Regulation of lipid accumulation using nitrogen for microalgae lipid production in Schizochytrium sp. ABC101

Journal Pre-proof Regulation of lipid accumulation using nitrogen for microalgae lipid production in Schizochytrium sp. ABC101 Jung-Hyun Ju, Dong-Jin Ko, Sun-Yeon Heo, Jong-Jea Lee, Young-Min Kim, BongSoo Lee, Min-Soo Kim, Chul-Ho Kim, Jeong-Woo Seo, Beak-Rock Oh PII:

S0960-1481(20)30233-0

DOI:

https://doi.org/10.1016/j.renene.2020.02.047

Reference:

RENE 13068

To appear in:

Renewable Energy

Received Date: 16 May 2019 Revised Date:

31 December 2019

Accepted Date: 11 February 2020

Please cite this article as: Ju J-H, Ko D-J, Heo S-Y, Lee J-J, Kim Y-M, Lee B-S, Kim M-S, Kim C-H, Seo J-W, Oh B-R, Regulation of lipid accumulation using nitrogen for microalgae lipid production in Schizochytrium sp. ABC101, Renewable Energy (2020), doi: https://doi.org/10.1016/ j.renene.2020.02.047. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.

Credit Author Statement: Jung-Hyun Ju: Writing-Original draft preparation, Data Curation. Dong-Jin Ko: Investigation, Data Curation. Sun-Yeon Heo: Investigation, Validation. Jong-Jea Lee: Resources. Young-Min Kim: Validation, Visualization. Bong-Soo Lee: Writing - Review & Editing. Min-Soo Kim Writing - Review & Editing. Chul-Ho Kim: Conceptualization, Project administration. Jeong-Woo Seo: Conceptualization, Funding acquisition. Beak-Rock Oh: Supervision, Writing- Reviewing and Editing

1

Regulation of lipid accumulation using nitrogen for microalgae lipid production in

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Schizochytrium sp. ABC101

3 4

Jung-Hyun Ju a, b, Dong-Jin Ko a, Sun-Yeon Heo a, Jong-Jea Lee a, Young-Min Kim b, Bong-

5

Soo Lee c, Min-Soo Kim a, Chul-Ho Kim a, Jeong-Woo Seo a, Beak-Rock Oh a, *

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a

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Institute of Bioscience and Biotechnology (KRIBB), Korea

9

b

Microbial Biotechnology Research Center, Jeonbuk Branch Institute, Korea Research

Department of Food Science & Technology and Functional Food Research Center,

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Chonnam National University, Korea

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c

Deportment of Microbial & Nano Materials, Mokwon University, Korea

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* Corresponding author. E-mail address: [email protected] (B.R. Oh).

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1

ABSTRACT

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Lipid-rich microalgae are a potential source for the bio-oil production. We isolated a novel

3

oleaginous Thraustochytrid microalga from seawater, Schizochytrium sp. ABC101, and tested

4

the effect of different growth conditions on its production of lipids. Batch fermentation

5

experiments indicated that depletion of nitrogen led to lipid accumulation, and lipid

6

accumulation led to increased cell size and decreased cell wall thickness. Based on these

7

results, we tested three types of fed-batch fermentations (carbon and no nitrogen feeding,

8

carbon and limited nitrogen feeding, carbon and sufficient nitrogen feeding) to examine the

9

effect of adjusting the nitrogen supply on lipid accumulation. The limited nitrogen feeding

10

provided the most efficient lipid production, with maxima after 60 h (dry cell weight [DCW]:

11

64.7 g/L, lipids: 25.4 g/L, docosahexaenoic acid [DHA]: 11.0 g/L). Use of corn steep liquor

12

(CSL) as the sole nitrogen source also led to excellent performance, with maxima at 84 h

13

(DCW: 86.0 g/L, lipids: 37.2 g/L, DHA: 16.7 g/L). In addition, use of CSL rather than yeast

14

extract reduced the lipid production cost by 50%. Our results suggest that regulation of the

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nitrogen source improves the efficiency of microalgal lipid production and use of a low-cost

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nitrogen source reduces production costs.

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Keywords:

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Microalgal bio-oil; Biodiesel; Docosahexaenoic acid; Nitrogen regulation; Lipid regulation;

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Corn steep liquor

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1. Introduction

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The demand for fossil fuels has increased because of economic development and

3

population growth, and this has led to problems such as the exhaustion of traditional fuel

4

supplies and pollution [1, 2]. Environmentally sustainable alternative energy sources are

5

needed to resolve these problems. Biodiesel is an environmentally friendly fuel that can be

6

produced sustainably [3] from fish oil [4] and from crops such as soybean [5] and corn [6].

