Biomass and Lipid Accumulation Kinetics and the Transcriptome of Oleaginous Microalga Tetradesmus Bernardii (Chlorophyceae) Under Heterotrophic Culture With Different Nitrogen Source Supply and Carbon/Nitrogen Ratio

Background: Heterotrophic cultivation of microalgae has been proposed as a viable alternative method for novel high-value biomolecules, enriched biomass and biofuel production because of their allowance of high cell density levels, as well as simple production technology. Tetradesmus bernardii, a newly isolated high-yielding oleaginous microalga under photoautotrophic conditions, is able to grow heterotrophically, meaning that it can consume organic carbon sources in dark condition. We investigated the effect of different carbon/nitrogen (C/N) ratios on the growth and lipid accumulation of T. bernardii in heterotrophic batch culture under two nitrogen sources (NaNO 3 , CO(NH 2 ) 2 ). In addition, we conducted time-resolved transcriptome analysis to reveal the metabolic mechanism of T. bernardii in heterotrophic culture. Results: T. bernardii can accumulated high biomass concentrations in heterotrophic batch culture which the highest biomass of 46.09 g/L was achieved at 100 g/L glucose concentration. The rate of glucose to biomass was exceed 55% when the glucose concentration was less than 80 g/L, and the C/N ratio was 44 at urea treatment. The culture was benecial to lipid accumulation at a C/N ratio between 110 and 130. NaNO 3 used as a nitrogen source enhanced the lipid content more than urea, and the highest lipid content was 45% of dry weight. We performed RNA-seq to analyze the time-resolved transcriptome of T. bernardii. As the nitrogen was consumed in the medium, nitrogen metabolism related genes were signicantly up-regulated to speed up the N metabolic cycle. As chloroplasts were destroyed in the dark, the metabolism of cells was transferred from chloroplasts to cytoplasm. However, storage of carbohydrate in chloroplast remained active, mainly the synthesis of starch, and the precursor of starch synthesis in heterotrophic culture may largely came from the absorption of organic carbon source (glucose). With regard to lipid metabolism, the related genes of fatty acid synthesis in low nitrogen concentration increased gradually with the extension of cultivation time. Conclusion: T. bernardii exhibited rapid growth and high lipid accumulation in heterotrophic culture. It may be a potential candidate for biomass and biofuel production. Transcriptome analysis showed that multilevel regulation ensured the conversion from carbon to the synthesis of carbohydrate and lipid. in chloroplasts and mitochondria, respectively. The expression of GDH1 and GDH2 was down-regulated in HN and LN. GS1 located in cytoplasm was gradually up-regulated with culture time, but GS2 located in chloroplast maintained a certain expression level in the early stage of culture and then was down-regulated, indicating that the reaction was transferred to cytoplasm in the late stage. The resulting glutamine (Gln) was then reduced to Glu catalyzed by glutamate synthase (or glutamate oxglutarate amino transferase, GOGAT). Similarly, GOGAT includes two types which were located in the cytoplasm (GOGAT1) and chloroplast (GOGAT2). The results showed that the gene of GOGAT1 was slightly up-regulated, and the gene of GOGAT2 was maintained. In heterotrophic culture, ammonia may be mainly metabolized by GO/GOGAT in T. bernardii.

organic substrates [21,22]. To evaluate the effect of different glucose concentrations on growth and lipid accumulation, T. bernardii was cultured in mEndo medium with a glucose concentration ranging from 10 to 80 g/L and nitrogen concentration of 18 mM under inorganic and organic nitrogen sources, which were NaNO 3 and urea, respectively. Biomass under different glucose concentrations showed that there was no signi cant difference in biomass on day 12 and day 18 (P>0.05) (Fig. 1a,b,c,d). In the culture with NaNO 3 used as nitrogen source and glucose concentration from 10 to 60 g/L, the biomass of T. bernardii increased with increasing initial glucose concentrations, and the highest biomass achieved was 21.55 g/L. When the glucose concentration was more than 60 g/L, biomass no longer increased but decreased. The decrease in biomass was likely due to high glucose inhibition, which is a common phenomenon in heterotrophic batch cultures [13]. The highest glucose-to-biomass conversion rate was 43.4%, which was obtained at a glucose concentration of 20 g/L. When urea was used as nitrogen source, the biomass of T. bernardii increased with increasing glucose concentrations from 10 to 40 g/L. There was no signi cant difference in biomass when the glucose concentration exceeded 40 g/L (P>0.05). The highest biomass after 12 days of culture was 17.90 g/L. The conversion rates of glucose to biomass in this treatment were 55.4%, 54.65%, 50.23%, 43.5%, 34.72% and 28.98% at the glucose concentration of 10,20,30,40,50, and 60 g/L, respectively. Compared with biomass under two nitrogen sources, the biomass under heterotrophic culture with urea was signi cantly higher than that of NaNO 3 when the glucose concentration was 10-40 g/L.
The biomass of culture with NaNO 3 used as nitrogen source, however, was signi cantly higher than that of urea when the glucose concentration was more than 50 g/L. The biomass of T. bernardii not only was affected by the initial glucose concentration, but also varied with nitrogen sources.
