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Bioresource Technology

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Comparative physiological and metabolomic analyses of the hyper-accumulation of astaxanthin and lipids in Haematococcus pluvialis upontreatment with butylated hydroxyanisole

Wei Dinga,1, Qingqing Lia,1, Benyong Hana, Yongteng Zhaoa, Shuxiang Gengb, Delu Ningb,Ting Mab, Xuya Yua,⁎

a Faculty of Life Sciences and Technology, Kunming University of Science and Technology, Kunming, Yunnan, Chinab Yunnan Academy of Forestry, Kunming 650051, China

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O

Keywords:Haematococcus pluvialisAstaxanthinLipidButylated hydroxyanisolePhysiologicalMetabolomics

A B S T R A C T

The major goal of this study was to explore the functions of butylated hydroxyanisole (BHA) combined withabiotic stress on the cultivation of the microalga Haematococcus pluvialis for astaxanthin and lipid production.Here, the effect of BHA on astaxanthin and lipid accumulation and physiological and metabolomic profiles wasinvestigated. These results suggested that astaxanthin content was increased by 2.17-fold compared to thecontrol. The lipid content was enhanced by 1.22-fold. BHA treatment simultaneously reduced carbohydrates andprotein and delayed the decay of chlorophyll. Furthermore, metabolomic analysis demonstrated that BHA up-regulated and activated the bioprocesses involved in cellular basal metabolism and signalling systems, such asglycolysis, the TCA cycle, amino acid metabolism and the phosphatidylinositol signalling system, thus enhancingastaxanthin and lipid accumulation. Altogether, this research shows the dramatic effects of BHA on algal me-tabolism in the regulation of key metabolic nodes and provides novel insights into microalgal regulation andmetabolism.

1. Introduction

Many bioactive compounds produced by microalgae have beenidentified as having nutritional value and medical applications (Rizwanet al., 2018). Among these compounds, the useful functions of

astaxanthin, which is a xanthophyll carotenoid composed of twoterminal six-membered ring structures and several conjugated un-saturated double bonds, have been widely investigated, and the resultssuggested that this compound can be synthesized de novo by someplants, microalgae, yeast and bacteria; this compound has several

https://doi.org/10.1016/j.biortech.2019.122002Received 20 June 2019; Received in revised form 9 August 2019; Accepted 10 August 2019

⁎ Corresponding author.E-mail address: xuya_yu@163.com (X. Yu).

1 These authors contributed equally to this work.

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activities, such as antioxidant and anti-inflammatory activities (Yanget al., 2013; Fakhri et al., 2018; Zhao et al., 2018). Currently, astax-anthin is widely used in various feed additives, pharmaceutical agentsand functional foods.

Haematococcus pluvialis (Chlorophyceae, Volvocales) is a unicellularfresh water microalga that is dispersed in many habitats worldwide andexhibits hyper-accumulation of astaxanthin, exceeding that in otherpublished sources such as plants and Chlorella zofingiensis; this organismis considered the best natural resource of astaxanthin and a majorproducer of this commercial compound (Panis and Carreon, 2016).Therefore, various strategies have been used to boost the accumulationof astaxanthin from H. pluvialis. It has been reported that under stress,such as salinity stress, high light intensity and nutrient depletion, H.pluvialis undergoes a series of physiological and biochemical changes,resulting in the biosynthesis of astaxanthin to respond to adversity(Ding et al., 2019a; Solovchenko, 2015). Moreover, the physiologicaland metabolic pathways of H. pluvialis can be regulated by variouschemical modulators and small molecules (Yu et al., 2015; Ding et al.,2018a). Although these approaches and strategies have enhanced theaccumulation of the desired metabolites, relatively few studies havereported the global profile of the physiological and metabolic pathwaysin H. pluvialis. Hence, the identification of the global profile regulatedby chemical modulators under stress conditions for the development ofpotential strategies is of utmost significance.

