Accepted Manuscript Impact of carbon and nitrogen feeding strategy on high production of biomass and docosahexaenoic acid (DHA) by Schizochytrium sp. LU310 Xueping Ling, Jing Guo, Xiaoting Liu, Xia Zhang, Nan Wang, Yinghua Lu, ISon Ng PII: DOI: Reference:
S0960-8524(14)01389-3 http://dx.doi.org/10.1016/j.biortech.2014.09.130 BITE 14020
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
24 August 2014 23 September 2014 25 September 2014
Please cite this article as: Ling, X., Guo, J., Liu, X., Zhang, X., Wang, N., Lu, Y., Ng, I-S., Impact of carbon and nitrogen feeding strategy on high production of biomass and docosahexaenoic acid (DHA) by Schizochytrium sp. LU310, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.09.130
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Original research to
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“Bioresource Technology on Special Issue of Bio/Chemicals from Algae”
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Impact of carbon and nitrogen feeding strategy on high production of
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biomass and docosahexaenoic acid (DHA) by Schizochytrium sp.
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LU310
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Xueping Linga, Jing Guo a, Xiaoting Liua, Xia Zhanga, Nan Wanga, Yinghua Lua, I-Son Nga,b*
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a
Department of Chemical and Biochemical Engineering, College of Chemistry and
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Chemical Engineering, Xiamen University, Xiamen 361005 PR China;
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b
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Taiwan.
Department of Chemical Engineering, National Cheng Kung University, Tainan 70101,
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*Corresponding author: I-Son Ng
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Tel: +886-62757575; Fax: +886-62344496;
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E-mail: [email protected]
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ABSTRACT: A new isolated Schizochytrium sp. LU310 from the mangrove forest of
2
Wenzhou, China, was found as a high producing microalga of docosahexaenoic acid
3
(DHA). In this study, the significant improvements for DHA fermentation by the batch
4
mode in the baffled flasks (i.e. higher oxygen supply) were achieved. By applied the
5
nitrogen-feeding strategy in 1000 mL baffled flasks, the biomass, DHA concentration
6
and DHA productivity were increased by 110.4%, 117.9% and 110.4%, respectively.
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Moreover, DHA concentration of 21.06 g/L was obtained by feeding 15 g/L of glucose
8
intermittently, which was an increase of 41.25% over that of the batch mode. Finally, an
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innovative strategy was carried out by intermittent feeding carbon and simultaneously
10
feeding nitrogen. The maximum DHA concentration and DHA productivity in the
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fed-batch cultivation reached to 24.74 g/L and 241.5 mg/L/h, respectively.
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Keywords: Docosahexaenoic acid (DHA); Schizochytrium sp.; Baffled flasks; Nitrogen
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feeding; Carbon feeding
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1. Introduction Docosahexaenoic acid (DHA, 22:6 n-3) has received worldwide attention due to its
3
beneficial physiological functions for humans, for instance, promotion effect for visual
4
acuity and neural development, lowering the incidence of cardiovascular diseases, and
5
reducing risk factors involved in diseases like hypertension, arthritis, arteriosclerosis
6
and thrombosis (Ren et al., 2013; Sijtsma and Swaaf, 2004; Sun et al., 2014). Due to its
7
widespread use, the global DHA market has grown significantly in the recent years and
8
expected to reach $34.7 billion in 2016.
9
Schizochytrium sp., a thraustochytrid, is an algae-like microorganism that is closely
10
related to heterokont algae (Barclay et al., 1994; Morita et al., 2006; Sijtsma and Swaaf,
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2004). It is well-known for producing polyunsaturated fatty acids (PUFAs), among
12
which DHA usually represents 30-40 % of the total fatty acids (Yokochi et al., 1998),
13
and thus is considered as a promising and commercially viable alternative DHA
14
feedstock. However, this source of DHA is limited by the high production costs in
15
comparison to the current fish oil prices. In order to reduce the costs of
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microalgae-based DHA production, higher volumetric productivity must be pursued by
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culture optimization. A yield of 90-100 g/L DCW with high lipid content (54-63%, w/w)
18
by Aurantiochytrium sp. strain T66 under N starvation and O2 limitation had been
19
reported (Jakobsen et al., 2008). In another case, an intermittent oxygen feeding method
20
to maintain a 50% dissolved oxygen level with a C:N ratio of 1.25 for Schizochytrium
21
limacinum SR21, to achieve DCW at 61.76 g/L and DHA concentration at 20.3 g/L had
22
also been reported (Huang et al., 2012). The literature showed that the cell growth rate,
23
DHA content and DHA productivity of oleaginous microorganisms were mainly
24
influenced by the culture conditions, such as nitrogen concentration, carbon 3
1
concentration and dissolved oxygen level (de Swaaf et al., 2002; Ganuza and Izquierdo,
2
2007; Ganuza et al., 2008; Jakobsen et al., 2008; Sun et al., 2014). It has been suggested
3
that nitrogen limitation could enhance the amount of acetyl-CoA and NADPH, which
4
were the necessary precursors for lipid accumulation (de Swaaf et al., 2002). Recently,
5
Ren et al. (2014) also found that the DHA percentage of TFAs would be decreased after
6
nitrogen exhaustion. However, the phenomenon could be relieved by monosodium
7
glutamate (MSG) addition, which could activate the enzyme of acetyl-CoA carboxylase
8
(Kowluru et al., 2001) and glucose-6-phosphate dehydrogenase (Lan et al., 2002), and
9
thus it ensures the sufficient supply of acetyl-CoA and NADPH for DHA synthesis to
10
enhance the accumulation of lipid and DHA.
11
The accumulated lipids and fatty acids within oleaginous microorganisms are,
12
therefore, dynamic storage materials and are produced in times of plenty when carbon is
13
sufficient or starvation when carbon is deficient (Ganuza et al., 2007; Ratledge 2002;
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Wu et al., 2005). Moreover, the high carbon to nitrogen ratio is beneficial to lipid
15
accumulation (Yokochi et al., 1998), while the DHA percentage fluctuated with the
16
glucose addition (Ren et al., 2014), indicating glucose feeding would be proper for
17
DHA production. In addition, it had been proved that low dissolved oxygen (DO) was
18
favored for DHA production (Jakobsen et al., 2008), and fermentation strategy was
19
performed by controlling DO for the optimization of DHA production (Chi et al., 2009).
20
A stepwise aeration control strategy was developed for efficient DHA production in a
21
1,500-L bioreactor using fed-batch fermentation (Ren et al., 2010), or an intermittent
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oxygen feeding method to maintain a 50% DO level (Huang et al., 2012). However, till
23
now, no efforts have been made to simultaneously evaluate MSG addition and glucose
24
feeding at nitrogen limitation condition for increasing DHA production of 4
1 2
Schizochytrium microalgae. In this study, a new isolated microalga, Schizochytrium sp. LU310 was investigated
3
for its ability to produce DHA in the baffled flasks. MSG addition and glucose feeding
4
were applied to optimize DHA productivity. Moreover, an innovative strategy as
5
combining MSG addition and glucose feeding at nitrogen limitation condition was
6
further assessed for its effectiveness in improving DHA production. This approach
7
would provide guidance for the large-scale production of DHA and other similar
8
polyunsaturated fatty acids, and give insight into the carbon and nitrogen metabolism in
9
oil-producing microorganisms.
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2. Materials and Methods
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2.1. Identification of strain
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The microalga, Schizochytrium sp. LU310, used in this work was isolated from the
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mangrove forest of Wenzhou, Zhejiang, China. It was identified by the morphology and
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18S rRNA sequencing using the primers as 18S-F:
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5'-TCTCAAAGAYTAAGSCATGC-3' and 18S-R:
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5'-TTCACCTTCCTCTAAACAATAAG-3'. The 18S rRNA sequence was deposited
18
into GenBank under accession number KM245569.
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2.2. Morphological analysis A drop of fresh strain LU310 was taken on a slide glass to be observed and recorded by light microscopy (OLYMPUS BX41, Japan) continuously.
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2.3. Culture conditions 5
1
The seed broth contained (g/L, pH 6.5): glucose, 30; Na2 SO4, 12; yeast extract, 10;
2
MgSO4, 2; (NH4) 2SO4, 1; KH2PO4, 1; K2 SO4, 0.65; KCl, 0.5; CaCl2·2H2O, 0.17. The
3
fermentation broth contained (g/L, pH=6.5): glucose, 120; Na2 SO4, 12; MSG, 5; corn
4
steep powder (Sigma Co), 5; MgSO4, 2; (NH4) 2SO4, 1; KH2 PO4, 1; K2 SO4, 0.65; KCl,
5
0.5; CaCl2·2H2O, 0.17 as well as trace element solution(1 mL/L) and vitamin solution
6
(2 mL/L). The trace element solution was composed of (mL/L): FeSO4·7H2O, 10;
7
Ca1/2Pantothenate, 3.2; MnCl2·4H2O, 3; ZnSO4·7H2O, 3; NiSO4·6H2O, 2; CuSO4·5H2O,
8
2; CoCl2·6H2O, 0.04; Na2MoO4·2H2O, 0.04. Vitamin solution consisted of (mg/L):
9
thiamine, 9.5; cyanocobalamin, 0.15. Before autoclaving (121 oC, 20 min) the pH of the
10
medium was adjusted to 6.5 with 2 M NaOH. Yeast extract was purchased from OXOID
11
Ltd., UK. If not specifically mentioned, all other chemicals were provided by
12
Sinopharm Chemical Reagent Co. Ltd., China. The trace element solution and the
13
vitamin solution were filter-sterilized (0.2 µm) and added after sterilization. The stored
14
cells were cultured at 28 oC, 170 rpm for 48 h in the seed broth. After three generations
15
of cultivation, the seed culture (4% v/v) was then transferred to fermentation broth and
16
incubated at 28 oC, 200 rpm for 120 h or more.
