Nitrogen Feeding Strategies and Metabolomic Analysis To Alleviate

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Omics Technologies Applied to Agriculture and Food

Nitrogen feeding strategies and metabolomic analysis to alleviate high-nitrogen inhibition on docosahexaenoic acid production in Crypthecodinium cohnii Liangsen Liu, Fangzhong Wang, Ji Yang, Xingrui Li, Jinyu Cui, Jing Liu, Mengliang Shi, Kang Wang, Lei Chen, and Weiwen Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03634 • Publication Date (Web): 18 Sep 2018 Downloaded from http://pubs.acs.org on September 19, 2018

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Journal of Agricultural and Food Chemistry

Innovation Center of Chemical Science and Engineering (Tianjin), PR China Zhang, Weiwen; Laboratory of Synthetic Microbiology, School of Chemical Engineering & Technology, Tianjin University, Tianjin, PR China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), PR China; Center for Biosafety Research and Strategy, Tianjin University, Tianjin, P.R. China

ACS Paragon Plus Environment


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Nitrogen feeding strategies and metabolomic analysis to

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alleviate high-nitrogen inhibition on docosahexaenoic acid

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production in Crypthecodinium cohnii

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Liangsen Liu,†,‡,§ Fangzhong Wang,# Ji Yang,# Xingrui Li,†,‡,§ Jinyu Cui,†,‡,§ Jing

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Liu,†,‡,§ Mengliang Shi,†,‡,§ Kang Wang,†,‡,§ Lei Chen,†,‡,§ Weiwen Zhang*,†,‡,§,#

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†

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Technology, Tianjin University, Tianjin, PR China;

Laboratory of Synthetic Microbiology, School of Chemical Engineering &

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‡

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University, Tianjin, PR China;

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§

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and Engineering (Tianjin), PR China;

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#

Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin

SynBio Research Platform, Collaborative Innovation Center of Chemical Science

Center for Biosafety Research and Strategy, Tianjin University, Tianjin, P.R. China

15 16 17 18 19 20 21 22 23 24 25 26 27

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ABSTRACT: It is well known that high-nitrogen content inhibits cell growth and

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docosahexaenoic

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Crypthecodinium cohnii. In this study, two nitrogen feeding strategies, pulse-feeding

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and continuous-feeding, were evaluated to alleviate high-nitrogen inhibition effects on

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C. cohnii. The results showed that continuous-feeding with a medium solution

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containing 50% (w/v) yeast extract at 2.1 mL/h during 12-96 h was the optimal

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nitrogen feeding strategy for the fermentation process, when glucose concentration

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was maintained at 15-27 g/L during the same period. With the optimized strategy,

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71.2 g/L of dry cell weight and DHA productivity of 57.1 mg/L/h were achieved. In

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addition, metabolomic analysis was applied to determine the metabolic changes

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during different nitrogen feeding conditions, and the changes in amino acids,

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polysaccharides, purines and pentose phosphate pathway were observed, providing

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valuable metabolite-level information for exploring mechanism of high-nitrogen

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inhibition effect, and further improving DHA productivity in C. cohnii.

acid

(DHA)

biosynthesis

in

heterotrophic

microalgae

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KEYWORDS: Docosahexaenoic acid, high nitrogen inhibition, continuous-feeding

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nitrogen, metabolomic analysis, Crypthecodinium cohnii

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INTRODUCTION

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The ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) has been

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demonstrated beneficial to human health.1 Early studies have shown that DHA plays

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an important role in the development of the nervous and visual systems of infants, and

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in improving brain and vision function in adults.2-4 It also has positive effects in

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preventing cardiovascular, and asthma diseases.5-6 According to a previous report, the

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market value of ω-3 polyunsaturated fatty acid continues to increase and is expected

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to reach 4.3 billion dollars by 2019.7

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The traditional source of DHA is marine fish oil. However, fish oil has a fishy

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smell, and its sustainable supply has been challenged due to the reduction of fish

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resources worldwide.8 Heterotrophic fermentation such as using Schizochytrium sp.

