Transcriptomic survey reveals multiple adaptation mechanisms in response to nitrogen deprivation in marine Porphyridium cruentum.

Single-cell red microalga Porphyridium cruentum is potentially considered to be the bioresource for biofuel and pharmaceutical production. Nitrogen is a kind of nutrient component for photosynthetic P. cruentum. Meanwhile, nitrogen stress could induce to accumulate some substances such as lipid and phycoerythrin and affect its growth and physiology. However, how marine microalga Porphyridium cruentum respond and adapt to nitrogen starvation remains elusive. Here, acclimation of the metabolic reprogramming to changes in the nutrient environment was studied by high-throughput mRNA sequencing in the unicellular red alga P. cruentum. Firstly, to reveal transcriptional regulation, de novo transcriptome was assembled and 8,244 unigenes were annotated based on different database. Secondly, under nitrogen deprivation, 2100 unigenes displayed differential expression (1134 upregulation and 966 downregulation, respectively) and some pathways including carbon/nitrogen metabolism, photosynthesis, and lipid metabolism would be reprogrammed in P. cruentum. The result demonstrated that nitrate assimilation (with related unigenes of 8-493 fold upregulation) would be strengthen and photosynthesis (with related unigenes of 6-35 fold downregulation) be impaired under nitrogen deprivation. Importantly, compared to other green algae, red microalga P. cruentum presented a different expression pattern of lipid metabolism in response to nitrogen stress. These observations will also provide novel insight for understanding adaption mechanisms and potential targets for metabolic engineering and synthetic biology in P. cruentum.

In situ cryo-ET structure of phycobilisome-photosystem II supercomplex from red alga.

Phycobilisome (PBS) is the main light-harvesting antenna in cyanobacteria and red algae. How PBS transfers the light energy to photosystem II (PSII) remains to be elucidated. Here we report the in situ structure of the PBS-PSII supercomplex from Porphyridium purpureum UTEX 2757 using cryo-electron tomography and subtomogram averaging. Our work reveals the organized network of hemiellipsoidal PBS with PSII on the thylakoid membrane in the native cellular environment. In the PBS-PSII supercomplex, each PBS interacts with six PSII monomers, of which four directly bind to the PBS, and two bind indirectly. Additional three 'connector' proteins also contribute to the connections between PBS and PSIIs. Two PsbO subunits from adjacent PSII dimers bind with each other, which may promote stabilization of the PBS-PSII supercomplex. By analyzing the interaction interface between PBS and PSII, we reveal that αLCM and ApcD connect with CP43 of PSII monomer and that αLCM also interacts with CP47' of the neighboring PSII monomer, suggesting the multiple light energy delivery pathways. The in situ structures illustrate the coupling pattern of PBS and PSII and the arrangement of the PBS-PSII supercomplex on the thylakoid, providing the near-native 3D structural information of the various energy transfer from PBS to PSII.

Difference in light use strategy in red alga between Griffithsia pacifica and Porphyridium purpureum.

Phycobilisomes (PBSs) are the largest light-harvesting antenna in red algae, and feature high efficiency and rate of energy transfer even in a dim environment. To understand the influence of light on the energy transfer in PBSs, two red algae Griffithsia pacifica and Porphyridium purpureum living in different light environment were selected for this research. The energy transfer dynamics in PBSs of the two red algae were studied in time-resolved fluorescence spectroscopy in sub-picosecond resolution. The energy transfer pathways and the related transfer rates were uncovered by deconvolution of the fluorescence decay curve. Four time-components, i.e., 8 ps, 94 ps, 970 ps, and 2288 ps were recognized in the energy transfer in PBSs of G. pacifica, and 10 ps, 74 ps, 817 ps and 1292 ps in P. purpureum. In addition, comparison in energy transfer dynamics between the two red algae revealed that the energy transfer was clearly affected by lighting environment. The findings help us to understand the energy transfer mechanisms of red algae for adaptation to a natural low light environment.

Scaffolding proteins guide the evolution of algal light harvesting antennas.

Photosynthetic organisms have developed diverse antennas composed of chromophorylated proteins to increase photon capture. Cryptophyte algae acquired their photosynthetic organelles (plastids) from a red alga by secondary endosymbiosis. Cryptophytes lost the primary red algal antenna, the red algal phycobilisome, replacing it with a unique antenna composed of αß protomers, where the ß subunit originates from the red algal phycobilisome. The origin of the cryptophyte antenna, particularly the unique α subunit, is unknown. Here we show that the cryptophyte antenna evolved from a complex between a red algal scaffolding protein and phycoerythrin ß. Published cryo-EM maps for two red algal phycobilisomes contain clusters of unmodelled density homologous to the cryptophyte-αß protomer. We modelled these densities, identifying a new family of scaffolding proteins related to red algal phycobilisome linker proteins that possess multiple copies of a cryptophyte-α-like domain. These domains bind to, and stabilise, a conserved hydrophobic surface on phycoerythrin ß, which is the same binding site for its primary partner in the red algal phycobilisome, phycoerythrin α. We propose that after endosymbiosis these scaffolding proteins outcompeted the primary binding partner of phycoerythrin ß, resulting in the demise of the red algal phycobilisome and emergence of the cryptophyte antenna.

