Growth and agitation features, biomass composing, lipid word picture and metabolic profiling analysis of two different Schizochytrium sp. strains, the original strain and the industrial adaptative strain, were investigated in the fed-batch agitation procedure. The concluding cell biomass, entire lipoids content and docosahexaenoic acid ( DHA ) content of the adaptative strain were much higher than that of the original strain, and the DHA productiveness of the adaptative strain was 146.7 mg L-1h-1, which was the highest 1 in all the published value. The amylum and saccharide contents of the biomass in the original strain were higher than those in the adaptative strain and the protein contents in the biomass of both strains were comparatively low ( less than 2 % ) . The lipid word picture of the two different discolorations illustrated that the per centums of DHA in entire lipoids and each lipid category did non alter much in the adaptative strain while the per centums of DHA in impersonal lipoids was much less than that in polar lipoids in the original strain, and the contents of unsaponifiable affairs in the lipoid produced by the adaptative strain were clearly less than that by the original strain. The metabolic differentiation extensively existed between these two strains were revealed by the mark secret plan of Principal Component Analysis ( PCA ) . In add-on, possible biomarkers responsible for know aparting different strains were identified as myo-inositol, histidine, alanine, asparagine, cysteine and oxalic acid. These findings provided new penetrations into the industrial strain showing and farther betterment of DHA production by Schizochytrium sp.
Keywordsi?sSchizochytrium sp. ; docosahexaenoic acid ; lipid word picture ; metabolic profiling analysis
Polyunsaturated fatty acids ( PUFAs ) , the critical membrane constituents in most eucaryotes and the precursors of many lipid-derived signaling molecules, are of import foods and have many benefits on worlds [ 1 ] . Docosahexaenoic acid ( DHA ; 22:6, n-3 ) , a sort of long-chain PUFAs, had drawn increasing attending for its wellness benefits to both babies and grownups [ 2-5 ] . The traditional commercial beginning of DHA is fish or fish oil. However, the worsening fish stocks, typical olfactory property odor, unpleasant gustatory sensation, hapless oxidative stableness, seasonal fluctuation of oil composing, marine chemical pollution and high processing cost of fish oil bound its usage as a nutrient additive. Microalgae or marine Fungis may be interesting options for fish oil as in the marine nutrient concatenation they are the initial manufacturers of ?-3 PUFAs and had stable and dependable lipid composing [ 5 ] . Schizochytrium sp. , a sort of Marine thraustochytrid, has the capableness of synthesising important sums of entire lipid rich in DHA [ 6 ] . Previous researches on the production of DHA by Schizochytrium sp. had chiefly focused on accomplishing high cell denseness and high DHA content [ 7-15 ] , surveies focused on the lipid composing every bit good as the distribution of fatty acids in single lipid category for the industrial production of DHA were non much. In the present survey, the agitation public presentation of two different Schizochytrium sp. strains, the beginning strain and the industrial adaptative strain, were investigated in a 10-L bioreactor utilizing fed-batch agitation. In add-on, the biomass composing and lipid word picture of the agitation consequences of the two strains were besides studied. These consequences will supply utile information for the downstream processing of commercialised production of DHA -rich microbic lipoids.
Metabolomics, one of the freshly developed functional genomics tools, focused on analysing the systematic cellular behaviour at molecular degree and emerged as a utile tool competent to test a great measure of metabolites in biological samples and supply of import physiological information on assorted biological systems [ 16-19 ] . The intracellular metabolome reacts more quickly to environmental alterations than the transcriptome and proteome as the signaling can utilize present receptors and enzymes to change fluxes within a rigorous metabolic web [ 19, 20 ] . Up to now, research on the metabolomic profile analysis of the DHA bring forthing strain Schizochytrium sp. had non been reported yet ; small had been known about the metabolome characteristics of this buttery micro-organism consistently. In this paper, gas chromatography-mass spectroscopy ( GC-MS ) was applied to observe the alterations of intracellular metabolites during the fed-batch agitations of the beginning strain and the industrial adaptative strain. Chief constituents analysis ( PCA ) of intracellular metabolites was performed to separate the biomarkers during the fed-batch civilizations of these two different strains. Interpreting the metabolomic differentiation of the original and the industrial adaptative strains in agitation procedures would supply new penetrations into the industrial strain showing and optimisation of this commercial DHA-producing micro-organism.