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2.4. Dry cell weight (DCW), glucose, and nitrogen measurements 3 mL broth was used to determine DCW by gravimetric method. 3 mL broth was
20
transferred to a pre-weighted centrifuge tube and centrifuged at 7,000 x g for 10 min.
21
The cell pellet was washed twice with distillated water and dried at 60 oC until constant
22
(12 h) to analyze the total weight. The supernatant from centrifugation was collected to
23
measure the glucose concentration. Glucose concentration was determined by the 3,
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5-dinitrosalicylic acid (DNS) method (Miller, 1959). Nitrogen concentration in the broth 6
1
was determined using alkaline potassium persulfate digestion and UV
2
spectrophotometry according to the China Standard GB 11894-89 (Ren et al., 2010).
3 4
2.5. Total lipids (TLs) 3 mL fermentation broth was mixed with 4 mL HCl (12N) and incubated in water
5 6
bath at 65 oC for 45 min. TLs from the mixture were extracted by three times (15 min of
7
each time) with 5 mL n-hexane, and then the collected extracts were mixed and purified
8
in a rotary vacuum evaporator at 60 oC (Chang et al., 2013).
9 10
2.6. Fatty acid composition analysis Fatty acid methyl esters (FAMEs) were prepared regarding to previous methods
11 12
(Ren et al., 2009; Yeh and Chang, 2012) with modifications: 5 mL, 0.5 M
13
KOH-methanol was added to a tube containing TLs. The tubes were heated in a water
14
bath at 65 oC for 10 min and added 5 mL 30% BF3-ether then. The tubes were further
15
heated in a water bath at 65 oC for 30 min and followed of 5 mL hexane was added
16
when the tubes cooled down to room temperature. Arachidic acid methyl (4 g/L, Sigma,
17
USA) as an internal standard was added in the tubes and mixed through a vortex for 1
18
min, and then settled for separation of two phases after adding 1 mL saturated sodium
19
chloride solution for preventing emulsification. The upper phase containing FAMEs was
20
applied to a gas chromatograph (Agilent GC 7890, USA) equipped with a 100 m×0.25
21
mm capillary column (SP™-2560, USA). The column was increased from 140 to 240
22
o
23
injector and detector were both set at 260 oC. Nitrogen was used as the carrier gas at 20
24
cm/s. Peaks were identified using the authentic standards of fatty acid methyl esters
C at 3 oC /min and then maintained at 240 oC for further 30 min. The temperature of the
7
1
(Sigma, USA). Fatty acids were quantified from the peak areas relative to the peak of
2
the internal standard.
3 4
3. Results and discussion
5
3.1. Identification of new strain
6
Based on a phylogenetic tree analysis of the 18SrRNA sequence for the most
7
closely related species (Fig.1), this strain was identified and designated as
8
Schizochytrium sp. LU310 (accession number KM245569). The new strain was 99.5%
9
similar to the Schizochytriumsp.FJU-512 (accession number AY758384), 89.8% similar
10
to the Schizochytriumsp.ATCC20888 (accession number DQ 367050) with regard to its
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18S rRNA sequence.
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The morphology of cell was studied by light microscopy (data not shown). The
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strain LU310 were very similar to Schizochytrium limacinum SR21, especially in the
14
reproduction with continuous binary fission, which made it classified as Schizochytrium
15
instead of Traustochytrium (Honda et al., 1998; Morita et al., 2006). Reproduction with
16
continuous binary fission made the strain to be cultivated fast and easily, which can be
17
used as an enormous industrial potential strain.
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3.2. The effect of DO on strain LU310 As listed in Table 1, the DHA productivity of Schizochytrium sp. LU310 attained in
21
1000 mL baffled flasks was 194.38 mg/L/h, which was higher when compared with the
22
published value of 117 mg/L/h by Thraustochytrid strain 12B (Perveen et al., 2006),
23
125 mg/L/h by Schizochytrium sp. G13/2S (Ganuza et al. 2008), 43.7 mg/L/h by
24
Schizochytrium limacinum SR 21 (Chi et al., 2009) and 119 mg/L/h by Schizochytrium 8
1
sp. CCTCC M209059 (Ren et al., 2010), respectively. The productivity is to our best
2
knowledge to be the highest report for flask-cultured DHA-producing microorganisms.
3
Baffled flasks and normal flasks were used in fermentation of LU310, because
4
baffled flasks have higher oxygen transfer coefficient (kLa) than normal flasks under the
5
identical shaking speed (Gupta and Rao, 2003; Chang et al. 2013). As shown in Fig.2,
6
cells cultivated in baffled flasks achieved higher biomass, TLs and DHA concentration
7
than that in normal flasks. As indicated in Fig. 2a and 2e, the glucose and nitrogen
8
consumption rate of cells in baffled flasks were obviously increased compared to that in
9
normal flasks during the fermentation. Fig. 2b showed that cell growth enhanced to a
10
large extent in baffled flasks, especially before 48 h. The highest DCW and biomass
11
productivity up to 60.82 g/L and 1.21 g/L/h were obtained in the 1000 mL baffled flasks.
12
While in the normal flasks (250 or 1000 mL), the final DCW and biomass productivity
13
decreased (Table 1). As baffled flasks supplied more DO than normal flasks, the
14
Schizochytrium sp. processed higher DO supply (baffled flasks) and obtained faster cell
15
growth, which leaded to consume more carbon and nitrogen source. On the contrary,
16
culture in low DO supply (normal flasks), the activity of glucose and nitrogen
17
assimilation was suppressed so that low cell growth was observed. The high cell density
18
was concerned for high production of intracellular products (Ganuza et al., 2008),
19
therefore, baffled flasks culture with high DO was favorable for cell growth and more
20
carbon and nitrogen source used for cell respiration and energy metabolism.
21
As shown in Fig. 2c and 2d, lipids began to accumulate accompanied with the cell
22
growth. Previous report showed that lipid accumulation of Schizochytrium sp. was a
23
growth-associated process, but the nitrogen depletion enhanced the accumulation of
24
lipids (Ganuza and Izquierdo, 2007; Ganuza et al., 2008; Jakobsen et al., 2008). But the 9
1
lipid accumulation was subsequently induced by nitrogen depletion. This particular
2
pattern was also observed in Schizochytrium G13/2S (Ganuza and Izquierdo, 2007). In
3
addition, nitrogen utilization was also considerably affected by different oxygen supply
4
levels. During high DO cultures, nitrogen was consumed much faster which was almost
5
exhausted at 48 h, so that lipids and DHA synthesized much more quickly. However, in
6
low oxygen supply culture, nitrogen was assimilated more slowly which was almost
7
exhausted at 72 h (Fig. 2e), and the DHA production was also showed correspondingly
8
tardy (Fig. 2d).
9
As summary in Table 1, the highest DCW, lipid and DHA content in DCW were
10
achieved in the high oxygen supply culture (1000 mL baffled flasks), which increased
11
30.0%, 23.1% and 48.7% higher than the value in the 1000 mL normal flasks,
12
respectively. It indicated that oxygen supply had apparent enhancement on the cell
13
growth and DHA production of Schizochytrium sp. LU310. Previous studies also
14
showed the similar results (Chang et al., 2014; Qu et al., 2010; Qu et al., 2013). For the
15
scale-up of the aerobic fermentation processes by the organisms that are not sensitive to
16
shear force, the effect of oxygen transfer is normally the most important factor (Qu et al.,
17
2013).The mentionable points were 152.1% and 164.0% improvements in DCW and
18
DHA productivity were observed under 1000 mL baffled flasks than that of the best
19
results controlled by 1000 mL normal flasks. The culture time of this strain also was
20
shortened from 120 h to 84 h, which was quite favorable for the scale-up production of
21
DHA fermentation in the industry.
22
GC analysis showed that the major fatty acids extracted from Schizochytrium sp.
23
LU310 were tetradecanoic acid (C14:0), hexadecanoic acid (C16:0), stearic acid
24
(C18:0), docosapentenoic acid (C22:5), and docosahexaenoic acid (C22:6). And the 10
1
proportion of other FAs in the normal flasks reached to 23.4-28.2% (w/w), which
2
included pentadecylic acid (C15:0), palmitic acid (C16:1), margaric acid (C17:0),
3
oleinic acid (18:1), linolenic acid (C18:3), and ecosapeatanolic acid (C20:5). Fig. 3
4
illustrated the profiles of fatty acids composition of LU310 grown in the different flasks.
5
The composition of total fatty acids produced by LU310 was quite similar except DHA.
6
The DHA proportion of total fatty acids increased significantly with the increase of DO
7
supply levels. The highest DHA proportion of 40.9% was obtained in 1000 mL baffled
8
flasks, which was 25.8% improvement over the best results controlled by 1000 mL
9
normal flasks. Many reports indicated that low dissolved oxygen was favored for DHA
10
production (Jakobsen et al., 2008; Qu et al., 2010; Ren et al., 2010; Chang et al., 2014).
11
As a matter of fact, in baffled flasks cultures offered more oxygen, so all medium
12
ingredients were effectively employed to achieve high cell density at the initial
13
fermentation stage. In flasks, the higher the biomass was, the lower oxygen level
14
dissolved. Therefore, oxygen was also limited after 48 h in this study, which improved
15
the production of DHA. Our results were consistent with the previous studies but
16
provided an alternative feasible and convey approach by used the baffled flask.
