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Photosynthetic organisms have evolved photoprotective mechanisms to acclimate to light intensity fluctuations in their natural growth environments. Photosystem (PS) II subunit S (PsbS) and light-harvesting complex (LHC) stress-related proteins (LhcSR) are essential for triggering photoprotection in vascular plants and green algae, respectively. The activity of both proteins is strongly enhanced in the moss Physcomitrella patens under high-light conditions. However, their role in regulating photosynthesis acclimation in P. patens under fluctuating light (FL) conditions is still unknown. Here, we compare the responses of wild-type (WT) P. patens and mutants lacking PsbS (psbs KO) or LhcSR1 and 2 (lhcsr KO) to FL conditions in which the low-light phases were periodically interrupted with high-light pulses. lhcsr KO mutant showed a strong reduction in growth with respect to WT and psbs KO under FL conditions. The lack of LhcSR not only decreased the level of non-photochemical quenching, resulting in an over-reduced plastoquinone pool, but also significantly increased the PSI acceptor limitation values with respect to WT and psbs KO under FL conditions. Moreover, in lhcsr KO mutant, the abundance of PSI core and PSI-LHCI complex decreased greatly under FL conditions compared with the WT and psbs KO. We proposed that LhcSR in P. patens play a crucial role in moss acclimation to dynamic light changes.

The quality of light is an important abiotic factor that aff ects the growth and development of photosynthetic organisms. In this study, we exposed the unicellular green alga Dunaliella salina to red (660 nm) and blue (450 nm) light and analyzed the cell growth, total carotenoid content, and transcriptomes. The growth of D. salina was enhanced by illumination with red light, whereas blue light was not able to promote the algal growth. In contrast, the total carotenoid content increased under both red and blue light. The RNA of D. salina was sequenced and the transcriptomic response of algal cells to red and blue light was investigated. Six transcripts encoding for the blue light receptor cryptochrome were identifi ed, and transcripts involved in the carotenoid metabolism were up-regulated under both red and blue light. Transcripts encoding for photoprotective enzymes related to the scavenging of reactive oxygen species were up-regulated under blue light. The present transcriptomic study provides a more comprehensive understanding of carotenoid biosynthesis in D. salina under diff erent wavelengths of light.

Since 2007, Ulva prolifera, a green macroalgal species that blooms periodically in the Yellow Sea of China, has caused the world’s biggest green tide, which induced harmful ecological impacts and economic losses. When the alga is subject to prolonged exposure to air, it suffers abiotic stresses. To explore the physiological and molecular mechanisms of salt stress, the transcriptome data of U. prolifera at different salinities (30, 50, and 90 psu) were obtained by high-throughput sequencing using the Illumina HiSeq platform, and photosynthetic physiological parameters were also measured. The results showed that a total of 89,626 unigenes were obtained after de novo assembly, of which 60,441 unigenes were annotated in the databases (NR, NT, KO, SWISS-PROT, PFAM, GO, and KOG). GO functional enrichment analysis revealed that the enrichment of differentially expressed genes (DEGs) was mainly in cellular, cell, and binding processes. KEGG metabolic pathway enrichment analysis showed that the most frequently enriched pathways of DEGs included glycolysis, pyruvate metabolism, peroxisome, and fatty acid biosynthesis. In addition, resistance-associated proteins, such as heat-shock proteins, microtubule-associated proteins, ubiquitin-associated proteins, abscisic-acid-signaling-pathway-associated proteins, and antioxidant-related proteins are upregulated under salt stress. Genes associated with photosynthesis and carbon fixation pathways are also upregulated, accompanied by an increase in photosynthetic oxygen release rates. These findings provide a basis for understanding the molecular mechanisms of the response of U. prolifera to salinity change, thus providing a theoretical basis for the analysis of the green tide outbreak mechanism.

Cyanobacteria are rich in phytochemicals, which have beneficial impacts on the prevention of many diseases. This study aimed to comprehensively characterize phytochemicals and evaluate multifunctional bioactivities in the ethanolic extract of the cyanobacterium Leptolyngbya sp. KC45. Results found that the extract mainly contained chlorophylls, carotenoids, phenolics, and flavonoids. Through LC–ESI–QTOF–MS/MS analysis, 38 phenolic compounds with promising bioactivities were discovered, and a higher diversity of flavonoids was found among the phenolic compounds identified. The extract effectively absorbed the harmful UV rays and showed high antioxidant activity on DPPH, ABTS, and PFRAP. The extract yielded high-efficiency inhibitory effects on enzymes (tyrosinase, collagenase, ACE, and α-glucosidase) related to diseases. Interestingly, the extract showed a strong cytotoxic effect on cancer cells (skin A375, lung A549, and colon Caco-2), but had a much smaller effect on normal cells, indicating a satisfactory level of safety for the extract. More importantly, the combination of the DNA ladder assay and the TUNEL assay proved the appearance of DNA fragmentation in cancer cells after a 48 h treatment with the extract, confirming the apoptosis mechanisms. Our findings suggest that cyanobacterium extract could be potentially used as a functional ingredient for various industrial applications in foods, cosmetics, pharmaceuticals, and nutraceuticals.

Phycobiliproteins is a natural product with many biological activities in various seaweeds. Phycobiliproteins have been widely used for anti-oxidation, anti-tumor, anti-inflammatory and immune-enhancing activities as a functional factor. Phycobiliproteins with high purity are considerably more expensive than common. To provide with a systematic, deep and detailed information about those features of phycobiliproteins, we performed a relatively comprehensive analysis on structural composition, the application of phycobiliproteins in the fields of fluorescent probe and photodynamic therapy in this report.

BackgroundMacroalgae, particularly commercially grown seaweed, substantially contribute to CO2 removal and carbon storage. However, knowledge regarding the CO2 concentrating mechanism (CCM) of macroalgae is limited. Carbonic anhydrase (CA), the key component of biophysical CCM, plays important roles in many physiological reactions in various organisms. CA has been widely studied in microalgae and higher plants. However, few characteristics of CA in Pyropia yezoensis are known, particularly its intracellular location and responses to different concentrations of Ci.ResultsWe retrieved transcriptomic and genomic data for P. yezoensis and identified, amplified and characterized 11 putative genes encoding CA. The predicted corresponding proteins clustered into three subfamilies: α-, β- and γ-type. Transcriptomic and qRT-PCR results revealed different expression levels of these PyCA genes in the two life stages of P. yezoensis, and their expression levels varied under different Ci conditions. The intracellular localization of eight CA isoforms—one in the chloroplasts, four in the cytoplasm and three in the mitochondria—were elucidated with fusion proteins. One γCA isoform (PyCA10) was shown to be localized in the cytosol, and its localization is discussed. We also discuss the potential roles of the CA isoforms in development and the CCM in the two life stages. ConclusionsCA isoforms in P. yezoensis are widely distributed within cells, and higher PyCA expression, particularly of certain chloroplastic, cytosolic and mitochondrional CAs, is observed more often during the leafy stage. This expression results in stronger carbon sequestration ability in leafy thalli than filamentous thalli.