Grafting Robust Thick Zwitterionic Polymer Brushes via Subsurface-Initiated Ring-Opening Metathesis Polymerization for Antimicrobial and Anti-Biofouling.

In the present work, high-thickness zwitterionic polymer brushes based on imidazolium salts were successfully grafted via a novel subsurface-initiated ring-opening metathesis polymerization (subsurface-initiated ROMP) from polydimethylsiloxane (PDMS), and their antifouling performance was evaluated in detail. First, an initiator-embedded PDMS was prepared via copolymerization of PDMS prepolymer and ROMP initiator, and then zwitterionic polymer brushes were grafted via subsurface-initiated ROMP from surface to subsurface of the PDMS due to the implanted ROMP initiator. Results from a series of characterization methods such as infrared spectroscopy, X-ray photoelectron spectroscopy, contact angle, and atomic force microscopy proved the zwitterionic polymer brushes' successful grafting. The grafting thickness of zwitterionic polymer brushes via subsurface-initiated ROMP can reach the micron scale, and the as-prepared zwitterionic polymer based surfaces showed good lubricating properties compared to traditional surface-initiated ROMP, which hints that polymer brushes can be grafted not only on the surface but also on the subsurface of PDMS. The protein adhesion test and biofouling assay of zwitterionic polymer brushes were tested in the laboratory, and the results indicated that the zwitterionic polymer-functionalized PDMS can effectively resist the adhesion of bovine serum albumin and algae (Porphyridium and Dunaliella) and has good anti-bacterial activity against both Escherichia coli and Staphylococcus aureus.

Effects of nitrogen and phosphorous stress on the formation of high value LC-PUFAs in Porphyridium cruentum.

This study systematically examined the effect of nitrogen and phosphorous stress on the formation of linoleic acid (LA), arachidonic acid (ARA), and eicosapentaenoic acid (EPA) in Porphyridium cruentum gy-h56. P. cruentum was cultivated in six different media conferring different conditions of nitrogen (N) sufficiency/deprivation and phosphorous (P) sufficiency/limitation/deprivation. Over a 16-day cultivation process, the dry-weight content, proportion of total fatty acids (TFAs), and the concentration in the medium of linoleic acid (LA) were greatly improved by a maximum of 2.5-, 1.6-, and 1.1-fold, respectively, under conditions of N or P deprivation compared with N and P sufficiency. In contrast, levels of EPA or ARA were not enhanced under N or P stress conditions. Additionally, the results showed that N deprivation weakened the impact of P deficiency on the content and proportions of LA and EPA, while P deprivation enhanced the impact of N starvation on the content and proportions of LA and EPA. The conditions of N sufficiency and P deprivation (N+P-) were the optimal conditions for the production of LA, while the optimal conditions for EPA, ARA, and TFAs production were N sufficiency and P limitation (N+P-lim). This study suggests the potential application of combining N removal from saline wastewater with the production of LA, ARA, EPA, and biodiesel.

Electrochemical behavior of the 316L steel type in a marine culture of microalgae (Porphyridium purpureum) under the 12/12 h photoperiod and effect of different working electrode exposure conditions on the biofilm-metal interface.

The industrial crops of microalgae use processes calling upon the presence of parts of metal nature such as steel 316L type. The goal of this study is to test the electrochemical behavior of this material in a marine culture of microalgae. Porphyridium purpureum was used under a photoperiod of alternation darkness/light 12/12 h, in order to apprehend the problems of biocorrosion involved in the biofouling. The evolution of the free potential of corrosion, according to the position of the samples and for different surface roughness, observations of the surface quality under the electron microscope with sweeping were carried out. The results showed that, overall, the strain P. purpureum does not have a corrosive effect on the 316L. The free potential of corrosion lies between -0.307 and -0.005 V(SCE). The adhesion of the cells seems stronger on the interface air/solid of the half-plunged sample with surface grit polished 1,000, confirmed by the presence of biofilm on the air part. The photoperiod acts on the evolution of the generated free potential of corrosion of the one 24-h period oscillation. Furthermore, the samples plunged horizontally lead to a stabilizing effect on the potential of free corrosion.

Overcoming recalcitrance in Porphyridium aerugineum Geitler employing encapsulation-dehydration cryopreservation methods.

