Reconstruction of a Genome-Scale Metabolic Model of Scenedesmus obliquus and Its Application for Lipid Production under Three Trophic Modes.

Green microalgae have emerged as beneficial feedstocks for biofuel production. A systems-level understanding of the biochemical network is needed to harness the microalgal metabolic capacity for bioproduction. Genome-scale metabolic modeling (GEM) showed immense potential in rational metabolic engineering, utilizing biochemical flux distribution analysis. Here, we report the first GEM for the green microalga, Scenedesmus obliquus (iAR632), a promising biodiesel feedstock with high lipid-storing capability. iAR632 comprises 1467 reactions, 734 metabolites, and 632 genes distributed among 7 compartments. The model was optimized under three different trophic modes of microalgal cultivation, i.e., autotrophy, mixotrophy, and heterotrophy. The robustness of the reconstructed network was confirmed by analyzing its sensitivity to the biomass components. Pathway-level flux profiles were analyzed, and significant flux space expansion was noticed majorly in reactions associated with lipid biosynthesis. In agreement with the experimental observation, iAR632 predicted about 3.8-fold increased biomass and almost 4-fold higher lipid under mixotrophy than the other trophic modes. Thus, the assessment of the condition-specific metabolic flux distribution of iAR632 suggested that mixotrophy is the preferred cultivation condition for improved microalgal growth and lipid production. Overall, the reconstructed GEM and subsequent analyses will provide a systematic framework for developing model-driven strategies to improve microalgal bioproduction.

"DNA signaturing" database construction for Tetradesmus species identification and phylogenetic relationships of Scenedesmus-like green microalgae (Scenedesmaceae, Chlorophyta).

Species identification of Scenedesmus-like microalgae, comprising Desmodesmus, Tetradesmus, and Scenedesmus, has been challenging due to their high morphological and genetic similarity. After developing a DNA signaturing tool for Desmodesmus identification, we built a DNA signaturing database for Tetradesmus. The DNA signaturing tool contained species-specific nucleotide sequences of Tetradesmus species or strain groups with high similarity in ITS2 sequences. To construct DNA signaturing, we collected data on ITS2 sequences, aligned the sequences, organized the data by ITS2 sequence homology, and determined signature sequences according to hemi-compensatory base changes (hCBC)/CBC data from previous studies. Four Tetradesmus species and 11 strain groups had DNA signatures. The signature sequence of the genus Tetradesmus, TTA GAG GCT TAA GCA AGG ACCC, recognized 86% (157/183) of the collected Tetradesmus strains. Phylogenetic analysis of Scenedesmus-like species revealed that the Tetradesmus species were monophyletic and closely related to each other based on branch lengths. Desmodesmus was suggested to split into two subgenera due to their genetic and morphological distinction. Scenedesmus must be analyzed along with other genera of the Scenedesmaceae family to determine their genetic relationships. Importantly, DNA signaturing was integrated into a database for identifying Scenedesmus-like species through BLAST.

Characterization and RNA-seq transcriptomic analysis of a Scenedesmus obliqnus mutant with enhanced photosynthesis efficiency and lipid productivity.