Background Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation. Results In this study, G6PDH was identified as a target for algal strain improvement, wherein G6PDH gene was successfully overexpressed and antisense knockdown in P. tricornutum, and systematic comparisons of the photosynthesis performance, algal growth, lipid content, fatty acid profiles, NADPH production, G6PDH activity and transcriptional abundance were performed. The results showed that, due to the enhanced G6PDH activity, transcriptional abundance and NAPDH production, overexpression of G6PDH accompanied by high-CO2 cultivation resulted in a much higher of both lipid content and growth in P. tricornutum, while knockdown of G6PDH greatly decreased algal growth as well as lipid accumulation. In addition, the total proportions of saturated and unsaturated fatty acid, especially the polyunsaturated fatty acid eicosapentaenoic acid (EPA; C20:5, n-3), were highly increased in high-CO2 cultivated G6PDH overexpressed strains. Conclusions The successful of overexpression and antisense knockdown of G6PDH well demonstrated the positive influence of G6PDH on algal growth and lipid accumulation in P. tricornutum. The improvement of algal growth, lipid content as well as polyunsaturated fatty acids in high-CO2 cultivated G6PDH overexpressed P. tricornutum suggested this G6PDH overexpression-high CO2 cultivation pattern provides an efficient and economical route for algal strain improvement to develop algal-based biodiesel production.

The unicellular green alga Dunaliella salina is an attractive organism for studying photoacclimation responses. Changes in irradiance level during cell growth affect the organization and structure of the photosystem and the composition of pigments. In this study, D. salina was exposed to different irradiance levels, and cell growth, photosynthetic activity, pigments, and neutral lipids were analyzed. Photosystem II activity and chlorophyll content were reduced, carotenoids content and neutral lipids increased as light intensity increased, suggesting that photosynthetic apparatus was reduced and photoprotection mechanisms were activated. The RNA of D. salina was sequenced to investigate the transcriptomic response of the organism after transitioning from normal light conditions to higher light intensity. Genes encoding for enzymes involved in photosynthesis were downregulated, whereas genes involved in the metabolism of carotenoid and triacylglycerol were upregulated. Genes encoding for photoprotective enzymes related to reactive oxygen species scavenging and to the xanthophyll cycle were also upregulated at higher irradiance levels. The present transcriptomic study would assist in the comprehensive understanding of photoacclimation mechanisms of D. salina.

Mariculture wastewater generated from the mariculture industry has increased public concern due to its impact on the sustainability of aquatic environments and aquaculture practices. Herein, the Bacterial-Algal Coupling System was applied for mariculture wastewater treatment. Microalgae growth in heterotrophy and mixotrophy (2000–8000 lux) were first compared. The best microalgal growth and nutrient removal were obtained at 5000 lux, where biomass productivity of microalgae was 0.465 g L−1 d−1, and 98.1% of chemical oxygen demand, 70.7% of ammonia-nitrogen, and 90.0% of total phosphorus were removed. To further understand the nutrient removal through microalgae cultivation, the enzyme activities involved in the Calvin cycle and the Tricarboxylic Acid cycle at different light intensities were determined. Under mixotrophic cultivation, there was a coordination between photosynthesis and heterotrophic metabolism in agal cells, which resulted in a high algal biomass production and removal efficiency of nutrients. This study provided a novel insight into the bioremediation of mariculture wastewater and microalgae cultivation.

Background Pyropia yezoensis (Rhodophyta) is widely cultivated in East Asia and plays important economic, ecological and research roles. Although inorganic carbon utilization of P. yezoensis has been investigated from a physiological aspect, the carbon concentration mechanism (CCM) of P. yezoensis remains unclear. To explore the CCM of P. yezoensis, especially during its different life stages, we tracked changes in the transcriptome, photosynthetic efficiency and in key enzyme activities under different inorganic carbon concentrations. Results Photosynthetic efficiency demonstrated that sporophytes were more sensitive to low carbon (LC) than gametophytes, with increased photosynthesis rate during both life stages under high carbon (HC) compared to normal carbon (NC) conditions. The amount of starch and number of plastoglobuli in cells corresponded with the growth reaction to different inorganic carbon (Ci) concentrations. We constructed 18 cDNA libraries from 18 samples (three biological replicates per Ci treatment at two life cycles stages) and sequenced these using the Illumina platform. De novo assembly generated 182,564 unigenes, including approximately 275 unigenes related to CCM. Most genes encoding internal carbonic anhydrase (CA) and bicarbonate transporters involved in the biophysical CCM pathway were induced under LC in comparison with NC, with transcript abundance of some PyCAs in gametophytes typically higher than that in sporophytes. We identified all key genes participating in the C4 pathway and showed that their RNA abundances changed with varying Ci conditions. High decarboxylating activity of PEPCKase and low PEPCase activity were observed in P. yezoensis. Activities of other key enzymes involved in the C4-like pathway were higher under HC than under the other two conditions. Pyruvate carboxylase (PYC) showed higher carboxylation activity than PEPC under these Ci conditions. Isocitrate lyase (ICL) showed high activity, but the activity of malate synthase (MS) was very low. Conclusion We elucidated the CCM of P. yezoensis from transcriptome and enzyme activity levels. All results indicated at least two types of CCM in P. yezoensis, one involving CA and an anion exchanger (transporter), and a second, C4-like pathway belonging to the PEPCK subtype. PYC may play the main carboxylation role in this C4-like pathway, which functions in both the sporophyte and gametophyte life cycles.

The previous study revealed that the mutant strain E thalli of Pyropia yezoensis had growth advantage compared with the initial strain NA, and transcriptome analysis revealed the relevant bioprocesses for improved growth. To investigate the growth mechanism at the protein level, this study used isobaric tags for relative and absolute quantification and liquid chromatography-mass spectrometry to analyze proteome of both strains. In total, 277 proteins were identified, among which 41 differentially expressed proteins were screened, including 22 up-regulated proteins and 19 down-regulated proteins. Integrated analysis of proteome and previous transcriptome showed that the proteins related to photosynthesis, protein biosynthesis, glycolysis, and pentose phosphate pathway were enhanced in mutant strain E, which were supposed to facilitate growth of mutant strain E, and the proteins relevant to stress resistance were up-regulated, which might promote the growth of mutant strain E under abiotic stresses, while proteins related to methylation were decreased, which might enhance the expression of growth relevant genes in mutant strain E. This study revealed the growth mechanism in mutant strain E by integrated omics analysis, and also provided theoretical foundation for molecular breeding of Py. yezoensis.