7

However, there are economic and ethical arguments against using food crops for the

8

production of biodiesel.

9

Microalgae are regarded as a more promising feedstock for biodiesel production than

10

food crops because they can grow at a high rate, are easily harvested, and can be cultivated

11

under various conditions [7,8]. Many studies have reported the production of biodiesel from

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microalgal oils [9-11]. Especially, certain Thraustochytrid strains have great promise as

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alternative feedstocks for biodiesel production because they can accumulate more than 60%

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of dry cell weight (DCW) as lipids [12,13]. The resulting microalgal lipids are mainly

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docosahexaenoic acid (DHA) and palmitic acid (PA) [9-14]. DHA is an omega-3

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polyunsaturated fatty acid (PUFA) that is used as a biosynthetic material for signaling

17

substances that induce termination of inflammatory responses in humans [15]. PA is C16

18

saturated fatty acid (SFA) that is used as an emollient and diluent in the cosmetics industry

19

[16]. Microalgal lipids can be produced safely and stably using pure cultures, and the

20

resulting products have potential used in high-value-added industries, such as medicine and

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cosmetics, because of price competitiveness. In addition, microalgae can use waste, such as

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crude glycerol [17,18] as a carbon source and algal-residue [19,20] as a nitrogen source, and

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reduce the levels of these pollutants. 3

1

The industrialization of bio-oil production from lipids produced by heterotrophic

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microalgae has been limited because costs still exceed those of oil production from fish [20].

3

This problem can be resolved by use of more efficient fermentation processes [21] and low-

4

cost industrial substrates [20]. Fermentation of high cell densities of Thraustochytrids is

5

essential for the economic production of microalgal bio-oil, and the carbon-to-nitrogen ratio

6

is a key regulatory factor [22-24]. Organic nitrogen sources are especially important for the

7

growth of heterotrophic microalgae [25,26]. Regulation of the nitrogen source during the

8

fermentation of heterotrophic microalgae can control lipid accumulation and cell growth.

9

When carbon and nitrogen are both abundant, cells use both nutrients, but when nitrogen is

10

deficient, cells only use the carbon for lipid accumulation [27]. Also, the accumulation of

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lipids can lead to changes in cell morphology. Some studies have examined the effect of lipid

12

accumulation on the cell walls of microalgae [28]. However, no studies have yet examined

13

the morphological changes of the cell wall of Thraustochytrid microalgae during growth and

14

lipid accumulation. Generally, cell walls protect against external stress, and cell lysis is more

15

likely in cells with thin walls [29]. The lysis of heterotrophic microalgae due to external

16

stress or autolysis reduces cell density and recovery from fermentation. Therefore, it is

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necessary to develop fermentation processes that consider the morphological changes of

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heterotrophic microalgae for more efficient lipid production.

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Yeast extract is a common industrial source of nitrogen that is widely used to lipid

20

production by Schizochytrium sp. [30-31]. Recent studies have investigated other nitrogen

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sources to reduce costs, such as algal residue [16,17], soybean meal hydrolysate [32], orange

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peel extract [33], and corn steep liquor (CSL) [34-36]. CSL, a by-product from wet corn

23

milling, is an especially promising alternative nitrogen source. CSL is rich in nitrogen, and 4

1

also contains a variety of other nutrients, such as minerals, vitamins, reducing sugars, and

2

lactic acid [36]. Therefore, CSL is a low-cost material that that is well suited for production

3

of lipids by heterotrophic microalgae. Although some studies have reported the use of CSL

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for lipid production by heterotrophic microalgae [34-36], there is little known about the

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effects of the different CSL components on microalgal lipid production.

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In this study, we examined the morphological changes and effects of nitrogen

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addition during the growth and lipid accumulation of a newly isolated Thraustochytrid strain

8

as a first step toward development of a system for microalgal lipid production by fed-batch

9

fermentation. Based on initial batch fermentation results, we examined the effects of three

10

types of fed-batch fermentation on the regulation of cell growth and lipid accumulation by

11

using different nitrogen supplementation strategies. In addition, we evaluated the effect of

12

CSL as a low-cost nitrogen source for production of lipids by microalgae.

