Effects of Silver Nanoparticles on the Red Microalga Porphyridium purpureum CNMN-AR-02, Cultivated on Two Nutrient Media.

The purpose of this study was to examine the influence of 10 and 20 nm nanoparticles (AgNPs) on the growth and biochemical composition of microalga Porphyridium purpureum CNMN-AR-02 in two media which differ by the total amount of mineral salts (MM1 with 33.02 g/L and MM2 with 21.65 g/L). Spectrophotometric methods were used to estimate the amount of biomass and its biochemical composition. This study provides evidence of both stimulatory and inhibitory effects of AgNPs on different parameters depending on the concentration, size, and composition of the nutrient medium. In relation to the mineral medium, AgNPs exhibited various effects on the content of proteins (an increase up to 20.5% in MM2 and a decrease up to 36.8% in MM1), carbohydrates (a decrease up to 35.8% in MM1 and 39.6% in MM2), phycobiliproteins (an increase up to 15.7% in MM2 and 56.8% in MM1), lipids (an increase up to 197% in MM1 and no changes found in MM2), antioxidant activity (a decrease in both media). The composition of the cultivation medium has been revealed as one of the factors influencing the involvement of nanoparticles in the biosynthetic activity of microalgae.

Biochemical changes in microalga Porphyridium cruentum associated with silver nanoparticles biosynthesis.

For the first time, the microalga Porphyridium cruentum was tested for its ability to produce silver nanoparticles. To characterize formed silver nanoparticles UV-vis Spectrometry, Scanning Electron Microscopy, Energy-dispersive analysis of X-rays and X-ray diffraction were used. It was shown that after biomass exposure to silver nitrate solution the extracellular formation of spherical-like nanoparticles took place. Functional groups responsible for metal binding were determined by Fourier-transform infrared spectroscopy. The complex of biochemical tests was used for biomass characterization and assessment of the changes of its main components (proteins, lipids, carbohydrates, and phycobilin) during nanoparticle formation. Obtained data indicate a significant decrease of proteins, carbohydrates, phycobiliproteins, and lipids content as well as antiradical activity of biomass. The obtained results show the necessity of determination of optimal conditions for obtaining Porphyridium cruentum biomass enriched with silver nanoparticles for its further application in the pharmaceuticals industry.

Light Intensity and Nitrogen Concentration Impact on the Biomass and Phycoerythrin Production by Porphyridium purpureum.

Several factors have the potential to influence microalgae growth. In the present study, nitrogen concentration and light intensity were evaluated in order to obtain high biomass production and high phycoerythrin accumulation from Porphyridium purpureum. The range of nitrogen concentrations evaluated in the culture medium was 0.075-0.450 g L-1 and light intensities ranged between 30 and 100 µmol m-2 s-1. Surprisingly, low nitrogen concentration and high light intensity resulted in high biomass yield and phycoerythrin accumulation. Thus, the best biomass productivity (0.386 g L-1 d-1) and biomass yield (5.403 g L-1) were achieved with NaNO3 at 0.075 g L-1 and 100 µmol m-2 s-1. In addition, phycoerythrin production was improved to obtain a concentration of 14.66 mg L-1 (2.71 mg g-1 of phycoerythrin over dry weight). The results of the present study indicate that it is possible to significantly improve biomass and pigment production in Porphyridium purpureum by limiting nitrogen concentration and light intensity.

Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method.

A mutated phytoene desaturase (pds) gene, pds-L504R, conferring resistance to the herbicide norflurazon has been reported as a dominant selectable marker for the genetic engineering of microalgae (Steinbrenner and Sandmann in Appl Environ Microbiol 72:7477-7484, 2006; Prasad et al. in Appl Microbiol Biotechnol 98(20):8629-8639, 2014). However, this mutated genomic clone harbors several introns and the entire expression cassette including its native promoter and terminator has a length > 5.6 kb, making it unsuitable as a standard selection marker. Therefore, we designed a synthetic, short pds gene (syn-pds-int) by removing introns and unwanted internal restriction sites, adding suitable restriction sites for cloning purposes, and introduced the first intron from the Chlamydomonas reinhardtii RbcS2 gene close to the 5'end without changing the amino acid sequence. The syn-pds-int gene (1872 bp) was cloned into pCAMBIA 1380 under the control of a short sequence (615 bp) of the promoter of pds (pCAMBIA 1380-syn-pds-int). This vector and the plasmid pCAMBIA1380-pds-L504R hosting the mutated genomic pds were used for transformation studies. To broaden the existing transformation portfolio, the rhodophyte Porphyridium purpureum was targeted. Agrobacterium-mediated transformation of P. purpureum with both the forms of pds gene, pds-L504R or syn-pds-int, yielded norflurazon-resistant (NR) cells. This is the first report of a successful nuclear transformation of P. purpureum. Transformation efficiency and lethal norflurazon dosage were determined to evaluate the usefulness of syn-pds-int gene and functionality of the short promoter of pds. PCR and Southern blot analysis confirmed transgene integration into the microalga. Both forms of pds gene expressed efficiently as evidenced by the stability, tolerance and the qRT-PCR analysis. The molecular toolkits and transformation method presented here could be used to genetically engineer P. purpureum for fundamental studies as well as for the production of high-value-added compounds.

Terpene Biosynthesis in Red Algae Is Catalyzed by Microbial Type But Not Typical Plant Terpene Synthases.

Red algae (Rhodophyta) and land plants belong to the monophyletic clade Archaeplastida, and taxa of both groups are rich producers of terpene secondary metabolites. The terpene carbon skeletons of land plants are made by two types of terpene synthases: typical plant terpene synthases and microbial-type terpene synthases (MTPSLs); however, terpene biosynthesis in red algae is poorly understood. By systematic sequence analysis of seven genomes and 34 transcriptomes of red algae, MTPSL homologs were identified within one genome and two transcriptomes, whereas no homolog of typical plant terpene synthase genes was found. Phylogenetic analysis showed that red algae MTPSLs group with bacterial terpene synthases. Analysis of the genome assembly and characterization of neighboring genes demonstrated red algal MTPSLs to be bona fide red algal genes and not microbial contaminants. MTPSL genes from Porphyridium purpureum and Erythrolobus australicus were characterized via heterologous expression in Escherichia coli and demonstrated to have sesquiterpene synthase activities. We detected a number of volatile sesquiterpenes in the headspace of P. purpureum and E. australicus cultures, most identical to the in vitro products of the respective MTPSLs. Expression of the MTPSL gene in P. purpureum was found to be induced by methyl jasmonate, suggesting a role for this gene in host defense. In summary, this study indicates that the formation of terpene carbon skeletons in red algae is carried out by MTPSLs that are phylogenetically unrelated to typical plant terpene synthases and most likely originated in Rhodophyta via horizontal gene transfer from bacteria.

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.

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.