Material and methods
Schizochytrium sp. CCTCC M209059, the same strain as our old survey [ 13 ] which was isolated from saltwater and stored in China Center for Type Culture Collection ( CCTCC ) , was used in the present survey as the original strain. This strain was preserved in 20 % ( v/v ) glycerin at -80a„? . The industrial adaptative strain was the industrialised strain which was screened and optimized in the research lab of Jiangsu TianKai Biotechnology Co. , Ltd.
The seed civilization medium and the conditions were the same as those used in our old survey [ 13 ] . After three coevalss of cultivation, the seed civilization ( 10 % , v/v ) was so transferred to a 10-L fermentor with a on the job volume of 7 L. The agitation conditions were the same as our old survey [ 21 ] .
Cell dry weight, entire lipoids, and fatty acid analysis
The measurings of cell dry weight, entire lipoids, single fatty acid, glucose, glutamate and pH used the same method as our old survey [ 21 ] .
Quantification of amylum and protein
The amylum content in Schizochytrium sp. was quantii¬?ed in extra utilizing a modii¬?ed version of the method used by Davis et Al. [ 22 ] .
Protein extraction was performed harmonizing to Weis et Al [ 23 ] with some modii¬?cations.
Analysis of lipid fractions
Analysis of lipid fractions was performed harmonizing to [ 24 ] with some modii¬?cations. The entire lipoid ( 2.5g ) was fractionated to impersonal lipoids ( NLs ) and polar lipoids ( PLs ) by elution on a silica column, ab initio with crude oil ether/diethyl quintessence ( 9:1 ) and so with methyl alcohol. After vaporization of the eluate, the sum of each lipid fraction was determined gravimetrically. The lipoid was fractionated by thin-layer chromatography ( TLC ) on a silicon oxide gel home base by developing with chloroform/acetone/methanol/acetic acid/water ( 50:20:10:10:5, v/v ) .
Isolation and analysis of unsaponifiable affairs, ?-carotene, squalene and cholesterin from lipid
Unsaponifiable affairs were isolated from lipid by saponification [ 25 ] . The ?-carotene content was analyzed harmonizing to Hart et Al [ 26 ] with some alterations. Dionex U3000 HPLC equipped with ultimate 3000 variable wavelength sensor and reversed-phase Superspher C18 columns ( 150-4.6 millimeter i.d. 5?m, Venusil ) at a column temperation of 30a„? 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 millimeter ) and flame ionisation sensor ( FID ) .
The measuring of cholesterin was harmonizing to the GB/T 5009.128-2003.
Sampling, slaking, and extraction of intracellular metabolites
In order to compare the metabolome profiles of Schizochytrium sp. at different agitation phases, cells were sampled every 12 hours during the agitation period. Cells were quenched and extracted harmonizing to Ding et Al [ 27, 28 ] with little alterations.
Metabolome analysis by GC-MS
The sample was analyzed by GC-MS as described antecedently [ 29 ] . 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 execute the Principal Components Analysis ( PCA ) to analyse the dataset [ 30 ] .
Time classs of fed-batch agitation of the original strain and adaptative strain ( Growth and agitation features )
Growth and agitation features of the two different Schizochytrium sp. strains were investigated by the fed-batch cultivations, severally. The consequences indicated that the agitation public presentations of the two strains were typical. As shown in Fig.1, the adaptative strain exhibited much better public presentation in growing and glucose metamorphosis than the original strain, and the concluding cell biomass, entire lipoids content and DHA content of the adaptative 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 productiveness of the adaptative 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 adaptative strain had a great betterment in the DHA bring forthing capableness than the original strain.
Comparison of biomass composing and lipid word picture of the original and adaptative strain
The differences of the biochemical composing of the two different Schizochytrium sp. strains were investigated in fed-batch civilization. Specific concentrations of amylum, saccharides, proteins, and lipoids in the original and adaptative strains were shown in Table 1. For the industrial adaptative strain, lipoid was the quantitatively most of import cell component, doing up over 60 % of the biomass, much more than the original strain ( about 46 % of the biomass ) . The amylum and saccharide contents of the biomass in the original strain were 49.1 and 135.1 milligram g-1, much higher than those in the adaptative strain ( 29.7 and 124.2 mg g-1 ) . The protein contents in the biomass of both strains were comparatively low ( less than 2 % ) .