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2. Materials and methods

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2.1. Isolation of Schizochytrium sp. ABC101

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Individual Schizochytrium sp. ABC101 cells were isolated by adding 9 mL of 0.1 M

17

PBS buffer (pH 7.0) to 1 mL of a seawater sample, followed by serial dilution, and then

18

spreading on agar plates with B1 medium (1 g/L yeast extract, 1 g/L peptone, 300 mg/L

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penicillin G, 500 mg/L streptomycin sulfate, 15 g/L agar, and seawater after passage through

20

a 0.2 µm filter) [37]. Single colony isolation was achieved by performing 3 subcultures, and

21

each isolated single colony was incubated at 28°C for 48 h in basal medium (20 g/L glucose,

22

10 g/L yeast extract, 9 g/L KH2PO4, 10 g/L sea salt, and 10 mg/L tetracycline). Each isolated 5

1

strain was stored in a 20% glycerol stock solution at –80°C.

2 3 4

2.2. Identification of Schizochytrium sp. ABC101 by 18S rRNA amplification Genomic DNA of Schizochytrium sp. ABC101 was isolated using a Genomic DNA

5

Purification Kit (Invitrogen, USA). The 18S rRNA gene was amplified by PCR using two

6

universal primers: 18-F (5′-ATGAACATCAAAAA-3′) and 18-R (5′-ATGAACATCAAAAA-

7

3′). The PCR protocol was: initial denaturation at 95°C for 5 min; 30 cycles of 95°C for 30 s,

8

55°C for 30 s, and 72°C for 90 s; and a final step at 72°C for 7 min. The sequence of the

9

amplified PCR fragment was confirmed by Solutions for Generic Technologies (SolGent,

10

Korea), and was compared with available 18S rRNA gene sequences from GenBank

11

(www.ncbi.nlm.nih.gov/blast) and EzTaxon (eztaxon-e.ezbiocloud.net) [38]. Phylogenetic

12

analysis was conducted using MEGA6 [39] with the neighbor-joining method [40]. Bootstrap

13

values were computed from 1000 replicates [41].

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2.3. Fermentation conditions Cultures were grown in 500 mL baffled flasks that contained 100 mL of modified

17

basal medium (60 g/L glucose, 10 g/L yeast extract, 9 g/L KH2PO4, 35 g/L sea salt, 10 mg/L

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tetracycline, and 1 mL/L vitamin stock solution [9.5 g/L thiamin, 0.2 g/L biotin and 1 g/L

19

cyanocobalamin]). Seed cells were cultured in 2 L baffled flasks that contained 600 mL of

20

modified basal medium for batch fermentation and fed-batch fermentation. Flasks were

21

incubated at 28°C for 24 h, and cultures (20% v/v) were subsequently inoculated into the 6

1

fermentor. Batch fermentation and fed-batch fermentation was performed using 3 L modified

2

basal medium at 28°C, initial pH 6.0, agitation speed 200 rpm, and aeration rate 1 vvm in a 5

3

L stirred-vessel system (CNS, Korea) that had a microbubble sparger (Aerowaters, Korea). A

4

solution of 900 g/L glucose, 300 g/L yeast extract, and 750 g/L CSL was used for intermittent

5

feeding. The glucose concentration was maintained at 10 to 40 g/L during fed-batch

6

fermentation. All presented results are averages of three independent experiments.

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2.4. Measurement of dry cell weight

9

Dry cell weight (DCW) was determined by freeze-drying fresh samples. The culture

10

broth (10 mL in a weighed 50 mL centrifuge tube) was centrifuged at 3500 rpm for 20 min at

11

4°C, the supernatant was removed by aspiration, and the cells were washed 2 times with 0.1

12

M sodium phosphate buffer (pH 7.0). The sample was frozen for 3 h, and lyophilized for 24 h,

13

and DCW was then measured.

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2.5. Measurement of total lipid Total lipid content was measured as described previously [42]. Briefly, 3 mL

17

chloroform, 6 mL methanol, and 3 mL 50 mM K2HPO4 buffer (pH 7.4) were added to dried

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cells from 10 mL culture broth. Samples were incubated at 28°C with agitation (200 rpm) for

19

1 h, after which 3 mL chloroform and 3 mL phosphate buffer were added to each tube. Each

20

tube was inverted 20 times, the samples were centrifuged at 3500 rpm for 10 minutes for

21

phase separation. Successively, the bottom layer (3 mL) was recovered and transferred to a

22

pre-weighed aluminum dish, and the solvent was evaporated for more than 30 min in a 90°C 7

1

drying oven. After cooling, the dish and contents were weighed, and total lipid was calculated

2

(to yield the weight of lipid extracted) using the following equation:

3

Total lipid (g/L) = (WL – WD) × VC / VP × 100,

4

where WD is the weight of an empty aluminum dish (g), WL is the weight of an aluminum

5

dish with dried lipid residue (g), VC is the total volume of chloroform in the sample (mL),

6

and VP is the volume of chloroform transferred to the aluminum dish (mL).

