Bioresource Technology 223 (2017) 141–148

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Bioresource Technology

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Enhancement of docosahexaenoic acid synthesis by manipulationof antioxidant capacity and prevention of oxidative damage inSchizochytrium sp.

http://dx.doi.org/10.1016/j.biortech.2016.10.0400960-8524/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: biotech@njtech.edu.cn (H. Huang).

1 These two authors contributed equally to this work.

Lu-Jing Ren 1, Xiao-Man Sun 1, Xiao-Jun Ji, Sheng-Lan Chen, Dong-Sheng Guo, He Huang ⇑Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), College of Biotechnology and Pharmaceutical Engineering, School of Pharmacy, NanjingTech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China

h i g h l i g h t s

� A new control strategy by manipulating antioxidant capacity was proposed.� Ascorbic acid enhances cellular antioxidant capacity to resist oxidative injury.� ROS decreased by 35.5% and T-AOC kept above zero until the end of fermentation.� Ascorbic acid increased PUFA content with specificity improvement in DHA content.� DHA yield was increased by 44% by two-point ascorbic acid addition strategy.

a r t i c l e i n f o

Article history:Received 16 August 2016Received in revised form 11 October 2016Accepted 13 October 2016Available online 15 October 2016

Keywords:Schizochytrium sp.Docosahexaenoic acidReactive oxygen speciesTotal antioxidant capacity

a b s t r a c t

Oxygen-mediated cell damage is an important issue in aerobic fermentation. In order to counteract theseproblems, effect of ascorbic acid on cell growth and docosahexaenoic acid (DHA) production was inves-tigated in Schizochytrium sp. Addition of 9 g/L ascorbic acid resulted in 16.16% and 30.44% improvementin cell dry weight (CDW) and DHA yield, respectively. Moreover, the total antioxidant capacity (T-AOC) ofcells decreased from 2.17 at 12 h to 0 at 60 h and did not recover, while ascorbic acid addition couldextend the time of arrival zero with the reduced intracellular ROS. However, ROS levels still increasedafter 72 h. Therefore, to further solve the problem of high ROS levels and low T-AOC of cells after 72 h,a two-point addition strategy was proposed. With this strategy, DHA yield was further increased to38.26 g/L. This work innovatively investigated the feasibility of manipulating Schizochytrium sp. cultiva-tion through ROS level and T-AOC.

� 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Docosahexaenoic acid (DHA) has gained increasing attentionover recent years due to its beneficial effects on human health(Ursin, 2003) . DHA acts as an important structural component ofcellular membranes and is closely associated with neural, cardio-vascular and reproductive conditions (Ji et al., 2015). It is impor-tant primarily due to its chain length and extraordinarily lowdegree of saturation. In the past, DHA was mainly produced fromfish oils. However, fish resources are affected by season and loca-tion, and are increasingly under pressure from overharvestingand environmental pollution, which creates the need to establish

sustainable sources of DHA (Tacon and Metian, 2008). Due largelyto their unique and diverse metabolic profiles, microalgae havebecome one of the most promising sources for the production ofnutritional supplements, cosmetics and biofuels (Brennan andOwende, 2010). The microalgae strain of Schizochytrium sp. hasbeen the subject of a substantial amount of research due to its highproduction of polyunsaturated fatty acids (PUFA), particularly withregard to DHA. Therefore, improving the growth and DHA accumu-lation of Schizochytrium sp. is extremely important for achievingsustainable commercial production of this important nutrient.

Most of the oleaginous microorganisms employed in DHA pro-duction were aerobic (Ren et al., 2010). Accordingly, Jakobsenet al. (2008) reported that, during the cultivation process for Schi-zochytrium sp., a higher oxygen supply was beneficial for cellgrowth and lipid accumulation. However, in an oxygen-rich envi-ronment, lipids, and among them especially the polyunsaturated

