Under low irradiation,the light regime modifies growth and metabolite production in various species of microalgae

The effects of continuous light exposure (24L:0D) and a 12 h:12 h light/dark regime (12L:12D) were compared on the growth and carotenoid, protein, sugar, lipid, and fatty acid contents in Chlorella vulgaris, Nannochloropsis sp., Isochrysis galbana, and Dunaliella salina cultured in a batchwise facility. These microalgae were grown axenically under a low photon flux density (PFD) of 27 μmol photons m^?2 s^?1. C. vulgaris, Nannochloropsis sp., and I. galbana exhibited the highest cell densities when cultured under 24L:0D, whereas D. salina grew better under the alternating light/dark regime. I. galbana accumulated high levels of proteins, sugars, and lipids and exhibited the highest carotenoid content under 24L:0D. Protein production was enhanced in C. vulgaris under 24L:0D. The highest total lipid content was recorded for D. salina, reaching 74.6 % of total proteins, sugars, and lipids in cells at the stationary phase when grown under 12L:12D. The light/dark regime at low PFD was sufficient to stimulate the accumulation of monounsaturated and polyunsaturated fatty acids in all four algae. Their levels, like those of saturated fatty acids, did not differ significantly under the two light regimes. D. salina was an important source of tetradecenoic acid 14:1(n-5). Nannochloropsis sp. produced a large amount of the essential eicosapentaenoic acid, which reached 20 % of total fatty acids under 12L:12D, while I. galbana exhibited the highest level of docosahexaenoic acid, which reached 21 % under both light regimes. This study demonstrated the feasibility of culturing microalgae under low PFD in order to produce large quantities of valuable metabolites, especially various lipids with neutraceutical value.     

Expression of soybean Kunitz trypsin inhibitor gene SKTI in Dunaliella salina

Recently, microalgae have attracted much attention as a new bioreactor system for producing high-value heterologous proteins. In this paper, we investigated the expression of soybean Kunitz trypsin inhibitor gene SKTI in the chlorophyte Dunaliella salina. Using D. salina genomic DNA as template, the entire coding region of SKTI was amplified by PCR as a 654-bp DNA fragment. It had 100 % identity with the published sequence of SKTI. The entire SKTI fragment was cloned into the expression vector pCAM2201 at a site just downstream of 35S promoter of to yield pCAMSKTI. D. salina cells transfected with pCAMSKTI by means of the lithium acetate/polyethylene glycol-mediated method expressed a protein of 20.1 kDa as detected by anti-SKTI antibody. In addition, SDS-PAGE analysis of the cell extract also revealed an intense protein band indicative of the recombinant SKTI. SKTI was therefore successfully expressed in D. salina, and the expression could be detected for at least 35 generations.     

The termination of the synthesis of proteins in vitro with extracts from the undeveloped cyst of Artemia salina

Soluble fractions (S-100) from both undeveloped cysts and developing embryos of Artemia salina promoted elongation of polypeptides initiated in vivo on polysomes of developing embryos or nauplius larvae. The ability of the extract from the undeveloped cyst to terminate correctly the synthesis of polypeptides has been determined indirectly from the distribution of polysomes before and after in vitro translation and, more directly, from the nature of the protein product released from rabbit reticulocyte polysomes. The extract from the undeveloped cyst and also, as expected, that from the developing embryo catalyzed a reduction in the amount of the polysomes of larger size and an increase in the amount of 80 S ribosomes. The soluble extract from the undeveloped cyst can terminate the synthesis of rabbit globin on reticulocyte polysomes. The major polypeptide product released from the polysomes had an electrophoretic mobility identical with that of the subunit of isolated rabbit globin. This indicated that the cyst contained the components necessary to complete and terminate the synthesis of polypeptides correctly and that the released protein product was not predominantly as a result of premature chain termination. The size distribution of Artemia salina proteins released from polysomes from developing embryos was similar when the synthesis was directed by the S-100 at each stage of development.     