Phenols are a numerous and ubiquitous group of low-molecular-weight antioxidants that are commonly present in the human diet(Bravo, 1998). Butylated hydroxyanisole (BHA) is a common phenolthat is widely used in a variety of foods and pharmaceuticals and iswidely recognized for its well-defined antioxidant and anticancerproperties (Williams et al., 1999). In microalgae, antioxidant activityhas been attributed to the direct reciprocity of the compounds with thequenching of the reactive oxygen species (ROS) generated, leading toregulation of the oxidative signalling pathway and modification of theantioxidant system (Goiris et al., 2012; Ding et al., 2018a). Previousreports have demonstrated that BHA enhances lipid productivity andmay play a role as an oxidative signalling molecule to reinforce pho-toprotection in Nannochloropsis salina (Franz et al., 2013). Similarly, itwas observed that BHA could enhance lipid content by 8.80% at aconcentration of 30 μM in Crypthecodinium cohnii (Sui et al., 2014).Moreover, Shang et al. (2016) demonstrated that BHA enhanced as-taxanthin and fatty acid (FA) biosynthesis in H. pluvialis under nitrogenlimitation and high light intensity. Although the results presented inthese studies showed the close association of BHA with increasing mi-croalgal astaxanthin and/or lipid biosynthesis levels via a previouslyunknown regulatory mechanism, the key physiological parameters,cellular components and metabolic nodes remain to be investigated.

Metabolomics is recognized as being more closely reflective of thephysiology of organisms than all other “omics” technologies becausemetabolites are terminal products biosynthesized after complex tran-scriptional, translational, and regulatory processes. Thus, metabolomicshas been applied to obtain a global understanding of the regulatorynetworks involved in microalgae metabolism (Kokabi et al., 2019). Theeffects of BHA on H. pluvialis cultures have been examined in previousworks; however, (Shang et al., 2016), little information is available onthe potential molecular mechanisms involved in microalgal metabolicnetworks.

In this study, the physiological responses and metabolic events as-sociated with the adaptation of H. pluvialis to BHA induction under highlight intensity combined with nitrogen deficiency were compared bymetabolomics. The study provided a global profile of the metabolomicbasis of the effect of BHA-mediated induction of astaxanthin bio-synthesis in H. pluvialis and provided novel insights into the functionaldeterminants of astaxanthin accumulation.

2. Materials and methods

2.1. Algal strain and growth conditions

Haematococcus pluvialis LUGU (18S GenBank: KM115647.1) wasobtained from Lake Lugu (27°42′00″N and100°47′00″E) and was usedin this study (Shang et al., 2016). The seed strain was conserved andgrown in BBM at 25 ± 1 °C in a bubbling column photobioreactor(0.2 m diameter, 0.3 m height, 3 L) illuminated with cool-white fluor-escent light at 30 μmolm−2 s−1 for 14 days.

For the BHA treatment culture, BHA was dissolved in di-methylsulfoxide (DMSO). The microalgae in the late exponentialgrowth stage were centrifuged and washed with aseptic water. Greenvegetative H. pluvialis LUGU cells were cultivated at a density of ca.2.5× 105 cells mL−1 in a 500-mL Erlenmeyer flask containing 300mLof nitrogen-deficient BBM with 2mg L−1 BHA (Shang et al., 2016) andwith filtered air bubbled through the culture at a rate of 0.4 vvm. Thecontrol was supplemented with an equal amount of DMSO. The lightintensity was 50 μmolm−2 s−1, and the cultivation temperature was25 ± 1 °C. Each group was examined in quadruplicate, with samplesharvested every three days.

2.2. Quantification of biomass, astaxanthin and lipid levels

Microalgal cells were isolated by centrifugation (5min, 3800×g),washed three times with distilled water, and dried to constant weight ina vacuum freeze dryer at−80 °C. Astaxanthin and lipids were extractedand detected according to a previously described method (Ding et al.,2019a; Zhao et al., 2014).

2.3. Measurement of the chlorophyll, carbohydrate, protein and ROS levelsof H. pluvialis

The chlorophyll content of H. pluvialis was isolated using themethod described by Wellburn (1994). Lyophilized microalgal cellsfrom the BHA treatment groups and the control group were used forextraction and measurement of the total carbohydrate and proteincontent as previously described, with glucose and bovine serum al-bumin (BSA) as standards (Ma et al., 2016; Berges et al., 1993). TheROS levels were monitored using 2′,7′-dichlorodihydrofluorescein dia-cetate (Beyotime; Shanghai, China) as a probe, as previously described(Ding et al., 2019a).