Dunaliella salina is a unicellular green alga extensively studied due to its capability of salt stress tolerance and high economic value for β-carotene production. Glycerol is synthesized to regulate intracellular osmotic pressure during salt stress in D. salina. In order to demonstrate the electron and reducing power distribution pattern in this process, photosynthetic performance and oxidative pentose phosphate pathway (OPPP) operation during salt stress were investigated. Glycerol was accumulated massively when D. salina was subjected to salt stress. Meanwhile, photosystem II (PSII) activity was increased while photosystem I (PSI) activity was decreased. This suggests that a small portion of the electrons generated in PSII were leaked and were transferred to generate reactive oxygen species (ROS), while most electrons subsequently used to PSI to generate ATP and NADPH. Furthermore, the OPPP was enhanced in order to provide reducing power and carbon skeleton for glycerol synthesis. In conclusion, during salt stress, the imbalance of photosynthetic electron distribution between PSII and PSI in D. salina resulted in a shortage of NADPH and ATP for glycerol biosynthesis, which also led the enhancement of the OPPP and ROS generation.

Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde-3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that α-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.

Microalgae are rich in various high-value compounds and thus have been proposed for use in biodiesel production and other industrial processes. Mixotrophy of microalgae can generate a high biomass and a high cell content of desirable compounds. To elucidate the probable relationship between pH drift, carbon source utilization, light intensity, and biomass accumulation in Chlorella cultivation, we compared the growth, pH drift, total biomass, and lipid and starch contents of Chlorella sorokiniana GXNN01 under different culture conditions. We also labeled the cell with 13C-labeled substrates. The results of pH drift and 13C labeling showed that Chlorella preferred acetate under high light, but glucose under low light. Glucose utilization resulted in decrease of pH, while acetate had the opposite effect. A combination of acetic acid and glucose as carbon source maintained the pH close to the initial value and significantly increased total biomass production. Cultures using acetic acid as carbon source or addition of sodium acetate under low light appeared to enhance total lipid content, while pH adjustment, addition of two carbon sources, or addition of sodium acetate under normal light (20 μmol photons m−2 s−1) resulted in high starch content. This knowledge will guide future Chlorella cultivation.

Light drives photosynthesis but may also cause photodamage. Oxygenic photosynthetic organisms have evolved various photoprotective responses to adapt to excessive light. Zeaxanthin and its precursor, β-carotene, play key photoprotective roles in higher plants and in most algae; however, it remains unclear if they are also involved in photoprotective responses in red algae. Here, we report that the concentrations of β-carotene and zeaxanthin increased significantly in response to high light conditions in Pyropia yezoensis (Bangiales, Rhodophyta) sporophytes. However, the response was relatively minor, and the accumulated zeaxanthin did not reduce when the stress was eliminated. The accumulation of β-carotene and zeaxanthin in P. yezoensis was accompanied with variations in non-photochemical quenching kinetics when the sporophytes were subjected to high light stress. We cloned the cDNAs encoding lycopene ϵ-cyclase and β-carotene hydroxylase in P. yezoensis, and quantified their expression levels. Our results suggested that the accumulation of β-carotene and zeaxanthin was attributed to the reduced expression levels of lycopene ϵ-cyclase and increased expression levels of β-carotene hydroxylase. Therefore, we propose that although red algae have evolved a common response to high light stress by accumulating β-carotene and zeaxanthin, the mechanism is not as flexible as in higher plants and other algae using the xanthophyll cycle. Rather than binding to PSII as in higher plants and other algae, the accumulated zeaxanthin may bind to PSI-light harvesting complex I in P. yezoensis, and in turn protect PSI against high light-induced photodamage.

Crocetin is one of the major active ingredients of the rare medicinal herb saffron (Crocus sativus L.), also known as crocus. Crocetin has a wide range of effects including antitumor, anticancer, antioxidant, and antiatherosclerotic activity. Crocetin is currently only obtained via extraction from crocus stigmas, with limited yields. This study developed a new method of production of crocetin from genetically engineered Chlorella vulgaris, a unicellular, fast-growing, and heterotrophically cultured green microalga. A plant expression vector carrying the genes crtRB and ZCD1, which encode enzymes controlling critical steps in crocetin biosynthesis, was introduced into C. vulgaris using the Agrobacterium tumefaciens-mediated transformation method. After hygromycin screening, resistant C. vulgaris strains were achieved. Polymerase chain reaction amplification confirmed that the crtRB and ZCD1 were successfully integrated into the C. vulgaris genome. High-performance liquid chromatography analysis of methanol extraction showed that the transgenic C. vulgaris was able to produce crocetin, while wild-type C. vulgaris was not. These results strongly suggest that introducing crtRB and ZCD1 into C. vulgaris can successfully produce crocetin. This is a new method of crocetin production in C. vulgaris to compensate for the shortage of crocetin.

Malate dehydrogenase (MDH) is an enzyme that catalyzes the interconversion of malate and oxaloacetate substrates and is widely distributed from prokaryotes to eukaryotes. It plays crucial roles in many important metabolic pathways and includes different isoforms based on coenzyme specificity and cellular localization. To study MDH in rhodophytes, we obtained a full-length cDNA clone (here designated PhMDH) encoding an NAD-malate dehydrogenase in the marine red alga Pyropia haitanensis. The nucleotide sequence of PhMDH was 1521 bp, including an open reading frame (ORF) of 984 bp. The amino acid sequence showed 73% identity with other MDHs in proteobacteria. Two MDH-like domains were detected in the 5–145 and 156–320 regions. Real-time fluorescent quantitative PCR (qPCR) was used to examine mRNA expression levels during the gametophyte and sporophyte phases. The transcription of PhMDH in the gametophyte was barely detectable, whereas PhMDH in the sporophyte showed a much higher expression level. The activity of PhMDH in the filamentous sporophyte was approximately double that of the leafy gametophyte. Considering these results, we suggest that PhMDH may be localized in the cytosol and play a role in carbon fixation in the sporophyte stage.