Oil production by six microalgae: impact of flocculants and drying on oil recovery from the biomass

Oil production in batch photoautotrophic cultures of the following microalgae is reported: the freshwater microalgae Chlorella vulgaris, Choricystis minor, and Neochloris sp.; the marine microalgae Nannochloropsis salina and Cylindrotheca fusiformis; and C. vulgaris grown in a full-strength seawater medium. In all cases, the solvent extraction of lipids from freeze-dried biomass is compared with extraction from the fresh biomass paste. For all algae, the oils could be extracted equally effectively from freeze-dried samples and the paste samples (67–88 % moisture by weight). Moisture content determinations of the biomass using the freeze-drying method and the high-temperature oven drying were found to be equivalent for all algae. The biomass recovered by flocculation with metal salts (aluminum sulfate, ferric chloride) followed by centrifugation had a certain amount of the flocculant irreversibly bound to it. Washing failed to remove the adsorbed flocculants. For all algae, the adsorbed flocculants did not interfere with oil recovery by solvent extraction. The solvent system of chloroform–methanol–water proved highly effective for quantitative extraction of the lipids from all algae.     

Isolation and proteomic analysis of the halotolerant alga <Emphasis Type="Italic">Dunaliella salina</Emphasis> flagella using shotgun strategy

Previous studies have demonstrated that flagella/cilia are critical organelles and play diverse roles of motility, sensory perception and development in many eukaryotic cells. However, there is very little information available about flagella composition in Dunaliella salina, a halotolerant, unicellular biflagellate green alga. In the present study, we used strategy of shotgun proteomics to identify flagella proteins after flagella were released and collected from D. salina. A total of 520 groups of proteins were identified under a stringent filter condition (Xcorr ≥1.9, ≥2.2 and ≥3.75; ΔCn ≥ 0.1). In addition to six kinds of known flagella proteins, the putative flagella proteins of D. salina identified by one or more peptides are abundant in signaling, cell division, metabolism, etc. The findings provide guidance for further studies to elucidate the roles of these proteins in the function and assembly of this organelle in microalgae.     

Microalgal cryo-preservation using dimethyl sulfoxide (Me2SO) coupled with two freezing protocols: Influence on the fatty acid profile

Procedures for determining the optimal pre-freezing protocol for cryo-preservation of microalgae are discussed. Three algal species were used (Chlorella vulgaris, Isochrysis galbana and Dunaliella salina) and cryo-stored using two different methods: the slow cooling and the fast freezing. In the slow cooling, each algae batch was treated with or without cryo-protectant (dimethyl sulfoxide: Me₂SO 5% v/v). After 20 min at 4 °C, the midi-straws were filled and cooled slowly (1.5 °C min⁻¹) to −140 °C, by a programmable freezer (Digitcool—IMV), before putting them directly into liquid nitrogen. Fast freezing was performed with 10% or 15% Me₂SO prior to plunging into liquid nitrogen. The three algal species followed the same re-growth pattern as that of the controls. The post-thawed viability with Me₂SO was good for all the selected algae (C. vulgaris >95%, I. galbana and D. salina >70% of the control), applying the slow cooling. The post-thawed viability without Me₂SO was 60% for I. galbana, 52% for D. salina and 33% for C. vulgaris. Fast freezing was not suitable for cryo-storage of I. galbana but gave good post-thawing viability for D. salina (70%). The decrease in fatty acid content of the cryo-stored algae was influenced by the temperature. The rapid decrease in temperature induced by fast freezing can explain the low level of fatty acid content of the three cryo-stored algae. Fatty acid profiles show that the nutritional values of the three cryo-stored micro-algae were not significantly affected especially when treated with slow cooling protocols.     

Evaluation of the effects of Chlorella vulgaris,Nannochloropsis salina,and Enterobacter cloacae on growth,yield and active compound compositions of Moringa oleifera under salinity stress

Application of Chlorella vulgaris, Nannochloropsis salina and Enterobacter cloacae has been reported to improve the growth of multiple plant species. Moringa oleifera is a medicinal plant found in Saudi Arabia. Its leaves, flowers and fruit have been used as food. Moringa oleifera is rich in rutin and gallic acid and many other bioactive compounds, which collectively contribute to its demonstrated range of pharmacological activities. In Saudi Arabia, the semi-arid and arid weather presents a significant challenge to agriculture. High salinity in cultivated land is a particular threat. We applied Chlorella vulgaris, Nannochloropsis salina, and Enterobacter cloacae at multiple salinities to Moringa oleifera to investigate their effects on the growth, yield, and photosynthetic pigment content. We also examined possible changes in the phytochemical composition. The application of Chlorella vulgaris, Nannochloropsis salina and Enterobacter cloacae enhanced plant growth and yield, while inhibition was observed at high (6000 ppm) salinity. The presence of Chlorella vulgaris and Nannochloropsis salina altered plant growth and yield and rutin and gallic acid content of Moringa oleifera plants grown in saline conditions. Microalgae species were recommended for use as a bio-fertiliser alternative to mainstream synthetic fertilisers.     