142 L.-J. Ren et al. / Bioresource Technology 223 (2017) 141–148

fatty acids, could be oxidized (Guichardant et al., 2011). In additionto causing unstable DHA production, lipid peroxidation can alsolead to the accumulation of high levels of reactive oxygen species(ROS) (Johansson et al., 2016). The resulting ROS can react withand cause damage to biological macromolecules such as DNA,lipids and proteins, which in turn can result in the loss of proteinfunction or even cell death (Ruenwai et al., 2011). In recent years,ROS levels have been frequently reported in association with fer-mentation control strategies. Li et al. (2011) found that a highROS level at low cultivation temperature was beneficial to increaselipid accumulation in Scenedesmus sp. Landolfo et al. (2008) foundthat cell viability and ethanol production in Saccharomyces cere-visiae depends on cellular ROS accumulation and scavenging sta-tus. In addition to environmental factors, such as temperature,PH and oxygen supply, which directly affect ROS levels, the exter-nal addition of antioxidants has become a method of choicemethod for the regulation of cellular ROS levels. It has beenreported that specific antioxidants, such as ascorbic acid, hydrogensulfide, ginsenosides and sesamol, can protect against lipid perox-idation (Huang et al., 2014; Molina et al., 2014). Such antioxidantshave a great potential to enhance the proliferation capacity of abroad range of cells, most likely directly due to their high abilityto scavenge intracellular ROS species. In recent years, there havebeen a number of attempts to improve cell growth or the produc-tion of target compound by external supplementation of antioxi-dants. Liu et al. (2015) increased the cell proliferation and fattyacid production capacity of Crypthecodinium cohnii by the externaladdition of sesamol. Xiao et al. (2006) found that ascorbic acid isbeneficial for cell viability and hirudin production. However, suchstudies typically only showed that antioxidants had an obviouseffect on the regulation of fermentation processes, but lackedessential analysis from the standpoint of cellular oxygen toxicityand antioxidant capacity. It was thus urgent to undertake studiesof the relationship of ROS, total antioxidant capacity (T-AOC), cellgrowth and DHA synthesis at a cellular level, in order to providea toolset for the understanding of macro-scale events in industrialfermentation processes.

Considering that DHA is used in infant food formulas, an addi-tive that is both safe and approved was selected for this study inthe form of ascorbic acid. Ascorbic acid, a well-known antioxidantused in the food industry as a nutrient or preservative, was knownto enhance T-AOC and reduce ROS production in cells (Molinaet al., 2014). ROS are classically thought of as cytotoxic inducersof oxidative stress, but recent evidence suggests that certain ROSspecies can also have signal transduction functions and as suchcan participate in cell proliferation, apoptosis and cell senescence(Arnold et al., 2001). It has been found that a strong T-AOC is indis-pensable for many essential biological functions, including protec-tion against membrane-lipid peroxidation of, especially ofessential polyunsaturated fatty acids, as well as against DNA dam-age (Aruoma, 1999). In addition to its potent antioxidant activity,ascorbic acid is also reported to enhance the enzyme activity ofglucose-6-phosphate dehydrogenase (Molina et al., 2014).

In this work, different concentrations of ascorbic acid weresupplemented to the fermentation medium in order to improvecell growth and DHA production of Schizochytrium sp. Inconjunction with this, we investigated the effects of ROS andT-AOC on microalgal growth and lipid accumulation. Finally, atwo-point ascorbic acid addition strategy was proposed to furtherimprove DHA production. This work is to our best knowledge thefirst effort to investigate the feasibility of manipulating cellgrowth and DHA production via manipulation of T-AOC and ROSlevels. The strategy proposed here may provide a new and alter-native direction for the industrial cultivation of oil-producingmicroalgae.

2. Materials and methods

2.1. Microorganism and culture conditions

Schizochytrium sp. HX-308(CCTCC M 209059), isolated fromseawater and stored in China Center for Type Culture Collection(CCTCC) (Huang et al., 2009), was used in this study. The strainwas preserved in 20%(v/v) glycerol at �80 �C.