2.4. Metabolite extraction and LC-MS/MS-based metabolomic analysis

Fifty milligrams of algal sample were homogenized with 1mL ofextraction solvent (containing an internal standard, acetonitrile-me-thanol-water at 2:2:1) at 45 Hz for 4min and sonicated in an ice-waterbath for 5min. Then, the samples were incubated for 1 h at −20 °C andcentrifuged at 12,000×g for 15min at 4 °C. The obtained supernatantswere aliquoted to vials and stored at −80 °C for UHPLC-QE Orbitrap/MS analysis. A mixture of equal amounts of the supernatants from all ofthe samples was prepared as a quality control (QC) sample.

A UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3column (2.1 mm×100mm, 1.8 μm) coupled to a Q Exactive system(Orbitrap MS, Thermo) was used for LC-MS/MS analyses. Mobile phaseA was 0.1% formic acid and 5mM ammonium acetate, and mobilephase B was acetonitrile. The elution gradient was set as follows:0–1min, 99% A; 1–8min, 1% A; 8–10min, 1% A; 10–12min, 99% A.The flow rate was 0.5 mLmin-1. The injection volume was 2 μL. The QEmass spectrometer was used for its ability to acquire MS/MS spectra onan information-dependent basis (IDA) during LC/MS experiments. Inthis mode, the acquisition software (Xcalibur 4.0.27, Thermo) con-tinuously evaluates the full-scan survey MS data as it collects andtriggers the acquisition of MS/MS spectra depending on preselectedcriteria. ESI source conditions were as follows: sheath gas flow rate, 45

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Arb; aux gas flow rate, 15 Arb; capillary temperature, 320 °C; full MSresolution, 70,000; MS/MS resolution, 17,500; collision energy, 20/40/60 eV in the NCE model; spray voltage, 3.8 kV (positive) or −3.1 kV(negative).

2.5. Multivariate statistical analysis

All raw data profiles were normalized to the quality control and thenumbers of the samples, and then, the unsupervised dimensionalityreduction method principal component analysis (PCA) was applied toall samples using R package models (http://www.r-project.org/). Torefine this analysis, a variable importance in projection (VIP) score ofthe (O)PLS model was applied to rank the metabolites that best dis-tinguished the two groups. The VIP threshold was set to 1. In addition,the T-test was also used as a univariate analysis for screening differ-ential metabolites. Metabolites with T-test P-values < 0.05 andVIP≥ 1 were considered differential metabolites between the twogroups. A heatmap was generated using MultiExperiment Viewer soft-ware, which is publicly available at http://www.tm4.org/.

KEGG is the major pathway-related public database that includesnot only genes but also metabolites. Metabolites were mapped to KEGGmetabolic pathways for pathway analysis and enrichment analysis.Pathway enrichment analysis identified significantly enriched meta-bolic pathways or signal transduction pathways in differential meta-bolites compared with the whole background. The calculations wereperformed according to a previously described method (Kanehisa et al.,2007).

2.6. RNA isolation and real-time quantitative RT-PCR

Total RNA was isolated from H. pluvialis, and cDNA was synthesizedas previously described (Ding et al., 2018b). The mRNA levels of the lcy,bkt, fad, acp and 18s genes were analyzed with an ABI 7500 Real-TimePCR System with SYBR Green Master Mix (TaKaRa; Shanghai, China)using the primers listed in the Supplementary data. The expression le-vels were normalized against the H. pluvialis 18S rRNA gene (an internalcontrol), which was expressed at a constant level under all experimentalconditions. The relative gene expression was determined according tothe 2−ΔΔCT method (Livak and Schmittgen, 2001).

3. Results and discussion

3.1. Effect of BHA on the biomass, astaxanthin and lipid levels of H.pluvialis

During the 13 d of cultivation, the H. pluvialis biomass increasedsignificantly from 0.26 and 0.24 g L−1 at day 1 to 0.59 and 0.52 g L−1

in the control and BHA treatment groups (Fig. 1A), respectively. No-tably, the highest biomass concentration was not markedly differentbetween the control and BHA treatment groups.

Detection of intracellular astaxanthin levels in H. pluvialis showedthat the astaxanthin content gradually increased from 3.97 at day 1 to29.67mg g−1, indicating a nearly 2.17-fold improvement comparedwith the control (13.66 mg g−1) after 13 d (Fig. 1B). Concurrently, amarked increase in lipid content was observed after exposure to BHAtreatment. The peak lipid content was 42.84%, which was 1.22-foldhigher than that observed without BHA treatment (35.19%) (Fig. 1C).These results indicate that the addition of BHA under nitrogen defi-ciency and high light intensity, despite not having an effect on micro-algal growth, can affect the concurrent accumulation of astaxanthin andlipids.

