Chengxu Zhou

Global characterization of the photosynthetic glycerolipids from a marine diatom Stephanodiscus sp. by ultra performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight mass spectrometry

The photosynthetic glycerolipids composition of algae is crucial for structural and physiological aspects. In this work, a comprehensive characterization of the photosynthetic glycerolipids of the diatom Stephanodiscus sp. was carried out by ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry (UPLC-ESI-Q-TOF MS). By use of the MS(E) data collection mode, the Q-TOF instrument offered a very viable alternative to triple quadrupoles for precursor ion scanning of photosynthetic glycerolipids and had the advantage of high efficiency, selectivity, sensitivity and mass accuracy. Characteristic fragment ions were utilized to identify the structures and acyl compositions of photosynthetic glycerolipids. Comparing the abundance of fragment ions, it was possible to determine the position of the sn-glycerol-bound fatty acyl chains. As a result, four classes of photosynthetic glycerolipid in the extract of Stephanodiscus sp. were unambiguously identified, including 16 monogalactosyldiacylglycerols (MGDGs), 9 digalactosyldiacylglycerols (DGDGs), 23 sulfoquinovosyldiacylglycerols (SQDGs) and 8 phosphatidylglycerols (PGs). As far as our knowledge, this is the first report on global identification of photosynthetic glycerolipids, including lipid classes, fatty acyl composition within lipids and the location of fatty acids in lipids (sn-1 vs. sn-2), in the extract of marine microalgae by UPLC-ESI-Q-TOF MS directly.

Effect of unialgal diets on the composition of fatty acids and sterols in juvenile ark shell Tegillarca granosa Linnaeus

This study has investigated the effects of six different unialgal diets ( Chaetoceros calcitrans , Platymonas helgolandica , Chlorella sp., Isochrysis galbana , Nannochloropsis oculata , and Pavlova viridis ) on the composition of fatty acids and sterols in juvenile ark shell Tegillarca granosa Linnaeus. The best feeding effects on the growth of shellfish were found in C. calcitrans, followed by I. galbana and P. viridis, whereas Chlorella sp. and N. oculata exhibited relatively poor effects. The fatty acid and sterol compositions in the six microalgae and the juvenile ark shell after feeding were analyzed, and 39 fatty acids and 18 sterols were identified. Although the results demonstrate a close correlation between the sterol compositions in algal species and juvenile ark shell, a similar correlation was not observed between fatty acids. In the juvenile ark shell fed microalgae, the ratio of total saturated fatty acids (SFA) rapidly decreases, whereas the proportion of total polyunsaturated fatty acids (PUFAs) increases considerably. The abundances of AA, EPA, and DHA increase most significantly in shellfish with better growth (fed C. calcitrans, I. galbana, and P. viridis). The number of sterol species is reduced, but the total sterol content in groups fed corresponding microalgae increases, and abundant plant sterols, instead of cholesterol, are accumulated in juvenile ark shell fed appropriate microalgae I. galbana and P. viridis. Therefore, to be more conducive to human health, I. galbana and P. viridis, of the six experimental microalgae, are recommended for artificial ark shell culture.

Profiles of photosynthetic glycerolipids in three strains of Skeletonema determined by UPLC-Q-TOF-MS

An exhaustive qualitative and quantitative profiling of the photosynthetic glycerolipids in three strains of the marine diatom Skeletonema sp. was carried out by ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry. In the diatom thylakoid membrane, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) account for about 45–70% and 5–15% of the total membrane lipids, respectively. The anionic sulfoquinovosyldiacylglycerol (SQDG) as well as the likewise anionic phosphatidylglycerol (PG) contribute between 10–40% and 4–10% each. The predominant species of MGDG were those with C16:3/C16:3, C20:5/16:1, and C20:5/C16:3. Three main molecular species of DGDG contained C20:5/C16:1, C20:5/C16:2, and C16:1/C16:1. The major molecular species of SQDG were those containing combinations of C14:0/C14:0, C14:0/C16:0, C14:0/C16:1, and C14:0/C16:3. All the PG classes contained the C18:1/C18:1 as the main molecular species. Based on the fatty acid species in sn-2 position, it is indicated that MGDG and DGDG are biosynthesized through prokaryotic pathway exclusively within the chloroplast, whereas PG and SQDG have a typical mixed biosynthetic pathway (both prokaryotic pathway and eukaryotic pathways). The chemical characteristics of photosynthetic glycerolipids related with ecological physiology are discussed.

Lipidomic analysis can distinguish between two morphologically similar strains of Nannochloropsis oceanica.

