•Different performances in photosystem I and photosystem II in D. salina under salt stress were detected.•The oxidative pentose phosphate pathway was enhanced in D. salina under salt stress.•The imbalance between PSI and II was resolved by the enhanced oxidative pentose phosphate pathway.•The leaked electrons were used in reactive oxygen species generation.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.

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.

•A marine photosynthetic bacterium was isolated and identified as Marichromatium purpuratum.•Property analysis of M. purpuratum LC83 was performed.•M. purpuratum LC83 improved the fermentative liquid conditions of P. agglomerans BH-18.•Hydrogen production and glucose consumed by coupled fermentation were higher than pure control.•The optimum inoculation ratio was 1/8 (strains BH-18/LC83) in coupled dark and photofermentation.A marine photosynthetic bacterial strain LC83 was isolated from bathing beach sludge and identified as Marichromatium purpuratum using light microscopic examination, Biolog tests, and 16S rRNA gene sequence analysis. The strain is a salt- and alkali-tolerant, vitamin-independent facultative anaerobe with an optimum sodium chloride (NaCl) concentration of 30‰, initial optimum pH of 7.0–8.0, optimum temperature of 30 °C, and optimum light intensity of 80–160 μmol/m2/s. The addition of M. purpuratum LC83 to the dark fermentation of Pantoea agglomerans BH-18 for 8 h clearly improved the pH and the oxidation–reduction potential of the fermentative liquid, retarded the reduction in glucose, and maintained the balance between the glucose generated photosynthetically by the photosynthetic bacteria and those consumed by the fermentative bacteria in the early stage of coupled fermentation. The addition of photosynthetic bacteria to dark fermentation at 8 h improved the fermentative liquid conditions in favor of hydrogen production. The yield of hydrogen and the amount of glucose consumed by the coupled fermentation were also both higher than during pure fermentation by P. agglomerans BH-18. The optimum inoculation ratio was 1/8 (strains BH-18/LC83). The coupled dark and photofermentation of M. purpuratum LC83 and P. agglomerans BH-18 is a feasible way of improving the level of hydrogen produced in marine culture.

Sargassum fusiforme, a species of brown seaweed with economic importance, inhabits lower intertidal zones where algae are often exposed to various stresses. In this study, changes in the photosynthetic performance of S. fusiforme under saline stress were investigated. The PSII performance in S. fusiforme significantly improved, when the thalli were exposed to 0% salinity, and remained high with prolonging treatment time. In contrast, the PSII activity declined considerably under salinities of 4.5 and 6%. The PSI activity did not change remarkably under saline stress, thus demonstrating higher tolerance to saline stress than PSII. In addition, the PSI activity could be also restored after saline treatments, when PSII was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. It might be as a result of changes in the NAD(P)H content in the thalli under saline stress. Our results suggested that PSI was much more tolerant to different saline stress than PSII in S. fusiforme. We demonstrated that S. fusiforme was much more tolerant to hyposaline than to hypersaline stress.

Flocculation harvesting of the fucoxanthin-rich marine microalga Isochrysis galbana has received little attention. Therefore, we attempted to screen for an optimal chemical flocculant and optimize flocculation conditions from five chemical flocculants—ferric chloride (FC), aluminum sulfate (AS), polyaluminum chloride (PAC), aluminum potassium sulfate (APS), and zinc sulfate (ZS)—for effective flocculation of I. galbana. The growth rate, photosynthetic performance, and fucoxanthin content were determined in re-suspended flocculated algal cells and in the flocculation supernatant cultured algal cells. The results showed that high growth rate and fucoxanthin accumulation were observed when FC was used as the flocculant in I. galbana cultures, which indicated that FC may cause less harm to I. galbana than the other aluminum-based flocculants. Furthermore, satisfactory flocculation efficiency was also observed when FC was used to flocculate I. galbana, and the FC dosage was less than that required for flocculation of I. galbana using PAC, APS, and AS. Thus, we selected FC as the optimal flocculant for harvesting I. galbana based on its flocculation efficiency together with algal physiological performance, growth rate, and fucoxanthin content.