H. pluvialis has been widely investigated because of the capacity ofthis organism to produce the highly valuable ketocarotenoid astax-anthin under inductive conditions and various stress conditions. Therehas been much research aimed at elucidating the astaxanthin biosyn-thetic pathway and the regulation of this pathway (Li et al., 2019; Ma

et al., 2018; Sun et al., 2018; Ding et al., 2018a,b). Nevertheless, pri-mary astaxanthin biosynthesis utilizes the carotene component as aprecursor, and bulk accumulation of astaxanthin also occurs via de novoFA and lipid synthesis (Schoefs et al., 2001; Chen et al., 2015). In H.pluvialis, the free astaxanthin biosynthesized under adversity was es-terified with FA and deposited in triacylglycerol (TAG)-rich cytosoliclipid bodies (LBs) (Holtin et al., 2009). In this study, BHA treatmentconcurrently stimulated hyper-accumulation of lipids and astaxanthinin H. pluvialis, astaxanthin and lipids in the BHA treatment group ac-cumulated to levels that were 2.17 and 1.22 times higher, respectively,than those in the untreated group (Fig. 1B and C). This result indicatesthat the synthesis of astaxanthin is closely correlated with the accu-mulation of lipids and the formation of LB for astaxanthin accom-modation. Moreover, as an antioxidant, BHA can block the peroxidationof lipids and alleviate product inhibition of carotenogenesis by freeastaxanthin. Similar results were reported by Zhao et al. (2018), Dinget al. (2018b) and Chen et al. (2015). Furthermore, observable changesin the linoleic acid metabolic pathway and biosynthesis of unsaturatedfatty acids were observed by metabolomic pathway enrichment (Fig. 4).As reported by Shang et al. (2016), the major acids, namely, palmitic(16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and linolelaidic(18:3) acids, were up-regulated during BHA treatment. Furthermore, denovo synthesis of astaxanthin and lipids via the front-end precursorsoccurs via a rapid flux through the intermediates. However, the role ofBHA in algal physiology and metabolic networks remains unclear.

3.2. Comparative analysis of the physiological effects of concurrentaccumulation of astaxanthin and lipids in H. pluvialis due to exogenous BHAtreatment

When exposed to inductive and/or stress conditions, the physiologyof algal cells often changes in response to environmental changes(Boussiba, 2000; Zhao et al., 2018). While many studies on H. pluvialishave detailed the culture method for astaxanthin biosynthesis, fewstudies have concentrated on physiological changes during the bio-synthesis of astaxanthin in H. pluvialis under inductive and stress con-ditions. Including two major categories: changes in microalgal activityand cellular components. The most significant physiological variationin H. pluvialis during the biosynthesis of astaxanthin is the reduction inchlorophyll content, as described in previous research (Ding et al.,2018a,b; Zhao et al., 2018). After cultivation of microalgae underabiotic stress, the chlorophyll content in the BHA treatment group andin the control were analyzed during induction. As illustrated in Fig. 2A,the chlorophyll content concomitantly decreased as the astaxanthinlevel increased. However, the chlorophyll content of the control de-creased sharply by 38%, which was a greater decrease than that ob-served for the experimental group (33%) during cultivation. This de-crease was attributed mainly to the reduction in O2 evolution rate orproduction of ROS (reactive oxygen species) (Boussiba, 2000; Dinget al., 2018b). Similar to previous studies, the carotenoid/chlorophyllratio (Car/Chl), representing the status of photoprotective functionagainst oxidative stress under nutrient-deprived conditions, exhibited acontinuous increase with the addition of NaHCO3 relative to the non-addition ratio (Qi et al., 2019). Matching results are shown in Fig. 2B.The intracellular ROS level was surged in cells exposed to nitrogenstarvation and high light in both the control and BHA groups. At 5 d,the highest level of ROS in BHA-treated cells was decreased by 18.7%compared to the control. Subsequently, the ROS level significantly de-creased with the accumulation of astaxanthin compared with the con-trol. Ding et al. (2018b) also found that in the microalga H. pluvialis,treatment with melatonin, which is an antioxidant, can suppress ROSbursts and prevent damage to the photosynthetic system.