Cultures of the recalcitrant microalga Porphyridium aerugineum were cryopreserved. A two-step, uncontrolled rapid freezing protocol, using methanol as cryoprotectant resulted in 23.8 percent viable cells. Cultures in the exponential growth phase, grown under low light intensity to prevent vacuole formation in cells, cryopreserved using a passive freezer, showed 22.4 percent viability. This value was enhanced to 31.5 percent when a controlled-rate freezer was employed. Optimized cultures in the exponential growth phase, cultivated in medium supplemented or not with vitamin B12, were then tested for freezing using the encapsulation-dehydration protocol. High cell loss was observed early during the sorbitol dehydration steps, but 63.6 percent of the remaining encapsulated cells were viable after thawing. This study confirmed the potential of encapsulation-dehydration as a method allowing to improve the low viability obtained with two-step freezing protocols. It also showed the importance of monitoring the response of algal cells to bead osmotic and evaporative dehydration pretreatments before freezing.

Ultrasound effects on the antioxidative defense systems of Porphyridium cruentum.

Ultrasound is a special physical stimulus that has a variety of biological effects. This study provides a first systemic investigation on the ultrasound-induced oxidation and protection actions of the antioxidant defense system in Porphyridium cruentum. The hydroxyl radical and superoxide anion radical scavenging ability of the cells and the electrolyte leakage of the cell membrane were examined. The change of glutathione and carotenoids produced with/without ultrasonic processing were measured; the enzyme activities of superoxide dismutase, catalase, and membrane bound ATPases (Na(+)/K(+)-ATPase, Ca(2+)/Mg(2+)-ATPase) were evaluated for either ultrasound-treated or untreated P. cruentum. The hydroxyl radical and superoxide anion radical scavenging ability of ultrasound-treated P. cruentum increase 49.8 and 76.0%, respectively, of which the electrolyte leakage and malonyldialdehyde accumulation are also found increased 48.6 and 48.0%, respectively, indicating a state of oxidative stress. A significant enhancement of the activities of superoxide dismutase by 53.5%, catalase, membrane bound ATPases (Na(+)/K(+)-ATPase, Ca(2+)/Mg(2+)-ATPase increased by 67.7 and 69.3%, respectively), and the increment of glutathione and carotenoids production are also observed. These results suggested that oxidative stress manifested by elevated reactive oxygen species levels and malonyldialdehyde contents might be resulted from the biophysical responses of P. cruentum to the physical stimuli, and most likely the enhanced antioxidation ability of the algal cells stimuli by ultrasonic comes from the enhancement of enzymatic and nonenzymatic preventive substances as observed in this work.

Variation in storage alpha-polyglucans of red algae: amylose and semi-amylopectin types in Porphyridium and glycogen type in Cyanidium.

Red algae are widely known to produce floridean starch but it remains unclear whether the molecular structure of this algal polyglucan is distinct from that of the starch synthesized by vascular plants and green algae. The present study shows that the unicellular species Porphyridium purpureum R-1 (order Porphyridiales, class Bangiophyceae) produces both amylopectin-type and amylose-type alpha-polyglucans. In contrast, Cyanidium caldarium (order Porphyridiales, class Bangiophyceae) synthesizes glycogen-type polyglucan, but not amylose. Detailed analysis of alpha-1,4-chain length distribution of P. purpureum polyglucan suggests that the branched polyglucan has a less ordered structure, referred to as semi-amylopectin, as compared with amylopectin of rice endosperm having a tandem-cluster structure. The P. purpureum linear amylose-type polyglucan, which has a lambda(max) of 630 nm typical of amylose-iodine complex and is resistant to Pseudomonas isoamylase digestion, accounts for less than 10% of the total polyglucans. We produced and isolated a cDNA encoding a granule-bound starch synthase (GBSS)-type protein of P. purpureum, which is probably the approximately 60-kDa protein bound tightly to the starch granules, resembling the amylose-synthesizing GBSS protein of green plants. The present investigation indicates that the class Bangiophyceae includes species producing both semi-amylopectin and amylose, and species producing glycogen alone.

Effects of agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum.

The effect of mechanical agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum was investigated in aerated continuous cultures with and without the added shear protectant Pluronic F68. Damage to cells was quantified through a decrease in the steady state concentration of the biomass in the photobioreactor. For a given aeration rate, the steady state biomass concentration rose with increasing rate of mechanical agitation until an upper limit on agitation speed was reached. This maximum tolerable agitation speed depended on the microalgal species. Further increase in agitation speed caused a decline in the steady state concentration of the biomass. An impeller tip speed of >1.56 m s(-1) damaged P. tricornutum in aerated culture. In contrast, the damage threshold tip speed for P. cruentum was between 2.45 and 2.89 m s(-1). Mechanical agitation was not the direct cause of cell damage. Damage occurred because of the rupture of small gas bubbles at the surface of the culture, but mechanical agitation was instrumental in generating the bubbles that ultimately damaged the cells. Pluronic F68 protected the cells against damage and increased the steady state concentration of the biomass relative to operation without the additive. The protective effect of Pluronic was concentration-dependent over the concentration range of 0.01-0.10% w/v.