Growth and fermentation characteristics, biomass composition, lipid characterization and metabolic profiling analysis of two different Schizochytrium sp. strains, the original strain and the industrial adaptive strain, were investigated in the fed-batch fermentation process. The final cell biomass, total lipids content and docosahexaenoic acid (DHA) content of the adaptive strain were much higher than that of the original strain, and the DHA productivity of the adaptive strain was 146.7 mg L-1h-1, which was the highest one in all the published value. The starch and carbohydrate contents of the biomass in the original strain were higher than those in the adaptive strain and the protein contents in the biomass of both strains were relatively low (less than 2%). The lipid characterization of the two different stains illustrated that the percentages of DHA in total lipids and each lipid class did not change much in the adaptive strain while the percentages of DHA in neutral lipids was much less than that in polar lipids in the original strain, and the contents of unsaponifiable matters in the lipid produced by the adaptive strain were distinctly less than that by the original strain. The metabolic distinction extensively existed between these two strains were revealed by the score plot of Principal Component Analysis (PCA). In addition, potential biomarkers responsible for discriminating different strains were identified as myo-inositol, histidine, alanine, asparagine, cysteine and oxalic acid. These findings provided new insights into the industrial strain screening and further improvement of DHA production by Schizochytrium sp.
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Keywordsï¼šSchizochytrium sp.; docosahexaenoic acid; lipid characterization; metabolic profiling analysis
Polyunsaturated fatty acids (PUFAs), the critical membrane components in most eukaryotes and the precursors of many lipid-derived signaling molecules, are important nutrients and have many benefits on humans . Docosahexaenoic acid (DHA; 22:6, n-3), a kind of long-chain PUFAs, had drawn increasing attention for its health benefits to both infants and adults [2-5]. The traditional commercial source of DHA is fish or fish oil. However, the declining fish stocks, typical odor smell, unpleasant taste, poor oxidative stability, seasonal variation of oil composition, marine chemical pollution and high processing cost of fish oil limit its use as a food additive. Microalgae or marine fungi may be interesting alternatives for fish oil as in the marine food chain they are the initial producers of ω-3 PUFAs and had stable and reliable lipid composition . Schizochytrium sp., a kind of marine thraustochytrid, has the capability of synthesizing significant amounts of total lipid rich in DHA . Previous researches on the production of DHA by Schizochytrium sp. had mainly focused on achieving high cell density and high DHA content [7-15], studies focused on the lipid composition as well as the distribution of fatty acids in individual lipid class for the industrial production of DHA were not much. In the present study, the fermentation performance of two different Schizochytrium sp. strains, the origin strain and the industrial adaptive strain, were investigated in a 10-L bioreactor using fed-batch fermentation. In addition, the biomass composition and lipid characterization of the fermentation results of the two strains were also studied. These results will provide useful information for the downstream processing of commercialized production of DHA -rich microbial lipids.
Metabolomics, one of the newly developed functional genomics tools, focused on analyzing the systematic cellular behavior at molecular level and emerged as a useful tool competent to screen a great quantity of metabolites in biological samples and provide important physiological information on various biological systems [16-19]. The intracellular metabolome reacts more rapidly to environmental changes than the transcriptome and proteome as the signaling can use present receptors and enzymes to alter fluxes within a strict metabolic network [19, 20]. Up to now, research on the metabolomic profile analysis of the DHA producing strain Schizochytrium sp. had not been reported yet; little had been known about the metabolome features of this oleaginous microorganism systematically. In this paper, gas chromatography-mass spectrometry (GC-MS) was applied to detect the changes of intracellular metabolites during the fed-batch fermentations of the origin strain and the industrial adaptive strain. Principal components analysis (PCA) of intracellular metabolites was performed to distinguish the biomarkers during the fed-batch cultures of these two different strains. Interpreting the metabolomic distinction of the original and the industrial adaptive strains in fermentation processes would provide new insights into the industrial strain screening and optimization of this commercial DHA-producing microorganism.