The seed culture medium and conditions of propagation werethe same as those used in our previous study (Qu et al., 2010). Afterthree generations, the seed cultures (1% v/v) were transferred to500-mL shake flasks containing 100 mL of medium and culturedat 28 �C under constant orbital shaking at 170 rpm, and the result-ing cell suspension was used as inoculant for the fermentation. Thefermentation medium was the same as reported in our previousstudy (Lian et al., 2010). The basic medium contained 50 g/L glu-cose and 0.4 g/L yeast extract, which were dissolved in artificialsea water. The artificial sea water contained (g/L): Na2SO4 10;(NH4)2SO4 0.8; KH2PO4 4; KCl 0.2; MgSO4 2; Monosodium gluta-mate 20; CaCl2 0.1 and the trance elements (g/L): Na2EDTA 6,FeSO4 0.29, MnCl2�4H2O 0.86, ZnSO4 0.8, CoCl2�6H2O 0.01,Na2MoO4�2H2O 0.01, NiSO4�6H2O 0.06 and CuSO4�5H2O 0.6.

2.2. Cell dry weight and total lipids

Cell dry weight was determined gravimetrically by harvesting10-mL culture aliquots by centrifugation at 4500g for 5 min. Thecells were subsequently transferred to a weighed filter paperand dried at 60 �C to constant weight (�12 h). The yield oftotal lipids (TLs), extracellular glucose and glutamate weredetermined the same way as published in our previous study(Qu et al., 2010).

2.3. Fatty acid and squalene analysis

Fatty acid methyl esters (FAMEs) preparation were performedas previously described products. FAMEs were prepared from0.2 g of dried cells and analyzed by gas system (GC-2010, Shi-madzu, Japan), equipped with a capillary column (DB-23,60 m * 0.22 mm) and flame ionization detector (FID). Nitrogenwas used as the carrier gas. The injector was maintained at250 �C with an inject volume of l lL. The column was raised from100 �C to 200 �C at 25 �C/min and then increased to 230 �C at4 �C/min, keeping this temperature for 9 min. The FID detectortemperature was set at 280 �C. FAMEs and squalene was identifiedthrough comparison with related external standards (Sigma, USA).The quantities of individual FAMEs and squalene were estimatedfrom the peak areas on the chromatogram using nonadecanoic acid(C19:0) as the internal standard.

2.4. Determination of total antioxidant capacity

Intracellular total antioxidant capacity (T-AOC) was determinedusing the T-AOC assay kit (Angle Gene, China) according to themanufacturer’s instructions. Cells were harvested by centrifuga-tion for 5 min, and washed with physiological saline in order toremove the excess of ascorbic acid in the medium. After being dis-rupted by ultrasonic disrupter for 15 min, 200 lL of crude extractwas mixed with 0.2 mM DPPH radicals. The mixture was incubatedfor 30 min at 37 �C and the absorbance at 517 nm. The radical scav-enging ability was calculated based on the difference in the mea-sured decrease of absorbance at 517 nm between the blank andthe respective sample, whereby a unit of T-AOC was defined asthe increase of OD517 nm caused by per mg cell dry weight, per

L.-J. Ren et al. / Bioresource Technology 223 (2017) 141–148 143

minute, at 37 �C. The value of T-AOC was calculated from the fol-lowing equation.

T-AOC ðU=mg CDWÞ ¼Ar�Ac0:01�30CDW

where Ar is the absorbance of the reaction sample (with DPPH solu-tion) and Ac is the absorbance of the control sample (without DPPHsolution).

2.5. Determination of intracellular reactive oxygen species

Intracellular ROS levels in collected cells were determinedaccording to Li et al. (2011), which use an oxidation-sensitiveindicator of the 20,70-Dichlorofluorescein diacetate (DCFH-DA).This probe is not fluorescent in its original form and can freelycross cell membranes. Into the living cells, two acetate groups(DA) are removed from the indicator forming DCFH, which is stillnot fluorescent. In the presence of ROS, DCFH is oxidized to fluo-rescent 20,70-dichlorofluorescein (DCF), which can be measured byfluorometry. DCFH-DA was added for an in-well concentration of10 Mm to collected cells, which were incubated for 30 min at37 �C. After a full half hour of exposition to the probe, the excessof indicator in the medium was washed with PBS, to make surethat only the intracellular oxidation was measured. Fluorescenceintensity was measured using a fluorescence spectrophotometerat an excitation wavelength of 485 nm and an emission wave-length of 530 nm.

Fig. 1. Effects of ascorbic acid on substrate consumption (glucose and MSG) and cell drydeviations of three replicates for each measurement).