17 18 19
3.3. Effects of feeding nitrogen on the fermentation In order to obtain high cell concentrations with high DHA content, several modes
20
of nitrogen addition were performed during fed batch cultivation. MSG was selected as
21
an effective nitrogen source because MSG addition could accelerate the substrate
22
consumption rate and enhance the accumulation of lipid and DHA, which has a positive
23
influence in the growth of microalgal culture (Ren et al., 2014; Sun et al., 2014). Herein,
24
the feeding concentration of nitrogen (MSG) ranged from 0.4 to 0.8 g/L according to its 11
1
nitrogen-consuming rate during fermentation. The MSG-feeding time was chosen as the
2
following: (1) 24 h, (before nitrogen depletion), (2) 48 h, (around nitrogen depletion), or
3
(3)72 h, (after nitrogen depletion for some time). Detailed information and results of
4
MSG-feeding culture were listed in Fig. 4 and Table 2. It demonstrated that MSG
5
feeding could enhance the DCW and DHA production. The 0.4 g/L MSG-feeding on
6
fermentation was better than that of 0.8 g/L MSG-feeding at the same time. When fed
7
with 0.4 g/L MSG at different time, as indicated in Table 2, the DHA concentration and
8
the lipid content in DCW were higher (ranging from 5.5% ~ 8.6% and 2.8% ~ 4.8%,
9
respectively) than that in the culture fed with 0.8 g/L MSG at the same time. According
10
to literature reports, nitrogen limitation may increase the intracellular content of fatty
11
acid acyl-CoA and activate diacylglycerol acyltransferase, which converts fatty acid
12
acyl-CoA to triglyceride (Hsieh and Wu, 2009; Takagi et al., 2000). This is why low
13
nitrogen concentrations increased not only the intracellular lipid content but also the
14
DHA concentration.
15
When fed with 0.4 g/L MSG at 72 h, as indicated in Table 2, the DHA
16
concentration and the TLs concentration were higher (ranging from 7.5%~18.4% and
17
11.6%~19.3%, respectively) than that in the culture fed with 0.4 g/L of MSG at 48 h and
18
24 h. The highest DCW of 55.9 g/L was obtained in the culture fed with 0.4 g/L of MSG
19
at 72 h, which was 6.3% and 10.3% more than that fed with 0.4 g/L of MSG at 48 h and
20
24 h, respectively. The possible reason for producing more DHA and biomass with
21
nitrogen feeding during 72 h than the feedings during other phases was that when fed
22
with MSG at 72 h, which not only offered nitrogen source for further cell growth but
23
also yielded more DHA at the end of fermentation, by activating more acetyl CoA
24
carboxylase to accumulate lipids (Hsieh et al., 2009). 12
1
Sun et al. (2014) reported that the limitation of organic and inorganic nitrogen had
2
different influences on cell growth. Organic nitrogen limitation inhibited the cell growth
3
obviously, but inorganic nitrogen did not. MSG addition could accelerate the substrate
4
consumption rate and enhance the accumulation of lipid and DHA, which has a positive
5
influence in the growth of microalgal culture (Kowluru et al., 2001; Lan et al., 2002;
6
Ren et al., 2014). Thus, MSG-feeding used for culturing Schizochytrium sp. in this
7
study.
8
Although the MSG-feeding strategy was favorable to the DHA production, DCW
9
and DHA productivity were only increased 10.0% and 10.4%. This might be caused by
10
the exhaustion of glucose at 48 h. The deficiency of carbon source made acetyl-CoA not
11
continuously supplied in the cytosol of the cell as a necessary precursor for fatty acid
12
synthetase (FAS), which is the pathway of DHA synthesis relies on (Ren et al., 2009).
13
Therefore, the DHA production was not apparently enhanced. It indicated that MSG
14
addition could relieve the drop tendency of DHA but not deal with the root of the
15
problem. Next, we employed intermittent addition of glucose to improve cultivation of
16
Schizochytrium sp. for higher biomass and DHA content.
17 18
3.4. Effects of feeding carbon source on the fermentation
19
According to the Fig. 2a, glucose was used up at 48 h, so the glucose feeding time
20
was chosen at 48h. The feeding concentration of carbon (glucose) ranged from 15 to 35
21
g/L according to the glucose-consuming rate during fermentation was used. The glucose
22
feeding was employed when glucose concentration in the flasks was lower than 1 g/L.
23
The time courses of DCW, DHA concentration, and glucose concentration in the
24
intermittent glucose feeding cultivation were demonstrated in Fig. 5. Compared with the 13
1
control group, the experimental groups achieved much higher biomass and DHA. The
2
highest DCW of 80.9 g/L, DHA concentration of 21.06 g/L and DHA productivity of
3
231.36 g/L/h were obtained by feeding 15 g/L of glucose. These results were 139.7%,
4
141.3% and 112.2% improvements over the best results controlled by fed 0 g/L glucose
5
(Table 3). The acetyl CoA, a metabolic product of glucose, is the starting material for
6
cell synthesis of FAS, thus to increase the proportion of TLs in cells. The results also
7
showed that the influence of feeding 15 g/L of glucose on the fermentation was better
8
than that of feeding with 35 g/L of glucose. Although the DCW in the intermittent
9
feeding of 35 g/L glucose, also increased obviously (reached to 78.6 g/L), the DHA
10
content in DCW was only 23.47 % (w/w), which was much less than that intermittent
11
feeding 15 g/L of glucose. It has reported that too high concentration of carbon source
12
would inhibit cell growth as well as lipid accumulation in DHA cultures (de Swaaf et al.,
13
2002; Ren et al., 2013; Sijtsma and Swaaf, 2004). Wu et al. (2005) also reported when
14
the glucose concentration was more than 40 g/L in the medium, the microorganism
15
synthesized fatty acids and secreted acids into the broth. In the present experiment,
16
glucose was used up at 48 h, which inhibited cell growth. Finally, the best condition was
17
applied of intermittent feeding 15 g/L of glucose at 48 h.
18 19 20
3.5. Effects of couple feeding carbon and nitrogen on DCW and DHA content Based on preliminary fed-batch results, the highest lipid and DHA content were
21
obtained by cultivation with feeding 0.4 g/L nitrogen during 72 h, while the highest
22
DCW and DHA concentration and DHA productivity were achieved by cultivation with
23
intermittent feeding 15 g/L glucose at 48 h. Thus, an innovative strategy was carried out
24
by intermittent feeding 15 g/L glucose when glucose concentration was < 1 g/L and 14
1
simultaneously feeding 0.4 g/L nitrogen at 72 h. As shown in Fig.6, the highest DCW of
2
88.6 g/L, DHA concentration of 24.74 g/L and DHA productivity of 241.1mg/L/h were
3
achieved in this couple feeding cultivation, which were an increase of 53.3%, 65.9%
4
and 16.5% over the control, respectively. DHA percentage of TLs reached 47.5% when
5
using the novel fermentation strategy, which was an increase of 16.7% over than that of
6
the control. Meanwhile, in this process, the DHA concentration and DHA productivity
7
were 51.5% and 24.0% higher than the best results of MSG-feeding, respectively.
8
Similarly, the DHA concentration and DHA productivity were 17.5% and 3.8% higher
9
than the best values obtained in culture with glucose-feeding, respectively. The results
10
clearly indicated that the optimal process with the strategy of nitrogen and carbon
11
addition can promote to produce lipids and DHA effectively. To our best knowledge, it
12
was the highest producing result in the flask-cultured of DHA-producing microalgae.
13
Regarding to the comparison results in Table 4, although high algae biomass had
14
been achieved in other researches (Huang et al., 2012; Ganuza et al., 2008; Ren et al.,
15
2010), the obtained DHA concentration and DHA productivity were much lower than
16
that of this study (i.e. 24.74 g/L and 241.5mg/L/h). Obvious differences existed between
17
the reports on the DHA productivity as listed in Table 4, and this probably roots in the
18
diversity of Schizochytrium sp. and the adoption of the culture modes. Our effective and
19
efficient nitrogen and carbon addition strategy is expected to facilitate the cultivation of
20
Schizochytrium sp. LU310. By intermittent feeding moderate glucose in the culture, the
21
cells can be maintained at nutrient-replete growth. The cells could instantly begin
22
increasing accumulating DHA because of small amounts of nitrogen supplied after
23
nitrogen deficiency. Therefore, the fermentation of Schizochytrium sp. combined with
24
carbon and nitrogen supply to increase DHA productivity would provide an alternative 15
1
strategy for other microalgae or yeast to obtain PUFAs.
2 3 4 5
4. Conclusion The cell growth rate and DHA accumulation of Schizochytrium sp. LU310 were
6
strongly related to carbon and nitrogen concentration. Based on the experimental
7
observations, the highest DCW and DHA concentration were obtained by cultivation
8
with feeding of 0.4 g/L nitrogen at 72 h and intermittent feeding of 15 g/L glucose when
9
glucose concentration was < 1 g/L, which was the highest production in the
10
flask-culture of DHA-producing microalgae. Consequently, DHA production from
11
microalgae can be successfully accomplished by using an innovative strategy with
12
intermittent feeding moderate glucose combined with limited amounts of MSG during
13
the cultivation.
14 15 16
16
1
Acknowledgements
2
The authors are grateful to the National Natural Science Foundation of China
3
(No.41206115), the Natural Science Foundation of Fujian Province of China (No.
4
2013J01060) and the National High-Tech R&D Program of China (No.
5
2014AA021701).
6 7 8
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Technol. 98, 281-287. 20
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[30] Wu, S.T., Yu, S.T., Lin, L.P., 2005. Effect of culture conditions on docosahexaenoic
2
acid production by Schizochytrium sp. S31. Process. Biochem. 40, 3103-3108.
3
[31] Yokochi, T., Honda, D., Higashihara, T., Nakahara, T., 1998. Optimization of
4
docosahexaenoic acid roduction by Schizochytrium limacinum SR21. Appl.
5
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[32] Yeh, K.L., Chang, J.S., 2012. Effects of cultivation conditions and media
7
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8
Chlorella vulgaris ESP-31. Bioresour. Technol. 105, 120-127.
9
[33] Zhang, L., Zhao, H.Y., Lai, Y.Q., Wu, J.P., Chen, H. L., 2013. Improving
10
docosahexaenoic acid productivity of Schizochytrium sp. by a two-stage
11
AEMR/shake mixed culture mode. Bioresour. Technol. 142, 719-722.
12
13
21
1
Figure legends:
2 3
Fig.1. Phylogenetic tree analyzed by 18S rRNA sequences with the neighbor joining
4
methods constructed with the MEGA 6.0 software.