Material and methods
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Schizochytrium sp. CCTCC M209059, the same strain as our previous study  which was isolated from seawater and stored in China Center for Type Culture Collection (CCTCC), was used in the present study as the original strain. This strain was preserved in 20% (v/v) glycerol at -80â„ƒ. The industrial adaptive strain was the industrialized strain which was screened and optimized in the laboratory of Jiangsu TianKai Biotechnology Co., Ltd.
The seed culture medium and the conditions were the same as those used in our previous study . After three generations of cultivation, the seed culture (10%, v/v) was then transferred to a 10-L fermentor with a working volume of 7 L. The fermentation conditions were the same as our previous study .
Cell dry weight, total lipids, and fatty acid analysis
The measurements of cell dry weight, total lipids, individual fatty acid, glucose, glutamate and pH used the same method as our previous study .
Quantification of starch and protein
The starch content in Schizochytrium sp. was quantiï¬ed in duplicate using a modiï¬ed version of the method used by Davis et al. .
Protein extraction was performed according to Weis et al  with some modiï¬cations.
Analysis of lipid fractions
Analysis of lipid fractions was performed according to  with some modiï¬cations. The total lipid (2.5g) was fractionated to neutral lipids (NLs) and polar lipids (PLs) by elution on a silica column, initially with petroleum ether/diethyl ether (9:1) and then with methanol. After evaporation of the eluate, the amount of each lipid fraction was determined gravimetrically. The lipid was fractionated by thin-layer chromatography (TLC) on a silica gel plate by developing with chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5, v/v).
Isolation and analysis of unsaponifiable matters, β-carotene, squalene and cholesterol from lipid
Unsaponifiable matters were isolated from lipid by saponification . The β-carotene content was analyzed according to Hart et al  with some modifications. Dionex U3000 HPLC equipped with ultimate 3000 variable wavelength detector and reversed-phase Superspher C18 columns (150-4.6 mm i.d. 5μm, Venusil) at a column temperation of 30â„ƒ were used.
Squalene was analyzed by GC system (GC- 2010, Shimadzu, Japan). The GC was equipped with a capillary column (DB-23, 60m-0.22 mm) and flame ionization detector (FID).
The measurement of cholesterol was according to the GB/T 5009.128-2003.
Sampling, quenching, and extraction of intracellular metabolites
In order to compare the metabolome profiles of Schizochytrium sp. at different fermentation stages, cells were sampled every twelve hours during the fermentation period. Cells were quenched and extracted according to Ding et al [27, 28] with slight modifications.
Metabolome analysis by GC-MS
The sample was analyzed by GC-MS as described previously . The GC-MS system consisted of a Finnigan Trace gas chromatograph and a Trace mass spectrometer (Thermo Finnigan, San Jose, USA).
Multivariate statistical analysis
STATISTICA was used to perform the Principal Components Analysis (PCA) to analyze the dataset .
Time courses of fed-batch fermentation of the original strain and adaptive strain (Growth and fermentation characteristics)
Growth and fermentation characteristics of the two different Schizochytrium sp. strains were investigated by the fed-batch cultivations, respectively. The results indicated that the fermentation performances of the two strains were distinctive. As shown in Fig.1, the adaptive strain exhibited much better performance in growth and glucose metabolism than the original strain, and the final cell biomass, total lipids content and DHA content of the adaptive strain were 67.21, 42.56 and 17.02 g L-1, much higher than that of the original strain (56.01, 25.82 and 8.19 g L-1). Furthermore, the DHA productivity of the adaptive strain was 146.7 mg L-1h-1, over two times of that of the original strain (60.2 mgL-1h-1). The observations described above indicated that the industrial adaptive strain had a great improvement in the DHA producing capability than the original strain.
Comparison of biomass composition and lipid characterization of the original and adaptive strain
The differences of the biochemical composition of the two different Schizochytrium sp. strains were investigated in fed-batch culture. Specific concentrations of starch, carbohydrates, proteins, and lipids in the original and adaptive strains were shown in Table 1. For the industrial adaptive strain, lipid was the quantitatively most important cell constituent, making up over 60% of the biomass, much more than the original strain (about 46% of the biomass). The starch and carbohydrate contents of the biomass in the original strain were 49.1 and 135.1 mg g-1, much higher than those in the adaptive strain (29.7 and 124.2 mg g-1). The protein contents in the biomass of both strains were relatively low (less than 2%).