The effects of high light (HL) stress on the photosynthetic proteins of green tide-forming macroalga were studied. The naturally grown Ulva prolifera was collected from an intertidal habitat and subjected to 1000 mu E m(-2) s(-1) HL for 12 h. The thylakoid membrane proteins were subsequently extracted and qualitatively analyzed using LC/MS. Quantitative analysis of the thylakoid membrane proteins showed that both PSII reaction center proteins and PSII chlorophyll antenna proteins were decreased. Comparatively, the main PSI reaction center proteins were well preserved. The cytochrome f subunit was significantly decreased, while the other subunits of cytochrome b (6) /f complex were maintained. These results demonstrated that the HL-induced cytochrome b (559) might play a role in photoacclimation of U. prolifera by involving in cyclic photosynthetic electron transfer (PET) flow around PSII. The LhcSR and PsbS proteins were also identified in U. prolifera, and PsbS was noted to be significantly increased under HL stress. The long-term photoacclimation mechanism of U. prolifera under HL stress might not depend on cyclic PET flow around PSI.

Cell penetrating peptides (CPPs) are effective molecular tools that can deliver various exogenous cargoes into different types of cells. However, no study to date has evaluated the use of CPPs to import nucleic acid substances into microalgae, which would be advantageous for numerous research and commercial applications. In this study, we tested the ability of nona-(L)-arginine (R9) to conduct CPP-mediated double-stranded RNA (dsRNA) translocations into four microalgal species (Chlamydomonas reinhardtii, Chlorella vulgaris, Phaeodactylum tricornutum, and Dunaliella salina). R9 successfully transported dsRNA into D. salina, whereas dsRNA internalizations were not detected in the other three algal species. Calcofluor white staining showed that the cell wall structure of D. salina differs from that of the other three species. These results indicate that the efficiency of CPP-mediated dsRNA transductions is closely related to algal cell wall structures, which vary in their ability to block entry of the CPP/ dsRNA complex. (C) 2015 Elsevier B.V. All rights reserved.

Based on the transposon-mutagenized library of Pantoea agglomerans BH18, mutant screens were conducted to obtain the strain with the highest Fe (III) reduction and hydrogen production. Of these transposon-mutagenized mutants, the mutant strain TB230 was screened for high Fe (III)-reducing efficiency and hydrogen production. The PCR amplification and kanamycin resistance selection results indicated that the transposon insertion of the mutant strain TB230 was stable. Hydrogen production of the mutant strain TB230 was (2.21 +/- 0.34) mol H-2/mol glucose, which increased hydrogen production by over 40% compared with that of the wild type strain. The accumulation concentration of Fe (II) in the medium of the mutant strain TB230 with Fe (OH)(3) as the sole electron acceptor was (7.39 +/- 0.49) mmol/l, which was approximately 3-fold greater than that of the wild type strain. The mutant strain TB230 showed high Fe (III)-reducing activity and hydrogen production by adopting glucose and pyruvate as the carbon source. In addition, the mutant strain TB230 was capable of Fe (III) reduction and hydrogen production under-fresh or marine conditions. This result indicates that the mutant strain with high microbial Fe (III) reduction and hydrogen production is beneficial for the improvement of anaerobic performance. (C) 2015 Elsevier Ltd. All rights reserved.

Hydrogen is recognized as a promising fuel for the future because it is renewable, environmentally harmless and cost-effective. Systematic research on the process of hydrogen production is important for increasing hydrogen yield and identification of suitable strains of bacteria. We investigated the hydrogen production pathways of Enterobacter cloacae. The maximum hydrogen yield was -707 ml L-1, the H-2 content of the gasphase was in the range 30-35% and the overall rate of hydrogen production was -70.70 ml h(-1) L-1. Ethyl alcohol and 2,3-butanediol were the major fermentative products, indicating the hydrogen-producing fermentation pathway of E. cloacae was the 2,3butanediol pathway. Proteins and enzymes involved in the 2,3-butanediol pathway were generally detected by proteomics analysis, providing further evidence for the presence of the 2,3-butanediol pathway in E. cloacae. Both C-13 labeling experiments and proteomics analysis indicated the Embden-Meyerhof-Pamas (EMP) pathway was the major pathway and the hexose monophosphate (HMP) pathway had a weaker role, or did not participate, in glucose catalysis in E. cloacae. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Biohydrogen production that utilizes high-salt organic wastewater as substrate not only could treat organic waste but also could generate green energy-hydrogen. Investigation of the hydrogen-producing mechanism will lay the foundation for the industrialization of biohydrogen production. In this study, analysis revealed that mixed-acid fermentation occurred in Pantoea agglomerans. However, the amount of hydrogen that would be produced by mixed-acid fermentation (50%) was found to be less than the actual total hydrogen content in the fermenter (67.4%). Hence, a proteomic study was conducted to detect other possible pathways of hydrogen production. The results showed a nitrogenase participated in hydrogen production as a hydrogenase under anaerobic conditions. The results of both proteomics and C-13 metabolic flux ratio analysis proved the substrates consumed during hydrogen production were supplied by EMP and PPP. All of these results revealed that hydrogen production was generated by the interaction of mixed-acid fermentation and nitrogenase in the marine bacterium P. agglomerans. EMP and PPP provided pyruvate, NAD(P)H, and ATP for hydrogen production. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Single-stage hydrogen production from glucose was investigated using the marine photosynthetic bacterium Rhodovulum sulfidophilum TH-79, a Tn7 transposon mutant of strain P5. The mutation in strain TH-79 did not affect its cell growth in glucose medium compared with the parent strain. TH-79 displayed improved photoheterotrophic hydrogen production performance when the medium contained glucose or galactose as the sole carbon source. The mutant produced about 7.07 mol H-2/mol glucose, which is similar to the yields of more complicated integration systems. A one-stage photofermentation system using a seawater culture medium appears to be a promising alternative to the integration of dark- and photofermentation systems. Copyright (c) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Phaeodactylum tricornutum is an economically important species of microalgae that is widely used in aquaculture, and it is rich in bioactive substances including eicosapentaenoic acid and fucoxanthin. The major bottleneck for industrialization of this species is harvesting. Flocculation is used to harvest microalgae, thus the selection of flocculants is of great importance. In this study, we compared the flocculation effect of four different chemicals (ferric chloride, aluminum sulphate, polyaluminum chloride, and aluminum potassium sulphate) on P. tricomutum. Microexamination showed that ferric and aluminum salts had similar flocculation effects on the algae. Growth and chlorophyll fluorescence measurements showed that P. tricornutum can be re-cultured after flocculation. Pigment analysis showed that flocculation did not result in degradation of fucoxanthin, which suggests that the four flocculants tested may be useful for industrial applications. The results also showed that ferric chloride was the best flocculant for harvesting P. tricomutum when the target product was fucoxanthin, as it had the least influence on the physiological activity of P. tricornutum and it did not lead to degradation of cell components. In contrast, aluminum is poisonous to the nervous system of animals and humans. In addition, the culture medium can be recycled after flocculation by ferric chloride. (C) 2014 Elsevier Ltd. All rights reserved.