5 6
Fig.2.Time course of fermentation profiles of Schizochytrium sp. LU310 in four
7
independent batch cultures with 1000 mL baffled flasks (Square symbols), 1000 mL
8
normal flasks (triangle symbols), 250 mL baffled flasks (circle symbols), and 250 mL
9
normal flasks (diamond symbols): (a) glucose exhaustion (g/L); (b) DCW (g/L); (c) TLs
10
concentration (g/L); (d) DHA concentration (g/L). Experiments were carried out in
11
duplicate trials.
12 13
Fig.3. Fatty acids composition of Schizochytrium sp. LU310 grown in different flasks
14
(250 mL normal flasks, 1000 mL normal flasks, 250 mL baffled flasks and 1000 mL
15
baffled flasks). Data shown are the average of two replicates (±SD standard deviation).
16 17
Fig.4. Effects of nitrogen feeding on cell growth and DHA production of
18
Schizochytrium sp. LU310 at different feeding concentrations and timing. Closed
19
symbols and black bar charts are 0.4 g/L nitrogen feeding, and open symbols and green
20
bar charts are 0.8 g/L nitrogen feeding. (a) Feeding of 0.4 or 0.8 g/L nitrogen at 24 h; (b)
21
Feeding of 0.4 or 0.8 g/L nitrogen at 48 h; (c) Feeding of 0.4 or 0.8 g/L nitrogen at 72 h.
22 23
Fig.5. Time courses of cell growth and carbon consumption for Schizochytrium sp.
24
LU310 at different glucose feeding concentration. DCW (Square symbols), TLs 22
1
concentration (unfilled bar charts), DHA concentration (filled bar charts), glucose
2
concentration (triangle symbols), nitrogen concentration (circle symbols). (a) Feeding
3
glucose of 0 g/L; (b) Feeding glucose of 15 g/L; (c) Feeding glucose of 35 g/L.
4 5
Fig.6. Time courses of cell growth, nitrogen and carbon consumption for
6
Schizochytrium sp. LU310 in the optimal mode. Open symbols and black bar charts are
7
control, closed symbols and green bar charts are feeding.
8
23
Table 1.
1 2
Parameters of biomass, TLs and DHA production during fermentation of
Schizochytrium sp. LU310 in the normal and baffled flasks. Parameters
Culture modes 250 mL
1000 mL
250 mL
1000 mL
normal
normal
baffled
baffled
flasks DCW (g/L)
42.48±1. 12
TLs concentration (g/L)
(w/w, %) Biomass
70
9
97
3
71
71
43 17.15±1.
37
58
53
39.92±0.
53.0
16.33±0.
88.6
67.25±1.
23.1
27.51±1.
48.7
78 67.30±3.
18.50±0.
30.0
85 12.22±0.
54.64±1.
60.82±0. 95
34.47±1.
8.66±0.4
52.75±5.
DHA in DCW
13
ment (%)*
flasks
52.30±0.
26.10±1.
7.27±0.3
TLs in DCW (w/w, %)
46.80±0.
21.05±1.
DHA
flasks
97
38
concentration (g/L)
flasks
Incre
15 23.87±1.
30
21
0.44
0.48
1.11
1.21
152.1
60.58
73.64
162.76
194.38
164.0
productivity (g/L/h) DHA productivity (mg/L/h) 3
* The values were calculated at the cultivation time when DHA was maxima in 1000 mL flasks.
4
24
Table 2. Effect of feeding nitrogen on the cell growth and DHA content in
1 2
MSG-feeding culture Ori ginal
2 4h-1
2 4h-2
4 8h-1
4 8h-2
7 2h-1
7 2h-2
Incre ment (%)*
Nitrogen feeding
0
concentration (g/L)
0. 4
DCW (g/L)
50.8
8 5
0.7 TLs concentration (g/L)
30.7 3
DHA concentration (g/L) TLs in DCW (w/w, %)
58.2
DHA in DCW (w/w, %)
23.4 2
DHA productivity (mg/L/h)
3
6
191. 15
2 8.28
1
6
6
2 6.73
1
6
2
9.65
3.44
10.0
3
26.7
1
17.9
6
19.7
2
33.0
10.4
4.60 6
2
5
5.93
5.41
0.34
4.75
3
1
--
3.7
8.93
2.09
2.04
5
3
0. 8
5.9
1.13
3.02
5.59
5
3
1
0. 4
1.6
2.63
3.20
8.77
5
3
1
0. 8
2.6
2.40
4.34
0
4
3
13.0
0. 4
9.4
4.87
7
0.
6.91 3
1.16
7.19
2
1
1
1
2
2
06.29
99.60
95.29
90.15
11.00
05.00
*Compared the control with the best result in fed-batch culture.
4
26
1 2
Table 3. Effect of feeding carbon on the cell growth and DHA content in glucose-feeding culture Contro
48h-1
48h-2
l
Incre ment (%)*
Glucose feeding
0
15
35
--
DCW (g/L)
57.8
80.9
78.6
39.7
TLs concentration (g/L)
31.73
48.10
43.57
47.1
DHA concentration (g/L)
14.91
21.06
17.84
41.3
TLs in DCW (w/w, %)
55.78
66.24
50.82
18.8
DHA in DCW (w/w, %)
25.79
28.77
23.47
11.6
DHA productivity (mg/L/h)
207.02
232.36
194.30
12.2
concentration (g/L)
3
*Compared control with the best result in fed-batch culture.
4 5
27
1 2
Table 4. Biomass, DHA yield and DHA productivities in Schizochytrium sp. from the literatures and this study. Strain in
Device
DC W (g/L)
Schizochytriu
D HA
m sp.
(g
SR21
Shake-fl ask
S31
0 Shake-fl
ask
Strain 12B ask G13/2S ask
ge in
117
8
3
125
01
9
Perveen et al., 2006
6.
37.
Unagul et al., 2007
6.
63.
Two-sta
Limacinum
63
0
9
Wu et al., 2005
6.
30.
Shake-fl
3.42
328
0
Yokochi et al., 1998
0.
28.
Shake-fl
35
2
59
or
(mg/L/ h)
4.
7.4
Bioreact
mangrovei
SR21
36.
Reference
productivity
/L)
Sk-02
DHA
Ganuza et al., 2008
6.
43.7
56
Chi et al., 2009
shake-flask HX-308 (CCTCC
Stepwis e
71. 0
17 .50
28
119
Ren et al., 2010
M209059)
aeration in flask
HX-308 M
Shakeflask after
45. 44
5.
56.22 %a
2
Lian et al., 2010
mutagenesis Bioreact
A. limacinum SR21 (ATCC
or
61. 8
20
123
.3
Huang et al., 2012
MYA-1381) ATCC 20888
AEMR/s hake
LU310
2
a
00 Shake-fl
ask 1
12.
2.
21.26
04 88.
6
Zhang et al., 2013
24
241.5
This study
.74
DHA productivity is not available in the paper and DHA percentage in the lipids
used instead.
3 4 5 6
29
1 2 3 4
Fig.1
5 6 7 8 9 10 11
30
a
b 70
1000-mL bafferled flasks 250-mLbafferled flasks 1000-mLnoral flasks 250-mLnoral flasks
120
1000-mL bafferled flasks 250-mLbafferled flasks 1000-mLnoral flasks 250-mLnoral flasks
60
100
80
DCW (g/L)
Glucose (g/L)
50
60
40 30
40
20
20
10 0
0 0
24
48
72
96
0
120
24
48
72
96
120
Time (h)
Time (h)
d 20
c 40
30
DHA (g/L)
Total lipids (g/L)
16
20
10
8
4 1000-mL bafferled flasks 250-mLbafferled flasks 1000-mLnoral flasks 250-mLnoral flasks
0 0
24
48
72
96
e 1.6
0
1.2 1.0 .8 .6 .4 .2 0.0 0
24
48
72
96
24
48
72
Time (h)
1000-mL bafferled flasks 250-mLbafferled flasks 1000-mLnoral flasks 250-mLnoral flasks
1.4
1000-mL bafferled flasks 250-mLbafferled flasks 1000-mLnoral flasks 250-mLnoral flasks
0
120
Time (h)
Nitrogen (g/L)
12
120
Time (h)
31
96
120
1
Fig. 2
2 3
Fatty acid composition (% of each FA per TLs)
100
Others DHA DPA C18:0 C16:0 C14:0
80
60
40
20
0
250 mL 1000 mL 250 mL 1000 mL normal flasks normal flasks baffled flasks baffled flasks
Culture methods
4 5 6
Fig. 3.
7 8
32
1
a 60
50
120 1.4 100
1.2
80
1.0
30
60
20
.8 .6
40 .4
10
20
0
.2
0 0
24
48
72
96
0.0
120
Time (h)
b
60
1.6
DCW Glucose TLs DHA Nitrogen
50
120
1.4 1.2
40 80
30 60
20
Glucose (g/L)
DCW (g/L) TLs and DHA (g/L)
100
1.0 .8 .6
40
.4 10
20
0
0
0
3
24
48
72
96
Time (h)
33
120
.2 0.0
Nitrogen (g/L)
2
Nitrogen(g/L)
40
Glucose (g/L)
DCW (g/L) TLs and DHA (g/L)
1.6
DCW Glucose TLs DHA Nitrogen
c 60
50
120 1.4 100
1.2
80
1.0
30
60
20
.8 .6
40 .4
10
20
0
0 0
24
48
72
96
Time (h)
1 2 3
Fig. 4
4
34
120
.2 0.0
Nitrogen (g/L)
40
Glucose (g/L)
DCW (g/L) TLs and DHA (g/L)
1.6
DCW Glucose TLs DHA Nitrogen
1 2
35
1 2
36
1 2
Fig. 5
3 4
37
100 90 80
120 1.4 100
1.2
80
60 50
60
40
1.0 .8 .6
40
30
.4 20 20 10 0
0 0
24
48
72
96
120
144
Time (h)
1 2 3
Fig. 6
4 5 6 7 8 9
38
168
192
.2 0.0
Nitrogen (g/L)
70
Glucose (g/L)
DCW (g/L) TLs and DHA (g/L)
1.6
DCW TLs DHA Glucose Nitrogen
Highlights
1 2 3 4
1.