In the culture with NaNO 3 used as nitrogen source and the nitrogen concentration of 18 mM, there was no signi cant difference between the lipid content of T. bernardii at day 18 and day 12 at the glucose concentration of 10 and 20 g/L (Fig. 1e, f). When the glucose concentration was more than 30 g/L, the lipid content of T. bernardii at day 18 was signi cantly higher than that at day 12 (P<0.01). The lipid content at day 18 showed an increasing effect of glucose concentration on lipid accumulation as glucose concentration increased from 10 to 70 g/L, and the highest lipid content reached 46.06% of dry weight. When the glucose concentration was more than 70 g/L, the lipid content decreased. In the culture with urea used as nitrogen source and the nitrogen concentration of 18 mM, there was no signi cant difference between the lipid content of T. bernardii at day 18 and day 12 at the glucose concentration of 10 g/L. When the glucose concentration was more than 20 g/L, the lipid content of T. bernardii at day 18 was signi cantly higher than that of day 12. According to the lipid content at day 18, the lipid content increased with the increase of glucose concentration in glucose concentration from 10 to 40 g/L, and the maximum lipid content was 32.27% of dry weight. When the glucose concentration exceeded 40 g/L, the lipid content no longer increased (Fig. 1g, h). In photoautotrophic culture, stress conditions commonly increase lipid content but decrease the biomass, whereas heterotrophic culture simultaneously accumulate lipids and biomass [13]. It was veri ed in T. bernardii cultured heterotrophically both NaNO 3 or urea as a nitrogen source. High lipid content and high biomass were both obtained at high glucose concentration (70 g/L of glucose at NaNO 3 group, 60 g/L of glucose at urea group). Notably, the effect of inorganic nitrogen source, NaNO 3 , on the increase of lipid content, was much better than that of organic nitrogen source, urea. Similar results were found in Crypthecodinium cohnii, where NaNO 3 supported much higher lipid content (26.9% of dry weight) than other nitrogen sources, including urea [23]. In Scenedesmus, NaNO 3 was also reported to yield the highest lipid content among other nitrogen sources [24]. In addition to the glucose concentration and nitrogen source, culture time also had a signi cant effect on lipid accumulation.
Ordinarily, lipid content attained its maximum value at stationary growth phase in heterotrophic culture, and the cells should be harvested at early stationary phase because of lipid degradation [18]. In heterotrophic culture of T. bernardii, the cell reached stationary growth phase on day 12, while the lipid content on day 18 was much higher than it was on day 12 at high glucose concentration. Lipid accumulation could increase with the extension of culture time, which indicated that lipid synthesis was still active at the stationary phase in T. bernardii.
2.2 Effects of different NaNO 3 concentrations on the growth and lipid accumulation of T. bernardii at a glucose concentration of 60 g/L According to the result that T. bernardii reached the highest biomass at a glucose concentration of 60 g/L, we investigated the effects of different NaNO 3 concentrations on the biomass and lipid content of T. bernardii at the glucose concentration of 60 g/L (Fig. 2). When the nitrogen concentration of NaNO 3 increased from 4.5 mM to 36 mM, the biomass of T. bernardii increased with the nitrogen concentration, and the highest biomass of 24.72 g/L was achieved, with the highest glucose to biomass conversion rate of 40.37%. When nitrogen concentration was more than 36 mM, the biomass decreased with increasing nitrogen concentration. The lipid content of T. bernardii at day 18 was signi cantly higher than that at day 12 under different nitrogen concentrations. When the nitrogen concentration was 4.5-15 mM, the lipid content increased with an increasing nitrogen concentration. The lipid content decreased, however, with the increase of nitrogen concentration when the nitrogen concentration was above 15 mM. The highest lipid content of 45.35% of dry weight was obtained with a C/N molar ratio of 133, which was most suitable for lipid accumulation with NaNO 3 used as nitrogen source.
2.3 Effects of different initial C/N ratio on the growth and lipid accumulation of T. bernardii under the culture with urea used as nitrogen source We noticed that the conversion rates of glucose to biomass with urea used as nitrogen source was much higher than it with NaNO 3 , and the highest conversion rate reached to 55.4%. To identify the highest biomass that the culture with urea used as nitrogen source could achieve, the culture with varying glucose concentrations (20,40,60,80,100, and 120 g/L) and different nitrogen concentration of urea (3 mM to 120 mM) in T. bernardii, was conducted. There was no signi cant difference in biomass between day 12 and day 18 at glucose concentrations of 20, 40, 60, 80, and 100 g/L ( Fig. S1; S2). Biomass had a similar trend with the change in nitrogen concentration as that under different glucose concentrations, where biomass increased with increasing nitrogen concentration but then plateaued. At the glucose concentration of 20 g/L, the biomass of T. bernardii increased with enhancing nitrogen concentration from 3 to 18 mM, and the highest biomass of 10.87 g/L was obtained (Fig. S1a, b). When the nitrogen concentration was more than 18 mM, biomass decreased with the increase of nitrogen concentration. At the glucose concentration of 40 g/L and nitrogen concentration increased from 4.5 mM to 36 mM, the biomass of T. bernardii increased with nitrogen concentration (Fig. S1c, d). The highest biomass obtained was 23.07 g/L, and the glucose to biomass conversion rate was 56.43%. At the glucose concentration of 60 g/L, the biomass of T. bernardii increased with the increase of nitrogen concentration from 4.5 mM to 45 mM (Fig. S1e, f). The highest biomass was 34.48 g/L, and the glucose conversion rate was 56.63%. At the glucose concentration of 80 g/L and the nitrogen concentration of 4.5-60 mM, the highest