3. Results and discussion

3.1. Effects of ascorbic acid on cell growth and substrate consumption

The addition of different concentration of ascorbic acid (0, 3, 6,9, 12 g/L) to the media caused significant changes in the substrateconsumption and growth profiles of Schizochytrium sp. As shown inFig. 1a, a significant difference in glucose consumption was identi-fied between non-supplemented cultures and those supplementedwith ascorbic acid after 24 h. The difference in consumed glucoseamounts continued to grow until 108 h, with yields of 326 g/L inthe cultures where 9 g/L ascorbic acid was added, compared to260 g/L in cultures without supplementation. Compared with othergroups, the addition of 12 g/L ascorbic acid actually decreased theglucose consumption rate, which indicated that high ascorbic acidaddition concentration inhibited substrate consumption. Similarly,as shown in Fig. 1c, an increase in the ascorbic acid concentrationresulted in a substantial increase of cell dry weight (CDW). Theoptimum concentration of ascorbic acid appears to be 9 g/L, andthis concentration resulted in the largest CDW of 106.72 g/L, whichwas 16.16% higher than that of the non-supplemented group. Thisresult is in line with previous works in which it has been reportedthat antioxidants have the potential to enhance cell growth byreducing intracellular ROS levels (Liu et al., 2015). However, a fur-ther increase in ascorbic acid concentration to 12 g/L resulted in adrop in CDW to 85.3 g/L, indicating that high ascorbic acid concen-tration (over 12 g/L) can actually hinder the growth of Schizochy-trium sp. The biochemical basis for this is as of yet not clear, but

weight (CDW) of Schizochytrium sp. (data represent the mean values and standard

144 L.-J. Ren et al. / Bioresource Technology 223 (2017) 141–148

previous studies have for example shown that high concentrationsof vitamin E inhibited cell proliferation in human mesangial andglomerular microvascular endothelial cells (Zhang et al., 2001).However, while both vitamin E and ascorbic acid are antioxidants,the system is not readily comparable to Schizochytrium sp., and aseparate study would be necessary to elucidate the exact mecha-nism of ascorbate-mediated growth inhibition, which is beyondthe scope of this paper.

3.2. Effects of ascorbic acid on lipid accumulation, lipid compositionand DHA production

As shown in Fig. 2, significant improvements of lipid accumula-tion were achieved by the addition of ascorbic acid and the effect

Fig. 2. Effects of ascorbic acid on lipid accumulation, fatty acid composition, DHA yieldstandard deviations of three replicates for each measurement).

was observed to occur in a dose- dependent manner. Lipid accu-mulation in the group supplemented with 3 g/L ascorbic acid wassimilar to the control group. With a further increase of ascorbicacid concentration to 6 g/L or 9 g/L, lipid accumulation alsoincreased (Fig. 2a). Maximum lipid yield of 65.50 g/L was obtainedwith 9 g/L of added ascorbic acid, and this yield was 14.46% higherthan that of non-supplemented group (Table 1). It has been docu-mented that ascorbic acid has ability to enhance glucose-6-phosphate dehydrogenase enzyme activity which was the mainresource of intracellular NADPH (Molina et al., 2014). And a suffi-cient supply of NADPH by glucose-6-phosphate dehydrogenaseenzyme is thought to be positively linked to cell growth and fattyacid biosynthesis at the early fermentation stage, which might bethe key reason for the observed enhancement of lipid production

and squalene content of Schizochytrium sp. (data represent the mean values and

Table 1Fermentation parameters of Schizochytrium sp at 108 h in a 5 L bioreactor.

Ascorbic acid concentration (g/L)

0 g/L 3 g/L 6 g/L 9 g/L 12 g/L

CDW (g/L) 91.87 ± 1.5 92.13 ± 1.9 95.34 ± 1.6 106.7 ± 1.5 85.30 ± 1.4TLs concentration (g/L) 56.03 ± 1.8 55.98 ± 1.2 60.32 ± 1.4 65.50 ± 1.3 47.08 ± 1.2DHA percentage in TFAs (%) 47.42 ± 0.3 50.61 ± 0.8 51.67 ± 0.6 52.81 ± 0.5 50.20 ± 0.4DHA yield (g/L) 26.57 ± 0.9 28.33 ± 0.9 31.17 ± 0.8 34.59 ± 1.0 23.63 ± 0.7SFA (%TFAs) 27.55 ± 0.2 24.05 ± 0.6 22.36 ± 0.6 20.64 ± 0.8 22.50 ± 0.8PUFA (%TFAs) 64.63 ± 0.3 68.18 ± 0.3 69.18 ± 0.5 71.79 ± 0.3 68.24 ± 1.2Squalene content (g/100 g lipids)(g/100 g lipids) 0.1 ± 0 0.32 ± 0.0 0.24 ± 0 1.18 ± 0 1.96 ± 0.01