Schizochytrium sp. LU 310 is an ideal stain for DHA-producing as its
impressive productivity.
5 6
2.
Baffled flask enhanced in high cell density cultivation of DHA.
3.
MSG-feeding combined with glucose feeding strategy was extremely useful
7 8 9
for DHA production.
10 11 12
4. DHA production was linearly correlated with the concentrations of carbon and nitrogen.
13 14
39
S0960-8524(14)01389-3 http://dx.doi.org/10.1016/j.biortech.2014.09.130 BITE 14020
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
24 August 2014 23 September 2014 25 September 2014
Please cite this article as: Ling, X., Guo, J., Liu, X., Zhang, X., Wang, N., Lu, Y., Ng, I-S., Impact of carbon and nitrogen feeding strategy on high production of biomass and docosahexaenoic acid (DHA) by Schizochytrium sp. LU310, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.09.130
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Original research to
2
“Bioresource Technology on Special Issue of Bio/Chemicals from Algae”
3 4
Impact of carbon and nitrogen feeding strategy on high production of
5
biomass and docosahexaenoic acid (DHA) by Schizochytrium sp.
6
LU310
7 8 9 10
Xueping Linga, Jing Guo a, Xiaoting Liua, Xia Zhanga, Nan Wanga, Yinghua Lua, I-Son Nga,b*
11 12 13
a
Department of Chemical and Biochemical Engineering, College of Chemistry and
14
Chemical Engineering, Xiamen University, Xiamen 361005 PR China;
15
b
16
Taiwan.
Department of Chemical Engineering, National Cheng Kung University, Tainan 70101,
17 18 19
*Corresponding author: I-Son Ng
20
Tel: +886-62757575; Fax: +886-62344496;
21
E-mail: [email protected]
22
23
1
1
ABSTRACT: A new isolated Schizochytrium sp. LU310 from the mangrove forest of
2
Wenzhou, China, was found as a high producing microalga of docosahexaenoic acid
3
(DHA). In this study, the significant improvements for DHA fermentation by the batch
4
mode in the baffled flasks (i.e. higher oxygen supply) were achieved. By applied the
5
nitrogen-feeding strategy in 1000 mL baffled flasks, the biomass, DHA concentration
6
and DHA productivity were increased by 110.4%, 117.9% and 110.4%, respectively.
7
Moreover, DHA concentration of 21.06 g/L was obtained by feeding 15 g/L of glucose
8
intermittently, which was an increase of 41.25% over that of the batch mode. Finally, an
9
innovative strategy was carried out by intermittent feeding carbon and simultaneously
10
feeding nitrogen. The maximum DHA concentration and DHA productivity in the
11
fed-batch cultivation reached to 24.74 g/L and 241.5 mg/L/h, respectively.
12 13 14
Keywords: Docosahexaenoic acid (DHA); Schizochytrium sp.; Baffled flasks; Nitrogen
15
feeding; Carbon feeding
16 17
2
1 2
1. Introduction Docosahexaenoic acid (DHA, 22:6 n-3) has received worldwide attention due to its
3
beneficial physiological functions for humans, for instance, promotion effect for visual
4
acuity and neural development, lowering the incidence of cardiovascular diseases, and
5
reducing risk factors involved in diseases like hypertension, arthritis, arteriosclerosis
6
and thrombosis (Ren et al., 2013; Sijtsma and Swaaf, 2004; Sun et al., 2014). Due to its
7
widespread use, the global DHA market has grown significantly in the recent years and
8
expected to reach $34.7 billion in 2016.
9
Schizochytrium sp., a thraustochytrid, is an algae-like microorganism that is closely
10
related to heterokont algae (Barclay et al., 1994; Morita et al., 2006; Sijtsma and Swaaf,
11
2004). It is well-known for producing polyunsaturated fatty acids (PUFAs), among
12
which DHA usually represents 30-40 % of the total fatty acids (Yokochi et al., 1998),
13
and thus is considered as a promising and commercially viable alternative DHA
14
feedstock. However, this source of DHA is limited by the high production costs in
15
comparison to the current fish oil prices. In order to reduce the costs of
16
microalgae-based DHA production, higher volumetric productivity must be pursued by
17
culture optimization. A yield of 90-100 g/L DCW with high lipid content (54-63%, w/w)
18
by Aurantiochytrium sp. strain T66 under N starvation and O2 limitation had been
19
reported (Jakobsen et al., 2008). In another case, an intermittent oxygen feeding method
20
to maintain a 50% dissolved oxygen level with a C:N ratio of 1.25 for Schizochytrium
21
limacinum SR21, to achieve DCW at 61.76 g/L and DHA concentration at 20.3 g/L had
22
also been reported (Huang et al., 2012). The literature showed that the cell growth rate,
23
DHA content and DHA productivity of oleaginous microorganisms were mainly
24
influenced by the culture conditions, such as nitrogen concentration, carbon 3
1
concentration and dissolved oxygen level (de Swaaf et al., 2002; Ganuza and Izquierdo,
2
2007; Ganuza et al., 2008; Jakobsen et al., 2008; Sun et al., 2014). It has been suggested
3
that nitrogen limitation could enhance the amount of acetyl-CoA and NADPH, which
4
were the necessary precursors for lipid accumulation (de Swaaf et al., 2002). Recently,
5
Ren et al. (2014) also found that the DHA percentage of TFAs would be decreased after
6
nitrogen exhaustion. However, the phenomenon could be relieved by monosodium
7
glutamate (MSG) addition, which could activate the enzyme of acetyl-CoA carboxylase
8
(Kowluru et al., 2001) and glucose-6-phosphate dehydrogenase (Lan et al., 2002), and
9
thus it ensures the sufficient supply of acetyl-CoA and NADPH for DHA synthesis to
10
enhance the accumulation of lipid and DHA.
11
The accumulated lipids and fatty acids within oleaginous microorganisms are,
12
therefore, dynamic storage materials and are produced in times of plenty when carbon is
13
sufficient or starvation when carbon is deficient (Ganuza et al., 2007; Ratledge 2002;
14
Wu et al., 2005). Moreover, the high carbon to nitrogen ratio is beneficial to lipid
15
accumulation (Yokochi et al., 1998), while the DHA percentage fluctuated with the
16
glucose addition (Ren et al., 2014), indicating glucose feeding would be proper for
17
DHA production. In addition, it had been proved that low dissolved oxygen (DO) was
18
favored for DHA production (Jakobsen et al., 2008), and fermentation strategy was
19
performed by controlling DO for the optimization of DHA production (Chi et al., 2009).
20
A stepwise aeration control strategy was developed for efficient DHA production in a
21
1,500-L bioreactor using fed-batch fermentation (Ren et al., 2010), or an intermittent
22
oxygen feeding method to maintain a 50% DO level (Huang et al., 2012). However, till
23
now, no efforts have been made to simultaneously evaluate MSG addition and glucose
24
feeding at nitrogen limitation condition for increasing DHA production of 4
1 2
Schizochytrium microalgae. In this study, a new isolated microalga, Schizochytrium sp. LU310 was investigated
3
for its ability to produce DHA in the baffled flasks. MSG addition and glucose feeding
4
were applied to optimize DHA productivity. Moreover, an innovative strategy as
5
combining MSG addition and glucose feeding at nitrogen limitation condition was
6
further assessed for its effectiveness in improving DHA production. This approach
7
would provide guidance for the large-scale production of DHA and other similar
8
polyunsaturated fatty acids, and give insight into the carbon and nitrogen metabolism in
9
oil-producing microorganisms.
10 11
2. Materials and Methods
12
2.1. Identification of strain
13
The microalga, Schizochytrium sp. LU310, used in this work was isolated from the
14
mangrove forest of Wenzhou, Zhejiang, China. It was identified by the morphology and
15
18S rRNA sequencing using the primers as 18S-F:
16
5'-TCTCAAAGAYTAAGSCATGC-3' and 18S-R:
17
5'-TTCACCTTCCTCTAAACAATAAG-3'. The 18S rRNA sequence was deposited
18
into GenBank under accession number KM245569.
19 20 21 22
2.2. Morphological analysis A drop of fresh strain LU310 was taken on a slide glass to be observed and recorded by light microscopy (OLYMPUS BX41, Japan) continuously.
23 24
2.3. Culture conditions 5
1
The seed broth contained (g/L, pH 6.5): glucose, 30; Na2 SO4, 12; yeast extract, 10;
2
MgSO4, 2; (NH4) 2SO4, 1; KH2PO4, 1; K2 SO4, 0.65; KCl, 0.5; CaCl2·2H2O, 0.17. The
3
fermentation broth contained (g/L, pH=6.5): glucose, 120; Na2 SO4, 12; MSG, 5; corn
4
steep powder (Sigma Co), 5; MgSO4, 2; (NH4) 2SO4, 1; KH2 PO4, 1; K2 SO4, 0.65; KCl,
5
0.5; CaCl2·2H2O, 0.17 as well as trace element solution(1 mL/L) and vitamin solution
6
(2 mL/L). The trace element solution was composed of (mL/L): FeSO4·7H2O, 10;
7
Ca1/2Pantothenate, 3.2; MnCl2·4H2O, 3; ZnSO4·7H2O, 3; NiSO4·6H2O, 2; CuSO4·5H2O,
8
2; CoCl2·6H2O, 0.04; Na2MoO4·2H2O, 0.04. Vitamin solution consisted of (mg/L):
9
thiamine, 9.5; cyanocobalamin, 0.15. Before autoclaving (121 oC, 20 min) the pH of the
10
medium was adjusted to 6.5 with 2 M NaOH. Yeast extract was purchased from OXOID
11
Ltd., UK. If not specifically mentioned, all other chemicals were provided by
12
Sinopharm Chemical Reagent Co. Ltd., China. The trace element solution and the
13
vitamin solution were filter-sterilized (0.2 µm) and added after sterilization. The stored
14
cells were cultured at 28 oC, 170 rpm for 48 h in the seed broth. After three generations
15
of cultivation, the seed culture (4% v/v) was then transferred to fermentation broth and
16
incubated at 28 oC, 200 rpm for 120 h or more.