Most of the photosynthetic bacterial strains exhibit optimum hydrogen production at neutral initial pH, and lower initial pH resulted in a sharp decrease in hydrogen yield. Thus, screening of acid-tolerant hydrogen-producing photosynthetic bacteria is very important. To obtain acid tolerant mutants, a Tn7-based transposon was randomly inserted into the genomic DNA of Rhodovulum sulfidophilum P5. An acid tolerant mutant strain TH-102 exhibited increased hydrogen production in acidic environment (pH 4.5-6.5) and at higher temperatures (35 and 37 degrees C) than the wild-type strain. At pH 5.5 and 35 degrees C, the mutant strain TH-102 continuously produced hydrogen. The hydrogen yield and average rate were 2.16 +/- 0.10 mol/mol acetate and 10.06 +/- 0.47 mL/L h, which was about 17.32 and 15.37-fold higher than that of the wild-type strain, respectively. This acid-and temperature-tolerant mutant strain TH-102 could be used in a cost-effective hydrogen production process employing both dark fermentative and photosynthetic bacteria. (C) 2014 Elsevier Ltd. All rights reserved.

Inducing lipid accumulation in microalgae cells without suppressing cell growth is vital to the economical production of biodiesel from microalgae. In two experiments, we demonstrate that the cell concentration and lipid content of marine microalgae Isochrysis galbana depend upon the iron concentration in the growth media. In Experiment I, adding chelated FeCl3 to the medium at the late exponential growth phase prolonged this phase and increased the lipid content in I. galbana cells. The final cell density and lipid content of I. galbana supplemented with chelated FeCl3 was approximately 2 and 1.65 times higher than that of non-supplemented cultures, respectively. In Experiment II, I. galbana cells in the late exponential phase were collected and re-inoculated into new media containing Fe3+ at various concentrations. The final cell concentration and lipid content were maximized at the highest iron concentration (38% biomass by dry weight at 1.2x10(-5) mol/L Fe3+). In this study, intracellular neutral lipid storage was evaluated by fluorescent spectrophotometry using fluorochrome Nile red, and the measurement conditions were optimized.

Diatoms and dinoflagellates not only have extensive distribution and a huge biomass in marine ecosystems, but also have high lipid accumulation in nature or after physiological and genetic modification, which indicates that these organisms may be optimal candidate algal strains for biodiesel production. In this study, we determined the content of intracellular neutral lipids (triacylglycerol [TAG]) in the dinoflagellate Prorocentrum micans and in the diatom Phaeodactylum tricornutum using NR and BODIPY 505/515 staining. The freshwater green alga Scenedesmus obliquus was used as a control. Optimum concentrations of 1.000 and 1.500 μg mL−1 were determined for neutral lipid Nile red (NR) staining in P. micans and P. tricornutum. Unlike NR staining, the optimal concentrations of BODIPY 505/515 staining in P. micans and P. tricornutum were lower, at 0.100 and 0.075 μg mL−1, respectively. High correlation coefficients of R2 = 0.990 and R2 = 0.989 were obtained for P. micans and P. tricornutum intracellular neutral lipid content and the relative fluorescence intensity with NR staining, while the reference alga, S. obliquus, had a relatively low correlation coefficient of R2 = 0.908 when stained with NR. The neutral lipid content determined by thin-layer chromatography-flame ionization detector matched the analytical data from fluorescence measurements. These results indicated that NR and BODIPY 505/515 staining can be used as an excellent high-throughput approach to screen marine diatoms and dinoflagellates.

Based on the generation of Pantoea agglomerans BH18 library using Tn7 transposon system, mutant screens were conducted for improvement the ability of hydrogen production of this strain. These mutants were used to test for ability of hydrogen production in the initial pH of 5.0. In contrast to wild type strain BH18, a transposon mutant, named as strain TB108, was screened for high hydrogen-producing capability and acid tolerance in the initial pH of 5.0. The factors required for hydrogen production of the aciduric transposon-mutagenized strain TB108 were determined. The mutant strain TB108 similar as wild type strain BH18 was able to produce hydrogen over a wide range of salt concentration from 0.4% to 6%. Under the marine conditions with the initial pH of 5.0 and glucose concentration of 10 g/L, the total hydrogen production of the mutant TB108 was (1.36 +/- 0.04) mol H-2/mol glucose (mean +/- SE.), increasing by 55% compared with wild type. In addition, the mutant strain TB108 could produce hydrogen using many carbon sources such as fructose, glucose, sucrose, sorbitol and so on. This result demonstrated that the mutant strain with high acid tolerance is beneficial for improvement of hydrogen production. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Prorocentrum donghaiense is the most common bloom-forming species in the East China Sea, causing serious damage to regional marine ecosystems, marine fisheries, public health, and aquatic environment. To minimize fisheries losses caused by this harmful alga, a simple and accurate detection method need to be developed to provide adequate early warning for P. donghaiense blooms. In this study, we report the development and application of reverse transcription-coupled loop-mediated isothermal amplification (RT-LAMP) in the field detection of P. donghaiense. The partial large subunit rDNA (LSU D1-D2), small subunit rDNA, and internal transcribed spacers of P. donghaiense were first sequenced to design species-specific amplification primers. Primer set screen showed that the LSU D1-D2 was the best target for LAMP in terms of sensitivity and stability. The amplification conditions including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time, and temperature, were optimized. The specificity of RT-LAMP for P. donghaiense was also confirmed through tests using a few common harmful algae. RT-LAMP targeting RNA exhibited a detection limit of 0.6 cells, which is more sensitive than LAMP and PCR targeting DNA and reverse transcription-polymerase chain reaction (RT-PCR) targeting RNA. Finally, an improved protocol for natural samples was applied to the field material. The optimized detection protocol could be completed within 1 h. In addition, positive RT-LAMP results could be confirmed through the production of white magnesium pyrophosphate precipitate or through mixing the fluorescent dye GeneFinder (TM) with the amplification products. In summary, the established RT-LAMP is specific, sensitive, and rapid method that is promising for the field detection of P. donghaiense. (c) 2013 Elsevier B.V. All rights reserved.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes carboxylation of phosphoenolpyruvate or decarboxylation of oxaloacetate, which plays an important role in some C-4 plants and algae. The full-length cDNA of the PEPCK gene cloned from Porphyra haitanensisT. J. Chang et B. F. Zheng (Bangiales, Rhodophyta), was denoted Phpepck. It had a nucleotide sequence of 2300 bp, including a 5-untranslated region (UTR) of 147 bp, a 3-UTR of 329 bp and an open reading frame of 1824 bp. The expression of PEPCK in P.haitanensis sporophytes and gametophytes was examined at the levels of transcription, translation and enzyme activity. Both PEPCK abundance and activity were higher in sporophytes than those in gametophytes, suggesting that a C-4-like carbon-fixation mechanism might be functional in P.haitanensis sporophytes.