biomass was 44.99 g/L, with glucose conversion rate of 55.61% (Fig. S2a, b). When the glucose concentration was 100 g/L and the nitrogen concentration of urea was 22.5-75 mM, the biomass of T. bernardii increased gradually, and the highest biomass was 46.09 g/L which the glucose to biomass conversion rate was 45.59% (Fig. S2c,d). At the glucose concentration of 120 g/L, it was markedly different from that the glucose concentration of 20-100 g/L in heterotrophic culture (Fig. S2e,f). At all nitrogen concentrations, the biomass at day 12 was signi cantly different to that at day 18, and the highest biomass was only 24.13 g/L, at which the glucose conversion rate was 20%. This indicated that the glucose concentration of 120 g/L had a signi cant inhibitory effect on the growth of T. bernardii. Although glucose is a common organic carbon source in heterotrophic culture, the speci c effects of glucose on the metabolism of microalgae vary greatly with species. For Chlorella vulgaris, due to the glucose absorption, the hexose/H + transport system alkalinized the medium when there was su cient glucose [25]. Thus, high levels of glucose have been shown to inhibit microalgal growth, at least for a while. In order to promote cell growth of C. vulgaris and Scenedesmus acutus, initial glucose concentrations should be limited to 10 g/L and 1 g/L, respectively [26]. For C. saccharophila, the glucose concentration for optimal growth rate was 2.5 g/L, and it would inhibit growth when glucose concentration exceeded 25 g/L. Inhibition of C. sorokiniana appeared at glucose concentration of 5 g/L [27]. C. protothecoides achieved the optimal yield at a glucose concentration of 85 g/L [28]. In Nitzschia laevis, biomass yield gradually decreased with the increase of glucose concentration from 1 to 40 g/L [29]. G. sulphuraria can grow at a glucose concentration of 166 g/L, but their growth was inhibited at a high glucose concentration [30]. The highest biomass of T. bernardii was obtained at glucose concentration of 100 g/L, but the conversion rate of glucose to biomass at 100 g/L glucose was lower than it at glucose concentration of 80 g/L, which indicated that high glucose concentration of 100 g/L had a certain degree of inhibition on the growth of T. bernardii, but T. bernardii could still obtain high biomass concentration at a high glucose concentration of 100 g/L, which demonstrated that T. bernardii had a relatively high glucose tolerance. This inhibition on the growth of T. bernardii was even more pronounced at a glucose concentration of 120 g/L when biomass was greatly decreased. An appropriate glucose concentration for microalgal growth in heterotrophic culture may be related to a variety of factors, of which microalgae species is the main factor, followed by the culture conditions [3].
The biomass on day 12 of the above culture was summarized and presented in Fig. 3a. The biomass of T. bernardii in heterotrophic culture was closely related to nitrogen concentration and glucose concentration. At a relatively low nitrogen concentration, the biomass was mainly determined by nitrogen concentration. For instance, at a nitrogen concentration of 9 mM, the biomass was about 10 g/L, regardless of whether the initial glucose concentration was 20, 40, 60, or 80 g/L. Furthermore, the correlation between nitrogen concentration and biomass revealed a strong linear dependence. When the glucose concentration was 40, 60, and 80 g/L and the nitrogen concentration was less than 48 mM, the relationship between nitrogen concentration and biomass was y (biomass) = 0.834x (nitrogen concentration) + 2.50 (R 2 =0.991, P<0.01). Speci cally, at the glucose concentration of 40 g/L and nitrogen concentration ≤24 mM, it was y = 0.772x + 3.69 (R 2 =0.981, P<0.01). There was y = 0.841x + 2.54 (R 2 =0.991, P<0.01) (nitrogen concentration ≤36 mM), and y = 0.857x + 1.41 (R 2 =0.996, P<0.01) (nitrogen concentration ≤48 mM), which was at the glucose concentration of 60 and 80 g/L, respectively. When the glucose concentration was 100 g/L and nitrogen concentration was less than 75 mM, the relationship between nitrogen concentration and biomass was y = 0.523x + 9.49 (R 2 =0.955, P<0.01). At this time, the adjusted R 2 (0.955) was lower than that at the glucose concentration of 40, 60 and 80 g/L, indicating that the linear relationship between biomass and nitrogen concentration was inhibited to some extent by the high glucose concentration. Safdar et al. [23] also reported that the growth of Crypthecodinium cohnii in heterotrophic culture was linearly correlated with N supply to a certain level and then showed a negative impact. At the glucose concentration of 120 g/L in T. bernardii, the biomass was signi cantly affected by the high glucose concentration. Meanwhile, at a high nitrogen concentration, the biomass of T. bernardii was mainly determined by glucose concentration. For example, at the glucose concentration of 20 g/L, the highest biomass was 10.87 g/L, and at the glucose concentration of 80 g/L, the highest biomass was 44.99 g/L.
The carbon molar concentration of glucose per gram was 33 mM, so the relationship between C/N ratio and biomass was calculated (Fig. 3b). A high or low C/N ratio in the medium was not conducive to the accumulation of biomass and the biomass reached the maximum under an appropriate C/N. This was consistent with Auxenochlorella protothecoides, in which the highest dry weight was achieved at a C/N (g/g) ratio of 40 and the biomass decreased at both higher and lower C/N ratios [21]. By associating biomass with glucose concentration in the medium, the relationship between the conversion rate of glucose to biomass and C/N ratio in the medium was also calculated (Fig. 3c). When the glucose concentration was 20-80 g/L, the conversion rate of glucose to biomass reached more than 50% in the culture with a C/N ratio from 37-55, and when the C/N ratio was 44, the conversion rate of glucose to biomass exceeded 55%. The high conversion rate of glucose to biomass of T. bernardii showed its great potential for application in biomass production of heterotrophic culture.