L.-J. Ren et al. / Bioresource Technology 223 (2017) 141–148 145

(Sun et al., 2016). The lowest lipid accumulation of 47.08 g/L wasreported in cultures the addition of 12 g/L ascorbic acid, whichwas most likely directly caused by the low biomass obtained underthese conditions.

Interestingly, ascorbic acid exhibited significant effects not onlyon the total lipids, but also on fatty acid composition. Saturatedfatty acids (SFA) in Schizochytrium sp. are mainly composed ofC14:0 and C16:0, whereas PUFA mainly consist of docosapentenoicacid (DPA) and DHA. With increasing culture time, SFA and PUFAbiosynthesis followed opposite trajectories (Fig. 2c and d). SFA per-centage in TFAs in TFAs decreased over time from 24–31% at 48 hto 20–27% at 108 h (Table 1). It is important to note that SFA per-centage in TFAs of cultures supplemented with ascorbic acid wereall lower than that of the control. Although SFA percentage in TFAsdecreased in all supplemented cultures up to 108 h, the decreasewas more prominent in cultures having 9 g/L ascorbic acid. In thatcondition, the SFA percentage in TFAs was only 20.64% in TFAs at108 h, which was 25.08% lower than that of the control (Fig. 2c).Interestingly, an opposite trend was observed for PUFA percentagein TFAs (Fig. 2d). PUFA percentage in TFAs increased significantly incultures having 9 g/L ascorbic acid, resulting in PUFA of 71.79% ofTFAs, which was 11.07% higher than that of control (Table 1). Moreimportantly, the addition of ascorbic acid showed little influenceon the percentage of DPA in TFAs, so that most of the increase inPUFA was attributed to DHA. Accordingly, DHA percentage of TFAsincreased significantly and reached to 52.81% in the presence of9 g/L ascorbic acid at 108 h, which was 11.36% higher than thatof the unsupplemented group (Fig. 2e). Interestingly, because ofthe concomitant considerable increase of both biomass and DHApercentage, a final DHA yield of 34.66 g/L was obtained in the pres-ence of 9 g/L ascorbic acid, which was significantly 30.44% higherthan that of the control cultures (Fig. 2b).

PUFA are well-known antioxidants, and a considerable amountof research has been conducted surrounding their production anduse (Richard et al., 2008). After prolonged fermentation (60 h), cellsmight begin to consume PUFA to protect themselves from oxida-tive damage, and this secondary consumption can result in reducedPUFA percentage in TFAs in the non-supplemented group. In addi-tion to PUFA, squalene, which contains six isoprene units, is also animportant antioxidant in Schizochytrium sp. (Kohno et al., 1995).Consequently, the squalene contents of all the cultures supple-mented with ascorbic acid was higher than that of cultures withoutsupplementation (Fig. 2f). And increasing concentration of ascorbicacid resulted in increased squalene content, with a peak value cor-responding to 1.96 g in 100 g lipids achieved with the addition of12 g/L of ascorbic acid. On the other hand, in the cultures grownwithout supplementation, the squalene contents amounted to only0.1 g in 100 g lipids (Table 1). In aerobic fermentation, squalenemight be oxidized or consumed by organisms and converted toother compounds. The addition of ascorbic acid evidently pro-tected squalene from oxidation, which resulted in increase of squa-lene accumulation.

