0.51
Gly
0.70±0.07
C17:0
0.45±0.09
Ala
1.03±0.04
C18:0
1.63±0.36
Cys
0.22±0.02
C18:1 n-6
0.24±0.07
Val
1.85±0.17
C18:3 n-3
0.49±0.06
Ile
0.46±0.06
C18:3 n-6
0.37±0.12
Leu
0.77±0.09
C20:0
0.34±0.05
Tyr
0.35±0.08
C20:3 n-6
0.25±0.10
Phe
0.54±0.04
C20:4 n-6
0.47±0.06
Lys
0.77±0.10
C22:0
0.18±0.03
His
0.39±0.05
C20:5 n-3
0.56±0.16
Arg
0.63±0.02
C22:5 n-6
9.81±0.34
Pro
0.77±0.03
C22:6 n-3
44.54±0.51
Total
11.81
Saturated
41.55
Unsaturated
56.73
Values are expressed as mean ± s .d . (n=3) the biomass was cultivated with a medium based on starch and soybean hydrolysates.
Table 5 T he biomass and DHA yield by the strains Schizochytrium and Aurantiochytrium using low-cost feedstock. Biomass （g/L）
DHA yield （g/L）
Strain
Feedstock
Schizochytrium limacinum SR21
Sweet sorghum juice
2.5
13
Schizochytrium mangrovei Sk-02
coconut water
28
6
14
Schizochytrium sp. KH105
Shochu Distillery Wastewater
30
3.4
15
Schizochytrium limacinum SR21
crude glycerol
11.78
1.74
24
Schizochytrium sp. G13/2S
ammonia
63.3
6
16
Aurantiochytrium sp. KRS101
Spent yeast
31.8
4.15
17
Schizochytrium limacinum OUC88
maize starch hydrolysate and soybean meal hydrolysate
81.84
in China（the price are shown in Table 6） with a production approximately 26 million tons of maize starch and 32 million tons of soybean meal in 2009 （Agricultural Statistics of China crop year 2009- 2010）. From the results of this study, these hydrolysates can provide all the nutrients required for high-density cultivation of S. limacinum OUC88 and DHA production, thus utilization of these low-cost substrates will improve the economical and competitive efficiency of commercial DHA production.
9.4
19.2
Ref.
This study
4 Conclusion This study showed that maize starch and soybean meal, raw materials from food industry, can serve as low-cost substrates for cultivation of Schizochytrium limacinum OUC88 for producing DHA. These low-cost substrates provided sufficient nutrients for high-density cultivation of S. limacinum OUC88, and produced high amount of DHA. Thus, the use of maize starch hydrolysate and soybean meal hydrolysate will significantly reduce the cost of DHA
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Production of High Docosahexaenoic Acid by Schizochytrium sp. Using Low-cost Raw Materials from Food Industry
Table 6 Potential price and fermentation cost of different fermentation substrates. Exp. C-source dosage (W/W)a a
N-source dosage (W/W)
c
Price of C-source ($/ton)
b
Cost of C-source ($/ton powder) Price of N-source ($/ton)c
b
Cost of N-source ($/ton powder)
b
Cost of other auxiliary material ($/ton powder)
Cost of total fermentation process ($/ton powder)
b
Tradition craft
Our experiment
2.5
2.3
0.42
0.67
555-667
445-524
1387.5-1667.5
1023.5-1205.2
6200-10000
480-720
2604-4200
321.6-482.4
619.1-952.4
619.1-952.4
4610.6-6819.9
1964.2-2640
a
: The dosage for producing per gramme powder of S.limacinum OUC88. b : The cost for producing per ton powder of S.limacinum OUC88. c : The data were according to information published on www.alibaba.com. production.
Supporting Information This material is available free of charge via the Internet at http://dx.doi.org/jos.64.10.5650/jos.ess.14164
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