At glucose concentrations of 20, 40, 60, and 80 g/L, the lipid content of T. bernardii in the heterotrophic culture with different urea concentrations was determined. The lipid content of T. bernardii at day 18 was much higher than the lipid content at day 12 ( Fig. S3; S4). In culture with the glucose concentration of 20 g/L, and nitrogen concentration from 3 to 9 mM, the highest lipid content was 30% of the dry weight obtained at day 12. When the culture time was prolonged, the lipid content was increased. The highest lipid content was obtained at the nitrogen concentration of 6 mM, which was 37.72% of the dry weight ( Fig. S3). At the glucose concentration of 40, 60 and 80 g/L, the highest lipid content in T. bernardii after 18 days was 35.35, 36.66 and 35% of the dry weight, respectively which was obtained at nitrogen concentration of 12, 18 and 24 mM, respectively ( Lipid accumulation in T. bernardii under heterotrophic culture was closely related to C/N molar ratio (Fig. 3d). With an increase of the C/N ratio, the lipid content in the cells increased and then decreased. The C/N molar ratio of 110-130 was most bene cial to the accumulation of lipids in the heterotrophic culture of T. bernardii. This nding was in accordance with the report by Papanikolaou and Aggelis [14], in which the highest lipid production need initial C/N ratio of greater than 20 in oleaginous microorganisms. The highest lipid concentration in Auxenochlorella protothecoides was observed at a C/N (g/g) ratio of 60 [21]. Reportedly, however, the lipid content in C. sorokiniana was lowest at a C/N ratio of approximately 20 and increased at higher or lower C/N values [16]. The response of microalgal lipid accumulation to C/N ratio varied with species.
2.4 Change in the growth, glucose uptake, and cellular C and N content of T. bernardiiin different culture conditions over time To further analyze the in uence of NaNO 3 and urea on the growth and lipid accumulation of T. bernardii in heterotrophic culture, time-course change of glucose and nitrogen uptake rate, and cellular C and N content in the culture with nitrogen concentrations of 15 mM and 45 mM at glucose concentration of 60 g/L were investigated (Fig. 4a). When NaNO 3 was used as nitrogen source, the growth rate of T. bernardii in the early phase of culture was much higher than that in the urea treatment. The growth rate at nitrogen concentration of 45 mM was higher than the nitrogen concentration of 15 mM during the exponential growth phase after the third day of culture. The treatment of 45 mM NaNO 3 reached the stationary phase on the sixth day of culture. The nal biomass was 25.1 g/L and the conversion rate of glucose to biomass was 41%. When urea was used as a nitrogen source and the nitrogen concentration was 45 mM, the biomass was higher than that of NaNO 3 treatment after seven days of culture. The biomass was 33.71 g/L on the ninth day of cultivation, and the conversion rate of glucose to biomass was 55%. At low nitrogen concentration, the growth was always higher when NaNO 3 used as nitrogen source rather than urea. Interestingly, the effect of NaNO 3 and urea on biomass in T. bernardii was dependent on the nitrogen concentrations. At high nitrogen concentration, urea resulted in higher biomass accumulation, while at low nitrogen concentration, T. bernardii grew faster with NaNO 3 than with urea. In Scenedesmus bijugatus, the highest speci c growth rate was presented in the culture with NaNO 3 and KNO 3 and urea exhibited less of an effect on biomass [31]. A similar result was observed in Crypthecodinium cohnii [23].
Conversely, in Chlorella protothecoides, the highest yield of biomass was obtained in the culture with urea as nitrogen sources, when compared with nitrate and ammonium [28].
The concentration of residual glucose in the medium showed that the glucose uptake rate was closely correlated with growth ( Fig. 4b). At the initial stage of culture, the glucose uptake rate in the culture with NaNO 3 used as nitrogen source was much higher than that of the urea treatment. In the middle and late stages of culture, the glucose uptake rate of the high nitrogen concentration (45 mM) was higher than that of the low nitrogen concentration (15 mM). There was almost no glucose remaining in the high nitrogen concentration treatment. At low nitrogen concentration, there were 37% and 55% of the original glucose concentration remaining in the culture of NaNO 3 and urea treatment, respectively. It was indicated that NaNO 3 used as nitrogen source was more conducive to the rapid absorption of glucose by T. bernardii compared with the urea treatment under the same nitrogen concentration. Under the high nitrogen concentration, the biomass of the urea treatment was much higher than that of the NaNO 3 treatment.
Urea used as nitrogen source was more bene cial to the transformation of glucose into biomass by T. bernardii meaning high conversion rate of glucose to biomass.