Harmful algal blooms recently have been under the spotlight throughout the world, because of their negative impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning andmonitoring of blooms, among which the techniques based on taxonomic probes are themost favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were taken into consideration. The partial large subunit rDNA (D1-D2) of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization (FISH) tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K. mikimotoi, respectively. Both the specific (taxonomic) (Pmin0443 and Kmik0602) and the control probes (UniC0512 and UniR0499) were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the techniques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P. minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant difference (p > 0.05) was observed in the cell densities of the samples determined by FISH and light microscopy (LM). All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae.

The culture of microalgae using organic carbon sources decreases the cost of operation in closed systems. The effect of carbon sources on microalgae is thus an interesting problem in not only theoretical research but also practical production. The short-term effects of acetate and microaerobic conditions on the growth, photosynthesis, and respiration of the green microalga Chlorella sorokiniana I. Shihira & R.W. Krauss GXNN 01 were described after acetate addition to autotrophic cultures. As the acetate concentration increased, cells needed a longer lag phase to grow, and 243.8 mM acetate completely inhibited growth. Acetate addition induced an immediate response in photosynthesis and respiration. The activity of PS II and PS I were impaired and declined with different rates, and then recovered compared with autotrophic cells. Carbonic anhydrase and Rubisco activities were also inhibited at the beginning, and respiration was increased. We propose that ATP consumption for acetate assimilation results in surplus NADPH, and then accumulated reducing power over-reduces inter-photosystem components and raises the transthylakoid proton gradient, which redistributes energy between PS I and PS II, and leads to a decrease in the PS II/PS I ratio and O2 evolution. An apparent cyclic electron flow was also observed, which may be mainly mediated by NAD(P)H dehydrogenase-dependent pathway since NADPH was in excess. These observations pointed to an acclimation process after acetate addition, and suggested the interaction between photosynthesis and respiration involving ATP and reducing power.

A newly enriched marine phototrophic bacterial consort was studied for its capability of hydrogen production in batch cultivations using butyrate as the sole carbon source. Analyses of denaturing gradient gel electrophoresis (DGGE) profiles showed that the mixed bacterial consort consisted mainly of Ectothiorhodospira, Sporolactobacillus, and Rhodovulum. Important parameters investigated include temperature, light intensity, initial pH, and butyrate concentration. The pH of the culture medium significantly increased as fermentation proceeded. Optimal cell growth was observed at temperature of 25-35 degrees C, light intensity of 80-120 mu mol photons/m(2) s, initial pH of 8, butyrate concentration of 20-40 mmol/l. Optimal conditions for hydrogen production were 30 degrees C, light intensity of 80 mu mol photons/m(2) s, initial pH 8. The increase of butyrate concentration (10-50 mmol/l) resulted in higher hydrogen production, but the yield of hydrogen production (mol H-2/mol butyrate) gradually decreased with increasing butyrate concentration. The maximal hydrogen yield and hydrogen production rate were estimated to be 2.52 +/- 0.12 mol H-2/mol butyrate and 19.40 +/- 2.32 ml/l h, respectively. These results indicate that optimization of the culture conditions resulted in a simultaneous increase in biohydrogen production and cell growth. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Cells of Haematococcus pluvialis Flot. et Will were collected in four different growth phases. We quantified the initial and total enzyme activity of ribulose-1,5-bisphosphate carboxylase (Rubisco) in crude extracts, and the relative expression of large-subunit ribulose-1,5-bisphosphate caboxylase / oxygenase (rbcL) mRNA. We measured the ratio of photosynthetic rate to respiration rate (P/R), maximal effective quantum yield of photosystem II (Fv/Fm), electron transport rate (ETR), actual photochemical efficiency of PSII in the light (PSII), and non-photochemical quenching (NPQ). Green vegetative cells were found to be in the most active state, with a relatively higher P/R ratio. These cells also displayed the lowest NPQ and the highest Fv/Fm, ETR, and PSII, indicating the most effective PSII. However, both Rubisco activity and rbcL mRNA expression were the lowest measured. In orange resting cysts with relatively lower P/R and NPQ, Rubisco activity and rbcL expression reached a peak, while Fv/Fm, ETR, and ΦPSII were the lowest measured. Taking into account the methods of astaxanthin induction used in industry, we suggest that Rubisco may participate in astaxanthin accumulation in H. pluvialis. A continuous and sufficient supply of a carbon source such as CO2 may therefore aid the large scale production of astaxanthin.

A group of coenocytic marine algae differs from higher plants, whose totipotency depends on an intact cell (or protoplast). Instead, this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals. It is thought that lectins play a key role in the aggregation process. We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides. The lectin was ca. 27 kDa with a pI between pH 5 and pH 6. The absence of carbohydrate suggested that the lectin was not a glycoprotein. The hemagglutinating activity (HA) of the lectin was not dependent on the presence of divalent cations and was inhibited by N-Acetylgalactosamine, N-Acetylglucosamine, and the glycoprotein bovine submaxillary mucin. The lectin preferentially agglutinated Gram-negative bacterium. The HA of this lectin was stable between pH 4 to pH 10. Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution (0.5 mg ml−1). Our results suggest that the regeneration of B. hypnoides is mediated by this lectin. We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater (pH 8.0). Given that nigericin dissipates proton gradients across the membrane, we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

Acid, alkali, heat-shock, KNO3 and control pretreatment methods applied to anaerobic sludge were evaluated for their ability to selectively enrich the marine hydrogen-producing mixed microflora. Seawater culture medium was used as the substrate. The hydrogen yield of pretreated microflora was higher than that of the un-pretreated control (P < 0.05). Among the pretreatment methods studied, heat-shock pretreatment yielded the greatest hydrogen production, which was 14.6 times that of the control. When the effect of initial pH on hydrogen production of heat-shock pretreated samples was studied, hydrogen was produced over the entire pH range (pH 4–10). The hydrogen yield peaked at initial pH 8 (79 mL/g sucrose) and then steadily decreased as the initial pH increased. Sucrose consumption was high at neutral initial pH. During the process of hydrogen production, pH decreased gradually, which indicated that the acquired microflora consisted of acidogenic bacteria.