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and Crypthecodinium cohnii to produce DHA has been considered as a sustainable,

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high-quality DHA alternative source.9-11 It has been reported that the lipid content of

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C. cohnii can reach more than 50% of its dry cell weight (DCW)12 and the proportion

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of DHA in total fatty acids (TFAs) can be rough 40-50%, respectively.13 In addition,

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the cost of subsequent separation and purification is one of the major challenges for

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producing DHA using microalga,1 which can be greatly reduced because other types

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of polyunsaturated fatty acids were less than 1% of the total lipid in C. cohnii.14

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Moreover, C. cohnii containing no eicosapentaenoic acid (EPA) which is undesirable

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in infant diets,8 is the first DHA-producing microalgae for infant formulas approved

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by the U.S. Food and Drug Administration.15

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Significant efforts have been carried out to improve DHA productivity in C. 3


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cohnii in recent years.11, 16 For example, studies found that the DCW was significantly

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improved using ethanol or acetic acid as carbon source by 199.6% and 293.5%

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compared that with using glucose as carbon source, respectively.11, 17-18 The highest

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DCW reported to date is 109 g/L by feeding acetic acid in a 400-h C. cohnii

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fermentation process.11 However, since acetic acid is easy to volatile and also

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corrosive to the equipment, it is not the best strategy for large-scale DHA

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production.19

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Besides carbon sources, nitrogen sources also play an important role in the

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growth of microorganism and metabolite accumulation. High-nitrogen inhibition is a

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well-known phenomenon in microorganisms.20-22 For example, Suzuki, et al.23 found

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that excessive nitrogen sources not only inhibited the growth but also reduced the

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accumulation of poly-β-hydroxybutyric acid (PHB) in Pseudomonas sp. K. The

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impact of high nitrogen concentration on growth and lipid content was also studied in

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microalgae. The DCW of Schizochytrium sp. S31 began to drop when nitrogen

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content exceeded 0.4% (w/v), and the lipid in biomass was reduced when nitrogen

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content exceeded 0.2% (w/v).21 High nitrogen increased DCW, but reduced lipid

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content, leading to an overall lower lipid productivity in Chlorella vulgaris.24 Studies

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have been previously carried out to solve or alleviate high nitrogen inhibition

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effect.24-26 PHB production was increased by using a constant feeding rate of nitrogen

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source or a constant feeding ratio of carbon/nitrogen (C/N) in Protomonas extorquens

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sp. K.25-26 A strategy of shifting high nitrogen to low nitrogen concentration

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throughout the fermentation phase was adopted to increase lipid productivity in

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Chlorella vulgaris.24 By adopting feeding nitrogen at a fixed amount of 0.4 g/L

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nitrogen, a recent study achieved high DCW with high DHA content of 88.6 g/L and

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24.74 g/L in Schizochytrium sp. LU310.27 For C. cohnii, high initial nitrogen

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concentration has been reported to decrease both final DCW and lipid content.16, 18

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But so far, no systematic analysis on high nitrogen inhibition effects and nitrogen

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feeding strategy has been carried out in C. cohnii.

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In the present study, to alleviate high-nitrogen inhibition effect, different

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nitrogen feeding strategies were examined in C. cohnii M-1-2, a mutant with high

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DHA productivity.28 After optimization, a DHA productivity of 57.1 mg/L/h was

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achieved, which is the highest reported so far for C. cohnii. In addition, metabolomic

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analysis that has been demonstrated as an effective way to analyze metabolic changes

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in C. cohnii,29-30 was used to analyze the possible mechanism of high nitrogen

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inhibition. The findings can be valuable for industrial DHA production in C. cohnii,

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and also provide useful reference for optimizing other high-value products from other

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microalgae.

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MATERIALS AND METHODS

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Strain and chemicals

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Crypthecodinium cohnii M-1-2 is a laboratory-maintained mutant strain, which

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was obtained by atmospheric and room-temperature plasma mutagenesis of C. cohnii

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ATCC 30556.28 Sea salt and antifoam SE-15 were obtained from Sigma-Aldrich (St

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Louis, USA). Yeast extract was obtained from OXOID (Basingstoke, UK). All the 5


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other chemicals were purchased from Jiang Tian Chemical Technology Co., Ltd.

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(Tianjin, China).