The analysis of cellular C and N content is presented in Table 1. The N absorption rate of the cells was calculated by the cellular N content combined with the biomass concentration. In the culture with NaNO 3 used as nitrogen source, the cellular N content on the rst day of culture was higher than that of the urea treatment. The absorption rate of NaNO 3 -N in the early stage of heterotrophic culture was higher than the rate of urea-N uptake by T.
bernardii. With the prolongation of culture time, the cellular N content was decreased and the cellular C content was increased when NaNO 3 was used as nitrogen source, so the cellular C/N ratio increased gradually. On the last day of culture, 15 mM NaNO 3 was absorbed completely by T. bernardii, but only 80% of total nitrogen from 45 mM NaNO 3 was absorbed. When urea was used as nitrogen source, cellular N content increased with culture time and then decreased. The cellular C content increased gradually, and cellular C/N ratio decreased and then increased. On the last day of culture, T. bernardii completely absorbed 15 and 45 mM of urea-N. Nitrate is a major source of nitrogen that has a strong in uence on microalgal growth and metabolism [1]. Nitrate assimilation was in uenced by environment, and most microalgae assimilate nitrate more rapidly in the light than in the dark [1].
Our previous study con rmed that the uptake of NaNO 3 in T. bernardii was faster in phototrophic culture than in heterotrophic culture (Gao et al. 2019).
In this study, we noticed that NaNO 3 assimilation was faster than urea assimilation in the early stage of culture in T. bernardii. Combined with higher glucose uptake rate in NaNO 3 treatment, it resulted in higher growth rate of T. bernardii in the culture with NaNO 3 used as nitrogen source than in the culture with urea. At the late stage of culture, high concentration of NaNO 3 (45 mM) and glucose was not absorbed completely by T. bernardii, and high concentration of urea and glucose was absorbed completely, and thus the biomass at high concentration of NaNO 3 was lower than that achieved in the urea treatment. The cellular C/N ratio showed that the value at low concentration of NaNO 3 treatment was higher than that in the urea treatment, which may lead to high lipid accumulation.

Transcriptome analysis of the T. bernardii under heterotrophic culture
In order to elucidate the metabolic mechanism of T. bernardii in heterotrophic culture, time-resolved transcriptome analysis under different C/N ratios was conducted. The heterotrophic culture of T. bernardii at glucose of 40 g/L with urea-N concentrations of 9 mM and 27 mM was performed, and then samples were collected at 0 h, 12 h, 2 d, 3 d, 5 d, 9 d, 12 d and 15 d for the measurements of RNA-seq data.
Under a high N concentration condition (27 mM, HN), the growth rate of T. bernardii was higher than under a low nitrogen concentration culture (9 mM, LN), and it reached the stationary phase on the fth day of culture, in which the highest biomass of 20.22 g/L was achieved (Fig. 5a). The biomass was increased gradually under LN and it reached 17.60 g/L. The cellular C content of T. bernardii increased with culture time, but the range of increase varied with nitrogen concentration (Fig. 5b). Cellular C content under LN was higher than it in HN culture after the seventh day. The content of cellular N under HN increased and then decreased, with 3.5%-4% of dry weight remaining; the variation range of the cellular C/N ratio was smaller compared with LN, in which cellular N content decreased to 1.6% of dry weight. In HN culture, the lipid content of T. bernardii was about 10% of dry weight, whereas in LN, the lipid content increased gradually with culture time and the lipid content reached 34% of dry weight (Fig. 5c). The carbohydrate content of T. bernardii was more than 45% of dry weight in the whole culture time (Fig. 5d). It peaked at 65% and 62% of dry weight on the fth day under HN and LN, respectively. We noticed that T. bernardii accumulated a high content of carbohydrate in heterotrophic culture, which was used as primary carbon storage compound. The fatty acid composition of T. bernardii was mainly palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3), which accounted for more than 80% of the total fatty acid content (Fig. 5e). Under LN, the content of C18:1 increased 1.6 times which made up 40% of the total fatty acids on day 17, and the content of C18:2 and C18:3 was decreased. Under HN, the change in fatty acid content of total fatty acids was relatively small, and the total fatty acid content of dry weight had no signi cant increase over culture time ( 7% to 9% of dry weight) (Fig.   5f). Under LN, the total fatty acid content increased gradually, and reached 26% of dry weight.
For reference transcriptome analysis, we collected 15 samples at different time points mentioned above in HN and LN culture and extracted total RNA. Illumina Hiseq 4000 platform was used for double-ended sequencing, and a total of 11.01 Gb of clean reads were obtained (Table S1). The number of unigenes after assembly was 40,697 with a GC content of 55.64%. For functional annotation, a total of 25,062 unigenes (61.58%) were annotated (Fig.   S5), of which the number of unigenes annotated by the NR database was the most (21,634), accounting for 53.16%. Unigenes annotated by NR database matched at least one species. The species distribution of the NR annotation of T. bernardii was statistically analyzed (Fig.S6). It was found that Volvox carteri f. nagariensis had the highest matching degree with the T. bernardii at 23.17% (5,012). There were 3809 genes in T. bernardii matched with Chlamydomonas reinhardtii and, 2,214 and 1,890 genes were matched with Chlorella variabilis and Coccomyxa subellipsoidea c-169, respectively. These four species of microalgae belong to the same family and same class of chlorophyta, which indicated that the results of this gene annotation had very high reliability.