Microalgae have received significant attention as potential next-generation microbiologic cell factories for biofuels. However, the production of microalgal biofuels is not yet sufficiently cost-effective for commercial applications. To screen higher lipid-producing strains, heavy carbon ion beams are applied to induce a genetic mutant. An RNA-seq technology is used to identify the pathways and genes of importance related to photosynthesis and biofuel production. The deep elucidation of photosynthesis and the fatty acid metabolism pathway involved in lipid yield is valuable information for further optimization studies. This study provided the photosynthetic efficiency and transcriptome profiling of a unicellular microalgae, Scenedesmus obliqnus mutant SO120G, with enhanced lipid production induced by heavy carbon ion beams. The lipid yield (52.5 mg L-1) of SO120G mutant were enhanced 2.4 fold compared with that of the wild strain under the nitrogen deficient condition. In addition, the biomass and growth rate were 57% and 25% higher, respectively, in SO120G than in the wild type, likely owing to an improved maximum quantum efficiency (Fv/Fm) of photosynthesis. As for the major pigment compositions, the content of chlorophyll a and carotenoids was higher in SO120G than in the wild type. The transcriptome data confirmed that a total of 2077 genes with a change of at least twofold were recognized as differential expression genes (DEGs), of which 1060 genes were up-regulated and 1017 genes were down-regulated. Most of the DEGs involved in lipid biosynthesis were up-regulated with the mutant SO120G. The expression of the gene involved in the fatty acid biosynthesis and photosynthesis of SO120G was upregulated, while that related to starch metabolism decreased compared with that of the wild strain. This work demonstrated that heavy-ion irradiation is an promising strategy for quality improvement. In addition, the mutant SO120G was shown to be a potential algal strain for enhanced lipid production. Transcriptome sequencing and annotation of the mutant suggested the possible genes responsible for lipid biosynthesis and photosynthesis, and identified the putative target genes for future genetic manipulation and biotechnological applications.

A multi-omic characterization of temperature stress in a halotolerant Scenedesmus strain for algal biotechnology.

Microalgae efficiently convert sunlight into lipids and carbohydrates, offering bio-based alternatives for energy and chemical production. Improving algal productivity and robustness against abiotic stress requires a systems level characterization enabled by functional genomics. Here, we characterize a halotolerant microalga Scenedesmus sp. NREL 46B-D3 demonstrating peak growth near 25 °C that reaches 30 g/m2/day and the highest biomass accumulation capacity post cell division reported to date for a halotolerant strain. Functional genomics analysis revealed that genes involved in lipid production, ion channels and antiporters are expanded and expressed. Exposure to temperature stress shifts fatty acid metabolism and increases amino acids synthesis. Co-expression analysis shows that many fatty acid biosynthesis genes are overexpressed with specific transcription factors under cold stress. These and other genes involved in the metabolic and regulatory response to temperature stress can be further explored for strain improvement.

Identification of microalgae cultured in Bold's Basal medium from freshwater samples, from a high-rise city.

This study aimed at exploring microalgal heterogeneity from fresh water samples collected from inland water bodies in the heavily built city of Singapore. Culturable pure isolates (n = 94) were subject to an in-house microalgal DNA extraction method and LSU rDNA sequencing. Isolates were analysed for their predominance and distribution. A total of 17 different algal genera were identified (H = 2.8, EH = 0.6), of which Scenedesmus spp. and Chlorella spp. constituted 27.5% and 21.3% of isolates respectively, followed by Micractinium spp. (18.8%) and Chlamydomonas spp. (12.5%). We also report 16 new microalgal strains from this region. The data is important from an ecological and biotechnological perspective.

Role of DNA methylation in the chromium tolerance of Scenedesmus acutus (Chlorophyceae) and its impact on the sulfate pathway regulation.

DNA methylation is a very important epigenetic modification that participates in many biological functions. Although many researches on DNA methylation have been reported in various plant species, few studies have assessed the global DNA methylation pattern in algae. Even more the complex mechanisms by which DNA methylation modulates stress in algae are yet largely unresolved, mainly with respect to heavy metal stress, for which in plants, metal- and species- specific responses were instead evidenced. In this work, we performed a comparative Whole-Genome Bisulfite Sequencing (WGBS) on two strains of the green alga Scenedesmus acutus with different Cr(VI) sensitivity. The pattern of distribution of 5-mC showed significant differences between the two strains concerning both differentially methylated local contexts (CG, CHG and CHH) and Differentially Methylated Regions (DMRs) as well. We also demonstrated that DNA methylation plays an important role in modulating some genes for sulfate uptake/assimilation confirming the involvement of the sulfate pathway in the Cr-tolerance. Our results suggest that DNA methylation may be of particular importance in defining signal specificity associated with Cr-tolerance and in establishing new epigenetic marks which contribute to the adaptation to metal stress and also to transmit the epigenomic traits to the progeny.