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2.6. Measurement of fatty acid composition

9

The fatty acid composition was analyzed by gas chromatography (GC). The obtained

10

dried cells were placed in screw-cap test tubes, was transferred to a glass vial, after which

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3 mL of 4% (v/v) methanolic sulfuric acid was added and the sample was heated at 90°C for

12

1 h. After allowing samples to cool at room temperature. 1 mL distilled water was added and

13

samples were mixed by briefly vortexing. Fatty acid methyl esters were extracted into 0.3 ~ 1

14

mL of n-hexane and analyzed by an Agilent 6890N Network Gas Chromatography (Agilent

15

Technologies, USA) equipped with a flame ionization detector and an HP-5 column (30 m ×

16

0.32 mm × 0.25 mm; Agilent Technologies, USA). After holding at 150°C for 2 min, the

17

column temperature was raised to 270°C at a rate of 7°C per min, and held at 270°C for 2

18

min.

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2.7. Measurement of glucose, lactic acid, total nitrogen and total phosphorus The concentration of residual glucose and lactic acid were determined using a high 8

1

performance liquid chromatography (HPLC) system (1200 series, Agilent, USA) that had a

2

refractive index detector (RID) and an ion-exchange column (300 × 78 mm; Aminex HPX-

3

87H; Bio-Rad, USA). The mobile phase was 2.5 mM H2SO4, the flow rate was 0.6 mL/min,

4

the column temperature was 65°C, and the RID was maintained at 45°C [43]. Total nitrogen

5

and total phosphorus concentrations were measured using a HS-TN (CA)-L kit (concentration

6

range: 1 to 50 mg/L) and a HS-TP-H kit (concentration range: 1 to 15 mg/L) (Humas, Korea)

7

with a HS-2300 plus water analyzer (Humas, Korea).

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3. Results and discussion 3.1. Isolation and identification of Schizochytrium sp. ABC101 We collected seawater samples from the coral reef area near Jeju Island in Korea, and

12

isolated and identified a strain of Schizochytrium sp. based on NCBI blast results of the 18S

13

rRNA gene sequence. The resulting neighbor-joining tree (Supplementary Fig. 1) shows that

14

Schizochytrium sp. ABC101 clustered with all other strains of Schizochytrium, and was in a

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tight cluster (high bootstrap value) with Schizochytrium limacinum OUC166 (HM042907).

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3.2. Metabolic and morphological changes during batch fermentation We initially examined the fermentation characteristics of Schizochytrium sp. ABC101

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by performing batch fermentation in a 5 L fermentor (Fig. 1). The increase of DCW and

20

glucose consumption had similar time courses. In particular, the cells had exponential growth

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from 6 h to 21 h, and the consumption of glucose increased rapidly from 6 h and was

22

completely exhausted at 21 h. At that time, the DCW was 33.5 g/L and the DCW productivity 9

1

was 1.6 g/L·h. Lipid accumulation had a different time course, in that it began rapidly at 12 h

2

and continued to 21 h. The` cells consumed most of the nitrogen during the initial 12 h, and

3

the level then remained below 100 mg/L (Fig. 1b). At 21 h, the lipid yield was 12.7 g/L and

4

the productivity was 1.4 g/L·h. After depletion of nitrogen (12 h to21 h), glucose

5

consumption was 50.9 g/L, the lipid level was 11.8 g/L, and pure DCW (excluding lipids)

6

was 4.8 g/L. At that time, the conversion yield of lipid was 0.23 g/g glucose and that of pure

7

DCW was 0.09 g/g glucose. These results indicate that the cells used most of the glucose for

8

lipid production after depletion of the nitrogen source. Nitrogen depletion can induce the

9

formation of acetyl-CoA and NADPH, which are used for lipid biosynthesis [44]. In addition,

10

a decrease in adenosine monophosphate (AMP)-dependent isocitrate dehydrogenase (ICDH)

11

activity by deamination of AMP due to nitrogen consumption can also promote lipid

12

accumulation [45]. These results thus confirmed that cells used glucose for growth when

13

nitrogen was abundant, but used glucose to produce lipids after nitrogen depletion. The DHA

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content increased up to 12 h, and then remained relatively constant at 40 to 42%.