To investigate the response and adaptation of T. bernardii to different C/N ratios in heterotrophic culture, the cells grown under HN and LN were collected at 0 h, 12 h, 2 d, 3 d, 5 d, 9 d, 12 d and 15 d in culture, and RNA-seq for each sample were conducted. RSEM was used to calculate the expression of each sample, and the different expression genes (DEGs) in each sample was compared with 0 h as the control (Fig. S7). A total of 9,100 genes were up-regulated and 15,690 genes were down-regulated under HN. Under LN, a total of 10,058 genes were up-regulated and 9,964 genes were down-regulated. With the extension of culture time, the nutrients in the culture medium were consumed, and a large number of genes were downregulated under stationary phase. Enrichment analysis of KEGG metabolic pathways was carried out for all up-regulated and down-regulated genes under HN and LN. The top 20 metabolic pathways that were signi cantly up-regulated and down-regulated were presented in Fig. S8. Under HN, with the consumption of nutrients in the culture medium, the genes associated with growth, such as ATP binding cassette (ABC) transporters that hydrolyze ATP to transport sugar, lipids and peptides, were signi cantly down-regulated. There were 39 unigenes encoding ABC transporters which were downregulated. The related genes of RNA transport and carbon xation (22 unigenes) were down-regulated. Meanwhile, the related gene of fatty acid elongation, trans-2-enoyl-CoA reductase (Unigene11017), 3-ketoacyl-CoA synthase (Unigene6926, CL562.Contig2, Unigene1412), very-long-chain 3oxoacyl-CoA reductase (Unigene947) and very-long-chain 3-hydroxyacyl-CoA dehydratase (Unigene28343) were continuously down-regulated after the 9th day, resulting in low fatty acid content and no signi cant change in the content of long-chain unsaturated fatty acid. In addition, the related genes of carotenoid biosynthesis (17 unigenes) were signi cantly down-regulated. The up-regulated genes in the LN were mainly enriched in proteasome, fatty acid metabolism, glycerophopholipid metabolism, and N metabolism. The down-regulated genes in the LN were mainly enriched in the pathway of photosynthesis-antenna proteins (ko00196), porphyrin and chlorophyll metabolism (ko00860), and photosynthesis (ko00195).
Fatty acid biosynthesis is often limited by the supply of NADPH, as the elongation of fatty acids during their biosynthesis by two carbon units requires two NADPH molecules [11]. Oxidative pentose phosphate pathway provides NADPH to the cell through the oxidation of glucose. Its key enzymes include glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Under their co-catalysis, glucose-6-phosphate is oxidized to ribulose 5-phosphate (Ru5P) accompanied by the production of NAPDH. Under HN and LN, the expression of genes encoding G6PDH (Unigene3815) and 6PGDH (Unigene30058) did not change signi cantly in the early stage of culture, but were signi cantly up-regulated in the late stage of culture (Fig. 6a). This result suggested that with the extension of culture time, more NADPH is needed to synthesize storage substances.
Three key enzymes of the glycolysis pathway, including hexokinase (HK), 6-phosphofructokinase (PFK, rate-limiting enzyme) and pyruvate kinase (PK) [32], catalyzed the process which is irreversible, as well as the corresponding enzymes in the glucogenesis pathway, including glucose 6-phosphatase, fructose 1, 6-bisphosphatase (FBP), pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PCK). At the early stage of heterotrophic culture, the gene encoding HK (Unigene18767) was up-regulated rstly, and then was down-regulated. This was consistent with the high glucose content obtained by cells that absorb glucose from the medium at the early stage of culture. In LN culture, the genes encoding PFK (Unigene24519) and PK (Unigene12413) were up-regulated with the culture time. Compared with HN, the variation range in LN was relatively small. This result indicated that the glycolysis pathway was more signi cant at the late stage in LN. The gene encoding FBP was down-regulated over culture time, and the expression changes of the genes encoding PC and PCK in the glucogenesis pathway were relatively moderate in HN and LN. Changes in their expression levels indicated the direction of the carbon ow within the cell. ADP-glucose pyrophosphorylase (AGP) catalyzes the synthesis of ADP-glucose (ADPG) which is the precursor of starch synthesis. Starch synthase (SS) catalyzed the transfer of glucose from ADPG to the non-reductive end of α-1, 4-glucan. The branching enzyme (BE) catalyzed the formation of an α-1, 6glycosidic bond. These three enzymes were the key enzymes for starch synthesis (Fig. 6a). The gene encoding AGP (Unigene27221) was up-regulated in the early period, and then was down-regulated, which was consistent with the changes in cellular carbohydrate content (Fig. 5). Not that genes in the gluconeogenic pathway were not signi cantly up-regulated, so glucose, the precursor for the synthesis of starch, was likely to be directly derived from the external organic carbon source absorbed by the cell.
Synthesis of fatty acids begins with acetyl-coenzyme A (CoA), which produces malonyl-CoA under the catalysis of acetyl-coenzyme A carboxylase (ACCase). ACCase is the rst enzyme in the fatty acids synthesis pathway and is also a key rate-limiting enzyme [2]. In algae, ACCase has two different forms, a heteromeric and homomeric form [33]. It is generally believed that heteromeric ACCase, also known as prokaryotic ACCase, exists in plasmids, and homomeric ACCase, also known as eukaryotype ACCase, exists in the cytoplasm. Heteromeric ACCase has four functional subunits, including the biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and α-and β-carboxyltransferases (CT). The gene of α-CT was encoded by the chloroplast genome, and the other three subunits were encoded by nuclear genes. Homomeric ACCase is a single homologous dimer protein; these four subunits are located on one polypeptide and it is encoded by the nuclear gene acc1, which is highly conserved. The change in gene expression of the three subunits encoding heteromeric ACCases in T. bernardii was consistent over time. The expression of genes encoding BCCP (Unigene18839), BC (Unigene8052), and CT (Unigene30882) were signi cantly up-regulated at the late stage in LN compared with those of HN. The change in the gene encoding homomeric ACCase (Unigene29953) was relatively moderate. This result indicated that the changes of ACCase were mainly regulated by the heteromeric form.