Conversely, the carbohydrate content increased from 35.14% DW atday 1 to 51.56% DW at day 5 (Fig. 2C). However, after 5 d, the car-bohydrate content decreased significantly (ca. 15.32 and 7.5% in theBHA and control groups, respectively), and the BHA treatment group

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Fig. 1. (A) Effect of exogenous butylated hydroxyanisole (BHA) on the biomass of H. pluvialis during induction. (B) Effect of exogenous butylated hydroxyanisole(BHA) on the astaxanthin content of H. pluvialis during induction. (C) Effect of exogenous butylated hydroxyanisole (BHA) on the lipid content of H. pluvialis duringinduction. Vertical bars represent the means ± SD (n=4). *indicates statistical significance at p < 0.05, **indicates statistical significance at p < 0.01 comparedwith the control.

Fig. 2. Effect of exogenous butylated hydroxyanisole (BHA) on physiological of H. pluvialis. (A) Chlorophyll concentration, (B) ROS levels, (C) carbohydrate content,(D) protein content. Vertical bars represent the means ± SD (n= 4). * indicates statistical significance at p < 0.05, ** indicates statistical significance at p < 0.01compared with the control.

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maintained a low level throughout the remainder of the culture period.H. pluvialis mainly relies on photosynthetically fixed carbon, which isstored in carbon-containing molecules and diverted to numerouspathways for the synthesis of major macromolecules. After BHA treat-ment, the conversion of green motile cells to red aplanospores occursconcomitantly with a substantial enhancement in carbohydrate levels,which may reach up to 61.56% of the cellular dry weight. This findingindicates that exogenous BHA treatment played a positive role incarbon fixation during microalgal photosynthesis compared with thecontrol. Additionally, transformation to a resting phase (lower meta-bolism) and biosynthesis of compounds do not require high energyinput. Subsequently, the carbohydrate content began to decrease andwas lower than that of the control (Fig. 2C). The acceleration of thedepletion of carbohydrates may be due to the accumulation of sec-ondary metabolites, such as lipids and astaxanthin, under abiotic stress.Fernandes et al. (2013) indicated that the starch levels of the algaParachlorella kessleri exhibited a transitory increase concurrent with arise in lipid accumulation, followed by a piecemeal decrease in starchcontent under nutrient-deficient conditions. Furthermore, an improve-ment in lipid accumulation associated with astaxanthin biosynthesiswas also observed in the BHA treatment group (Fig. 1C).

The protein content (Fig. 2D) continued to increase after the first 5 dof the experiment; however, as the result of the increase in biomass, theprotein content in the biomass of the BHA treatment and control groupsdecreased greatly from 36.36 to 9.05mg g−1 and 35.06 to22.23mg g−1, respectively, after 13 d. As configurational componentsand functional biomacromolecules in microalgae, proteins are vulner-able to denaturation under cultivation conditions (Brikis et al., 2018).Under nitrogen starvation conditions, proteins, as an intracellular ni-trogen pool, play an important role in the regulation of nitrogen me-tabolism. The results showed the distinct activation effect of BHA on Nmetabolism in H. pluvialis. Significant upregulation of numerous aminoacid (AA) pathways was observed, including the tyrosine, phenylala-nine, tryptophan, methionine, threonine and GABA pathways (Fig. 5).Under N deficiency, the cell protein levels decreased significantly, andN was perhaps recycled into AAs to alleviate stress. Moreover, cata-bolism-related proteins could be re-distributed as sugars and lipidsfollowing shifting of carbon skeletons during biosynthesis of carbon-containing compounds. A similar phenomenon to changing proteincontent was observed in H. pluvialis upon treatment with butylatedhydroxytoluene (Zhao et al., 2018). The current result also shows acentral role of the tricarboxylic acid cycle (TCA cycle) in astaxanthinand lipid accumulation upon BHA treatment (Fig. 5). The enhancedactivity of the TCA cycle could provide a rich substrate for astaxanthinand lipid biosynthesis in the form of additional malate. Interestingly,the levels of the substrates (pyruvate, glycerol-3-phosphate and mal-onyl-CoA) used to synthesize astaxanthin and lipids also simultaneouslyincreased. These results suggested that BHA treatment could dominatephysiological processes and indirectly regulate metabolic partitioningof astaxanthin and lipids biosynthesis in H. pluvialis.