Mechanisms protect airborne green microalgae during long distance dispersal.

Viable microalgae occur in the air. Whether they can survive the stresses such as UV, desiccation and freezing temperatures at high altitudes during long distance dispersal is rarely studied. If yes, what mechanisms confer the tolerance? Four freshwater airborne green microalgae were isolated from Dongsha Atoll in the South China Sea, classified as Scenedesmus sp. DSA1, Coelastrella sp. DSA2, Coelastrella sp. DSA3 and Desmodesmus sp. DSA6 based on their morphologies and ITS sequences. Their survival rates under UV stress were tightly correlated with their cell wall thickness. All the four airborne green microalgae survived the air-dry stress on benchtop followed by - 20 °C freeze-desiccation stress for 4 weeks, but not the two waterborne green microalgae Desmodesmus sp. F5 and Neodesmus sp. UTEX 2219-4 used as controls. Three of the four airborne microalgae survived the lyophilization treatment, excluding Desmodesmus sp. DSA6 and the two waterborne microalgae. The four airborne microalgae produced carotenoids under prolonged stress conditions, which might help detoxify the reactive oxygen species generated under environmental stresses and shield from the high-light stress in the air. Characterization of these airborne microalgae may help answer how the descendants of green algae survived on the land about 450 MYA.

Algal Lymphadenitis in a Dog Caused by Scenedesmus Species.

A 6-year-old, spayed female Labrador/Weimaraner cross-breed dog that had previously lived in Arizona presented in Montana for an annual examination with an incidentally enlarged popliteal lymph node, which was subsequently biopsied. Histologically, the lymph node was expanded by eosinophil-rich granulomas with both extracellular and intrahistiocytic green algae. These algae had intracytoplasmic, birefringent, and refractile granules; readily formed 2 to 3 mm green colonies on Columbia blood agar medium; and ultrastructurally had a multilayered cell wall and intracytoplasmic chloroplasts. Amplified product from the internal transcribed spacer and D1/D2 regions of the 28S ribosomal RNA gene had high sequence identity to Scenedesmus sp. Despite similar infection in the retropharyngeal lymph node 1 year later, the animal remained otherwise healthy with no clinical signs. To the authors' knowledge, this is the first case of Scenedesmus species infection in a dog and is a differential diagnosis for Coccidioides immitis.

Mechanistic understanding towards the effective lipid production of a microalgal mutant strain Scenedesmus sp. Z-4 by the whole genome bioinformation.

Currently, the complex mechanism of lipid production in microalgal cells is still unclear, and the platform suitable for microalgal genetic transformation is urgent to be established. In this study, the whole genome of a lipid-rich microalgal mutant strain Scenedesmus sp. Z-4 and a lipid-poor wild strain Scenedesmus sp. MC-1 were sequenced, and results revealed that the sequences of 1,256 genes were changed and 148 differential genes related to glucose and lipid metabolism were identified. Especially, gene differentiation of acetyl-CoA carboxylase (ACCase) and phosphoenolpyruvate carboxylase (PEPC) in mutant strain Z-4 and wild strain MC-1, which played key roles in lipid synthesis, were evaluated. Furthermore, to investigate whether mutated ACCase and PEPC genes affect the lipid production, two genes from mutant strain Z-4 were transformed into the expression system of wild strain MC-1. Nine transformants with higher lipid content were successfully obtained, in which the optimal transformant with 28.6% more intracellular lipid than wild strain MC-1 was isolated by overexpression of mutated ACCase gene, demonstrating the important role of ACCase in lipid accumulation of microalgal cells. These results could provide a better understanding of the superior lipid production of mutant strain Scenedesmus sp. Z-4.

Carbohydrates and polyphenolics of extracts from genetically altered plant acts as catalysts for in vitro synthesis of silver nanoparticle.