3.3. Effects of ascorbic acid on cellular ROS and T-AOC

In order to further explore the mechanism of the observedeffects of ascorbic acid on cell growth and lipid production, wedetermined the intracellular amounts of reactive oxygen species(ROS) and total antioxidant capacity (T-AOC) of Schizochytriumsp. intervals of 12 h during the entire fermentation process. Thetime profiles of ROS levels during cultivations are presented inFig. 3a. Interestingly, at the period from 12 h to 60 h, ROS reachedhighest value at 12 h and then gradually declined. This phe-nomenon was also observed in other microorganism (Menonet al., 2013). One reason for the apparent peak in ROS levels at12 h might be the stress experienced by the cells when the cellsare shifted from a medium with depleted nutrients in the seed cul-ture to a nutrient rich medium. It has documented that the effect ofnitrogen starvation could induce oxidative stress, but the effect onexponential phase is far less than stationary phase (Liu et al., 2012),which attributed to that the stress mechanism of cells in exponen-tial phase is so active that cells can product a lot of stress factors toresist the negative effect of nitrogen starvation. In this study, nitro-gen (MSG) was depleted at 36 h (Fig. 1b), but the decrease in ROSlevels was observed at 36–60 h, which might attributed to the pro-duction of squalene and pigment. As shown in Fig. 2f, squalenemaintained high content before 60 h. Moreover, in our previousstudy(Ren et al., 2014), the date showed that the content ofb-carotene was high before 72 h. Squalene and b-carotene asantioxidant could remove cellular ROS levels (Sun et al., 2016;Gupta et al., 2013). In stage of 36–60 h, Schizochytrium sp. belongsto exponential phase and could product lots of squalene andb-carotene to resist ROS levels induced by nitrogen starvation,which could result that ROS levels decreased at 36–60 h. After72 h, however, ROS levels significantly increased, which may bedue to growth inhibition caused by the high cell concentrationsreached by this time. The following period, from 72 to 108 h, coin-cides with a phase of rapid lipid accumulation, and it is in thisstage that PUFA peroxidation is likely to cause high levels of ROS(Ruenwai et al., 2011). In this stage, the cells start to experienceoxidative stress because the amount of ROS starts to exceed theability of the antioxidant defense systems to detoxify them. Thebuildup of ROS could cause oxidative damage to cellular compo-nents and have adverse effect on cell growth, which might explainthe lower CDW in the group without supplementation. Ascorbicacid acts as a scavenger which directly neutralizes different radi-cals. As indicated in Fig. 3a, ROS level in the group grown with9 g/L of ascorbic acid was reduced by 34.59% at 12 h of cultivationand remained significantly lower at 108 h compared to the non-supplemented control group. This environment was beneficial tocell metabolism, resulting in the highest CDW and best lipidaccumulation of all groups. However, a similar improvement wasnot observed in the cultures grown with 12 g/L of ascorbic acid,even though these had the lowest ROS level of 73.27 at 108 h. Thisphenomenon implies that an excessive reduction of ROS levels may

Table 2Fermentation results of strategy III in a 5 L bioreactor and % changes compared to thesame results from strategies I and II. (Strategy I: no-addition of ascorbic acid; strategyII: initial addition of 9 g/L of ascorbic acid; strategy III: two-point addition of 9 g/Lascorbic acid at each point).

StrategyStrategy III

Increase (%)compared to

StrategyI

StrategyII

CDW (g/L) 112.3 ± 2.2 22.23 5.3TLs concentration (g/L) 70.2 ± 1.3 25.29 7.18DHA percentage in TFAs (%) 54.5 ± 0.4 14.93 3.2DHA yield (g/L) 38.26 ± 0.7 44.0 10.61SFA (%TFAs) 19.4 ± 0.3 �29.6 �6.01PUFA (%TFAs) 72.1 ± 0.2 11.56 0.43Squalene content (g/100 g lipids) (g/

100 g lipids)2.13 ± 0.01 2030 80.5

Strategy III compared strategy I: increase (%)= the value of strategy III�the value of strategy I

the value of strategy I � 100%Strategy III compared strategy II: increase (%)= the value of strategy III�the value of strategy II

the value of strategy II � 100%

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in fact not be conducive to cell growth and lipid accumulation. Ithas been reported that higher ROS level could positively influencelipid accumulation (Li et al., 2011). In other words, the negativeeffects of exceedingly high ascorbic acid concentration outweighedthe positive effects of oxidative protection.