The development of microalgae on an industrial scale largely depends on the economic feasibility of mass production. High light induces productive suspensions during cultivation in a tubular photobioreactor. Herein, we report that high light, which inhibited the growth of Chlorella sorokiniana under autotrophic conditions, enhanced the growth of this alga in the presence of acetate. We compared pigments, proteomics and the metabolic flux ratio in C. sorokiniana cultivated under high light (HL) and under low light (LL) in the presence of acetate. Our results showed that high light induced the synthesis of xanthophyll and suppressed the synthesis of chlorophylls. Acetate in the medium was exhausted much more rapidly in HL than in LL. The data obtained from LC-MS/MS indicated that high light enhanced photorespiration, the Calvin cycle and the glyoxylate cycle of mixotrophic C. sorokiniana. The results of metabolic flux ratio analysis showed that the majority of the assimilated carbon derived from supplemented acetate, and photorespiratory glyoxylate could enter the glyoxylate cycle. Based on these data, we conclude that photorespiration provides glyoxylate to speed up the glyoxylate cycle, and releases acetate-derived CO2 for the Calvin cycle. Thus, photorespiration connects the glyoxylate cycle and the Calvin cycle, and participates in the assimilation of supplemented acetate in C. sorokiniana under high light.

Photosynthetic performances and glucose-6-phosphate dehydrogenase (G6PDH) activity in Physcomitrella patens changed greatly during salt stress and recovery. In P. patens, the cyclic electron flow around photosystem (PS) I was much more tolerant to high salt stress than PSII. After high salt stress, the PSII activity recovered much more slowly than that of PSI, which was rapidly restored to pretreatment levels even as PSII was almost inactivate. This result suggested that after salt stress the recovery of the cyclic electron flow around PSI was independent of PSII activity. In addition, G6PDH activity and NADPH content increased under high salt stress. When G6PDH activity was inhibited by glucosamine (Glucm, a G6PDH inhibitor), the cyclic electron flow around PSI and the NADPH content decreased significantly. Additionally, after recovery in liquid medium containing Glucm, the PSI activity was much lower than in liquid medium without Glucm. These results suggested the PSI activity was affected significantly by G6PDH activity and the NADPH content. Based on the above results, we propose that G6PDH in P. patens has a close relationship with the photosynthetic process, possibly providing NADPH for the operation of the cyclic electron flow around PSI during salt stress and promoting the restoration of PSI.

Background Microalgae are potential sources of biofuels and high-value compounds. Mixotrophic conditions usually promote growth of microalgae. The pennate diatom Phaeodactylum tricornutum, with its short life cycle, completely sequenced genome, and ease of transformation, can be used as a model for studying carbon metabolism in microalgae. Results We compared the growth rate of P. tricornutum (IOCAS-001) under different conditions and labeled the cells using [13C]glycerol (GL). The results revealed GL promoted the growth of P. tricornutum. Ser and Gly were synthesized via photorespiration. The 13C enrichment of Ser and Gly under nitrogen-limited conditions was much higher compared to other amino acids, indicating the enhancement of photorespiration. Addition of sodium acetate decreased the growth rate of P. tricornutum under nitrogen-limited conditions. Our results indicated that the GL carbon backbone enters the Calvin cycle in the form of dihydroxyacetone phosphate (DHAP), producing xylulose 5-phosphate (X5P) with a GL2_3-generated carbon backbone distributed at X5P1_2 and ribose 5-phosphate (R5P) with GL1-derived carbon atoms at R5P1 and R5P2. Both R5P and X5P can be converted into ribulose-1,5-bisphosphate (RuBP). By oxygenation of RuBP carboxylase/oxygenase (Rubisco) and metabolism through photorespiration, these RuBPs generate Ser and Gly with GL1 or GL2-derived carbon atoms at position 1 and GL1 or GL3-derived carbon atoms at other positions, resulting in a low level of 13C enrichment of Gly1 and Ser1. Conclusion Our results indicated different strains of P. tricornutum have different mechanisms for organic carbon metabolism. Photorespiration is involved in GL metabolism and is important for the nitrogen-limited response in P. tricornutum. Classification Metabolic flux analysis, microalgae Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0256-5) contains supplementary material, which is available to authorized users.

Thermal analysis and thermolysis kinetics of three kinds of seaweeds and fir wood (M. glyptostriboides Huet Cheng), a kind of typical land plant, had been conducted. The results showed that thermal stability follows the order of Grateloupia filicina < Ulva lactuca < Dictyopteris divaricata < fir wood. A notable difference on heat flow between seaweeds and fir wood during thermolysis was that the former were mainly connected with exothermic processes at relatively lower temperature regimes. while the latter was connected with an apparent endotherm at a relatively higher temperature regime followed by a maximum exothermic peak. This suggested that the heat coupling might be realized if co-thermolysis of seaweeds and fir wood were carried out. The main devolatilization phase of each seaweed could be described by Avrami-Erofeev equation, which indicated that thermolysis of seaweeds follows the mechanism of random nucleation and nuclei growth, whereas that of fir wood by Z-L-T equation and its thermolysis mechanism was three-dimensional diffusion. The activation energies calculated for both seaweeds and fir wood increase as conversion increases. However, those for the former have wider distribution. (c) 2006 Elsevier Ltd. All rights reserved.

Newly regenerated thalli were used to study the phototropism of Bryopsis hypnoides Lamouroux under different qualities of light. Positive phototropism in the thalli and negative phototropism in the rhizoids of B. hypnoides were investigated and analyzed in terms of bending. Both thalli and rhizoids developed from thallus segments exhibited typical tip growth, and their photoreceptive sites for phototropism were also restricted to the apical hemisphere. The bending curvature of rhizoids and thalli were determined with unilateral lights at various wavelengths and different fluence rates after a fixed duration of illumination. The trends of bending from the rhizoid and thallus were coincident, which showed that the action spectrum had a large range, from ultraviolet radiation (366.5 nm) to green light (524 nm). Based on the bending curvatures, blue light had the highest efficiency, while the efficiency of longer wavelengths (> 500 nm) was significantly lower. External Ca2+ had no effect on the bending curvature of thalli and rhizoids. Blue light (440 nm) induced thallus branching from rhizoids, while red light (650 nm) had no such effect. Fast-occurring chloroplast accumulation in the outermost cytoplasmic layer of the blue light (440 nm)-irradiated region in the rhizoid was observed, from which protrusions (new thalli) arose after 4 h of the onset of illumination, and this action was thought to be driven by the dynamics of actin microfilaments.