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Lipid fractions of the two different Schizochytrium sp. strains were analyzed by elution on a silica column. As shown in Figure 2, the neutral lipids made up over 85% of the total lipids in both strains. The difference in the lipids fractions between the two different strains was not significant. The polar lipids content in the original strain was 13.09%, a little more than that in the adaptive strain (11.32%). The fatty acids composition of total lipids and individual lipid class of the two strains was further analyzed and the results were listed in Table 2. In the adaptive strain, the percentages of DHA in total lipids and each lipid class did not change much (45.53% in total lipids, 45.47% in neutral lipids and 44.12% in polar lipids). However, in the original strain, the percentages of DHA in total lipids and each lipid class change much among different lipid class (41.45% in total lipids, 35.51% in neutral lipids and 52.01% in polar lipids). In addition, the ratios of the total unsaturated fatty acids divided total saturated fatty acids in the adaptive strain were obviously higher than that in the original strain, indicating that the fluidity of the lipid produced by the adaptive strain was much better than that produced by the original strain.
Three main unsaponifiable matters, β-carotene, squalene and cholesterol, were isolated from lipid by saponification and analyzed by HPLC and GC-MS, respectively. Table 3 illustrated that the contents of the main unsaponifiable matters in the lipid produced by the adaptive strain were distinctly less than that by the original strain. In the adaptive strain, the contents of β-carotene, squalene and cholesterol were 34.91μg g-1, 19.99 and 40.93 mg g-1 while in the original strain, these contents were 50.28μg g-1, 26.58 and 49.59 mg g-1.
Metabolic profiling and Multivariate statistical analysis of the fed-batch cultures of the adaptive strain and the original strain
The metabolites of the adaptive Schizochytrium sp. strain and the original Schizochytrium sp. strain were analyzed by GC-MS to compare their metabolic differeneces in the fed-batch cultures. More than 60 putative intracellular metabolites were detected and 39 of them were identified and quantified in all samples from different fermentation stages. The identified metabolites included a range of intermediates which belonged to several chemical classes including organic acids, amino acids, alcohols as well as sugars.
Unsupervised evaluation and independent t-test were used to observe global trends which were conspicuously different between the two different strains, offering comparative interpretation of the metabolic alterations for the adaptive Schizochytrium sp. strain. The metabolic profile data was analyzed by PCA to gain perception into the nature of the multivariate data and evaluate biological change (Fig. 3a and 3b). PCA score plot (Figs. 3b) demonstrated that the maximum variability in the data set clearly differentiated between different strains (precisely, between the adaptive strains and the original strain), with the first component (PC1) covering 46.45% of the data variance and the second principal component (PC2) explained 16.15% of the total detected metabolites pools variance.
PCA loading plot (Fig. 3a) showed the potential biomarkers for distinguishing samples from the two different Schizochytrium sp. strains. The potential biomarkers which were significant for distinguishing the two different strains mainly included myo-inositol, histidine, alanine, asparagines, cysteine and oxalic acid.
Besides the multivariate data analysis to examine the global variations between the two different strains, the differences of individual compounds were also analyzed in greater detail. The distinctions of the content of several intracellular metabolites, which were the potential biomarkers analyzed by the PCA loading plots, myo-inositol, histidine, alanine, asparagine, cysteine and oxalic acid were measured and the results were showed in Fig 4. It is illustrated that the content of myo-inositol in the adaptive strain was higher than that in the original strain during the early stage of the fermentation, but in the late stage the situation was the reverse. The trends of the contents of the four amino acids, histidine, alanine, asparagines and cysteine were similar that the adaptive strain accumulated lower contents of these amino acids than the original strain during the whole fermentation process. In addition, the content of oxalic acid in the adaptive strain was lower than the original during the whole culture period, the same trend as the amino acids.