Photoprotection mechanisms protect photosynthetic organisms, especially under stress conditions, against photodamage that may inhibit photosynthesis. We investigated the effects of short-term immersion in hypo- and hypersalinity sea water on the photosynthesis and xanthophyll cycle in Sargassum fusiforme (Harvey) Setchell. The results indicated that under moderate light [110 μmol(photon) m−2 s−1], the effective quantum yield of PSII was not reduced in S. fusiforme fronds after 1 h in hyposalinity conditions, even in fresh water, but it was significantly affected by extreme hypersalinity treatment (90‰ sea water). Under high light [HL, 800 μmol(photon) m−2 s−1], photoprotective mechanisms operated efficiently in fronds immersed in fresh water as indicated by high reversible nonphotochemical quenching of chlorophyll fluorescence (NPQ) and de-epoxidation state; the quantum yield of PSII recovered during the subsequent relaxation period. In contrast, fronds immersed in 90‰ sea water did not withstand HL, barely developed reversible NPQ, and accumulated little antheraxanthin and zeaxanthin during HL, while recovery of the quantum yield of PSII was severely inhibited during the subsequent relaxation period. The data provided concrete evidence supporting the short-term tolerance of S. fusiforme to immersion in fresh water compared to hypersalinity conditions. The potential practical implications of these results were also discussed.

•Fermentative products, proteomics and flux ratio were analyzed in Pantoea agglomerans.•The results indicated hydrogen was generated by fermentation and nitrogenase.•Proteomics and 13C metabolic flux ratio analysis indicated participation of PPP.•EMP and PPP provided pyruvate, NAD(P)H, and ATP for hydrogen production.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 13C 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.

•We perform research on the hydrogen production pathways of Enterobacter cloacae.•The maximum hydrogen yield was ∼707 ml L−1.•The hydrogen-producing pathway of E. cloacae was the 2,3-butanediol pathway.•EMP is the major pathway and HMP has a weaker role in glucose catalysis.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 H2 content of the gas-phase 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 2,3-butanediol 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 13C labeling experiments and proteomics analysis indicated the Embden-Meyerhof-Parnas (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.

•Mutant strain TH-79 of Rhodovulum sulfidophilum was created by transposon insertion.•H2 yield of strain TH-79 from glucose was 58.72% higher than its parent strain.•H2 Yield from glucose was similar to those of more complicated integration systems.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 H2/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.

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.

In order to characterize the structure and function of the alternative oxidase (AOX) gene in Pyropia haitanensis, full-length complementary DNA (cDNA) of the locus (denoted as PhAOX) was obtained by in silico cloning, rapid amplification of cDNA ends, and reverse transcription PCR. The PhAOX sequence was 3,192 bp including a 5′-untranslated region (UTR) of 1,484 bp, a 3′-UTR of 274 bp, and an open reading frame of 1,434 bp predicted to encode a protein of 477 amino acids with a molecular mass of 50.47 kDa and a putative isoelectric point of 9.75. PhAOX is predicted to be a precursor protein with mitochondrial-targeting peptides. The deduced amino acid sequence of PhAOX was most similar to PyAOX from Porphyra (= Pyropia) yezoensis (77 % identity); however, gene sequences from PhAOX and PyAOX showed obvious divergence. PhAOX had a 315-bp intron but PyAOX had no intron. Expression of mRNA in filamentous thalli and the foliose thallus, which are different life cycle stages, was examined by real-time fluorescent quantitative PCR (qPCR). The highest PhAOX expression was in filamentous thalli and was about 10 times the expression in the foliose thallus. We showed that an alternative respiration pathway also existed in P. haitanensis using a noninvasive microsensing system. The contribution of the alternative pathway to total respiration in filamentous thalli was greater than the contribution in the foliose thallus. This result was consistent with the level of PhAOX gene expression observed in different life cycle stages.

Gloiopeltis furcata (Postels & Ruprecht) J. Agardh, a macroalga, which grows in an upper, intertidal zone, can withstand drastic environmental changes caused by the periodic tides. In this study, the photosynthetic and morphological characteristics of G. furcata were investigated. The photosynthetic performance and electron flows of the thalli showed significant variations in response to desiccation and salinity compared with the control group. Both PSII and PSI activities declined gradually when the thalli were under stress. However, the electron transport rate of PSI showed still a low value during severe conditions, while the rate of PSII approached zero. Furthermore, PSI activity of the treated thalli recovered faster than PSII after being submerged in seawater. Even though the linear electron flow was inhibited by DCMU [3-(3, 4-dichlorophenyl)-1,1-dimethylurea], the cyclic electron flow could still be restored. The rate of cyclic electron flow recovery declined with the increasing time of dark treatment, which suggested that stromal reductants from starch degradation played an important role in the donation of electrons to PSI. This study demonstrated that PSII was more sensitive than PSI to desiccation and salinity in G. furcata and that the cyclic electron flow around PSI played a significant physiological role. In addition, G. furcata had branches, which were hollow inside and contained considerable quantities of funoran. These might be the most important factors in allowing G. furcata to adapt to adverse intertidal environments.