Lipid fractions of the two different Schizochytrium sp. strains were analyzed by elution on a silica column. As shown in Figure 2, the impersonal lipoids made up over 85 % of the entire lipoids in both strains. The difference in the lipoids fractions between the two different strains was non important. The polar lipoids content in the original strain was 13.09 % , a little more than that in the adaptative strain ( 11.32 % ) . The fatty acids composing of entire lipoids and single lipid category of the two strains was farther analyzed and the consequences were listed in Table 2. In the adaptative strain, the per centums of DHA in entire lipoids and each lipid category did non alter much ( 45.53 % in entire lipoids, 45.47 % in impersonal lipoids and 44.12 % in polar lipoids ) . However, in the original strain, the per centums of DHA in entire lipoids and each lipid category alteration much among different lipid category ( 41.45 % in entire lipoids, 35.51 % in impersonal lipoids and 52.01 % in polar lipoids ) . In add-on, the ratios of the entire unsaturated fatty acids divided entire concentrated fatty acids in the adaptative strain were evidently higher than that in the original strain, bespeaking that the fluidness of the lipoid produced by the adaptative strain was much better than that produced by the original strain.
Three chief unsaponifiable affairs, ?-carotene, squalene and cholesterin, were isolated from lipid by saponification and analyzed by HPLC and GC-MS, severally. Table 3 illustrated that the contents of the chief unsaponifiable affairs in the lipoid produced by the adaptative strain were clearly less than that by the original strain. In the adaptative strain, the contents of ?-carotene, squalene and cholesterin were 34.91?g g-1, 19.99 and 40.93 milligrams g-1 while in the original strain, these contents were 50.28?g g-1, 26.58 and 49.59 mg g-1.
Metabolic profiling and Multivariate statistical analysis of the fed-batch civilizations of the adaptative strain and the original strain
The metabolites of the adaptative Schizochytrium sp. strain and the original Schizochytrium sp. strain were analyzed by GC-MS to compare their metabolic differeneces in the fed-batch civilizations. More than 60 putative intracellular metabolites were detected and 39 of them were identified and quantified in all samples from different agitation phases. The identified metabolites included a scope of intermediates which belonged to several chemical categories including organic acids, aminic acids, intoxicants every bit good as sugars.
Unsupervised rating and independent t-test were used to detect planetary tendencies which were conspicuously different between the two different strains, offering comparative reading of the metabolic changes for the adaptative Schizochytrium sp. strain. The metabolic profile informations was analyzed by PCA to derive perceptual experience into the nature of the multivariate informations and measure biological alteration ( Fig. 3a and 3b ) . PCA mark secret plan ( Figs. 3b ) demonstrated that the maximal variableness in the information set clearly differentiated between different strains ( exactly, between the adaptative strains and the original strain ) , with the first constituent ( PC1 ) covering 46.45 % of the informations discrepancy and the 2nd chief constituent ( PC2 ) explained 16.15 % of the entire detected metabolites pools discrepancy.
PCA burden secret plan ( Fig. 3a ) showed the possible biomarkers for separating samples from the two different Schizochytrium sp. strains. The possible biomarkers which were important for separating the two different strains chiefly included myo-inositol, histidine, alanine, asparagines, cysteine and oxalic acid.
Besides the multivariate informations analysis to analyze the planetary fluctuations between the two different strains, the differences of single compounds were besides analyzed in greater item. The differentiations of the content of several intracellular metabolites, which were the possible biomarkers analyzed by the PCA lading secret plans, myo-inositol, histidine, alanine, asparagine, cysteine and oxalic acid were measured and the consequences were showed in Fig 4. It is illustrated that the content of myo-inositol in the adaptative strain was higher than that in the original strain during the early phase of the agitation, but in the late phase the state of affairs was the contrary. The tendencies of the contents of the four amino acids, histidine, alanine, asparagines and cysteine were similar that the adaptative strain accumulated lower contents of these amino acids than the original strain during the whole agitation procedure. In add-on, the content of oxalic acid in the adaptative strain was lower than the original during the whole civilization period, the same tendency as the amino acids.