The two morphologically similar microalgae NMBluh014 and NMBluh‐X belong to two different strains of Nannochloropsis oceanica. They possess obviously different feeding effects on bivalves, but are indistinguishable by 18S rRNA and morphological features. In this work, lipidomic analysis followed by principal component analysis and orthogonal projections to latent structures discriminant analysis provided a clear distinction between these strains. Metabolites that definitively contribute to the classification were selected as potential biomarkers. The most important difference in polar lipids were sulfoquinovosyldiacylglycerol (containing 18:1/16:0 and 18:3/16:0) and monogalactosyldiacylglycerol (containing 18:3/16:3 and 20:5/14:0), which were detected only in NMBluh‐X. Additionally, an exhaustive qualitative and quantitative profiling of the neutral lipid triacylglycerol (TAG) in the two strains was carried out. The predominant species of TAG containing 16:1/16:1/16:1 acyl groups was detected only in NMBluh‐X with a content of ~93.67 ± 11.85 nmol · mg−1 dry algae at the onset of stationary phase. Meanwhile, TAG containing 16:0/16:0/16:0 was the main TAG in NMBluh014 with a content of 40.25 ± 3.92 nmol · mg−1. These results provided the most straightforward evidence for differentiating the two species. The metabolomic profiling indicated that NMBluh‐X underwent significant chemical and physiological changes during the growth process, whereas NMBluh014 did not show such noticeable time‐dependent metabolite change. This study is the first using Ultra Performance Liquid Chromatography coupled with Electrospray ionization‐Quadrupole‐Time of Flight Mass Spectrometry (UPLC‐Q‐TOF‐MS) for lipidomic profiling with multivariate statistical analysis to explore lipidomic differences of plesiomorphous microalgae. Our results demonstrate that lipidomic profiling is a valid chemotaxonomic tool in the study of microalgal systematics.

Precise identification of photosynthetic glycerolipids in microalga Tetraselmis chuii by UPLC-ESI-Q-TOF-MS

Precise structural identification of photosynthetic polar glycerolipids in microalga Tetraselmis chuii has been established using Ultra Performance Liquid Chromatography-Electrospray ionization-Quadrupole-Time of Flight Mass Spectrometry (UPLC-ESI-Q-TOF-MS) by direct analysis of the total lipids extract. The mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in both positive and negative modes. The structural determination was based on the characteristic product ions yielded by different glycerolipids under ESI-MS/MS mode, and confirmed the molecular species by the carboxylate anions produced by glycerolipids in the negative mode. As a result, more than 40 lipid molecular species, including 11 monogalactosyldiacylglycerols (MGDG), 7 digalactosyldiacylglycerols (DGDG), 16 sulfoquinovosyldiacylglycerols (SQDG), and 9 phosphatidylglycerols (PG), were detected in Tetraselmis chuii, which had never been identified before in this microalga. Furthermore, some intact lipid molecules with hydroxylated fatty acids that could not be detected by the traditional GC-MS method were found this time, providing novel information for the photosynthetic lipidome of Tetraselmis chuii. Comparative studies on fatty acids at the sn-2 position showed that SQDG and MGDG are dominantly biosynthesized through the prokaryotic pathway, PG is a typically mixed biosynthetic pathway, while DGDG is somewhat peculiar with C14:0 and C16:0 at its sn-2 position. This method could provide a full structural profile of intact photosynthetic lipid molecular species, which may be applied to study the physiological and ecological functions of lipid by monitoring their individual changes.

Structural elucidation of co-eluted triglycerides in the marine diatom model organism Thalassiosira pseudonana by ultra- performance liquid chromatography/quadrupole time-of-flight mass spectrometry

RATIONALE The precise identification of fatty acids at the sn-2 position of triacylglycerols (TAGs), especially for positional regioisomers (AAB/ABA), needs to be established during mass spectrometry analysis. The detailed structural information about TAGs is significant not only for the assessment of biofuel quality, but also for the tracing of biosynthetic precursors. METHODS Total lipid was extracted from T. pseudonana by a modified Bligh and Dyer method. The qualitative analysis of TAGs in T. pseudonana was carried out using ultra-performance liquid chromatography/electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOF-MS). The raw LC/MS data were analyzed using MassLynx software (version 4.1, Waters). RESULTS The acyl group at the sn-2 position of the TAGs has been identified unequivocally by [M + Li-R1/3COOH-R2CH=CHCOOH](+) and the abundance of [M + Li-R1/3COOH-R2CH=CHCOOH](+) can be used to confirm whether the TAG isomers are co-eluted. In total, twelve TAGs were identified in T. pseudonana based on the fragmentation patterns discussed above. The data indicated that only C16 fatty acids were located at the sn-2 position, which was important to trace the biosynthetic precursors of TAGs. CONCLUSIONS We put forward a hypothesis that TAGs in T. pseudonana are only derived from lipids in chloroplasts through prokaryotic biosynthesis pathway based on the precise information of sn-2 fatty acids, which is significant not only for the assessment of biofuel quality, but also for the tracing of biosynthetic precursors.