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Batch cultivation of different initial yeast extract concentration

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C. cohnii M-1-2 was maintained in liquid static culture containing 9 g/L glucose,

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25 g/L sea salt, and 2 g/L yeast extract at 25 °C in the dark. They were sub-cultivated

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every two weeks with 10% (v/v) inoculum size. The seed was inoculated into a

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500-mL shake flask with 100 mL of basal medium (9 g/L glucose, 25 g/L sea salt, and

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2 g/L yeast extract) and incubated at 25 °C and 180 rpm for 48 h. Then cultures were

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inoculated into 1000-mL flasks containing 200 mL of medium consisted of 27 g/L

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glucose, 25 g/L sea salt, and 6 g/L yeast extract. They were cultivated at 25 oC and

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shaken at 180 rpm for 84 h and then inoculated into a 5-L fermenter (Demei, China)

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containing 3 L of medium consisted of 81 g/L glucose, 25 g/L sea salt, and varying

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concentrations of yeast extract (3, 6 or 12 g/L). The inoculum size was 10% (v/v), and

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the pH was maintained at 6.5±0.1 by automated addition of 1 M H2SO4. The

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dissolved oxygen was automatically controlled above 30% of air saturation by

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adjustment of the agitation speed. The air flow level was 1 vvm. The 10% (w/v) of

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antifoam SE-15 was used for controlling foam.

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Fed-batch cultivation with different nitrogen feeding strategies

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The fermentation condition of fed-batch cultivation was roughly the same as that

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of batch cultivation with only minor modification. The initial medium was 27 g/L

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glucose, 25 g/L sea salt, and yeast extract 6 g/L. For the pulse-feeding nitrogen

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strategies, glucose with 80% (w/v) solution was fed into medium to maintain the

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glucose concentration in the medium between 15 and 27 g/L, and yeast extract with

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50% (w/v) solution was added simultaneously according to a constant C/N (w/w) ratio

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of feeding medium. The C/N (w/w) ratios were set at 6:1, 4:1 and 3:1. And a fed-batch

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cultivation was fed only with glucose as control. For the continuous-feeding nitrogen

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strategies, 50% (w/v) yeast extract solution was fed at a constant flow rate of 1.5, 2.1,

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or 2.7 mL/h between 12 and 96 h, and the 80% (w/v) glucose stock was fed into the

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fermenter to maintain the glucose concentration in the medium between 15 g/L and 27

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g/L. And a fed-batch cultivation was fed with 50% (w/v) yeast extract solution at a

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flow rate of 2.1 mL/h during 12-96 h and 132-156 h.

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Analysis of dry cell weight, glucose concentration and ammonium nitrogen

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concentration

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Three milliliters of fermentation broth were extracted and centrifuged at 13000 ×

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g, and 4 °C for 5 min. The sediment was washed twice with distilled water and then

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freeze-dried for 16 h. It was used for determination of dry cell weight by gravimetric

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method. The specific growth rate of the logarithmic period based on the equation µ =

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(lnX2 −lnX1)/(t2 − t1), where X represents the DCW at the time t.31

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The supernatant was extracted to measure glucose content according to a previous 7


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study.29 Since yeast extract is a mixture, ammonium nitrogen concentration, the

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determination method of which is well established and widely used for the

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measurements in the fermentation broth,32-33 was used to characterize nitrogen

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concentration during the fermentation process. Ammonium nitrogen concentration

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was detected following the methods described previously.32

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Analysis of total lipid and fatty acid composition

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The analysis of total lipid was conducted according to a previous study with a

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minor modification.34 Briefly, Twenty-five milligrams of freeze-dried algae powder

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was used to extract total lipid with chloroform:methanol (2:1, v/v) adding 0.01%

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butylated hydroxytoluene, and the extraction was repeated three times. The extract

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was washed once by 1 M KCl solution and ddH2O respectively. The total lipid was

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dried in a vacuum concentrator (ZLS-1, Hunan, China). It was used for determination

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of total lipid content by gravimetric method. The fatty acids were analyzed by an

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Agilent 5975 MSD/7890 instrument according to previous studies.12,

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twenty-five mg of lyophilized powder was added with 2 mL of chloroform, 2 mL of

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methanol of 3% (v/v) sulfuric acid, and 100 µL of 6 mg/mL heptadecanoic acid (an

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internal standard) methanol solution. The reaction was carried out at 100 °C for 4 h.