Effects of various concentrations of two heavy metals, namely Cd and Cu, on gametophytes of Laminaria japonica Aresch were determined by recording morphological changes of gametophytes, determining pH values and the heavy metal content of the culture solution, calculating the germination rate of sporophytes, and observing heavy metal (Cd) distribution using a fluorescence microscope. The results showed that heavy metals damaged the gametophytes, and were even lethal, and that the higher the concentration of heavy metal ions, the greater the injury to gametophytes. Gametophytes could not survive in culture solutions containing more than 100 mg/L Cd and 50 mg/L Cu and were only able to survive in culture solution containing a mixture of Cd and Cu tip to a concentration of 10 mg/L, which indicates that gametophytes have a higher tolerance to Cd than Cu and that multiple heavy metal ions in solution markedly aggravate the damage to gametophytes compared with individual heavy metal ions. With increases in the concentration of the heavy metal, the burgeoning rate of sporophytes decreased acutely, and solutions containing multiple heavy metal ions caused even more marked harm to sporophytes than solutions containing a single heavy metal ion, because most sporophytes died in mixed solutions. The pH value of the culture medium dropped immediately at the beginning (the first day) of treatment, increased over the following days, and then decreased again. The pH of culture media containing multiple heavy metal ions showed greater variation than media containing a single heavy metal ion, with the extent of the decrease in pH of culture media containing multiple ions being greatest during the last period of the experiment. With increases in the concentration of heavy metals, the capacity of gametophytes to accumulate these ions increased. The blue fluorescent light emitted by the Cd- and Cd-binding protein complex existing in gametophytes in media containing different concentrations of Cd showed clearly the distribution of the ion in gametophytes and the results obtained were consistent with distribution determined using other methods. All results of the present study showed that gametophytes of L. japonica play a remarkable role as heavy metal decontaminators, especially with regard to Cd.

Fucoxanthin was extracted from the intact rhizoid of Laminaria japonica Aresch with dimethyl sulfoxide (DMSO), and then recovered from the DMSO extract by partitioning into ethyl acetate and subsequent evaporation. Some isolation conditions such as solvent volume and extraction time were screened. The quantity and quality of the extracted fucoxanthin were determined by spectral analysis (absorption spectra and fluorescence emission spectra). The results indicated that: (1) the average total content of fucoxanthin was 122.1 mu g in 1 g of fresh L japonica rhizoid; (2) in comparison with the widely used organic solvent, acetone, DMSO was much more effective for the extraction of fucoxanthin; (3) both DMSO volume and extraction time influenced extraction efficiency such as the recovery rate and purity of fucoxanthin (1 g of fresh L. japonica rhizoid treated with 4 mL DMSO for 60 min, yielded > 88% of the total fucoxanthin with purity 0.63); (4) when (NH4)(2)SO4 concentration was in the range of 0.5- 1.0 mol/L, the pigments rapidly and entirely moved from DMSO into the ethyl acetate phase; (5) the ethyl acetate and DMSO were recycled using a rotary evaporator.

Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6-11), and the protoplasts aggregated best at pH 8-9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH 8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to Delta pH-based translocation (Delta pH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.

The thylakoid membranes were isolated and purified from gametophyte of Porphyra yezoensis Ueda (P. yezoensis) by sucrose density gradient ultracentrifugation. After P. yezoensis gametophyte thylakoid membranes were solubilized with SDS, the photosystem II (PSII) particles were isolated and purified. The activity of PSII particles was determined with DCIP (2,6-dichloroindophenol) photoreduction reaction. The composition of purified PSII particles was detected by SDS-PAGE. As a result, seven proteins including 55 kD protein, 47 kD protein, 43 kD protein, 33 kD protein, 31 kD protein, 29 kD protein, and 18 kD protein were found. Compared with PSII particles of higher plants and other algae, they were identified as D1/D2 complex, CP47, CP43, 33 kD protein, D1, D2 and cyt c-550 respectively. Besides, other three new proteins of 20 kD, 16 kD and 14 kD respectively were found. Among these extrinsic proteins, the 16 kD and 14 kD proteins had not been reported previously, and the 20 kD protein was found for the first time in multicellular red algae.

R-phycoerythrin was isolated and purified from Gracilaria verrucosa on an expanded-bed adsorption column combined with ion-exchange chromatography, which can effectively solve the problem of blockage of chromatographic columns due to polysaccharides during isolation and purification of phycobiliproteins. 0.1 M (NH4)(2)SO4 proved best to elute R-phycoerythrin from the expanded-bed column, and desalted 0.1 M (NH4)(2)SO4 eluate was used on an ion-exchange column to purify the R-phycoerythrin. Using this two-stage chromatography, the purity (OD565/OD280) of the R-phycoerythrin from G. verrucosa is increased to 4.4, and the yield of purified R-phycoerythrin can reach 0.141 mg . g(-1) of the frozen alga.

Under high light (HL) stress, astaxanthin-accumulating Haematococcus pluvialis and β-carotene-accumulating Dunaliella salina showed different responsive patterns. To elucidate cellular-regulating strategies photosynthetically and metabolically, thylakoid membrane proteins in H. pluvialis and D. salina were extracted and relatively quantified after 0 h, 24 h and 48 h of HL stress. Proteomic analysis showed that three subunits of the cytochrome b6/f complex were greatly reduced under HL stress in H. pluvialis, while they were increased in D. salina. Additionally, the major subunits of both photosystem (PS) II and PSI reaction center proteins were first reduced and subsequently recovered in H. pluvialis, while they were gradually reduced in D. salina. D. salina also showed a greater ability to function using the xanthophyll-cycle and the cyclic photosynthetic electron transfer pathway compared to H. pluvialis. We propose a reoriented and effective HL-responsive strategy in H. pluvialis, enabling it to acclimate under HL. The promising metabolic pathway described here contains a reorganized pentose phosphate pathway, Calvin cycle and glycolysis pathway participating in carbon sink formation under HL in H. pluvialis. Additionally, the efficient carbon reorientation strategy in H. pluvialis was verified by elevated extracellular carbon assimilation and rapid conversion into astaxanthin.

A marine yeast was isolated from mangrove sludge and named Candida tropicalis BH-6. The optimum temperature and the initial pH value for growth of the isolated strain were 37 A degrees C and 5.0, respectively. The strain had high salt tolerance and could survive at NaCl concentrations of 0-6 %. Additionally, the yield of hydrogen production by C. tropicalis BH-6 was only 66.30 ml/l. However, when the yeast was mixed with Pantoea agglomerans BH-18, hydrogen production increased significantly to a maximum of 1707.5 ml/l, which was 36.94 and 247.54 % higher than the monoculture of P. agglomerans BH-18 and C. tropicalis BH-6, respectively. Taken together, these results revealed that in mixed culture, the yeast strain isolated from the same ecosystem as P. agglomerans BH-18 likely consumed the organic acids produced by fermentation, thus eliminating the factor inhibiting hydrogen production by P. agglomerans BH-18. As a result, the yield of hydrogen production during mixed culture increased significantly.