Comparison of ectoine synthesis regulation in secreting and non-secreting strains of Halomonas

Ectoine is an osmotic pressure compatible solute. It is synthesized by Halomonas and other microorganisms in a hypertonic environment. As a stabilizing agent of cells proteins, nucleic acids and other biological products, ectoine has wide applications. Therefore, an efficient production method for ectoine is in great demand. Ectoine is overproduced by Halomonas salina DSM 5928, an ectoine-secreting strain, in which the synthesis of ectoine is not limited by its intracellular threshold concentration. In order to explain the mechanism of secretion of ectoine, the response to NaCl stress, and the release and uptake kinetics of ectoine were compared between H. salina DSM 5928 and Halomonas elongata DSM 2581, a non-ectoine-secreting strain. Moreover, the ectoine binding protein TeaA from each of these two strains was cloned and expressed, and binding abilities were examined in vitro. The results indicated that H. salina DSM 5928 and H. elongata DSM 2581 respond to NaCl in the medium in different ways. Compared with H. elongata DSM 2581, the amount of ectoine released was higher and the uptake of ectoine under NaCl stress was lower in H. salina DSM 5928. In addition, the binding ability of TeaA to ectoine in H. salina DSM 5928 was also lower. These results reveal the secretion mechanism of ectoine as well as critical regulation and control factors involved in ectoine synthesis.     

Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors

Additional evidence about the influence of the cell wall physical and chemical characteristics on protein extractability was determined by calculating the conversion factors of five different microalgae known to have different cell wall composition, and their protein extracts. The conversion factors obtained for crude rigid cell walled Chlorella vulgaris, Nannochloropsis oculata and Haematococcus pluvialis were 6.35, 6.28 and 6.25, respectively, but for their protein extracts the values were lower with 5.96, 5.86 and 5.63. On the other hand, conversion factor obtained for fragile cell walled microalgae Porphyridium cruentum and Athrospira platensis was 6.35 for the former and 6.27 for the latter, with no significant difference for their protein extract with 6.34 for the former and 6.21 for the latter. In addition, the highest hydro-soluble protein percentage recovered from total protein was for P. cruentum 80.3 % and A. platensis 69.5 % but lower for C. vulgaris with 43.3 %, N. oculata with 33.3 % and H. pluvialis with 27.5 %. The study spotted the light on the influence of the cell wall on evaluating the conversion factor and protein extractability. In addition, it showed the necessity of finding the conversion factor every time accurate protein quantification is required, and proved that there is not a universal conversion factor that can be recommended.     

Algal carotenoids and chemotaxonomy

A qualitative and quantitative examination of the carotenoids of pure cultures of four marine microalgae, including Chroomonas salina, Vaucheria sessilis and Coccolithus huxleyi is reported. The latter contained a new, major carotenoid, and not fucoxanthin.     

Application of the microalga Plagioselmis prolonga for the assessment of water quality in the Amursky and Nakhodka Bays (Sea of Japan)

Based on biotesting, we carried out an estimation of the water quality in the Amursky and Nakhodka Bays (Sea of Japan) using Plagioselmis prolonga (Cryptophyta). The obtained data were compared with the data from water biotesting using Dunaliella salina (Chlorophyta). It was shown that water from the Amursky Bay produced more a negative effect on both microalgae than water from the Nakhodka Bay. It was established that sensitivity of the P. prolonga microalga exceeded that of D. salina. This was confirmed by a sharp decrease of the P. prolonga motile cell number in the studied water.     