17 18 19
2.4. Dry cell weight (DCW), glucose, and nitrogen measurements 3 mL broth was used to determine DCW by gravimetric method. 3 mL broth was
20
transferred to a pre-weighted centrifuge tube and centrifuged at 7,000 x g for 10 min.
21
The cell pellet was washed twice with distillated water and dried at 60 oC until constant
22
(12 h) to analyze the total weight. The supernatant from centrifugation was collected to
23
measure the glucose concentration. Glucose concentration was determined by the 3,
24
5-dinitrosalicylic acid (DNS) method (Miller, 1959). Nitrogen concentration in the broth 6
1
was determined using alkaline potassium persulfate digestion and UV
2
spectrophotometry according to the China Standard GB 11894-89 (Ren et al., 2010).
3 4
2.5. Total lipids (TLs) 3 mL fermentation broth was mixed with 4 mL HCl (12N) and incubated in water
5 6
bath at 65 oC for 45 min. TLs from the mixture were extracted by three times (15 min of
7
each time) with 5 mL n-hexane, and then the collected extracts were mixed and purified
8
in a rotary vacuum evaporator at 60 oC (Chang et al., 2013).
9 10
2.6. Fatty acid composition analysis Fatty acid methyl esters (FAMEs) were prepared regarding to previous methods
11 12
(Ren et al., 2009; Yeh and Chang, 2012) with modifications: 5 mL, 0.5 M
13
KOH-methanol was added to a tube containing TLs. The tubes were heated in a water
14
bath at 65 oC for 10 min and added 5 mL 30% BF3-ether then. The tubes were further
15
heated in a water bath at 65 oC for 30 min and followed of 5 mL hexane was added
16
when the tubes cooled down to room temperature. Arachidic acid methyl (4 g/L, Sigma,
17
USA) as an internal standard was added in the tubes and mixed through a vortex for 1
18
min, and then settled for separation of two phases after adding 1 mL saturated sodium
19
chloride solution for preventing emulsification. The upper phase containing FAMEs was
20
applied to a gas chromatograph (Agilent GC 7890, USA) equipped with a 100 m×0.25
21
mm capillary column (SP™-2560, USA). The column was increased from 140 to 240
22
o
23
injector and detector were both set at 260 oC. Nitrogen was used as the carrier gas at 20
24
cm/s. Peaks were identified using the authentic standards of fatty acid methyl esters
C at 3 oC /min and then maintained at 240 oC for further 30 min. The temperature of the
7
1
(Sigma, USA). Fatty acids were quantified from the peak areas relative to the peak of
2
the internal standard.
3 4
3. Results and discussion
5
3.1. Identification of new strain
6
Based on a phylogenetic tree analysis of the 18SrRNA sequence for the most
7
closely related species (Fig.1), this strain was identified and designated as
8
Schizochytrium sp. LU310 (accession number KM245569). The new strain was 99.5%
9
similar to the Schizochytriumsp.FJU-512 (accession number AY758384), 89.8% similar
10
to the Schizochytriumsp.ATCC20888 (accession number DQ 367050) with regard to its
11
18S rRNA sequence.
12
The morphology of cell was studied by light microscopy (data not shown). The
13
strain LU310 were very similar to Schizochytrium limacinum SR21, especially in the
14
reproduction with continuous binary fission, which made it classified as Schizochytrium
15
instead of Traustochytrium (Honda et al., 1998; Morita et al., 2006). Reproduction with
16
continuous binary fission made the strain to be cultivated fast and easily, which can be
17
used as an enormous industrial potential strain.
18 19 20
3.2. The effect of DO on strain LU310 As listed in Table 1, the DHA productivity of Schizochytrium sp. LU310 attained in
21
1000 mL baffled flasks was 194.38 mg/L/h, which was higher when compared with the
22
published value of 117 mg/L/h by Thraustochytrid strain 12B (Perveen et al., 2006),
23
125 mg/L/h by Schizochytrium sp. G13/2S (Ganuza et al. 2008), 43.7 mg/L/h by
24
Schizochytrium limacinum SR 21 (Chi et al., 2009) and 119 mg/L/h by Schizochytrium 8
1
sp. CCTCC M209059 (Ren et al., 2010), respectively. The productivity is to our best
2
knowledge to be the highest report for flask-cultured DHA-producing microorganisms.
3
Baffled flasks and normal flasks were used in fermentation of LU310, because
4
baffled flasks have higher oxygen transfer coefficient (kLa) than normal flasks under the
5
identical shaking speed (Gupta and Rao, 2003; Chang et al. 2013). As shown in Fig.2,
6
cells cultivated in baffled flasks achieved higher biomass, TLs and DHA concentration
7
than that in normal flasks. As indicated in Fig. 2a and 2e, the glucose and nitrogen
8
consumption rate of cells in baffled flasks were obviously increased compared to that in
9
normal flasks during the fermentation. Fig. 2b showed that cell growth enhanced to a
10
large extent in baffled flasks, especially before 48 h. The highest DCW and biomass
11
productivity up to 60.82 g/L and 1.21 g/L/h were obtained in the 1000 mL baffled flasks.
12
While in the normal flasks (250 or 1000 mL), the final DCW and biomass productivity
13
decreased (Table 1). As baffled flasks supplied more DO than normal flasks, the
14
Schizochytrium sp. processed higher DO supply (baffled flasks) and obtained faster cell
15
growth, which leaded to consume more carbon and nitrogen source. On the contrary,
16
culture in low DO supply (normal flasks), the activity of glucose and nitrogen
17
assimilation was suppressed so that low cell growth was observed. The high cell density
18
was concerned for high production of intracellular products (Ganuza et al., 2008),
19
therefore, baffled flasks culture with high DO was favorable for cell growth and more
20
carbon and nitrogen source used for cell respiration and energy metabolism.
21
As shown in Fig. 2c and 2d, lipids began to accumulate accompanied with the cell
22
growth. Previous report showed that lipid accumulation of Schizochytrium sp. was a
23
growth-associated process, but the nitrogen depletion enhanced the accumulation of
24
lipids (Ganuza and Izquierdo, 2007; Ganuza et al., 2008; Jakobsen et al., 2008). But the 9
1
lipid accumulation was subsequently induced by nitrogen depletion. This particular
2
pattern was also observed in Schizochytrium G13/2S (Ganuza and Izquierdo, 2007). In
3
addition, nitrogen utilization was also considerably affected by different oxygen supply
4
levels. During high DO cultures, nitrogen was consumed much faster which was almost
5
exhausted at 48 h, so that lipids and DHA synthesized much more quickly. However, in
6
low oxygen supply culture, nitrogen was assimilated more slowly which was almost
7
exhausted at 72 h (Fig. 2e), and the DHA production was also showed correspondingly
8
tardy (Fig. 2d).
9
As summary in Table 1, the highest DCW, lipid and DHA content in DCW were
10
achieved in the high oxygen supply culture (1000 mL baffled flasks), which increased
11
30.0%, 23.1% and 48.7% higher than the value in the 1000 mL normal flasks,
12
respectively. It indicated that oxygen supply had apparent enhancement on the cell
13
growth and DHA production of Schizochytrium sp. LU310. Previous studies also
14
showed the similar results (Chang et al., 2014; Qu et al., 2010; Qu et al., 2013). For the
15
scale-up of the aerobic fermentation processes by the organisms that are not sensitive to
16
shear force, the effect of oxygen transfer is normally the most important factor (Qu et al.,
17
2013).The mentionable points were 152.1% and 164.0% improvements in DCW and
18
DHA productivity were observed under 1000 mL baffled flasks than that of the best
19
results controlled by 1000 mL normal flasks. The culture time of this strain also was
20
shortened from 120 h to 84 h, which was quite favorable for the scale-up production of
21
DHA fermentation in the industry.
22
GC analysis showed that the major fatty acids extracted from Schizochytrium sp.
23
LU310 were tetradecanoic acid (C14:0), hexadecanoic acid (C16:0), stearic acid
24
(C18:0), docosapentenoic acid (C22:5), and docosahexaenoic acid (C22:6). And the 10
1
proportion of other FAs in the normal flasks reached to 23.4-28.2% (w/w), which
2
included pentadecylic acid (C15:0), palmitic acid (C16:1), margaric acid (C17:0),
3
oleinic acid (18:1), linolenic acid (C18:3), and ecosapeatanolic acid (C20:5). Fig. 3
4
illustrated the profiles of fatty acids composition of LU310 grown in the different flasks.
5
The composition of total fatty acids produced by LU310 was quite similar except DHA.
6
The DHA proportion of total fatty acids increased significantly with the increase of DO
7
supply levels. The highest DHA proportion of 40.9% was obtained in 1000 mL baffled
8
flasks, which was 25.8% improvement over the best results controlled by 1000 mL
9
normal flasks. Many reports indicated that low dissolved oxygen was favored for DHA
10
production (Jakobsen et al., 2008; Qu et al., 2010; Ren et al., 2010; Chang et al., 2014).
11
As a matter of fact, in baffled flasks cultures offered more oxygen, so all medium
12
ingredients were effectively employed to achieve high cell density at the initial
13
fermentation stage. In flasks, the higher the biomass was, the lower oxygen level
14
dissolved. Therefore, oxygen was also limited after 48 h in this study, which improved
15
the production of DHA. Our results were consistent with the previous studies but
16
provided an alternative feasible and convey approach by used the baffled flask.