3.3. Comparison of the metabolite changes in H. pluvialis under exogenousBHA treatment

To examine the metabolic profiles and metabolic division of astax-anthin and lipid metabolism in H. pluvialis under exogenous BHAtreatment, samples were detected by UHPLC. Altogether, 353 metabo-lites of central metabolism were identified (Supplementary data). Thesimilarities and differences between metabolomic profiles are shown inPCA plots (Fig. 3A). PCA plots of the metabolomic profiles 5 and13 days after supplementation with BHA in combination with stressconditions were generated. The score plots clearly suggested clusteringof each biological replicate sample. Additionally, a suitable distancebetween the metabolomic profiles of the control and BHA-treatedsamples was detected. Moreover, the metabolomic results of the BHAtreatment and control samples under nitrogen starvation and high light

intensity among all samples were distinct at both culture times. Amongall the groups, BHA treatment was the primary determinant factor, asprofiles with and without BHA treatment were well separated in thePCA plots at both 5 and 13 d, which is consistent with the accumulationof astaxanthin under these conditions presented above (Fig. 1B). Theseconsequences showed the overall high reproducibility of the metabo-lomics analysis.

Heat maps of 21 metabolites in microalgae with or without BHAtreatment at 5 and 13 d are presented (Fig. 3B). While the fold changeswere relatively small, the results showed distinct upregulation of mel-atonin at 5 d and pyruvic acid, tyrosine, glucose-6-phosphate, malicacid, glycerol-3-phosphate, gluconic acid, succinate, maltose, γ-ami-nobutyric acid (GABA) and nicotinamide adenine dinucleotide (NAD) at13 d. The level of uric acid was reduced. Among the metabolites in theBHA treatment group, pyruvic acid is an important zymolyte for as-taxanthin accumulation, and glycerol-3-phosphate is a necessary sub-strate for lipid biosynthesis, providing the C-skeleton required for theformation of astaxanthin and lipids from photosynthesis in microalgae.Interestingly, heat map analysis indicated that glucose, which is anessential precursor for astaxanthin and lipid synthesis, was not

Fig. 3. (A) PCA analysis of metabolomic profiles. Score plot of cell samplescollected at 5 and 13 d. (B) Heat maps of metabolomic profiles. The numberrepresents biological replicates.

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markedly regulated in the BHA-treated samples. This result indicatesthat glucose may maintain a metabolic balance throughout the meta-bolic process. The enhancement of astaxanthin and lipid biosynthesisby BHA treatment may not be directly associated with the increasedsupply of the precursor glucose.

3.4. Pathway enrichment and KEGG analyses

To further explore the dominant role of BHA in the changes in keymetabolic pathways underlying the function of this compound in theimprovement of astaxanthin and lipid accumulation, KEGG analyseswere performed for the BHA treatment and control groups. The resultsshowed that BHA supplementation differentially regulated mis-cellaneous metabolites involved in key metabolic pathways (Fig. 4) andmodified the cellular metabolites. The top 20 enriched pathways wereidentified by KEGG pathway analysis. Pathway enrichment analysisshowed that the pathways “Biosynthesis of unsaturated fatty acids”,“Alanine, aspartate and glutamate metabolism”, “Linoleic acid meta-bolism”, “Glucosinolate biosynthesis”, “Amino sugar and nucleotidesugar metabolism”, “Butanoate metabolism”, “Inositol phosphate me-tabolism”, and “Phosphatidylinositol signalling system” were enrichedwith statistical significance (p-value < 0.05). However, no significantpathway enrichment was observed for “Valine, leucine and isoleucinebiosynthesis”, “Folate biosynthesis”, “Aminoacyl-tRNA biosynthesis”,“Arginine and proline metabolism”, “Photosynthesis”, and “Valine,leucine and isoleucine degradation”.