Eco-friendly biosynthetic approach for silver nanoparticles production using plant extracts is an exciting advancement in bio-nanotechnology and has been successfully attempted in nearly 41 plant species. However, an established model plant system for systematically unraveling the biochemical components required for silver nanoparticles production is lacking. Here we used Arabidopsis thaliana as the model plant for silver nanoparticles biosynthesis in vitro. Employing biochemical, spectroscopic methods, selected mutants and over-expressor plants of Arabidopsis involved in pleotropic functions and sugar homeostasis, we show that carbohydrates, polyphenolics and glyco-proteins are essential components which stimulated silver nanoparticles synthesis. Using molecular genetics as a tool, our data enforces the requirement of sugar conjugated proteins as essentials for AgNPs synthesis over protein alone. Additionally, a comparative analysis of AgNPs synthesis using the aqueous extracts of some of the plant species found in a brackish water ecosystem (Gracilaria, Potamogeton, Enteromorpha and Scendesmus) were explored. Plant extract of Potamogeton showed the highest potential of nanoparticles production comparable to that of Arabidopsis among the species tested. Silver nanoparticles production in the model plant Arabidopsis not only opens up a possibility of using molecular genetics tool to understand the biochemical pathways and components in detail for its synthesis.

Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus.

BACKGROUND: Genetic transformation of microalgae has been hampered by inefficient transgene expression, limiting the progress of microalgal biotechnology. Many vector tools and strategies have been developed in recent years to improve transgene expression in the model microalga Chlamydomonas, but these were hardly applied to other microalgae. In this work, naturally-isolated oleaginous microalgae were accessed for genetic transformation, and various expression systems were evaluated in a selected microalga to circumvent inefficient transgene expression. RESULTS: Initially, a strain of Scenedesmus acutus was selected from the oleaginous microalgal collection based on its highest transformation rate and transgene stability. This strain, which had very low or no GFP reporter expression, was first tested to improve transgene expression by using intron-containing constructs and the transcript fusion using ble::E2A. The intron-containing constructs yielded 2.5-7.5% of transformants with 2-4-fold fluorescence signals, while the majority of the transformants of the transcript fusion had the fluorescence signals up to 10-fold. Subsequently, three UV-induced S. acutus mutants were isolated with moderate increases in the level and frequency of transgene expression (2-3-fold and 10-12%, respectively). Finally, a transcript fusion system was developed using psy white mutants with an expression vector containing PSY::E2A for complementation and light selection. Transformants with green colonies were selected under light exposure, and the transgene expression was detected at protein levels. Although the improvement using PSY::E2A was only minor (1-2-fold increase and ~ 7% of transformants), this system provides an alternative selectable marker that is compatible with large-scale culture. CONCLUSIONS: Here, the overall improvement of transgene expression using the Chlamydomonas tools was moderate. The most effective tool so far is the transcript fusion using ble::E2A system. This work demonstrates that, so far, genetic engineering of non-model microalgae is still a challenging task. Further development of tools and strategies for transgene expression in microalgae are critically needed.

Transcriptome analysis for phosphorus starvation-induced lipid accumulation in Scenedesmus sp.

Phosphorus stress was applied to enhance the lipid production in Scenedesmus sp. The highest lipid production (350 mg L-1) and lipid content (approximately 41.0% of dry weight) were obtained by addition of 2 mg L-1 NaH2PO4·2H2O every 2 days, which were higher than those in replete phosphorus. Correspondingly, carbohydrate content decreased significantly. We speculated that phosphorus limitation could block starch biosynthesis, and photosynthate flow tended to fatty acid biosynthesis to cope with stress. To investigate the mechanism that phosphorus stress triggers the carbon fixation to lipid biosynthesis, the transcriptome analysis was carried out by the Illumina RNA-seq platform. A total of 2897 genes were identified as differentially expressed genes. The observed overexpression of lipid production under phosphorus stress was bolstered by up-regulation of genes encoding for DGAT and pyruvate kinase, activation of carbohydrate metabolism pathway and fatty acid biosynthesis, and repression of carbohydrate synthesis-presumably to shunt the carbon flux toward TAG biosynthesis. The transcriptome will be useful to understand the lipid metabolism pathway and obtain the engineering economic algae species aimed at biodiesel production.