17 18 19
3.3. Effects of feeding nitrogen on the fermentation In order to obtain high cell concentrations with high DHA content, several modes
20
of nitrogen addition were performed during fed batch cultivation. MSG was selected as
21
an effective nitrogen source because MSG addition could accelerate the substrate
22
consumption rate and enhance the accumulation of lipid and DHA, which has a positive
23
influence in the growth of microalgal culture (Ren et al., 2014; Sun et al., 2014). Herein,
24
the feeding concentration of nitrogen (MSG) ranged from 0.4 to 0.8 g/L according to its 11
1
nitrogen-consuming rate during fermentation. The MSG-feeding time was chosen as the
2
following: (1) 24 h, (before nitrogen depletion), (2) 48 h, (around nitrogen depletion), or
3
(3)72 h, (after nitrogen depletion for some time). Detailed information and results of
4
MSG-feeding culture were listed in Fig. 4 and Table 2. It demonstrated that MSG
5
feeding could enhance the DCW and DHA production. The 0.4 g/L MSG-feeding on
6
fermentation was better than that of 0.8 g/L MSG-feeding at the same time. When fed
7
with 0.4 g/L MSG at different time, as indicated in Table 2, the DHA concentration and
8
the lipid content in DCW were higher (ranging from 5.5% ~ 8.6% and 2.8% ~ 4.8%,
9
respectively) than that in the culture fed with 0.8 g/L MSG at the same time. According
10
to literature reports, nitrogen limitation may increase the intracellular content of fatty
11
acid acyl-CoA and activate diacylglycerol acyltransferase, which converts fatty acid
12
acyl-CoA to triglyceride (Hsieh and Wu, 2009; Takagi et al., 2000). This is why low
13
nitrogen concentrations increased not only the intracellular lipid content but also the
14
DHA concentration.
15
When fed with 0.4 g/L MSG at 72 h, as indicated in Table 2, the DHA
16
concentration and the TLs concentration were higher (ranging from 7.5%~18.4% and
17
11.6%~19.3%, respectively) than that in the culture fed with 0.4 g/L of MSG at 48 h and
18
24 h. The highest DCW of 55.9 g/L was obtained in the culture fed with 0.4 g/L of MSG
19
at 72 h, which was 6.3% and 10.3% more than that fed with 0.4 g/L of MSG at 48 h and
20
24 h, respectively. The possible reason for producing more DHA and biomass with
21
nitrogen feeding during 72 h than the feedings during other phases was that when fed
22
with MSG at 72 h, which not only offered nitrogen source for further cell growth but
23
also yielded more DHA at the end of fermentation, by activating more acetyl CoA
24
carboxylase to accumulate lipids (Hsieh et al., 2009). 12
1
Sun et al. (2014) reported that the limitation of organic and inorganic nitrogen had
2
different influences on cell growth. Organic nitrogen limitation inhibited the cell growth
3
obviously, but inorganic nitrogen did not. MSG addition could accelerate the substrate
4
consumption rate and enhance the accumulation of lipid and DHA, which has a positive
5
influence in the growth of microalgal culture (Kowluru et al., 2001; Lan et al., 2002;
6
Ren et al., 2014). Thus, MSG-feeding used for culturing Schizochytrium sp. in this
7
study.
8
Although the MSG-feeding strategy was favorable to the DHA production, DCW
9
and DHA productivity were only increased 10.0% and 10.4%. This might be caused by
10
the exhaustion of glucose at 48 h. The deficiency of carbon source made acetyl-CoA not
11
continuously supplied in the cytosol of the cell as a necessary precursor for fatty acid
12
synthetase (FAS), which is the pathway of DHA synthesis relies on (Ren et al., 2009).
13
Therefore, the DHA production was not apparently enhanced. It indicated that MSG
14
addition could relieve the drop tendency of DHA but not deal with the root of the
15
problem. Next, we employed intermittent addition of glucose to improve cultivation of
16
Schizochytrium sp. for higher biomass and DHA content.
17 18
3.4. Effects of feeding carbon source on the fermentation
19
According to the Fig. 2a, glucose was used up at 48 h, so the glucose feeding time
20
was chosen at 48h. The feeding concentration of carbon (glucose) ranged from 15 to 35
21
g/L according to the glucose-consuming rate during fermentation was used. The glucose
22
feeding was employed when glucose concentration in the flasks was lower than 1 g/L.
23
The time courses of DCW, DHA concentration, and glucose concentration in the
24
intermittent glucose feeding cultivation were demonstrated in Fig. 5. Compared with the 13
1
control group, the experimental groups achieved much higher biomass and DHA. The
2
highest DCW of 80.9 g/L, DHA concentration of 21.06 g/L and DHA productivity of
3
231.36 g/L/h were obtained by feeding 15 g/L of glucose. These results were 139.7%,
4
141.3% and 112.2% improvements over the best results controlled by fed 0 g/L glucose
5
(Table 3). The acetyl CoA, a metabolic product of glucose, is the starting material for
6
cell synthesis of FAS, thus to increase the proportion of TLs in cells. The results also
7
showed that the influence of feeding 15 g/L of glucose on the fermentation was better
8
than that of feeding with 35 g/L of glucose. Although the DCW in the intermittent
9
feeding of 35 g/L glucose, also increased obviously (reached to 78.6 g/L), the DHA
10
content in DCW was only 23.47 % (w/w), which was much less than that intermittent
11
feeding 15 g/L of glucose. It has reported that too high concentration of carbon source
12
would inhibit cell growth as well as lipid accumulation in DHA cultures (de Swaaf et al.,
13
2002; Ren et al., 2013; Sijtsma and Swaaf, 2004). Wu et al. (2005) also reported when
14
the glucose concentration was more than 40 g/L in the medium, the microorganism
15
synthesized fatty acids and secreted acids into the broth. In the present experiment,
16
glucose was used up at 48 h, which inhibited cell growth. Finally, the best condition was
17
applied of intermittent feeding 15 g/L of glucose at 48 h.
18 19 20
3.5. Effects of couple feeding carbon and nitrogen on DCW and DHA content Based on preliminary fed-batch results, the highest lipid and DHA content were
21
obtained by cultivation with feeding 0.4 g/L nitrogen during 72 h, while the highest
22
DCW and DHA concentration and DHA productivity were achieved by cultivation with
23
intermittent feeding 15 g/L glucose at 48 h. Thus, an innovative strategy was carried out
24
by intermittent feeding 15 g/L glucose when glucose concentration was < 1 g/L and 14
1
simultaneously feeding 0.4 g/L nitrogen at 72 h. As shown in Fig.6, the highest DCW of
2
88.6 g/L, DHA concentration of 24.74 g/L and DHA productivity of 241.1mg/L/h were
3
achieved in this couple feeding cultivation, which were an increase of 53.3%, 65.9%
4
and 16.5% over the control, respectively. DHA percentage of TLs reached 47.5% when
5
using the novel fermentation strategy, which was an increase of 16.7% over than that of
6
the control. Meanwhile, in this process, the DHA concentration and DHA productivity
7
were 51.5% and 24.0% higher than the best results of MSG-feeding, respectively.
8
Similarly, the DHA concentration and DHA productivity were 17.5% and 3.8% higher
9
than the best values obtained in culture with glucose-feeding, respectively. The results
10
clearly indicated that the optimal process with the strategy of nitrogen and carbon
11
addition can promote to produce lipids and DHA effectively. To our best knowledge, it
12
was the highest producing result in the flask-cultured of DHA-producing microalgae.
13
Regarding to the comparison results in Table 4, although high algae biomass had
14
been achieved in other researches (Huang et al., 2012; Ganuza et al., 2008; Ren et al.,
15
2010), the obtained DHA concentration and DHA productivity were much lower than
16
that of this study (i.e. 24.74 g/L and 241.5mg/L/h). Obvious differences existed between
17
the reports on the DHA productivity as listed in Table 4, and this probably roots in the
18
diversity of Schizochytrium sp. and the adoption of the culture modes. Our effective and
19
efficient nitrogen and carbon addition strategy is expected to facilitate the cultivation of
20
Schizochytrium sp. LU310. By intermittent feeding moderate glucose in the culture, the
21
cells can be maintained at nutrient-replete growth. The cells could instantly begin
22
increasing accumulating DHA because of small amounts of nitrogen supplied after
23
nitrogen deficiency. Therefore, the fermentation of Schizochytrium sp. combined with
24
carbon and nitrogen supply to increase DHA productivity would provide an alternative 15
1
strategy for other microalgae or yeast to obtain PUFAs.
2 3 4 5
4. Conclusion The cell growth rate and DHA accumulation of Schizochytrium sp. LU310 were
6
strongly related to carbon and nitrogen concentration. Based on the experimental
7
observations, the highest DCW and DHA concentration were obtained by cultivation
8
with feeding of 0.4 g/L nitrogen at 72 h and intermittent feeding of 15 g/L glucose when
9
glucose concentration was < 1 g/L, which was the highest production in the
10
flask-culture of DHA-producing microalgae. Consequently, DHA production from
11
microalgae can be successfully accomplished by using an innovative strategy with
12
intermittent feeding moderate glucose combined with limited amounts of MSG during
13
the cultivation.
14 15 16
16
1
Acknowledgements
2
The authors are grateful to the National Natural Science Foundation of China
3
(No.41206115), the Natural Science Foundation of Fujian Province of China (No.
4
2013J01060) and the National High-Tech R&D Program of China (No.
5
2014AA021701).
6 7 8
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AEMR/shake mixed culture mode. Bioresour. Technol. 142, 719-722.
12
13
21
1
Figure legends:
2 3
Fig.1. Phylogenetic tree analyzed by 18S rRNA sequences with the neighbor joining
4
methods constructed with the MEGA 6.0 software.
5 6
Fig.2.Time course of fermentation profiles of Schizochytrium sp. LU310 in four
7
independent batch cultures with 1000 mL baffled flasks (Square symbols), 1000 mL
8
normal flasks (triangle symbols), 250 mL baffled flasks (circle symbols), and 250 mL
9
normal flasks (diamond symbols): (a) glucose exhaustion (g/L); (b) DCW (g/L); (c) TLs
10
concentration (g/L); (d) DHA concentration (g/L). Experiments were carried out in
11
duplicate trials.