Photosynthetic electron flow changed considerably during desiccation and re-hydration of the intertidal macroalgae Porphyra haitanensis. Activities of both photosystem (PSI) and photosystem (PSII) increased significantly at moderate desiccation levels. Whereas PSII activity was abolished at an absolute water content (AWC) <24 %, PSI remained active with progressive decreases in AWC to values as low as 16 %. This result suggested that cyclic electron flow around PSI was still active after inactivation of linear electron flow following severe desiccation. Moreover, the PSI activity was restored more rapidly than that of PSII upon re-hydration. Pretreatment of the blades with 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) suppressed PSII activity following desiccation to an AWC of ~16 % AWC. Cyclic electron flow around PSI decreased markedly in blades pretreated with DCMU than in blades without pretreatment of DCMU during re-hydration in seawater containing DCMU. All results suggested that the activity of PSII under desiccation conditions plays an important role in the operation of cyclic electron flow during desiccation and its recovery during re-hydration. Therefore, we proposed the PSII activity during desiccation could eventually lead to the accumulation of NADPH, which could serve as electron donor for P700+ and promote its recovery during re-hydration, thereby favoring the operation of cyclic electron flow.

Pseudogenes share sequence similarities with functional genes, but in general they have lost their protein-coding ability. The identification of pseudogenes is a very important step in genome annotation. Phaeodactylum tricornutum is a marine diatom that is rich in polyunsaturated fatty acids (PUFAs). The genome of P. tricornutum has been completely sequenced. To identify pseudogenes in P. tricornutum, we developed a pipeline to discover and characterize pseudogenes. We identified a total of 1654 ‘true’ processed pseudogenes, 714 duplicated pseudogenes and 4729 pseudogene fragments. The results of the bioinformatics analysis indicated that the genome sequence of P. tricornutum contained many pseudogenes and pseudogene fragments.

Pantoea agglomerans BH18, isolated from mangrove sludge, could produce hydrogen under marine culture condition. To improve the hydrogen-producing capacity of this strain, we constructed a stable transposon-mutagenized library of P. agglomerans BH18. A Tn7-based transposon was randomly inserted into genomic DNA of P. agglomerans BH18. Mutants were identified by kanamycin resistance and amplification of the inserted transposon sequences. A transposon mutant, named as strain TB212, was screened for the highest hydrogen production ability. The total volume of hydrogen gas evolved by this mutant strain TB212 was 60% higher than that of the wild type. The mutant strain TB212 was able to produce hydrogen over a wide range of initial pH from 5.0 to 10.0, with an optimum initial pH of 7.0, and hydrogen production was 2.52 ± 0.02 mol H2/mol glucose (mean ± S.E.) under marine culture condition. The mutant strain TB212 could produce hydrogen at the salt concentration from 3 to 6%. It was concluded that the transposon-mutagenized library may be a useful tool for investigation of high efficiency hydrogen-producing bacteria.Highlights► Transposon mutagenesis was utilized to enhance the hydrogen production capacity. ► A transposon mutagenesis library of Pantoea agglomerans BH18 was constructed. ► Mutant TB212 had good heredity stability and the highest hydrogen production ability. ► H2 yield of mutant TB212 was 2.52 ± 0.07, increased by 60% for wild type. ► The mutant TB212 could produce H2 at all pH and salt concentration ranges tested.

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 °C, light intensity of 80–120 μmol photons/m2 s, initial pH of 8, butyrate concentration of 20–40 mmol/l. Optimal conditions for hydrogen production were 30 °C, light intensity of 80 μmol photons/m2 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 H2/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 H2/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.Highlights► A marine phototrophic bacteria consort was enriched and used for H2 production. ► Butyrate, one of the main VFA produced by dark-acidogenesis process, was used for H2 production. ► The microbial community was analyzed using 16 S rDNA-based techniques.