Two new karlotoxins found in Karlodinium veneficum (strain GM2) from the East China Sea

The dinoflagellate Karlodinium veneficum is a harmful algal bloom species with a worldwide distribution. This small athecate dinoflagellate makes a family of polyketide toxins that are hemolytic, cytotoxic and ichthyotoxic. The first chemical structure for karlotoxins from East China Sea (ECS) is reported here. The two new karlotoxins, namely 4,5-dihydro-KmTx 2 (compound 1) and 4,5-dihydro-dechloro-KmTx 2 (compound 2), were isolated and purified from monoalgal cultures of K. veneficum strain GM2. Their structures were determined by spectroscopic analysis, including tandem mass spectrometry as well as 1D and 2D NMR experiments. These new karlotoxin congeners feature a saturated polyol arm different from previously reported for KmTx 2 that appears to increase hemolytic activity.

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Karlodinium veneficum

The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) combined with a chromatographic lateral flow dipstick (LFD) assay to rapidly and specifically detect the Karlodinium veneficum ITS gene. Four groups of LAMP primers were specially designed to target the K. veneficum ITS gene. The LAMP-LFD detection limit was 7.4pg/μL (approximately 6.5cells/mL) of K. veneficum genomic DNA and was 10 times more sensitive than standard PCR. The LAMP-LFD method exhibited high specificity and accurately identified K. veneficum algal isolates, but not other algal isolates. To test the assays accuracy, samples from positive results were further analyzed by sequencing and phylogenetic analysis, all of which were identified as K. veneficum. Over all, the LAMP-LFD assay established in this paper can be used as a reliable and simple method to detect the K. veneficum.

Change of Volatile Components in Six Microalgae With Different Growth Phases.

BACKGROUND Head space solid-phase microextraction-gas chromatography-mass spectrometry has been applied to analyze the volatile components of six marine microalgae (Thalassiosira weissflogii, Nitzschia closterium, Chaetoceros calcitrans, Platymonas helgolandica, Nannochloropsis spp. and Dicrateria inornata) from Bacillariophyta, Chlorophyta and Chrysophyta, respectively, in different growth phases. RESULTS All volatile compounds were identified by database searching in the NIST08 Mass Spectral Library and analyzed by principal component analysis with SIMCA-P software (Umetrics, Umea, Sweden). The results clearly revealed that the volatile components of the six microalgae were significantly different in the exponential, stationary and declining phases. Aldehydes, alkanes, some esters and dimethyl sulfide significantly changed in different growth phases. CONCLUSION This is the first report on the comprehensive characteristics of volatile components in different microalgae and in different growth phases. The results may provide reference data for studies on the flavor of cultivated aquatic organism, odor formation in nature water, choice of feeding period and microalgae species selection for the artificial rearing of marine organisms.

Lipid compositions in diatom Conticribra weissflogii under static and aerated culture conditions

The diatom Conticribra weissflogii is a microalga with high nutrition value, rich in docosahexaenoic acids (DHA) and eicosapentaenoic acids (EPA). In order to study the effect of culture conditions on the changes of lipid compositions, the intact lipid structural profiles and fatty acids in C. weissflogii were monitored under static and aerated culture conditions. The results showed that, lipids identified in C. weissflogii were neutral lipid triacylglycerols (TAG), betaine lipid diacylglycerylcarboxy‐N‐hydroxymethyl‐choline (DGCC), phosphatidylcholine (PC) and four classes of photosynthetic glycerolipids. The profiles of lipid metabolites of C. weissflogii were different between two culture modes, with the following characteristics under aerated conditions: TAGs increased significantly, whereas the levels of sulfoquinovosyl diacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), and DGCC decreased. Furthermore, higher contents of EPA‐rich TAG and EPA/DHA‐rich DGCC were detected at the end of stationary phase, while EPA/DHA‐rich PC, EPA‐rich MGDG and EPA‐rich digalactosyldiacylglycerol (DGDG) were obtained in the exponential phase under static conditions. Meanwhile, the contents of almost all classes of the essential fatty acids (EFAs)‐enriched lipids increased at onset of stationary phase under aerated conditions. Taken together, given that the high levels of EFAs are required for artificial rearing of marine organisms, aeration is critically important for increasing the production rate and the contents of EFA molecules and therefore increasing the nutritional value of the microalgae.