A new approach for detection and quantification of microalgae in industrial-scale microalgal cultures.

In industrial-scale microalgal cultures, non-target microalgae compete with the desired species for nutrients and CO2, thus reducing the growth rate of the target species and the quality of the produced biomass. Microalgae identification is generally considered a complicated issue; although, in the last few years, new molecular methods have helped to rectify this problem. Among the different techniques available, DNA barcoding has proven very useful in providing rapid, accurate, and automatable species identification; in this work, it is used to assess the genomic identity of the microalga species Scenedesmus sp. 'almeriensis', a common strain in industrial-scale cultures. Barcode markers rbcL and ITS1-5.8S-ITS2 were sequenced and the obtained genomic information was used to design a quantitative PCR assay to precisely quantify the S. almeriensis concentration in microalgal cultures of industrial interest. TaqMan chemistry was used to quantify down to 1 µg/L dry weight of S. almeriensis cells, including in the presence of concentrated mixed cultures of other microalgae. A simple direct qPCR approach was also investigated to avoid classic DNA extraction and to reduce total assay time to approximately 2 h. The objective was to design strain-specific tools able to confirm and quantify the presence of different strains in whatever microalgae culture so as to achieve maximal productivity and quality of the produced biomass.

The complete chloroplast genome of the green algae Hariotina reticulata (Scenedesmaceae, Sphaeropleales, Chlorophyta).

In this study, the chloroplast genome of Hariotina reticulata was fully sequenced and compared to other Sphaeropleales chloroplast genomes. It is 210,757 bp larger than most Sphaeropleales cpDNAs. It presents a traditional chloroplast structure, and contains 103 genes, including 68 protein-coding genes, six rRNA genes and 29 tRNA genes. The coding region constitutes of 43% of the whole cpDNA. Eighteen introns are found in 11 genes and six introns are unique for Hariotina. 11 open reading frames are identified among these introns. The synteny between Hariotina and Acutodesmus cpDNAs is in general identical, while within Sphaeropleales order, high variability in cpDNA architecture is indicated by general high DCJ distances. Ankyra judayi exhibits the greatest dissimilarity in gene synteny to the others and share some unique gene clusters with Treubaria triappendiculata. The phylogenomic analyses show that A. judayi is clustered with Treubariaceae species and sister to Chlorophyceae incertae sedis and other Sphaeropleales species. The monophyly of Sphaeropleales is rejected.

De novo transcriptome analysis and gene expression profiling of an oleaginous microalga Scenedesmus acutus TISTR8540 during nitrogen deprivation-induced lipid accumulation.

Nitrogen deprivation (-N) has been used as a technique to promote lipid accumulation in various microalgae. Scenedesmus acutus is a promising oleaginous green microalga that can be cultivated in organic wastewater for biodiesel production. Nevertheless, the molecular mechanisms controlling S. acutus lipid accumulation in response to -N remain unidentified. Physiological study determined that -N reduced cell growth and photosynthetic pigments. On the other hand, it promoted carbohydrate and neutral lipid accumulation. To find the mechanisms underlying lipid accumulation, we performed de novo transcriptome profiling of the non-model S. acutus in response to -N. The transcriptome analysis revealed that glycolysis and starch degradation were up-regulated; on the contrary, gluconeogenesis, photosynthesis, triacylglycerol (TAG) degradation and starch synthesis were down-regulated by -N. Under -N, the carbon flux was shifted toward fatty acid and TAG synthesis, and the down regulation of TAG lipase genes may contribute to TAG accumulation. A comparative analysis of the -N transcriptomes of oleaginous microalgae identified that the down-regulation of multiple lipase genes was a specific mechanism found only in the -N transcriptome of S. acutus. Our study unraveled the mechanisms controlling -N-induced lipid accumulation in S. acutus, and provided new perspectives for the genetic manipulation of biodiesel-producing microalgae.