In response to oxidative stress, cellular protection against oxi-dation and electrophile-mediated toxicities is provided by two dis-tinct types of antioxidative mechanisms (Dinkova-Kostova andTalalay, 2008; Halliwell, 2007). One is direct antioxidant activity,which scavenges reactive oxygen species directly and instanta-neously. The other is more complex and indirect, and is mediatedby the expression of antioxidant enzymes. In this study, completetime profiles of T-AOC levels were recorded during all cultivations.As shown in Fig. 3b, overall, the T-AOC of all cultures decreasedwith increasing fermentation time, whereby the T-AOC values ofthe cultures grown with added ascorbic acid were significantlyhigher, indicating that ascorbic acid can greatly improve theantioxidant capacity as intended. In the non-supplemented group,the T-AOC of Schizochytrium sp. reached 2.17 at 12 h and keptabove 0 before 60 h (Fig. 3b), which was most likely directly causedby the production of b-carotene. In our previous study, the contentof b-carotene was stable before 72 h and then began to decreaseafter entering the late stage of lipid accumulation until the endof the fermentation (Ren et al., 2014). As we known, b-carotenehas antioxidant activity and may thus provide a cellular defenseagainst active oxygen (Gupta et al., 2013). After 60 h, the T-AOCkept at a 0 level until the end of fermentation. This demonstratethat Schizochytrium sp. has a serious deficiency of T-AOC. Whenascorbic acid was added at a concentration of 9 g/L, the T-AOC ofcells was greatly increased and the cultures maintained an overallstronger antioxidant capacity during the entire fermentation per-iod. Although it has documented that ascorbic acid could inducethe pigment destruction (Choi et al., 2002), but the addition con-centration of ascorbic acid (9 g/L) is far more than the productionof b-carotene by Schizochytrium sp. Therefore, compared withascorbic acid, the effect of b-carotene on resisting cellular oxidativedamage was not worth mentioning. An improved T-AOC, especiallyduring later fermentation phases, can protect PUFA from oxidativedamage, and this is likely a key reason why the highest percentage(%TFAs) of PUFA was achieved with the addition of 9 g/L ascorbicacid supplementation. However, in the group of 9 g/L ascorbic acid,after 84 h, PUFA percentage (in %TFAs) began to decrease, whichaccompanied the rapid decrease of T-AOC (Fig. 3b). These illus-trated that the T-AOC shortage after 84 h has caused part of PUFAbeing oxidized. Though PUFA decreased after 84 h, but compare to

Fig. 3. Effects of ascorbic acid on T-AOC and ROS levels of Schizochytrium sp. (datameasurement).

DPA, DHA is comparatively recalcitrant to oxidation because theD4 double bond must first be removed by peroxisomalb-oxidation (Tocher, 2003). Thus, an apparent selective retentionof DHA resulted DHA percentage in TFAs was increased until to108 h. Li et al. (2007) quantified the antioxidant activity of extractsfrom 23 selected microalgae, using the ABTS assay with Trolox asstandard. And authors found that the algal strains tested had dis-parate antioxidant activities, with total values ranging from 1.3to 29.6 lmol Trolox/g. But not all groups of microalgae have stron-ger antioxidant capacity, and this is due to their general variabilityincluding their widely varied contents of target products, differentgrowth rates or yields, different ease of cultivation and other fac-tors (Li et al., 2007). In recent years, a number of various supple-ments have been added to growth media to improve theantioxidant capacity of microalgal cultures. For example, Gaffneyet al. (2014) improved the T-AOC of Schizochytrium sp. by the sup-plementation of flaxseed oil. Whereas, Burg and Oshrat (2015)found that addition salt to the media can enhance the T-AOC ofPorphyridium. Thus, these results indicate the possibility of manip-ulating cell growth and DHA production through ROS level and T-AOC. However, after 60 h under the described conditions, the ROSlevel of the cells was also too high and their T-AOC has rapidlydecreased, which might have directly caused damage to the cells

represent the mean values and standard deviations of three replicates for each

Fig. 4. Lipid accumulation, fatty acid composition, DHA yield and squalene content of Schizochytrium sp. grown using a two-point ascorbic acid addition strategy. Each datumis the mean ± standard deviations of three independent experiments replicates. The statistical significance between unsupplemented group and strategy III of ROS and T-AOCwas presented by t-test, respectively. *P < 0.05, **P < 0.01.