TAG synthesis includes the acyl-CoA dependent pathway, which is also called the Kennedy pathway and acyl-CoA independent pathway. For the Kennedy pathway, acyl-CoAs are sequentially added onto the sn-1, sn-2, and sn-3 positions of glycerol-3-phosphate by the catalyzation of glycero-3phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT), and diacylgycerol acyltransferase (DGAT), respectively (Fig. 6b). DGAT is the last and rate-limiting enzyme in TAG biosynthesis [2]. Two types of membrane-bound DGATs are known to have different protein sequences, functions, expression patterns and organelle sites [34]. The acyl-CoA independent pathway of TAG synthesis is catalyzed by phospholipid: diacylglcerol acyltransferase (PDAT). PDAT uses phospholipids as acyl donors and DAGs as the acyl receptors to produce TAGs. C. reinhardtii has ve genes encoding type II DGATs, one gene encoding type I DGAT and one PDAT gene. In T. bernardii, nine unigenes encode type 1 DGAT (Unigene22686, cl825. Contig1, Unigene23892, Unigene27837, cl1943.contig1, Unigene27236, Unigene3503, Unigene14224, and CL1495.Contig1). and two genes encode type I DGAT (Unigene10373, Unigene34540), and two PDAT genes (one type I (Unigene30932) and one type II (Unigene28641)). The DGAT2 gene was upregulated in the culture of T. bernardii, and its expression level was signi cantly higher than that of DGAT1, indicating that type II DGAT played a major role in the metabolic process. At the same time, the comparison between LN and HN showed that the genes related to lipid synthesis were signi cantly up-regulated at the late stage of culture in LN. Regarding fatty acid degradation, we found that the genes encoding triglyceride lipase were signi cantly up-regulated. This may have resulted in the slow rate of lipid accumulation under heterotrophic metabolism.
To investigate the effects of different C/N ratio on the nitrogen assimilation in T. bernardii, we analyzed the main nitrogen metabolism. The most commonly used nitrogen source in microalgae is nitrate. The nitrate ions (NO 3 -) are transported into microalgal cells by nitrate transporter proteins (Nrt).
After entering cells, nitrate is reduced to ammonium through sequential reactions catalyzed by nitrate reductase (NR) and ferredoxin-nitrite reductase (NiR) [3]. The genes encoding Nrt, NR and NiR in T. bernardii were signi cantly up-regulated with the extension of culture time (Fig. 6c). It indicated that with the consumption of nitrogen in the culture, the cells responded to the nitrogen stress by increasing the expression of the genes related to nitrogen uptake. Interestingly, T. bernardii used the urea as the nitrogen source, but related genes of urease were not signi cantly up-regulated, indicating that genes encoded urease were not regulated at the transcriptional level. Inorganic ammonia forms organic nitrogen compounds via the catalyzation by glutamate dehydrogenase (GDH) or glutamine synthetase (GS) to yield glutamate (Glu). Glu, as the most abundant amino acid in organisms, is the main nitrogen donor among nitrogenous compounds. GDH includes two types, NADPH-dependent (GDH1) and NADH-dependent (GDH2), which are located in chloroplasts and mitochondria, respectively. The expression of GDH1 and GDH2 was down-regulated in HN and LN. GS1 located in cytoplasm was gradually up-regulated with culture time, but GS2 located in chloroplast maintained a certain expression level in the early stage of culture and then was down-regulated, indicating that the reaction was transferred to cytoplasm in the late stage. The resulting glutamine (Gln) was then reduced to Glu catalyzed by glutamate synthase (or glutamate oxglutarate amino transferase, GOGAT). Similarly, GOGAT includes two types which were located in the cytoplasm (GOGAT1) and chloroplast (GOGAT2). The results showed that the gene of GOGAT1 was slightly up-regulated, and the gene of GOGAT2 was maintained. In heterotrophic culture, ammonia may be mainly metabolized by GO/GOGAT in T. bernardii.

Conclusion
In this study, Tetradesmus bernardii, a newly isolated high-yielding oleaginous microalgal strain under photoautotrophic conditions, also exhibited high biomass and lipid accumulation in heterotrophic batch culture. The effect of NaNO 3 on the increase of lipid content was much better than urea, and the highest lipid content of 45.0% of dry weight was achieved. Lipid accumulation could be further increased by extending the culture time. The biomass of T. bernardii in heterotrophic culture was closely related to nitrogen concentration and glucose concentration. A strong linear dependence was evident between the nitrogen concentration and biomass at certain concentrations of nitrogen and glucose. The rate of glucose to biomass exceeded 55% when the glucose concentration was less than 80 g/L in batch culture, which indicated T. bernardii had a relatively high glucose tolerance and great potential to be applied in biomass production in heterotrophic culture. Time-resolved transcriptome was conducted to elucidate C and N metabolism in heterotrophic culture of T. bernardii. As nitrogen was consumed in the medium, the nitrogen metabolism related genes were signi cantly up-regulated to speed up the N metabolic cycle. The synthesis of starch was active which was the main storage C compound in heterotrophic culture, and the precursor of starch synthesis largely came from the absorption of the organic carbon source (glucose). Regarding lipid metabolism, the related genes of fatty acid synthesis at a low nitrogen concentration increased gradually with the extension of the cultivation time, while fatty acid degradation was also active, which may have resulted in a slow rate of lipid accumulation under heterotrophic metabolism.