In microalgae, sugars are essential as respiratory substrates for thegeneration of energy and metabolic intermediates via the glycolysispathway that are then used for the synthesis of macromolecules. When

H. pluvialis is exposed to nitrogen starvation conditions, it leads tocarbon–nitrogen disequilibrium. Chen et al. (2017) reported that excesscarbon obtained from photosynthesis or glycolysis was re-distributedinto carbon-containing compounds, such as glucose-6-phosphate, fruc-tose-6-phosphate, citrate and leucine, and then diverted into lipid me-tabolism for the production of storage lipids via the GABA pathway,glycolysis, and the tricarboxylic acid cycle (TCA). In this study, meta-bolic profiling showed that BHA acceleration increased the contents ofglucose-6-phosphate and pyruvate (Fig. 5), which was produced bystarch through the glycolytic pathway. These results showed that theglycolytic pathway in the BHA treatment group maintained high ac-tivities, which may be related to the massive accumulation of carbo-hydrates. Moreover, global metabolite profiling also led to the fol-lowing observations (Fig. 5). In the BHA treatment group, the level ofmaltose, a disaccharide produced by starch degradation, increasedsignificantly at 13 d, indicating C flow from starch to glycolysis; mal-tose could be transformed into the carbon skeleton for astaxanthin andFA biosynthesis (Recht et al., 2014). Concomitantly, a large amount ofglycerol-3-phosphate, the backbone of TAG, was produced after BHAexposure. Similarly, a relationship between starch degradation andglycerol accumulation was observed after salt supplementation in Du-naliella tertiolecta (Goyal, 2007). Mannose levels also increased aftertreatment with BHA. This sugar has been reported to be correlated withstress response, carbon partitioning in the cell, and antioxidant sig-nalling (Hameed and Iqbal, 2014). Furthermore, an increase in malateand succinate levels was observed in the BHA treatment group. Malatefrom the TCA cycle could be directed to the fatty acid biosyntheticpathway through NADP-dependent malic enzyme, as observed byBender et al. (2014). This result may be due to the relatively strong shift

Fig. 4. KEGG pathways enrichment integrative analysis of butylated hydroxyanisole (BHA) vs control in H. pluvialis.

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from glycolysis to the TCA cycle for temporary storage of the carbonpool. Additionally, the TCA cycle could be upregulated in response tohigh levels of protein and amino acid degradation, which generatesTCA cycle intermediates and provides precursors for the resynthesis ofcertain amino acids, as observed by Hockin et al. (2012). Such as aGABA shunt, which is a means for generating C:N fluxes that enter theTCA cycle by assimilating carbons from glutamate. In this study, BHAtreatment upregulated the TCA cycle under abiotic stress and enhancedthe concentration of GABA and astaxanthin. Similar results were alsofound in our previous research that salt stress promoted the accumu-lation of GABA and astaxanthin in H. pluvialis (Ding et al., 2019b).Furthermore, Shaikh et al. (2019) reported that key metabolites, suchas malate and α-ketoglutarate, involved in the TCA cycle could beconverted into fatty acid biosynthesis by acetyl-CoA carboxylase

enzyme (ACCase). Thus, a coordinated upregulation of the TCA cycleand glycolysis might shift the flow of carbon skeletons towards fattyacid biosynthesis.

GABA, a non-protein amino acid, has been reported as a new plantgrowth regulator that is responsive to abiotic stress (Li et al., 2017). Onthe other hand, GABA metabolism occupies a crucial position betweenorganic acid and amino acid biosynthesis (Chen et al., 2017). Thisphenomenon is consistent with the increased GABA levels during BHAtreatment, and this change was accompanied by decreased glutamatelevels. Moreover, amino acid levels also increased in BHA-treated cells.Alipanah et al. (2015) reported that H. pluvialis might utilize its ownendogenous nitrogen for essential amino acids and NAD(P)H synthesis,which supplied NAD(P) H-dependent pathways such as astaxanthinbiosynthesis. Therefore, the upregulation of amino acid metabolism is

Fig. 5. Simplified scheme of the major carbon-nitrogen metabolism in H. pluvialis and the changes in identified metabolites throughout 13 d of butylated hydro-xyanisole (BHA) treatment under nitrogen starvation and high light. Increase (red) and decrease (green) in the cellular metabolite’s content are indicated; ( ) iscontrol and ( ) is BHA treatment group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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beneficial for astaxanthin biosynthesis. Inositol, a derivative of cyclo-hexane with six alcohol groups, plays an important role as a structuralcomponent of multiple secondary messengers in eukaryotic cells.Moreover, inositol is associated with the phosphatidylinositol signallingpathway involved in auxin transport, phytic acid biosynthesis, cell wallbiosynthesis, and the production of resistance-related molecules and isregarded as an important compound for normal plant growth and de-velopment (Stevenson et al., 2000). In this study, the key metaboliteinositol association with the phosphatidylinositol signalling system in-creased progressively in the BHA group. Cho et al. (2015) reported thatmyo-inositol treatment could enhance growth and lipid production inthe microalga Dunaliella salina. In this study, a significant change ininositol phosphate metabolism and the phosphatidylinositol signallingsystem was observed after BHA exposure (Fig. 4). Phosphoinositides arenecessary metabolites that regulate cellular physiology in eukaryotes.Previous results have suggested that phosphatidylinositol could serve asa key signalling lipid molecule with significant roles in lipid turnoverand serves as the precursor for lipogenesis (Hou et al., 2016). Althoughthe physiological changes, metabolites and metabolic pathways asso-ciated with the concurrent hyper-biosynthesis of astaxanthin and lipidsin H. pluvialis were observed and identified, these results require furtherverification.