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We also analyzed cell morphology at 9 h (cell growth stage, when the lipid content

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was 4.7% w/w) and 21 h (lipid accumulation stage, when the lipid content was 39.9% w/w)

17

by using transmission electron microscopy (TEM; Fig. 2). During the cell growth stage, cells

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diameter ranged from about 7 to 12 µm, lipids were hardly visible, and the cell walls were

19

approximately 120 to 130 nm thick. During the lipid accumulation stage, cell diameter ranged

20

from about 22 to 24 µm, there was a notable accumulation of lipids, and the cell walls were

21

approximately 50 to 70 nm thick. Thus, the accumulation of lipids was associated with

22

increased cell size and decreased cell wall thickness. Cells with thin walls due to lipid

23

accumulation are easily lysed by external stress, and this limits further lipid production. 10

1

Phototrophic microalgae such as Nannochloropsis sp. Chlorella sp. and Chlorococcum sp.

2

have been reported to increase cell wall thickness under nitrogen source deprivation

3

conditions [46]. However, to the best of our knowledge, there have been no reports of

4

changes in cell wall thickness due to lipid accumulation in heterotrophic microalgae.

5

As a result, sufficient nitrogen is required to support microalgal growth and to

6

prevent cell lysis during lipid accumulation, but nitrogen deficiency is required for

7

stimulation of lipid accumulation. Therefore, regulation of the nitrogen supply during each

8

production step may provide a more efficient production of microalgal lipids.

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3.3 Lipid accumulation using nitrogen regulation with fed-batch fermentation Fed-batch fermentation is a method that regulates the consumption of substrates, and

12

enables the more efficient production of high concentrations of products [47,48]. We

13

examined the effects of three types of fed-batch fermentations that used nitrogen to regulate

14

lipid accumulation: carbon feeding and no nitrogen feeding, carbon feeding and limited

15

nitrogen feeding, and carbon feeding and sufficient nitrogen feeding.

16

Fig. 3a and Fig. 3b show the results of the fed-batch fermentation with carbon

17

feeding and no nitrogen feeding. At 42 h, the maximal level of DCW was 44.2 g/L, lipid level

18

was 18.1 g/L, DHA was 7.5 g/L, and pure DCW was 26.1 g/L. At that time, the lipid content

19

was 41.0% and the DHA content was 41.5%. The cells had consumed most of the nitrogen by

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12 h, and the rapid accumulation of lipids began at about that time. The rate of glucose

21

consumption was remarkably decreased, and the cells entered the stationary phase after 42 h

22

(rather than 24 h). Although the glucose supply was sufficient, there was no more cell growth 11

1

and lipid accumulation after 42 h because nitrogen was limited. This is because nitrogen is an

2

essential structural element for cells [25]. Similar results were reported for Aurantiochytrium

3

sp. KRS101 [49].

4

Fig. 3c and Fig. 3d show the results of the fed-batch fermentation with carbon

5

feeding and limited nitrogen feeding. In this case, the nitrogen was supplied 2 times at 6 h

6

intervals beginning at 9 h (2000 mg/L total nitrogen). At 60 h, the maximum DCW was 64.7

7

g/L, lipid level was 25.4 g/L, DHA was 11.0 g/L, and pure DCW was 39.3 g/L. At that time,

8

the lipid content was 39.3% and the DHA content was 43.3%. The cells had consumed most

9

of the nitrogen by 24 h, and the rapid accumulation of lipids began at about that time. The

10

rate of glucose consumption was remarkably lower at 60 h. Interestingly, relative to fed-batch

11

fermentation with carbon feeding and no nitrogen feeding, the pure DCW was 1.5-fold

12

greater and the lipid content was 1.4-fold greater. When additional nitrogen was added with

13

limited feeding, there was a delay of lipid accumulation, and pure DCW increased until all of

14

the nitrogen was depleted. During the delay of lipid accumulation, glucose use was associated

15

with an increase of pure DCW, and this increase was ultimately responsible for the greater

16

lipid production. After the depletion of nitrogen, most of the remaining glucose was used for

17

lipid production. The amount of lipid accumulation was 16.8 g/L after nitrogen deficiency (24

18

h to 60 h), and most lipids (66.2% of maximum lipid yield) had accumulated at that time.