12 13
Fig.3. Fatty acids composition of Schizochytrium sp. LU310 grown in different flasks
14
(250 mL normal flasks, 1000 mL normal flasks, 250 mL baffled flasks and 1000 mL
15
baffled flasks). Data shown are the average of two replicates (±SD standard deviation).
16 17
Fig.4. Effects of nitrogen feeding on cell growth and DHA production of
18
Schizochytrium sp. LU310 at different feeding concentrations and timing. Closed
19
symbols and black bar charts are 0.4 g/L nitrogen feeding, and open symbols and green
20
bar charts are 0.8 g/L nitrogen feeding. (a) Feeding of 0.4 or 0.8 g/L nitrogen at 24 h; (b)
21
Feeding of 0.4 or 0.8 g/L nitrogen at 48 h; (c) Feeding of 0.4 or 0.8 g/L nitrogen at 72 h.
22 23
Fig.5. Time courses of cell growth and carbon consumption for Schizochytrium sp.
24
LU310 at different glucose feeding concentration. DCW (Square symbols), TLs 22
1
concentration (unfilled bar charts), DHA concentration (filled bar charts), glucose
2
concentration (triangle symbols), nitrogen concentration (circle symbols). (a) Feeding
3
glucose of 0 g/L; (b) Feeding glucose of 15 g/L; (c) Feeding glucose of 35 g/L.
4 5
Fig.6. Time courses of cell growth, nitrogen and carbon consumption for
6
Schizochytrium sp. LU310 in the optimal mode. Open symbols and black bar charts are
7
control, closed symbols and green bar charts are feeding.
8
23
Table 1.
1 2
Parameters of biomass, TLs and DHA production during fermentation of
Schizochytrium sp. LU310 in the normal and baffled flasks. Parameters
Culture modes 250 mL
1000 mL
250 mL
1000 mL
normal
normal
baffled
baffled
flasks DCW (g/L)
42.48±1. 12
TLs concentration (g/L)
(w/w, %) Biomass
70
9
97
3
71
71
43 17.15±1.
37
58
53
39.92±0.
53.0
16.33±0.
88.6
67.25±1.
23.1
27.51±1.
48.7
78 67.30±3.
18.50±0.
30.0
85 12.22±0.
54.64±1.
60.82±0. 95
34.47±1.
8.66±0.4
52.75±5.
DHA in DCW
13
ment (%)*
flasks
52.30±0.
26.10±1.
7.27±0.3
TLs in DCW (w/w, %)
46.80±0.
21.05±1.
DHA
flasks
97
38
concentration (g/L)
flasks
Incre
15 23.87±1.
30
21
0.44
0.48
1.11
1.21
152.1
60.58
73.64
162.76
194.38
164.0
productivity (g/L/h) DHA productivity (mg/L/h) 3
* The values were calculated at the cultivation time when DHA was maxima in 1000 mL flasks.
4
24
Table 2. Effect of feeding nitrogen on the cell growth and DHA content in
1 2
MSG-feeding culture Ori ginal
2 4h-1
2 4h-2
4 8h-1
4 8h-2
7 2h-1
7 2h-2
Incre ment (%)*
Nitrogen feeding
0
concentration (g/L)
0. 4
DCW (g/L)
50.8
8 5
0.7 TLs concentration (g/L)
30.7 3
DHA concentration (g/L) TLs in DCW (w/w, %)
58.2
DHA in DCW (w/w, %)
23.4 2
DHA productivity (mg/L/h)
3
6
191. 15
2 8.28
1
6
6
2 6.73
1
6
2
9.65
3.44
10.0
3
26.7
1
17.9
6
19.7
2
33.0
10.4
4.60 6
2
5
5.93
5.41
0.34
4.75
3
1
--
3.7
8.93
2.09
2.04
5
3
0. 8
5.9
1.13
3.02
5.59
5
3
1
0. 4
1.6
2.63
3.20
8.77
5
3
1
0. 8
2.6
2.40
4.34
0
4
3
13.0
0. 4
9.4
4.87
7
0.
6.91 3
1.16
7.19
2
1
1
1
2
2
06.29
99.60
95.29
90.15
11.00
05.00
*Compared the control with the best result in fed-batch culture.
4
26
1 2
Table 3. Effect of feeding carbon on the cell growth and DHA content in glucose-feeding culture Contro
48h-1
48h-2
l
Incre ment (%)*
Glucose feeding
0
15
35
--
DCW (g/L)
57.8
80.9
78.6
39.7
TLs concentration (g/L)
31.73
48.10
43.57
47.1
DHA concentration (g/L)
14.91
21.06
17.84
41.3
TLs in DCW (w/w, %)
55.78
66.24
50.82
18.8
DHA in DCW (w/w, %)
25.79
28.77
23.47
11.6
DHA productivity (mg/L/h)
207.02
232.36
194.30
12.2
concentration (g/L)
3
*Compared control with the best result in fed-batch culture.
4 5
27
1 2
Table 4. Biomass, DHA yield and DHA productivities in Schizochytrium sp. from the literatures and this study. Strain in
Device
DC W (g/L)
Schizochytriu
D HA
m sp.
(g
SR21
Shake-fl ask
S31
0 Shake-fl
ask
Strain 12B ask G13/2S ask
ge in
117
8
3
125
01
9
Perveen et al., 2006
6.
37.
Unagul et al., 2007
6.
63.
Two-sta
Limacinum
63
0
9
Wu et al., 2005
6.
30.
Shake-fl
3.42
328
0
Yokochi et al., 1998
0.
28.
Shake-fl
35
2
59
or
(mg/L/ h)
4.
7.4
Bioreact
mangrovei
SR21
36.
Reference
productivity
/L)
Sk-02
DHA
Ganuza et al., 2008
6.
43.7
56
Chi et al., 2009
shake-flask HX-308 (CCTCC
Stepwis e
71. 0
17 .50
28
119
Ren et al., 2010
M209059)
aeration in flask
HX-308 M
Shakeflask after
45. 44
5.
56.22 %a
2
Lian et al., 2010
mutagenesis Bioreact
A. limacinum SR21 (ATCC
or
61. 8
20
123
.3
Huang et al., 2012
MYA-1381) ATCC 20888
AEMR/s hake
LU310
2
a
00 Shake-fl
ask 1
12.
2.
21.26
04 88.
6
Zhang et al., 2013
24
241.5
This study
.74
DHA productivity is not available in the paper and DHA percentage in the lipids
used instead.
3 4 5 6
29
1 2 3 4
Fig.1
5 6 7 8 9 10 11
30
a
b 70
1000-mL bafferled flasks 250-mLbafferled flasks 1000-mLnoral flasks 250-mLnoral flasks
120
1000-mL bafferled flasks 250-mLbafferled flasks 1000-mLnoral flasks 250-mLnoral flasks
60
100
80
DCW (g/L)
Glucose (g/L)
50
60
40 30
40
20
20
10 0
0 0
24
48
72
96
0
120
24
48
72
96
120
Time (h)
Time (h)
d 20
c 40
30
DHA (g/L)
Total lipids (g/L)
16
20
10
8
4 1000-mL bafferled flasks 250-mLbafferled flasks 1000-mLnoral flasks 250-mLnoral flasks
0 0
24
48
72
96
e 1.6
0
1.2 1.0 .8 .6 .4 .2 0.0 0
24
48
72
96
24
48
72
Time (h)
1000-mL bafferled flasks 250-mLbafferled flasks 1000-mLnoral flasks 250-mLnoral flasks
1.4
1000-mL bafferled flasks 250-mLbafferled flasks 1000-mLnoral flasks 250-mLnoral flasks
0
120
Time (h)
Nitrogen (g/L)
12
120
Time (h)
31
96
120
1
Fig. 2
2 3
Fatty acid composition (% of each FA per TLs)
100
Others DHA DPA C18:0 C16:0 C14:0
80
60
40
20
0
250 mL 1000 mL 250 mL 1000 mL normal flasks normal flasks baffled flasks baffled flasks
Culture methods
4 5 6
Fig. 3.
7 8
32
1
a 60
50
120 1.4 100
1.2
80
1.0
30
60
20
.8 .6
40 .4
10
20
0
.2
0 0
24
48
72
96
0.0
120
Time (h)
b
60
1.6
DCW Glucose TLs DHA Nitrogen
50
120
1.4 1.2
40 80
30 60
20
Glucose (g/L)
DCW (g/L) TLs and DHA (g/L)
100
1.0 .8 .6
40
.4 10
20
0
0
0
3
24
48
72
96
Time (h)
33
120
.2 0.0
Nitrogen (g/L)
2
Nitrogen(g/L)
40
Glucose (g/L)
DCW (g/L) TLs and DHA (g/L)
1.6
DCW Glucose TLs DHA Nitrogen
c 60
50
120 1.4 100
1.2
80
1.0
30
60
20
.8 .6
40 .4
10
20
0
0 0
24
48
72
96
Time (h)
1 2 3
Fig. 4
4
34
120
.2 0.0
Nitrogen (g/L)
40
Glucose (g/L)
DCW (g/L) TLs and DHA (g/L)
1.6
DCW Glucose TLs DHA Nitrogen
1 2
35
1 2
36
1 2
Fig. 5
3 4
37
100 90 80
120 1.4 100
1.2
80
60 50
60
40
1.0 .8 .6
40
30
.4 20 20 10 0
0 0
24
48
72
96
120
144
Time (h)
1 2 3
Fig. 6
4 5 6 7 8 9
38
168
192
.2 0.0
Nitrogen (g/L)
70
Glucose (g/L)
DCW (g/L) TLs and DHA (g/L)
1.6
DCW TLs DHA Glucose Nitrogen
Highlights
1 2 3 4
1.
Schizochytrium sp. LU 310 is an ideal stain for DHA-producing as its
impressive productivity.
5 6
2.
Baffled flask enhanced in high cell density cultivation of DHA.
3.
MSG-feeding combined with glucose feeding strategy was extremely useful
7 8 9
for DHA production.
10 11 12
4. DHA production was linearly correlated with the concentrations of carbon and nitrogen.
13 14
39