L.-J. Ren et al. / Bioresource Technology 223 (2017) 141–148 147

and consequently inhibited DHA production. Therefore, the 60 htime-point was a key control point in Schizochytrium sp. culture.

3.4. Regulation of DHA production by two-point addition of ascorbicacid

The ultimate aim of this study was to improve DHA productionby regulating cellular oxidative damage. Though DHA yield hasbeen improved by strategy II, but cellular oxidative damage wasstill unsolved completely. Therefore, to further solve the problemof high ROS levels and low T-AOC of Schizochytrium sp. at the laterstages of fermentation, a two-point addition strategy was imple-mented. In this strategy, 9 g/L of ascorbic acid was added to the ini-tial medium and an equivalent amount was added a second time at60 h. Table 2 summarizes the comparison of the results of the non-addition (I), initial addition (II) and two-point addition (III) strate-gies. As shown in Fig. 4, the addition of ascorbic acid to the culturemedium after 60 h relieved the observed problems to a certaindegree, and exhibited a significant effect on cell growth. Unlikeexperiment II, the T-AOC in experiment III increased to 7.5 U/mgCDW at 72 h from 5.9 U/mg CDW at 60 h. Consequently, the ROSlevels in experiment III correspondingly decreased by 35.5% and10.3%, respectively, compared with experiment I and II by theend of fermentation (Fig. 4b). The degree of the effect of ascorbicacid on ROS and T-AOC at the period from 60 h to108 h was far lessthan that within initial 60 h, which might be ascribed to the factthat more PUFA were accumulated in the cells and that the cul-tures reached much higher cell concentrations during the later fer-mentation stages.

Notwithstanding, CDW of experiment III reached 112.3 g/L,which was 22.23% and 5.3% higher than experiment I and II,respectively (Table 2). Therefore, a maximum yield correspondingto 70.2 g/L of total lipids was obtained in experiment III, whichwas 25.29% and 7.17% higher than that of experiment I and II,respectively. In contrast, the two-point addition strategy had noobvious effect on PUFA and SFA percentage in TFAs (Fig. 4d). AndPUFA percentage in TFAs slightly decreased after 84 h in all exper-iments. But DHA percentage in TFAs of experiment III was higherthan what was observed in the other groups, with a maximum of54.5% obtained at 108 h (Table 2). Thus, there was an apparentselective retention of DHA in the cellular lipids. This could beexplained by the fact that DHA is comparatively recalcitrant to oxi-dation, since the D4 double bond must first be removed by perox-isomal b-oxidation (Tocher, 2003). Therefore, concomitant withhigher lipid accumulation and DHA percentage in TFAs, the highestDHA yield of 38.26 g/L was observed in the two-point additionstrategy, which was 43.9% and 10.61% higher than that of experi-ment I and II, respectively. In contrast, the two-point additionstrategy still exhibited adverse effects on squalene accumulation,with a peak of 2.13 g in 100 g lipids achieved with experiment III(Fig. 4f).

4. Conclusions

In this study, the effects of different concentrations of ascorbicacid on cell growth and DHA production were investigated in Schi-zochytrium sp. Addition of 9 g/L ascorbic acid greatly increased celldry weight and DHA yield. These major performance changes were

148 L.-J. Ren et al. / Bioresource Technology 223 (2017) 141–148

accompanied with enhanced T-AOC and reduced ROS. Two-pointascorbic acid addition strategy was employed to further increaseDHA yield. In addition, ascorbic acid was also beneficial for increas-ing polyunsaturated fatty acids content. For the first time the highDHA synthesis of Schizochytrium sp. was correlated with ROS levelsand T-AOC, which provided a new method for other oil-producingmicroalga.

Acknowledgement

This work was financially supported by the National ScienceFoundation for Distinguished Young Scholars of China (No.21225626), the National Natural Science Foundation of China(No. 21306085 and No. 21476111), the National High TechnologyResearch and Development Program of China (No.2014AA021701), the Outstanding Youth Foundation of JiangsuNature Science Foundation (BK20160092) and the SpecializedResearch Fund for the Doctoral Program of Higher Education (No.20133221120008).

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