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After cooling, 1 mL of distilled water was used for stratification, and 1 µL chloroform

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phase was used to detect.

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GC–MS based metabolomic analysis and statistical analysis 8


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Briefly,


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Metabolomic analysis was carried out for continuous-feeding nitrogen strategies

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with 50% (w/v) yeast extract solution at a constant flow rate of 2.7 and 2.1 mL/h

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between 12 and 96 h. The cells were harvested at 36, 60, and 84 h of 2.7 mL/h feeding

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strategy, and were harvested at 36, 84, and 132 h of 2.1 mL/h feeding strategy

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respectively, corresponding to the early, middle and late exponential phases of the

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fermentation. Each sample has five biological replicates. The analytical method was

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based on a previous study.30 For statistical analysis, metabolomic data was

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standardized by the internal standard and cell numbers. Metabolomic data was first

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evaluated by principal component analysis (PCA), and then the volcano plot which is

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a scatter plot of negative log10-transformed p-values against the log2 fold change was

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used for comparative metabolomic analysis.36 Fold change ≥2.0 and p-values ≤0.05

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were used as a cutoff for significant changes of metabolites.

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RESULTS AND DISCUSSION

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Effects of initial nitrogen concentration on the biomass accumulation and DHA

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productivity in C. cohnii M-1-2

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It was reported that the initial nitrogen content has significant effects on growth

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and lipid accumulation in many microbes, such as Moritella marina MP-1 and

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Schizochytrium sp. S31.21, 37 Therefore, the effects of initial nitrogen concentration on

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the growth and DHA productivity of C. cohnii M-1-2 were investigated. In this study,

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nitrogen in the form of yeast extract was used. The results showed that when 3 g/L of

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nitrogen was used, C. cohnii M-1-2 entered the exponential phase without delay 9


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(Figure 1A). However, when the nitrogen concentration was increased to 6 or 12 g/L,

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the growth of C. cohnii M-1-2 was delayed and the lag time was extended to 24 or 72

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h (Figure 1A). The final DCW was the highest of 25.10 g/L at initial nitrogen content

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of 6 g/L and lowest of 18.52 g/L at 3 g/L of nitrogen, respectively. The ammonium

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nitrogen content during fermentation was shown in Figure 1B. For 3 g/L of nitrogen,

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the ammonium nitrogen was quickly exhausted in the first 12 h. For 6 or 12 g/L of

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nitrogen, the ammonium nitrogen began to rise at 12 h and reached peak at 24 or 72 h.

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And then ammonium nitrogen was decreased to zero in the following 24 or 36 h,

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correlated to the fast biomass accumulation of C. cohnii M-1-2. The final DCW,

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specific growth rate, lipid content, DHA productivity, and DHA percent in TFAs were

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determined and shown in Table 1. Comparing with those of 3 g/L of nitrogen, the

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final DCW and specific growth rate were increased by 35.53% and 63.33% when 6

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g/L of nitrogen was used, and were increased by 25.11% and 50% when 12 g/L of

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nitrogen was used, respectively. The lipid content and DHA percentage in TFAs were

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decreased with the increased initial nitrogen, so the highest lipid content and DHA

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percentage in TFAs were achieved at 3 g/L of nitrogen. However, the DHA

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productivity was the highest at 6 g/L of nitrogen, and the lowest at 12 g/L of nitrogen.

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The results suggested that low initial nitrogen content was insufficient to support C.

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cohnii M-1-2 growth but was favorable for increasing lipid accumulation and DHA

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percentage in TFAs, while a high initial nitrogen content inhibited growth, lipid or

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DHA accumulation in C. cohnii M-1-2. A strategy of feeding nitrogen needs be

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established in order to obtain high biomass accumulation and DHA production in C.

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cohnii M-1-2.

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Ammonium nitrogen was a reliable indicator for the supplementation of

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nitrogen source in the complex environment of fermenter.32-33 The content of

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ammonium nitrogen was increased until C. cohnii M-1-2 began to grow. The rapid

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decline or increase of ammonia nitrogen was also found in other microbial cultures

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such as Aurantiochytrium limacinum SR21 and Protomonas extorquens sp. K.25, 38

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The lipid content (% DCW, w/w) was decreased with the increase of the initial

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nitrogen content through the batch fermentation (Table 1), consistent with the

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previous discoveries in C. cohnii.16, 18 Isleten-Hosoglu and Elibol16 also found that the

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DCW of C. cohnii was decreased in the case of high nitrogen. Safdar, et al.20 recently

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explored the changes of the DCW, TFAs in C. cohnii under different nitrogen

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concentrations of NaNO3. The results showed that the lowest initial nitrogen

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concentration led to the lowest biomass accumulation and the highest TFAs content.