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Fig. 3e and Fig. 3f show the results of fed-batch fermentation with carbon feeding

20

and sufficient nitrogen feeding. In these experiments, the nitrogen was supplied 4 times at 6 h

21

intervals beginning at 12 h (4000 mg/L total nitrogen). At 60 h, the maximum DCW was 68.5

22

g/L, lipid level was 26.5 g/L, DHA was 11.6 g/L, and pure DCW was 42.0 g/L. At that time,

23

the lipid content was 38.7% and the DHA content was 43.9%. Except for the nitrogen source, 12

1

the remaining results were similar to those from fed-batch fermentation with carbon feeding

2

and limited nitrogen feeding. The rate of nitrogen consumption decreased rapidly after 24 h,

3

and remained at about 2000 mg/L, and the rapid accumulation of lipids began at about that

4

time. Although sufficient nitrogen was available, cell growth no longer occurred. Also, the

5

amounts of DCW, lipid, and DHA were not significantly different from cells that received

6

fed-batch fermentation with carbon feeding and limited nitrogen feeding, although the

7

amount of nitrogen feeding was doubled. The similarity of these results is probably because

8

other important constituents of microalgae, such as phosphate and trace elements, must also

9

be supplied for cell growth [50]. Because we did not supply these other nutrients, additional

10

cell growth and lipid accumulation did not occur.

11

The maximum lipid content for the three types of fed-batch fermentations were all

12

about 38 to 40%, regardless of nitrogen feeding. These results imply there is a limit to lipid

13

accumulation by Schizochytrium sp. ABC101 cells. From these results, we conclude that

14

increasing DCW is essential for producing a large amount of lipids. The DCW and pure

15

DCW will increase if lipid accumulation is delayed by feeding the cells an adequate amount

16

of nitrogen at the initial stage of growth. In other words, it is important to regulate the amount

17

of nitrogen during the initial stages of growth for efficient lipid production. Thus, we

18

regulated lipid accumulation with carbon feeding and limited nitrogen feeding in subsequent

19

fed-batch fermentation experiments.

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3.4 Effect of alternative low cost nitrogen source for lipid production Initial cell growth and lipid accumulation were efficient when using fed-batch fermentation with carbon feeding and limited nitrogen feeding. However, yeast extract is an 13

1

expensive nitrogen source when used for the industrial production of bio-oil. Table 1 shows

2

the prices of substrates to produce 1 kg of lipid based on our fed-batch fermentation results

3

with carbon feeding and limited nitrogen feeding. When yeast extract was used as a nitrogen

4

source, it accounted for 57.5% of the total cost of substrates. Comparison of yeast extract and

5

CSL as nitrogen sources indicated that yeast extract contained 10.3% total nitrogen and CSL

6

contained 4.0% total nitrogen (Table 2). Therefore, for the efficient and economical

7

production of microalgal lipids, we used 25 g/L CSL (1000 mg/L total nitrogen) during fed-

8

batch fermentation instead of 10 g/L yeast extract (1030 mg/L total nitrogen).

9

Table 3 shows the effect of using CSL for batch fermentation of Schizochytrium sp.

10

ABC101 at 21 h. Interestingly, when CSL was the sole nitrogen source, the fermentation

11

results were better than when yeast extract was used. This may be because CSL also provides

12

additional vitamins and minerals that enhance growth and the production of lipids and DHA.

13

Similar results were reported for Aurantiochytrium limacinum SR21 [34].

14

The tricarboxylic acid (TCA) cycle and its intermediates (organic acids) are needed

15

for fatty acid biosynthesis [44,45]. Because our CSL contained 7.2% lactic acid, we examined

16

the effect of adding lactic acid at the same concentration (1.8 g/L) with yeast extract. This

17

modified basal medium led to slightly less growth and total lipids, but a similar amount of

18

DHA (Table 3). However, even in this modified medium, the lipid and DHA levels were

19

lower than in the CSL medium. A previous study reported that the addition of 4 g/L lactic

20

acid to the growth medium increased the DHA content and yield of Schizochytrium

21

limacinum SR21 [51]. In the present study, lactic acid slightly increased the DHA content, but

22

had no significant effect on DHA yield. Because the lactic acid concentration was only 1.8

23

g/L in these experiments, it might be necessary to add more lactic acid to improve 14

1

fermentation and lipid production. In addition, used CSL contained 0.6% phosphorus, and the

2

additional amount of phosphorus added using 25 g/L CSL instead of 10 g/L yeast extract was

3

0.1 g/L. It is reported that phosphorus deficiency promotes DHA synthesis, while increasing

4

phosphorus concentration is inhibiting DHA production [52]. However, the amount used in

5

the fed-batch fermentation was so small that it had no significant effect.