3.5. qRT-PCR analysis elucidated that BHA treatment orchestrated theexpression of genes involved in lipid and astaxantnin biosynthesis pathways

In previous research, almost all of the genes of H. pluvialis involvedin astaxanthin synthesis from pyruvate were shown to be simulta-neously upregulated in transcriptional expression in inductive andstress conditions (Li et al., 2019; Ding et al., 2018b). Moreover, thegenes involved in the lipid biosynthetic pathway also showed hightranscriptional expression levels (Ding et al., 2018a). To verify theupregulation of the astaxanthin and lipogenesis pathways after BHAtreatment, qRT-PCR was performed on five selected genes involved inastaxanthin and lipid biosynthesis (Fig. 6). The qRT-PCR results cor-related well with the metabolomic analysis. Increased transcription ofgenes encoding acyl carrier protein (ACP) and fatty acid desaturase(FAD) was observed 9 days after BHA treatment. Of particular note isthe higher expression of acp, which participated in the initial conden-sing reaction in fatty acid biosynthesis and the encoding of acyl carrierprotein as an important component in both fatty acid and polyketidebiosynthesis (Shang et al., 2016), with more than 1.9-fold than thecontrol. Fatty acids can be further desaturated to different levels by aseries of desaturases, such as fatty acid desaturase (FAD) and ketoacyl-ACP-synthase (KAS), which provide zymolyte for lipid synthesis in

Fig. 6. Effects of BHA treatments on the transcriptional levels and expression kinetics of lipid and astaxanthin biosynthetic genes in H. pluvialis (acp: acyl carrierprotein; fad: fatty acid desaturase; kas: ketoacyl-ACP-synthase; lcy: lycopene β-cyclase: bkt: β-carorene ketolase).

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microalgae (Goncalves et al., 2016).Lycopene β-cyclase (LCY) catalyzes the conversion of lycopene to β-

carotene, a key precursor in astaxanthin formation that is responsiblefor providing the substrate for astaxanthin accumulation. After BHAtreatment, the expression of lcy is gradually up-regulated, therebyproviding sufficient substrate for subsequent reactions. Similar to theupregulation of the lcy gene in H. pluvialis by melatonin, the astaxantnincontent of the cells also increased (Ding et al., 2018a). A major genedownstream of the astaxanthin synthesis pathway, β-carotene ketolase(BKT), was most highly expressed after 9 d of culture. This behaviour isin line with the result of an earlier study that explored a strategy forenhancing astaxanthin accumulation (Li et al., 2019). These results areconsistent with the concurrent hyper-accumulation of astaxanthin andlipids in H. pluvialis upon treatment with BHA.

4. Conclusions

The effects of BHA on microalgal physiological characteristics, cel-lular metabolites and metabolic networks in H. pluvialis under nitrogendeficiency and high light intensity were comprehensively discussed.Interestingly, the results demonstrated that BHA unprecedentedlygoverns the metabolites involved in glycolysis, the TCA cycle, aminoacid metabolism and the phosphatidylinositol signalling system, con-currently increasing the astaxanthin and lipid content. In summary, thisstudy provides details regarding the BHA-mediated increase in micro-algal astaxanthin and lipid biosynthesis via effective regulation of keymetabolic nodes, which will be beneficial for future optimization ofastaxanthin and lipid production in H. pluvialis.

Acknowledgments

This work was funded by the National Natural Science Foundationof China (21766012), Key Science and Technology Project of YunanProvince (2018ZG003).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biortech.2019.122002.

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