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In addition, with the increase of initial nitrogen concentration, growth was

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significantly increased, while TFAs were decreased. However, when the initial

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nitrogen content in the form of NaNO3 greater than 1.6 g/L was used, the final DCW

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was obviously decreased and the TFAs content were also the lowest,20 consistent with

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the results obtained in this study. This phenomenon was also found in other

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microorganisms.21, 39 For example, Patil and Gogate39 found the DCW and lipid of

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Schizochytrium limacinum SR21 was decreased, when the initial yeast extract content

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was above 10 g/L. However, similar to other microorganisms accumulating lipid such

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as Isochrysis zhangjiangensis and Moritella marina MP-1 which grew slowly under

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the condition of nitrogen limitation, the changes in terms of DCW and lipid

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productivity were small in C. cohnii M-1-2,37, 40 so a suitable nitrogen feeding strategy

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is necessary for high lipid productivity.

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Effects of pulse-feeding strategies with constant C/N ratio of feeding medium on

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biomass accumulation and DHA productivity in C. cohnii M-1-2

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In order to alleviate the negative effects of high nitrogen content on the biomass

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and DHA production in C. cohnii M-1-2, pulse-feeding nitrogen strategies based on

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constant C/N ratio of feeding medium were investigated. The initial concentrations of

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glucose and yeast extract were set at 27 g/L and 6 g/L, respectively. The glucose

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concentration was maintained at 15-27 g/L. When glucose was added, yeast extract

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was added simultaneously according to a constant C/N ratio of feeding medium, and

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the feeding of only glucose was set as control.

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The results showed that feeding glucose with yeast extract increased the biomass

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accumulation of C. cohnii M-1-2, comparing with feeding of glucose only (Figure

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2A). Regardless of C/N ratio of feeding medium, the exponential phase started after

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12 h and ended at 96 h for C. cohnii M-1-2 fermentation (Figure 2A). The results

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showed that at C/N ratio of 4:1, the final biomass accumulation and specific growth

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rate were the highest (Table 2), while C/N ratio of both 3:1 and 6:1 generated lower

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DCW and specific growth rate. In order to examine whether high nitrogen inhibition

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effect was present, the ammonium nitrogen content of C/N ratio 4:1 was determined. 12



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As shown in (Figure 2B), ammonium nitrogen was completely exhausted after 24 h

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fermentation, suggesting the utilization of nitrogen source was quickly in the early

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stage of fermentation and the feeding of nitrogen need be carried out before 24 h to

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meet the needs of microalgae growth. The results showed that after nitrogen feeding,

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ammonium nitrogen began to accumulate from 72 to 96 h fermentation, but still at a

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low level of less than 5 mg/L, suggesting that almost all the nitrogen source was

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assimilated during the exponential growth of C. cohnii M-1-2. The concentration of

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ammonium nitrogen exceeded its initial amount and C. cohnii M-1-2 entered the

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stable period with the addition of nitrogen after 96 h, which indicated that the

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phenomenon of high nitrogen inhibition may happen after 96 h.

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The lipid content, fatty acid composition and DHA productivity were shown in

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Table 2. The results showed that the DHA productivity was also the highest with C/N

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ratio of 4:1. The lipid content and DHA percentage in TFAs were the highest with

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glucose only feeding. With the increased initial yeast extract content of feeding

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medium, the lipid content and DHA percentage in TFAs were decreased, which were

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in accordance with the results of batch culture discussed above. The results suggested

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that the pulse-feeding strategy was able to alleviate the high nitrogen inhibition effect

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on the growth of C. cohnii M-1-2, but inhibition effects were still existing on lipid and

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DHA accumulation of C. cohnii M-1-2 with an increasing initial yeast extract content

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of feeding medium. And the pulse-feeding strategy resulted in nitrogen accumulated

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in the late fermentation stage, which were not supportive to the growth and lipid

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accumulation of C. cohnii M-1-2.