6

Fig. 4 shows the results of fed-batch fermentation using CSL instead of yeast extract.

7

At 84 h, the maximum DCW was 86.0 g/L, the lipid level was 37.2 g/L, and DHA was 16.7

8

g/L. At that time, the lipid content was 43.3% and the DHA content was 44.8%

9

(Supplementary Table 1). Although use of CSL increased fermentation time, the results were

10

better than from yeast extract. Especially, DHA yield increased by increase of DHA content.

11

Feeding with CSL gradually increased the lactic acid level to 5.4 g/L, and this was probably

12

responsible for the increased DHA content. Previous research suggested that addition of

13

organic acids later during fermentation (during lipid accumulation) increased the overall

14

DHA level [51]. There was used 2.5-fold more CSL than yeast extract, but CSL was 31-fold

15

cheaper, thus reducing cost of the nitrogen source by 12.1-fold (Table 1). Notably, the total

16

price of substrates needed to produce 1 kg of lipids was more than 50% lower when using

17

CSL (Table 1).

18 19

4. Conclusions

20

We isolated a novel Thraustochytrid microalga, Schizochytrium sp. ABC101, and

21

examined the effect of different nutrients on lipid accumulation during batch fermentation

22

and fed-batch fermentation in 5 L fermentors. Our analysis of the effect of three nitrogen

23

feeding strategies during fed-batch fermentation indicated that the lipid yield increased by 15

1

1.4-fold with nitrogen feeding (relative to no nitrogen feeding). When CSL (rather than yeast

2

extract) was used as the sole nitrogen source, the lipid yield increased by 2.1-fold.

3

Supplementation of yeast extract with lactic acid improved DHA content of lipids. Growth of

4

these microalgae using CSL led to efficient production of lipids, and reduced the total

5

substrate cost by about 50%. Our results indicate that fed-batch fermentation of

6

Schizochytrium sp. ABC101 using CSL is an efficient and economical system for the

7

production of microalgal feedstock for bio-oil production.

8 9 10

Acknowledgements This work was supported by the by the Advanced Biomass R&D Center (ABC) of

11

Global Frontier Project funded by the Ministry of Science, ICT and Future Planning and the

12

Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative

13

Program.

14 15 16

Conflict of interest The authors declare that there is no conflict of interest.

17

18 19

Statement of informed consent, human/animal rights No conflicts, informed consent, human or animal rights applicable.

20

16

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17

25

1 2

Figure legends

3

Fig. 1. Time courses of changes in (a) DCW (g/L, gray bars), total lipids (g/L, dotted bars),

4

DHA (%, closed circles), (b) glucose consumption (g/L, closed squares), and residual total

5

nitrogen (mg/L, open circles) during batch fermentation of Schizochytrium sp. ABC101.

6 7

Fig. 2. Transmission electron microphotographs of Schizochytrium sp. ABC101 during batch

8

fermentation at (a) 9 h and (b) 21 h.

9 10

Fig. 3. Time courses of changes in DCW (g/L, gray bars), total lipids (g/L, dotted bars), DHA

11

(%, closed circles), glucose consumption (g/L, closed squares), and residual total nitrogen

12

(mg/L, open circles) during fed-batch fermentation of Schizochytrium sp. ABC101 with (a, b)

13

carbon feeding but no nitrogen feeding, (c, d) carbon feeding and limited nitrogen feeding,

14

and (e, f) carbon feeding and sufficient nitrogen feeding.

15 16

Fig. 4. Time courses of changes in (a) DCW (g/L, gray bars), total lipids (g/L, dotted bars),

17

DHA (%, closed circles), (b) glucose consumption (g/L, closed squares), and residual total

18

nitrogen (mg/L, open circles) during fed-batch fermentation of Schizochytrium sp. ABC101

19

with carbon feeding and limited CSL feeding.

20 21

Supplementary Fig. 1. Phylogenetic relationship of Schizochytrium sp. ABC101 with other 26

1

microalgae (from GenBank and EzTaxon) based on 18S rRNA sequences. The number at

2

each node indicates the percentage of support based on 1000 bootstrap-resamples of the

3

dataset. Bar: 0.02 substitutions per nucleotide.