In the present survey, agitation public presentation, biomass composing, lipid word picture of two different Schizochytrium strains, the original strain and the industrial adaptative strain, were investigated in the fed-batch agitation civilization to analyze the difference of the industrial agitation potency of the two strains. The metabolic profiling analysis of the two strains was besides investigated to analyze the metamorphosis difference of the two strains to understand the adaptative mechanism of the industrial strain showing and optimisation of Schizochytrium sp.
It could be seen from the agitation consequences that the industrial adaptative strain revealed much better public presentation in bring forthing entire lipoids and DHA than the original strain. The DHA productiveness of the adaptative strain was 146.7 mgL-1h-1, which exceeded the highest published value of 134 mgL-1h-1 by Schizochytrium sp. SR21 [ 31 ] , 115 mgL-1h-1 for Schizochytrium mangrovei Sk-02 [ 14 ] , 117 mgL-1h-1 for strain 12B [ 32 ] , 123 mgL-1h-1 for Aurantiochytrium limacinum SR21 [ 33 ] and 93 mgL-1h-1 by utilizing Aurantiochytrium sp. T66 [ 34 ] . These indicated that the industrial adaptative strain had the commercialisation potency of bring forthing DHA-rich individual cell lipoids.
The biomass composings of the two different Schizochytrium strains were besides investigated in fed-batch cultivation. The industrial adaptative strain had more lipid component and less amylum and saccharide in its biomass than the original strain. These consequences were consistent with the agitation public presentations of the two strains. The biochemical composing in this survey was different from the reported biomass composing of another DHA-producing micro-organism Crypthecodinium cohnii CCMP 316 [ 35 ] . In that discoloration, amylum made up 50 % of the biomass, lipoids and proteins each made up 12-15 % of the biomass. It was besides an account of the increasing involvements of the research of the micro-organism Schizochytrium in DHA production other than Crypthecodinium cohnii. The biomass composing of the two strains provided utile information for the downstream processes such as lipid extraction and biomass recycling.
Fig 2 illustrated that the impersonal lipoids made up approximately 85 % of the entire lipoids in both strains and the polar lipoids content in the adaptative strain was less than the original strain. The lipid category distribution of the entire lipoids in both strains in our research was close to those in the literature. Schizochytrium limacinum was reported to hold approximately 82 % of impersonal lipoids and 14 % of polar lipoids [ 36 ] , and the lipoids in Schizochytrium mangrovei FB3 contained over 90 % of impersonal lipoids and 5 % of polar lipoids [ 24 ] . In the adaptative strain, the per centums of DHA in entire lipoids and each lipid category did non alter much, while in the original strain, the per centums of DHA in impersonal lipoids was much less than that in polar lipoids. As the polar lipoids were chiefly consisted of phospholipids, the indispensable constituents of cell membranes, and these polar lipoids would be removed from the extracted lipoids during the downstream processes. Therefore, during the downstream processes, the extracted lipoids from the original strain would lose more DHA than the adaptative strain. Additionally, the higher content of the entire unsaturated fatty acids in the adaptative strain demonstrated that the fluidness of the lipoid produced by the adaptative strain was much better than that produced by the original strain. Furthermore, the contents of three chief unsaponifiable affairs in the lipoid produced by the adaptative strain were clearly less than that by the original strain, and the issues of these unsaponifiable affairs would increase the trouble of oil refinement.
By the analysis of the agitation features, biomass composing, lipid word picture of the two strains, the industrial adaptive strain showed much better DHA productiveness every bit good as the better lipid quality and less treating cost of the concluding oils. The analysis of the biomass composing and lipid word picture besides provided of import information for the downstream procedure of future commercialized production of DHA -rich microbic lipoids.
In the present survey, the metabolic difference between the adaptative strain and the original strain of Schizochytrium sp. were studied by metabolic profiling. Clear distinction by PCA mark secret plan ( Fig. 3b ) showed that unusually metabolic differentiation extensively existed between the adaptative and the original strain during fed-batch agitation. Throughout the industrial agitation procedure, Schizochytrium sp. was subjected to a assortment of environmental emphasiss, including osmotic force per unit area, gradual nutritionary depletion and toxic metabolites accretion. Therefore, the strain needed to set its metamorphosis to the industrial agitation conditions.