Effects of temperature, genetic variation and species competition on the sensitivity of algae populations to the antibiotic enrofloxacin.

Primary producers are amongst the most sensitive organisms to antibiotic pollution in aquatic ecosystems. To date, there is little information on how different environmental conditions may affect their sensitivity to antibiotics. In this study we assessed how temperature, genetic variation and species competition may affect the sensitivity of the cyanobacterium Microcystis aeruginosa and the green-algae Scenedesmus obliquus to the antibiotic enrofloxacin. First, we performed single-species tests to assess the toxicity of enrofloxacin under different temperature conditions (20°C and 30°C) and to assess the sensitivity of different species strains using a standard temperature (20°C). Next, we investigated how enrofloxacin contamination may affect the competition between M. aeruginosa and S. obliquus. A competition experiment was performed following a full factorial design with different competition treatments, defined as density ratios (i.e. initial bio-volume of 25/75%, 10/90% and 1/99% of S. obliquus/M. aeruginosa, respectively), one 100% S. obliquus treatment and one 100% M. aeruginosa treatment, and four different enrofloxacin concentrations (i.e. control, 0.01, 0.05 and 0.10mg/L). Growth inhibition based on cell number, bio-volume, chlorophyll-a concentration as well as photosynthetic activity were used as evaluation endpoints in the single-species tests, while growth inhibition based on measured chlorophyll-a was primarily used in the competition experiment. M. aeruginosa photosynthetic activity was found to be the most sensitive endpoint to enrofloxacin (EC50-72h =0.02mg/L), followed by growth inhibition based on cell number. S. obliquus was found to be slightly more sensitive at 20°C than at 30°C (EC50-72h cell number growth inhibition of 38 and 41mg/L, respectively), whereas an opposite trend was observed for M. aeruginosa (0.047 and 0.037mg/L, respectively). Differences in EC50-72h values between algal strains of the same species were within a factor of two. The competition experiment showed that M. aeruginosa growth can be significantly reduced in the presence of S. obliquus at a density ratio of 75/25% M. aeruginosa/S. obliquus, showing a higher susceptibility to enrofloxacin than in the single-species test. The results of this study confirm the high sensitivity of cyanobacteria to antibiotics and show that temperature and inter-strain genetic variation may have a limited influence on their response to them. The results of the competition experiment suggest that the structure of primary producer communities can be affected, at least temporarily, at antibiotic concentrations close to those that have been measured in the environment.

Unraveling the molecular mechanism of photosynthetic toxicity of highly fluorescent silver nanoclusters to Scenedesmus obliquus.

While the discovery of numerous attractive properties of silver at the nanoscale has increased their demand in many sectors including medicine, optics, sensing, painting and cosmetics, it has also raised wide public concerns about their effect on living organisms in aquatic environment. Despite the continuous effort to understand the various aspects of the toxicity of silver nanomaterials, the molecular level understanding on their cytotoxicity mechanism to biological organisms has remained unclear. Herein, we demonstrated the underlying mechanism of the photosynthetic toxicity against green algae namely, Scenedesmus obliquus by using an emerging silver nanomaterial, called silver nanoclusters (defined as r-Ag NCs). By exploiting the unique fluorescence properties of r-Ag NCs along with various other analytical/biological tools, we proposed that the photosynthetic toxicity of r-Ag NCs was largely attributed to the "joint-toxicity" effect of particulate form of r-Ag NCs and its released Ag+, which resulted in the disruption of the electron transport chain of light reaction and affected the content of key enzymes (RuBP carboxylase/ oxygenase) of Calvin cycle of algae cells. We believe that the present study can also be applied to the assessment of the ecological risk derived from other metal nanoparticles.

Expression of Synthetic Phytoene Synthase Gene to Enhance ß-Carotene Production in Scenedesmus sp. CPC2.