In the present study, fermentation performance, biomass composition, lipid characterization of two different Schizochytrium strains, the original strain and the industrial adaptive strain, were investigated in the fed-batch fermentation culture to study the difference of the industrial fermentation potential of the two strains. The metabolic profiling analysis of the two strains was also investigated to study the metabolism difference of the two strains to understand the adaptive mechanism of the industrial strain screening and optimization of Schizochytrium sp.
It could be seen from the fermentation results that the industrial adaptive strain revealed much better performance in producing total lipids and DHA than the original strain. The DHA productivity of the adaptive strain was 146.7 mgL-1h-1, which exceeded the highest published value of 134 mgL-1h-1 by Schizochytrium sp. SR21 , 115 mgL-1h-1 for Schizochytrium mangrovei Sk-02 , 117 mgL-1h-1 for strain 12B , 123 mgL-1h-1 for Aurantiochytrium limacinum SR21  and 93 mgL-1h-1 by using Aurantiochytrium sp. T66 . These indicated that the industrial adaptive strain had the commercialization potential of producing DHA-rich single cell lipids.
The biomass compositions of the two different Schizochytrium strains were also investigated in fed-batch cultivation. The industrial adaptive strain had more lipid constituent and less starch and carbohydrate in its biomass than the original strain. These results were consistent with the fermentation performances of the two strains. The biochemical composition in this study was different from the reported biomass composition of another DHA-producing microorganism Crypthecodinium cohnii CCMP 316 . In that stain, starch made up 50% of the biomass, lipids and proteins each made up 12-15% of the biomass. It was also an explanation of the increasing interests of the research of the microorganism Schizochytrium in DHA production other than Crypthecodinium cohnii. The biomass composition of the two strains provided useful information for the downstream processes such as lipid extraction and biomass recycling.
Fig 2 illustrated that the neutral lipids made up about 85% of the total lipids in both strains and the polar lipids content in the adaptive strain was less than the original strain. The lipid class distribution of the total lipids in both strains in our research was close to those in the literature. Schizochytrium limacinum was reported to have about 82% of neutral lipids and 14% of polar lipids , and the lipids in Schizochytrium mangrovei FB3 contained over 90% of neutral lipids and 5% of polar lipids . In the adaptive strain, the percentages of DHA in total lipids and each lipid class did not change much, while in the original strain, the percentages of DHA in neutral lipids was much less than that in polar lipids. As the polar lipids were mainly consisted of phospholipids, the essential components of cell membranes, and these polar lipids would be removed from the extracted lipids during the downstream processes. Thus, during the downstream processes, the extracted lipids from the original strain would lose more DHA than the adaptive strain. Additionally, the higher content of the total unsaturated fatty acids in the adaptive strain demonstrated that the fluidity of the lipid produced by the adaptive strain was much better than that produced by the original strain. Furthermore, the contents of three main unsaponifiable matters in the lipid produced by the adaptive strain were distinctly less than that by the original strain, and the exits of these unsaponifiable matters would increase the difficulty of oil refining.
By the analysis of the fermentation characteristics, biomass composition, lipid characterization of the two strains, the industrial adaptive strain showed much better DHA productivity as well as the better lipid quality and less processing cost of the final oils. The analysis of the biomass composition and lipid characterization also provided important information for the downstream process of future commercialized production of DHA -rich microbial lipids.
In the present study, the metabolic difference between the adaptive strain and the original strain of Schizochytrium sp. were studied by metabolic profiling. Clear differentiation by PCA score plot (Fig. 3b) showed that remarkably metabolic distinction extensively existed between the adaptive and the original strain during fed-batch fermentation. Throughout the industrial fermentation process, Schizochytrium sp. was subjected to a variety of environmental stresses, including osmotic pressure, gradual nutritional depletion and toxic metabolites accumulation. Thus, the strain needed to adjust its metabolism to the industrial fermentation conditions.