Porphyra yezoensis has a macroscopic foliage gametophyte phase with only a single cell layer, and is ideally suited for the study of the sexual differentiation process, from the vegetative cell to the spermatia. Firstly, we compared variations in the responses of the vegetative and male sectors to desiccation. Later, cell tracking experiments were carried out during the formation of spermatia from vegetative cells. The two sectors showed similar tolerance to desiccation, and the formation of spermatia from vegetative cells was independent of the degree of desiccation. Both light and scanning electron microscopy (SEM) observations of the differentiation process showed that the formation of spermatia could be divided into six phases: the one-cell, two-cell, four-cell, eight-cell, pre-release and spermatia phases. Photomicrographs of Fluorescent Brightener staining showed that the released spermatia had no cell walls. Photosynthetic data showed that there was a significant rise in Y(II) in the four-cell phase, indicating an increase in photosynthetic efficiency of PSII during this phase. We propose that this photosynthetic rise may be substantial and provide the increased energy needed for the formation and release of spermatia in P. yezoensis.

To isolate a salt tolerant hydrogen-producing bacterium, we used the sludge from the intertidal zone of a bathing beach in Tianjin as inoculum to enrich hydrogen-producing bacteria. The sludge was treated by heat-shock pretreatment with three different temperature (80, 100 and 121°C) respectively. A hydrogen-producing bacterium was isolated from the sludge pretreated at 80°C by sandwich plate technique and identified using microscopic examination and 16S rDNA gene sequence analysis. The isolated bacterium was named as Bacillus sp. B2. The present study examined the hydrogen-producing ability of Bacillus sp. B2. The strain was able to produce hydrogen over a wide range of initial pH from 5.0 to 10.0, with an optimum at pH 7.0. The level of hydrogen production was also affected by the salt concentration. Strain B2 has unique capability to adapt high salt concentration. It could produce hydrogen at the salt concentration from 4 to 60‰. The maximum of hydrogen-producing yield of strain B2 was 1.65 ± 0.04 mol H2/mol glucose (mean ± SE) at an initial pH value of 7.0 in marine culture conditions. Hydrogen production under fresh culture conditions reached a higher level than that in marine ones. As a result, it is likely that Bacillus sp. B2 could be applied to biohydrogen production using both marine and fresh organic waste.

The seaweed Ulva prolifera exists in 2 different states; attached to rocks or free-floating. However, there is little difference between the structures of the 2 states. U. prolifera thalli show significant differences in growth rate, with the attached thalli growing at a normal rate and free-floating thalli growing at a much faster rate. This raised the possibility that the growth of the two states may be regulated differently. miRNAs are important post-transcriptional regulators. In higher plants and animals, miRNAs have been extensively studied but they have been rarely studied in algae. To identify U. prolifera miRNAs and to investigate their possible roles in proliferation, we constructed and sequenced small RNA (sRNA) libraries from U. prolifera. Our results show that U. prolifera has a complex small RNA system that might play important roles in various processes.

•This manuscript investigated the effects of the increase of atmospheric CO2 and acid rain to the community of intertidal macroalgae.•Corallina sp. survived extremely low pH conditions (pH 3) and could regulate the pH of their ambient seawater through the dissolution of CaCO3.•It clarified that many macro-algae have disappeared and coralline algae now dominate the intertidal zone.The increase in atmospheric CO2 and acid rain precipitation are serious global environmental problems that have had worldwide consequences, including the damage of biodiversity in intertidal zones. The decline in species richness in the intertidal zone of Wenzhou, China, is a typical example. In this study, we investigated the effects of CO2-enriched seawater, CO2-enriched air and acid rain on a dominant species, Corallina sp., and an inferior species, Ulva conglobata, in the intertidal zone of Wenzhou. The responses of Corallina sp. and U. conglobata to high-CO2 seawater are similar, demonstrating that both of them are tolerant of seawater acidification induced by aerating CO2-enriched air. The PSII activities of Corallina sp. declined markedly when exposed to CO2-free air, while they increased significantly with high-CO2 air. An increase of non-photochemical quenching (NPQ) was induced by CO2-free air, but it declined remarkably with CO2-enriched air, suggesting that Corallina sp. can use atmospheric CO2 as carbon source for photosynthesis. Corallina sp. survived extremely low pH conditions (pH 3) and could regulate the pH of their ambient seawater through the dissolution of CaCO3, while the photosynthetic properties of U. conglobata decreased drastically and even the thalli were damaged irreversibly under low pH conditions (pH 3). These results suggest that Corallina sp. is much more tolerant of extremely low pH than U. conglobata. Based on these results, we suggest that it is not the increase of atmospheric CO2 but acid rain at least in part contributed to the damage of the biodiversity in the intertidal zone, with the result that these areas are now mainly dominated by Corallina sp.