ß-carotene is a valuable pigment abundant in some microalgal species but the low ß-carotene productivity of microalgae has become the major obstacles against its commercialization. This work aims to improve the productivity of algae-based ß-carotene via genetic engineering approaches. First, a synthetic psy gene construct (891 bp) encoding 297 amino acids is expressed in Scenedesmus sp. CPC2 host to enhance the ß-carotene production. The synthetic psy gene is designed by considering the highest consensus of amino acids (i.e., 62% identity) from Chlamydomonas reinhardtii, Dunaliella salina, and Mariella zofingiensis. The original ß-carotene content in wild-type Scenedesmus sp. CPC2 is 10.8 mg g-1 -cell when grown on BG11 medium under 2% CO2 aeration, 150 µmol m-2 s-1 light intensity and 25°C. After transformation of the psy gene into the microalgal host, the ß-carotene content of the best recombinant strain (i.e., transformant CPC2-4) significantly increased to over 30 mg g-1 -cell. The optimal production of ß-carotene with the CPC2-4 recombinant strain was achieved when the strain is grown on BG11 medium amended with 0.075 g of MgSO4 , giving approximately 3-fold higher ß-carotene content than that of the wild-type strain. The best cellular ß-carotene content obtained (i.e., 31.8 mg g-1 ) is superior to most algae-based ß-carotene production performance reported in the literature.

Biological effect of aqueous C60 aggregates on Scenedesmus obliquus revealed by transcriptomics and non-targeted metabolomics.

This work evaluated biological effect of nC60 on Scenedesmus obliquus. The cells were exposed to various concentrations of nC60 for 7days. Low-dose of nC60 was found to have a minor growth inhibitory effect. The transcriptomics and metabolomics were integrated to examine intricate molecular and cellular effects of nC60 on Scenedesmus obliquus. We found that Scenedesmus obliquus cells exposed to nC60 had several significant alterations in cellular transcription and biochemical processes. During the 7-day exposure to nC60, 2234 and 2,448 unigenes were differentially expressed by 0.1mg/L and 1mg/L nC60-treated groups compared with the control, including 2085 or 2247 up-regulated genes and 149 or 201 down-regulated genes, respectively. We successfully identified 22 metabolites, including 6 significantly changed metabolites, such as sucrose, d-glucose, and malic acid. The citrate cycle (TCA cycle) (ko00020) was the main target of both differentially expressed genes and metabolic change. However, accumulation of sucrose (end-product) could have induced feedback inhibition of photosynthesis in Scenedesmus obliquus, explaining the slight growth inhibition observed. The results provided a mechanistic understanding of the growth inhibition of nC60 toxicity. These genes and metabolites are useful biomarkers for future studies and offer new insights into the early detectable changes in Scenedesmus obliquus with nC60 exposure.

How DNA barcoding can be more effective in microalgae identification: a case of cryptic diversity revelation in Scenedesmus (Chlorophyceae).

Microalgae identification is extremely difficult. The efficiency of DNA barcoding in microalgae identification involves ideal gene markers and approaches employed, which however, is still under the way. Although Scenedesmus has obtained much research in producing lipids its identification is difficult. Here we present a comprehensive coalescent, distance and character-based DNA barcoding for 118 Scenedesmus strains based on rbcL, tufA, ITS and 16S. The four genes, and their combined data rbcL + tufA + ITS + 16S, rbcL + tufA and ITS + 16S were analyzed by all of GMYC, P ID, PTP, ABGD, and character-based barcoding respectively. It was apparent that the three combined gene data showed a higher proportion of resolution success than the single gene. In comparison, the GMYC and PTP analysis produced more taxonomic lineages. The ABGD generated various resolution in discrimination among the single and combined data. The character-based barcoding was proved to be the most effective approach for species discrimination in both single and combined data which produced consistent species identification. All the integrated results recovered 11 species, five out of which were revealed as potential cryptic species. We suggest that the character-based DNA barcoding together with other approaches based on multiple genes and their combined data could be more effective in microalgae diversity revelation.