PCA loading plot (Fig. 3a) revealed the potential biomarkers that were significant for distinguishing the two different strains. The differences of the content of several potential biomarkers, myo-inositol, histidine, alanine, asparagine, cysteine and oxalic acid were also compared. These results would contribute to understand the adaptive features of Schizochytrium sp. strain in the industrial fed-batch fermentation process. It could be seen from Fig. 4 that the adaptive strain accumulated higher concentration of intracellular myo-inositol than the original strain during the early stage of the fermentation, but the original strain had a much higher content of myo-inositol in the late stage. Myo-inositol was a kind of polyols which was reported to function as reserves compounds to carbohydrate, and played key roles in regulating osmotic pressure, storing reducing power and regulating coenzyme in living organisms [37, 38]. Caridi  found that under simultaneous osmotic and thermal stress, myo-inositol evoked positive influences on wine yeast. Adler et al  reported the accumulation of polyols in P. chrysogenum and A. niger in response to raised salinity. It was also reported that the high ethanol tolerance of S. cerevisiae could be attributed to intracellular myo-inositol content to some extent . Myo-inositol was also found to be the vacuum stress protectant of Saccharomyces cerevisiae in the repeated vacuum fermentations . So in this study, higher level of myo-inositol accumulated in the adaptive strain during the early stage of fermentation could be due to its faster metabolic adjustment than the original strain to adapt to the fermentation environment.
The contents of the four amino acids, histidine, alanine, asparagine and cysteine, had the same trend that they were higher in the original strain than the adaptive strain during the whole fermentation course. This result implied that these amino acids were critical in the cellular response to the stress of fermentation environment, and the adaptive strain had been adapted to the environmental stresses in the amino acids metabolism. It was also found that amino acids accumulated in Escherichia coli as the general metabolic response to oxidative, cold and heat conditions . In addition, the increased content of amino acids in the original strain of Schizochytrium sp. could be the consequence of increased protein degradation [38, 42, 43]. The degradation of proteins could partly be due to the necessity to remove the formed abnormal proteins under stress condition, or it could be explained that the protein degradation was a way to increase the amount of amino acids required for synthesizing new proteins which were essential for survival under harsh condition [38, 42, 44]. Furthermore, in our results, higher levels of amino acids such as alanine, asparagine and cysteine in the original strain during the fermentation process revealed a more active nitrogen metabolism in the original strain than in the adaptive strain. Amino acids were critical parts of carbon and nitrogen metabolism, and precursors of various cell constituent including proteins, nucleotides, and other compounds containing nitrogen . Investigation on amino acids provided insights into metabolic regulation, as well as the connection between amino acid metabolism and carbon-nitrogen status [45, 46]. Additionally, higher content of amino acids in the original strain also reflected a reduction of the TCA cycle flux as these metabolites were formed either during the glycolytic pathway or from its intermediate by-passes [27, 47]. In the original strain, higher levels of valine and alanine, which were derived from pyruvic acid, could indicate the higher metabolic activity around the pyruvic acid branch point . In some species, asparagine played a part in nitrogen transport and storage . In this study, higher levels of asparagine in the original strain than the adaptive strain disclosed that the transport and storage of nitrogen were more required in the original strain during the fed-batch fermentation process .
The content of oxalic acid in the adaptive strain was also lower than the original during the whole culture period. Oxalic acid probably originated from hydrolysis of oxaloacetate [49, 50], which was an intermediate of the TCA cycle. Thus, the higher level of oxalic acid in the original strain than the adaptive strain revealed a decrease of the TCA cycle flux, and this speculation was in accordance with the deduction that mentioned above about the content difference of amino acids in the two strains.
In this study, the GC-MS-based metabolomics provided metabolic profiles of two different Schizochytrium sp. strains during the fed-batch processes. The variations of intracellular metabolites observed in this paper led to a deeper comprehension of different metabolic status of the adaptive and the original strain during fermentation course. Understanding the mechanisms of the adaption of Schizochytrium sp. strain to the industrial fermentation conditions provided significant information for the further regulation of the metabolism network of Schizochytrium sp. for the development of more efficient strains for industrial DHA producing processes.
This work was financially supported by the National Basic Research Program of China (no. 2011CBA00802), the Scientific Research Project for Post-graduate in Jiangsu Province (no. CXLX11_0366), the Natural Science Foundation of Jiangsu Province (no. BK2012424), the National Science and Technology Pillar Program (no. 2011BAD23B03), and the National High Technology Research and Development Program of China (no. SS2012AA021704).