4

27

1

Table 1. Amounts and prices of different nitrogen sources needed to produce 1 kg of lipids by

2

fed-batch fermentation of Schizochytrium sp. ABC101. Price Amount (kg) (USD/kg)

Price (USD)

Price (%)

Glucose

1.9

4.44

8.5

28.8

Yeast extract

25.5

0.67

17.0

57.5

Seasalt

3.5

0.78

2.8

6.8

KH2PO4

6.5

0.20

1.3

3.2

Nitrogen Substrate source

Yeast extract

Total price

CSL

29.6 Glucose

1.9

4.44

9.3

62.8

CSL

0.8

2.78

1.4

9.5

Seasalt

3.5

0.78

2.8

18.9

KH2PO4

6.5

0.20

1.3

8.8

Total price

14.8

3

28

1

2

Table 2. Chemical characteristics of yeast extract and CSL. Yeast extract

CSL

Total nitrogen (%)

10.3

4.0

pH

5.7

4.5

Lactic acid (%)

ND

7.2

Glucose (%)

ND

0.9

Phosphorus (%)

1.3

1.1

ND: Not detected

3

29

1

Table 3. Effect of nitrogen source during fed-batch fermentation on growth parameters of

2

Schizochytrium sp. ABC101.

Nitrogen source

Concentration (g/L)

DCW (g/L)

Lipid content (%)

Total Glucose DHA DHA lipid consumption (%) (g/L) (g/L) (g/L)

Yeast extract

10

33.5

38.0

12.7

41.1

5.8

58.5

Yeast extract (lactic acid)

10 (1.8)

31.4

38.2

12.0

43.2

5.8

56.6

CSL

25

35.2

40.3

14.2

42.5

6.2

61.2

3

30

(b)

60

Exponential phase

Stationary phase

1400

40 30 20

1200

60

1000 800

40

600 400

20

200

10

0

0

0 0

3

6

9

0

12 15 18 21 24 30 36 42 48

6

12

18

24

Time (h)

Time (h)

Fig. 1

30

36

42

48

Glucose consumption (g/L)

50

80

Lag phase

Total nitrogen (mg/L)

DCW (g/L), Total lipid (g/L), DHA (%)

(a)

(a) 7.26 µm 12.11 µm 124.32 nm 7.05 µm

8.63 µm

129.73 nm 11.16 µm

(b) 71.83 nm

45.07 nm

24.11 µm

Fig. 2

20

0 0

12

24

36

48

60

150 2000 1500

100

1000 50 500 0

0 36 Time (h)

48

12

24

60

72

36

48

60

2500 150 2000 1500

100

1000 50 500 0

0 24

36 Time (h)

Fig. 3

48

20

0 12

24

60

72

36

48

60

72

Time (h)

(f) 3000

200

12

40

0

Time (h)

0

60

72

3000 Total nitrogen (mg/L)

Total nitrogen (mg/L)

2500

24

0 0

200

12

20

(d)

3000

0

40

72

Time (h)

(b)

60

80

200

2500 150 2000 1500

100

1000 50 500 0

0 0

12

24

36 Time (h)

48

60

72

Glucose consumption (g/L)

40

DCW (g/L), Total lipid (g/L), DHA (%)

60

80

Total nitrogen (mg/L)

80

(e)

Glucose consumption (g/L)

DCW (g/L), Total lipid (g/L), DHA (%)

(c)

Glucose consumption (g/L)

DCW (g/L), Total lipid (g/L), DHA (%)

(a)

(a) 80

2000

60 40 20

200

150

1500 100 1000 50

500 0

0 0

12

24

36

48

60

72

0 0

84

12

24

36

48

Time (h)

Time (h)

Fig. 4

60

72

84

Glucose consumption (g/L)

2500

Total nitrogen (mg/L)

DCW (g/L), Total lipid (g/L), DHA (%)

(b) 100

Highlights

The accumulation of lipids was associated with decreased cell wall thickness in Schizochytrium sp. ABC101. Fed-batch fermentation produced 37.2 g/L of lipid with regulation of lipid accumulation using nitrogen. The use of CSL to replace yeast extract increase lipid production by 1.5-folds and reduce total lipid production costs by 50%.

Conflict of interest The authors declare that there is no conflict of interest.

Statement of informed consent, human/animal rights No conflicts, informed consent, human or animal rights applicable.