Antiviral compounds obtained from microalgae commonly used as carotenoid sources

Pressurized liquid extraction (PLE), an environmentally friendly technique, has been used to obtain antiviral compounds from microalgae commonly used as carotenoid sources: Haematococcus pluvialis and Dunaliella salina. The antiviral properties of PLE extracts (hexane, ethanol and water) were evaluated against herpes simplex virus type 1 (HSV-1) at different stages during viral infection. Pretreatment of Vero cells with 75?μg?mL^?1 of H. pluvialis ethanol extract inhibited virus infection by approximately 85%, whereas the same concentration of water and hexane extracts reduced the virus infectivity 75% and 50%, respectively. D. salina extracts were less effective than H. pluvialis extracts and presented a different behaviour since water and ethanol extracts produced a similar virus inhibition (65%). Moreover, H. pluvialis ethanol extract was also the most effective against HSV-1 intracellular replication. The antiviral activity of water PLE extracts was found to correlate with polysaccharides since the polysaccharide-rich fraction isolated from these extracts showed higher antiviral activity than the original water extracts. A gas chromatography-mass spectrometry (GC-MS) characterization of the H. pluvialis ethanol extract showed the antiviral activity of this extract could be partially related with the presence of short-chain fatty acids, although other compounds could be involved in this activity; meanwhile, in the case of D. salina ethanol extract other compounds seemed to be implied, such as: β-ionone, neophytadiene, phytol, palmitic acid and α-linolenic acid. The results demonstrate the use of PLE allows obtaining antiviral compounds from microalgae used as carotenoids sources, which gives the microalgae biomass an added value.     

Identification of Low Molecular Mass GTP-Binding Proteins in Membranes of the Halotolerant Alga Dunaliella salina

A family of specific guanine nucleotide-binding proteins in Dunaliella salina was studied. Polypeptides of different subcellular fractions were separated by electrophoresis and transferred to nitrocellulose or Immobilon membranes. Incubation of the transfer blots with [³⁵S]GTPγS or [α-³²P]GTP showed no evidence for GTP-binding proteins in the chloroplast and cytosol fractions. However, two GTP-binding proteins with molecular masses of 28 and 30 kilodaltons were present in the plasma membrane and microsomal fractions. An additional 29 kilodalton GTP-binding protein was detected in the plasma membrane. The mitochondrial fraction contained significant amounts of only the 28 kilodalton GTP-binding protein. Binding of [³²P]GTP to the protein blots was completely prevented by 10 micromolar GTP or guanosine 5′-O-(2-thiodiphosphate) (added in 3 × 10⁴-fold excess), whereas ATP or CTP had no effect on the binding. The 28 kilodalton GTP-binding protein was recognized by polyclonal antibodies to the ras-related YPT1 protein of yeast but not by the anti-ras Y13-259 monoclonal antibody. GTP-binding proteins present in the microsomal fraction could not be solubilized by incubation of microsomes with 1 molar NaCl or 0.2 molar Na₂CO₃, but some GTP-binding activity was solubilized when microsomes were treated with 6 molar urea. These results indicate that D. salina GTP-binding proteins are tightly associated with the membranes. The covalent attachment of fatty acids to these proteins was also investigated. Electrophoresis followed by fluorography of delipidated microsomal proteins extracted from [³H]myristic acid-labeled cells showed an intense labeling of a 28 kilodalton protein. We conclude that D. salina contains proteins resembling the ras-related proteins found in animal cells and higher plants.     

Promising microbial consortia for producing biofertilizers for rice fields

Two cyanobacterial cultures from rice paddies of Kyzylorda Provence, Kazakhstan were isolated and characterized: Anabaena variabilis and Nostoc calsicola. Based on these cultures, new consortia of cyanobacteria, microalgae and Azotobacter were developed: ZOB-1 (Anabaena variabilis, Chlorella vulgaris, and Azotobacter sp.) and ZOB-2 (Nostoc calsicola, Chlorella vulgaris, and Azotobacter sp.). High growth rate and photosynthetic activity of microalgae were observed in these consortia. The active consortium ZOB-1 was selected, which improved germination and growth of rice plants. ZOB-1 was recommended as a biostimulator and biofertilizer for crops.     