Algae strain and culture medium
Tetradesmus bernardii was isolated from south lake in Jinan University, Guangdong province, China (23°13.166′, 113°34.754′), and deposited in our laboratory. The strain was identi ed and presented in Gao et al. [19]. The stock culture was maintained in a 250 mL ask containing 100 mL culture medium. The medium used in this heterotrophic experiment was modi ed Endo medium which contained glucose (10.0 g/L), (NH 2 ) 2 CO (2.0 g/L),

Cultivation conditions
In the heterotrophic culture of T. bernardii, NaNO 3 and urea were used as nitrogen sources with nitrogen concentrations of 18 mM, and organic carbon source, glucose was added into the medium at different concentrations (10, 20, 30, 40, 50, and 60 g/L). The cells were cultured in 250 mL ask with working volume of 150 mL. The initial density of culture was 0.5 ± 0.05 g/L. Algae were kept in the dark at a constant culture temperature of 30℃ at 180 rpm. Algal culture was collected at day 12 and day 18 to prepare freeze-dried algal powder for subsequent lipid determination. All conditions were performed in triplicate.
At the glucose concentration of 60 g/L and NaNO 3 used as the nitrogen source, the nitrogen concentration At the glucose concentration of 60 g/L, nitrogen sources were NaNO 3 and urea, with nitrogen concentrations of 15 mM and 45 mM. The growth and the absorption rate of glucose and nitrogen were measured by sampling every day for 9 days. 4.3 Analysis method 4.3.1 Biomass measurement 10 mL culture sample was taken by ltration through pre-weighed glass ber lter membrane with 0.45-μm of pore size (dry weight, W1). The lter membranes containing algal cells were subsequently dried in an oven at 105°C to constant weight (dry weight, W2). The dry weight of the algal cells was the difference between W2 and W1, divided by 10 mL.

Lipid extraction
Total lipid extraction was conducted on 50-80 mg of freeze-dried algal powder according to the method described by  with some modi cations. Algal powder was extracted with 2 mL of dimethyl sulfoxide-methanol mixture (V:V = 1:9) in a 50°C water bath for 1.5 h and was re-extracted with 4 mL diethyl ether-hexane mixed solution (V:V = 1:1) in an ice bath for 1.5 h. The supernatant was collected into the same glass vial and the extraction process was repeated. The determination of lipid content was by gravimetric method and the detail was described in Gao et al. [19].

Total carbohydrate determination
Total carbohydrate was extracted from the algal residue after lipid extraction. 10 mg residue was hydrolyzed with 5 mL of 0.5 M sulfuric acid at 100°C for 4 h. The total carbohydrate concentration was assessed quantitatively by the phenol-sulfuric acid method using glucose as reference [36]. The levels of residual glucose in the medium were also determined by the phenol-sulfuric acid method.

Fatty acids analysis
A total of 25 mg dry weight of freeze-dried biomass was added to 2 mL methanol solution comprising 2% H 2 SO 4 (V/V) in a small vial, and 0.25 mg heptadecanoic acid (Sigma Chemical Co., USA) was added which was used as an internal standard. Then the vial was lled with argon gas. The mixture was incubated in a water bath at 80°C for 1.5 h in order to promote the transmethylation of fatty acids. The fatty acid methyl esters (FAMEs) were analyzed with gas chromatography-ame ionization detector (GC-FID) on an Agilent Gas Chromatograph (Agilent 6890N GC, Agilent Technologies, USA) and authentic standards. Detailed procedure has been described by Gao et al [19].

Element analysis
About 300 μg freeze-dried algae powder was folded into a tin cup (Elemental Microanalysis, Okehampton, UK) and analyzed on a FLASH 2000 NC elemental analyzer (Brechbuhler Incorporated, Interscience B.V., Breda, The Netherlands) for particulate C and N.

Quantitative real-time polymerase chain reaction (RT-qPCR) analysis
To validate the expression levels of some of the genes, quantitative real-time polymerase chain reaction (RT-qPCR) analysis was performed using HN and LN samples. The qPCR primers (Table S2) were designed by Primer Premier 6.0 software. The RT-qPCR was performed on CFX96 Touch (Bio-rad, Hercules, CA, USA) with PrimeScript™ RT reagent kit and TB Green™ Premix Ex Taq™ II (TaKaRa Biotech Co., Beijing, China) according to the manufacturer's protocols. Samples were performed in triplicate. RT-PCR reactions were performed using a Step One Plus Real-Time PCR. The relative mRNA levels were normalized to the level of 18S rRNA gene in each sample and expressed as values of relative expression compared to that of the day 0 group. Relative levels of target mRNAs were determined using the 2 -ΔΔCt method and normalization [38]. The result of RT-qPCR analysis compared with transcriptome was presented in Fig. S9.

Statistical analysis
Statistics were performed using SPSS (version 22.0) statistical software. Signi cant differences (P < 0.05) between treatments were tested by one-way ANOVA (analysis of variance) (P<0.05).