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Due to the high nitrogen inhibition, the amount of nitrogen added each period of

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time needs to be under careful control, so a fed-batch through constant C/N ratio of

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feeding medium strategy was evaluated. It has been demonstrated as an effective

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strategy to improve final product productivity by controlling the C/N ratio of feeding

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medium in various microorganisms.26, 41 With the increase of C/N ratio of the feeding

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medium, the lipid and DHA content increased (Table 2), which was consistent with

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the findings that high C/N ratio was beneficial to the accumulation of the lipid and

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DHA in A. limacinum SR21.41 Besides, some studies have found that high C/N ratio

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promoted lipid accumulation in marine microorganisms.39, 42 High C/N ratio was also

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beneficial to the accumulation of other products. For example, Suzuki, et al.26 found

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that the PHB content was increased in Protomonas extorquens sp. K with the increase

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of C/N ratio of the feeding medium. Although the growth and DHA productivities

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were improved using the pulse-feeding strategies, there was still room for

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improvement because ammonium nitrogen concentration was also exhausted in the

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middle exponential phase and then sharply increased after 96 h during fermentation.

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This phenomenon suggested that the nitrogen might be not high enough to support an

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optimal growth in the early stage of the fermentation, and it might be excessive

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around the end of the fermentation, leading to possible high nitrogen inhibition effects,

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consistent with previous studies that nitrogen supply needed be limited at the later

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stage of fermentation, in order to promote lipid accumulation.27

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Effects of continuous-nitrogen feeding strategies on biomass accumulation and

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DHA productivity in C. cohnii M-1-2

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As mentioned above, the exponential phase of C. cohnii M-1-2 using

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pulse-feeding strategies was approximately from 12 to 96 h. After 96 h, the growth of

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C. cohnii M-1-2 was inhibited by any additional nitrogen feeding. It has been reported

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that continuous-feeding nitrogen at a constant rate was an effective way to avoid high

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nitrogen inhibition.22, 25 Therefore, a strategy that feeding 50% (w/v) yeast extract at a

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constant rate from 12 to 96 h was carried out. Glucose was controlled at 15-27 g/L

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during the fermentation.

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Figure 3A-C showed the time courses of DCW, lipid content, glucose

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concentration, and ammonium nitrogen concentration during fed-batch cultivation of

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C. cohnii M-1-2 with 50% (w/v) yeast extract feeding at different constant flow rates.

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During the first 48 h of the fermentation, the specific growth rate was 0.961, 1.136, or

322

1.119 day-1 for feeding rates of 1.5, 2.1, or 2.7 mL/h, respectively, and it was

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decreased to 0.541, 0.537, and 0.381 day-1 in the next 48 h fermentation, respectively.

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The specific growth rate was always the highest at the feeding rate of 2.1 ml/h, and

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the biomass accumulation was also the highest at this feeding rate (Figure 3B). As the

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cells stopped growing after 96 h at the feeding rate of 2.7 mL/h, the lowest biomass

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accumulation was observed for this rate. Regardless of the yeast extract feeding rates,

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the lipid content was increased with the progress of fermentation (Figure 3A-C). The

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final lipid content of the feeding rate at 1.5 or 2.1 mL/h was over 50%, while it was

330

only 44.41% at a feeding rate of 2.7 mL/h. Figure 4 showed the time courses of DHA 15


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percentage in TFAs at a constant nitrogen feeding rate. DHA percentage in TFAs was

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gradually increased with the progress of fermentation at the feeding rate of 1.5 or 2.1

333

mL/h during 12-96 h. However, the DHA percentage in TFAs at a feeding rate of 2.7

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mL/h was maintained at a low level. The DHA percentage in TFAs of 2.7 mL/h during

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12-96 h was always the lowest, and the DHA percentage in TFAs of 2.1 mL/h during

336

12-96 h was always the highest at the same period of the fermentation.

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The ammonium nitrogen of all the fermentations was almost exhausted in the first

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24 h of the fermentation (Figure 3A-C). For the feeding rate of 1.5 mL/h, the

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ammonium nitrogen maintained at a low level (

Nitrogen Feeding Strategies and Metabolomic Analysis To Alleviate

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