Germination and ROS detoxification in bell pepper (Capsicum annuum L.) under NaCl stress and treatment with microalgae extracts

We evaluated the salt tolerance of hybrids of pepper (Capsicum annuum L.) during germination. Treatments were applied at 0, 25, and 50 mM NaCl with preparations of supplemental extracts of the microalgae Dunaliella salina and Phaeodactylum tricornutum to determine the percentage germination rate as well as measured indicators of oxidative stress caused by the salt treatments during seed germination. We found that root growth was favorably influenced by the microalgae leading to increased germination rate. Tissues were analyzed in terms of superoxide radical production, lipid peroxidation, and activity of antioxidant enzymes viz. superoxide dismutase, catalase, and glutathione peroxidase. Our results suggest that application of microalgae extracts significantly reduced (p?<?0.05) superoxide radical production, as well as lower lipid peroxidation in comparison to plants without extracts of microalgae. The antioxidant enzymes increased in the presence of microalgae showing a significant difference (p?<?0.05). The results suggest differences in oxidative metabolism in response to the magnitude of salt stress and concentrations of microalgae help mitigate salt stress in plants during the germination process.     

Life-cycle assessment of microalgae culture coupled to biogas production

Due to resource depletion and climate change, lipid-based algal biofuel has been pointed out as an interesting alternative because of the high productivity of algae per hectare and per year and its ability to recycle CO₂ from flue gas. Another option for taking advantage of the energy content of the microalgae is to directly carry out anaerobic digestion of raw algae in order to produce methane and recycle nutrients (N, P and K). In this study, a life-cycle assessment (LCA) of biogas production from the microalgae Chlorella vulgaris is performed and the results are compared to algal biodiesel and to first generation biodiesels. These results suggest that the impacts generated by the production of methane from microalgae are strongly correlated with the electric consumption. Progresses can be achieved by decreasing the mixing costs and circulation between different production steps, or by improving the efficiency of the anaerobic process under controlled conditions. This new bioenergy generating process strongly competes with others biofuel productions.     

The effect of ultrasound on the growth and viability of microalgae cells

Ultrasound has shown potential for both increasing microalgal lipid extraction yields and for the control of microalgal blooms through cell disruption. The effect of ultrasound on the viability of microalgae was investigated on the following species: Dunaliella salina, Chlamydomonas concordia and Nannochloropsis oculata. Sonication with a 20 kHz probe (0.086 W cm^?3) caused complete cell disruption of D. salina after 4 min. This microalgae species does not have a true cell wall. In the case of C. concordia which has a thin cell wall complete cell disruption under the same conditions took 16 min. Under the same conditions, there was no visible disruption of N. oculata, a species which has a thick cell wall. However spectro-fluorophotometer analysis of the sonicated suspension of N. oculata showed that although the cells were intact, the level of intracellular chlorophyll was reduced by ~10 %. This clearly indicated damage to the microalgal cell wall. After 16 min, treatment cultures of all three species remained viable. Programmed cell death (PCD) has been induced in some microalgal species to terminate algal blooms; ultrasonic application did not induce PCD in any species tested. The supernatant of sonicated D. salina and C. concordia has also been shown to be able to boost the growth of established cultures. These results provide important information concerning the uses of ultrasound in both the microalgal biofuels industry and for the control of microalgal blooms.     

Optimization of protein extraction from Chlorella Vulgaris via novel sugaring‐out assisted liquid biphasic electric flotation system

Microalgae biomass has been consumed as animal feed, fish feed and in human diet due to its high nutritional value. In this experiment, microalgae specie of Chlorella Vulgaris FSP‐E was utilized for protein extraction via simple sugaring‐out assisted liquid biphasic electric flotation system. The external electric force provided to the two‐phase system assists in disruption of rigid microalgae cell wall and releases the contents of microalgae cell. This experiment manipulates various parameters to optimize the set‐up. The liquid biphasic electric flotation set‐up is compared with a control liquid biphasic flotation experiment without the electric field supply. The optimized separation efficiency of the liquid biphasic electric flotation system was 73.999 ± 0.739% and protein recovery of 69.665 ± 0.862% compared with liquid biphasic flotation, the separation efficiency was 61.584 ± 0.360% and protein recovery was 48.779 ± 0.480%. The separation efficiency and protein recovery for 5 × time scaled‐up system was observed at 52.871 ± 1.236% and 73.294 ± 0.701%. The integration of simultaneous cell‐disruption and protein extraction ensures high yield of protein from microalgae. This integrated method for protein extraction from microalgae demonstrated its potential and further research can lead this technology to commercialization.