PCA burden secret plan ( Fig. 3a ) revealed the possible biomarkers that were important for separating the two different strains. The differences of the content of several possible biomarkers, myo-inositol, histidine, alanine, asparagine, cysteine and oxalic acid were besides compared. These consequences would lend to understand the adaptative characteristics of Schizochytrium sp. strain in the industrial fed-batch agitation procedure. It could be seen from Fig. 4 that the adaptative strain accumulated higher concentration of intracellular myo-inositol than the original strain during the early phase of the agitation, but the original strain had a much higher content of myo-inositol in the late phase. Myo-inositol was a sort of polyols which was reported to map as militias compounds to carbohydrate, and played cardinal functions in modulating osmotic force per unit area, hive awaying cut downing power and modulating coenzyme in life beings [ 37, 38 ] . Caridi [ 39 ] found that under coincident osmotic and thermic emphasis, myo-inositol evoked positive influences on vino barm. Adler et al [ 40 ] reported the accretion of polyols in P. chrysogenum and A. niger in response to raised salt. It was besides reported that the high ethyl alcohol tolerance of S. cerevisiae could be attributed to intracellular myo-inositol content to some extent [ 41 ] . Myo-inositol was besides found to be the vacuity emphasis protectant of Saccharomyces cerevisiae in the perennial vacuity agitations [ 27 ] . So in this survey, higher degree of myo-inositol accumulated in the adaptative strain during the early phase of agitation could be due to its faster metabolic accommodation than the original strain to accommodate to the agitation environment.
The contents of the four amino acids, histidine, alanine, asparagine and cysteine, had the same tendency that they were higher in the original strain than the adaptative strain during the whole agitation class. This consequence implied that these aminic acids were critical in the cellular response to the emphasis of agitation environment, and the adaptative strain had been adapted to the environmental emphasiss in the amino acids metamorphosis. It was besides found that aminic acids accumulated in Escherichia coli as the general metabolic response to oxidative, cold and heat conditions [ 42 ] . In add-on, the increased content of aminic acids in the original strain of Schizochytrium sp. could be the effect of increased protein debasement [ 38, 42, 43 ] . The debasement of proteins could partially be due to the necessity to take the formed unnatural proteins under emphasis status, or it could be explained that the protein debasement was a manner to increase the sum of aminic acids required for synthesising new proteins which were indispensable for endurance under rough status [ 38, 42, 44 ] . Furthermore, in our consequences, higher degrees of amino acids such as alanine, asparagine and cysteine in the original strain during the agitation procedure revealed a more active N metamorphosis in the original strain than in the adaptative strain. Amino acids were critical parts of C and N metamorphosis, and precursors of assorted cell component including proteins, bases, and other compounds incorporating N [ 45 ] . Probe on amino acids provided penetrations into metabolic ordinance, every bit good as the connexion between amino acid metamorphosis and carbon-nitrogen position [ 45, 46 ] . Additionally, higher content of aminic acids in the original strain besides reflected a decrease of the TCA rhythm flux as these metabolites were formed either during the glycolytic tract or from its intermediate by-passes [ 27, 47 ] . In the original strain, higher degrees of valine and alanine, which were derived from pyruvic acid, could bespeak the higher metabolic activity around the pyruvic acid subdivision point [ 45 ] . In some species, asparagine played a portion in N conveyance and storage [ 48 ] . In this survey, higher degrees of asparagine in the original strain than the adaptative strain disclosed that the conveyance and storage of N were more required in the original strain during the fed-batch agitation procedure [ 45 ] .
The content of oxalic acid in the adaptative strain was besides lower than the original during the whole civilization period. Oxalic acid likely originated from hydrolysis of oxaloacetate [ 49, 50 ] , which was an intermediate of the TCA rhythm. Therefore, the higher degree of oxalic acid in the original strain than the adaptative strain revealed a lessening of the TCA rhythm flux, and this guess was in conformity with the tax write-off that mentioned above about the content difference of aminic acids in the two strains.
In this survey, the GC-MS-based metabolomics provided metabolic profiles of two different Schizochytrium sp. strains during the fed-batch procedures. The fluctuations of intracellular metabolites observed in this paper led to a deeper comprehension of different metabolic position of the adaptative and the original strain during agitation class. Understanding the mechanisms of the adaptation of Schizochytrium sp. strain to the industrial agitation conditions provided important information for the farther ordinance of the metamorphosis web of Schizochytrium sp. for the development of more efficient strains for industrial DHA bring forthing procedures.
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 ) .