Molecular phylogeny of a green microalga isolated from White Sea sponge <Emphasis Type="Italic">Halichondria panicea</Emphasis> (Pallas, 1766)

Molecular identification of eukaryotic microalga 1Hp86E-2 isolated from White Sea sponge Halichondria panicea (Pallas, 1766) was conducted, and phylogenetic analysis was carried out using the nucleotide sequence of 18S rRNA gene (GenBank, no. JX437624). Isolated microalga was classified to the genus Desmodesmus. Microalga 1Hp86E-2 proved to be closely related to the algae Desmodesmus sp. 3Dp86E-1, Desmodesmus sp. 2C166E, and Desmodesmus sp. 1Pm66B isolated from White Sea invertebrates. Phylogenetic analysis showed that these closely related organisms belong to a monophyletic group.     

Desmodesmus sp. 3Dp86E-1—a Novel Symbiotic Chlorophyte Capable of Growth on Pure CO2

A novel chlorophyte Desmodesmus sp. 3Dp86E-1 isolated from a White Sea hydroid Dynamena pumila was cultivated at CO₂ levels from atmospheric (the ‘low-CO₂’ conditions) to pure carbon dioxide (the 5, 20, and 100 % CO₂ conditions) under high (480 μE/(m² s) PAR) light. After 7 days of cultivation, the ‘100 % CO₂’ (but not 5 or 20 % CO₂) cells possessed ca. four times higher chlorophyll content per dry weight (DW) unit than the low-CO₂ culture. The rate of CO₂ fixation under 100 % CO₂ comprised ca. 1.5 L/day per L culture volume. After a lag period which depended on the CO₂ level, biomass accumulation and volumetric fatty acid (FA) content of the Desmodesmus sp. 3Dp86E-1 bubbled with CO₂-enriched gas mixtures increased and was comparable to that of the culture continuously bubbled with air. Under the low-to-moderate CO₂ conditions, the FA percentage of the algal cells increased (to 40 % DW) whereas under high-CO₂ conditions, FA percentage did not exceed 15 % DW. A strong increase in oleate (18:1) proportion of total FA at the expense of linolenate (18:3) was recorded in the ‘100 % CO₂’ cells. Electron microscopy and pulse–amplitude-modulated chlorophyll fluorescence investigation revealed no damage to or significant downregulation of the photosynthetic apparatus in ‘100 % CO₂’ cells grown at the high-PAR irradiance. Possible mechanisms of high-CO₂ tolerance of Desmodesmus sp. 3Dp86E-1 are discussed in view of its symbiotic origin and possible application for CO₂ biomitigation.     

Simultaneous enhancement of CO2 fixation and lutein production with thermo-tolerant Desmodesmus sp. F51 using a repeated fed-batch cultivation strategy

This study aimed to improve lutein production using a thermo-tolerant lutein-rich microalga Desmodesmus sp. F51. To achieve this goal, four fed-batch cultivation strategies were investigated for CO₂ fixation and lutein production of Desmodesmus sp. F51. Among them, Fed-batch IV showed the best performance, giving the highest CO₂ fixation rate and lutein productivity of 1582.4 mg/L/d and 3.91 mg/L/d, respectively. Both increasing the light intensity and limiting the nutrients led to a lower carotenoids content in the microalga, with a higher proportion of lutein and lower proportion of β-carotene being obtained in the carotenoids. The carotenoid present in the biomass was mainly lutein, accounting for 50–66% of total carotenoids. Repeated operations of the Fed-batch IV strategy could effectively improve CO₂ fixation and lutein production of Desmodesmus sp. F51, giving the best results of 1826.0 mg/L/d, and 4.61 mg/L/d, respectively. This performance is better than most of the previously reported values.     

Effects of illumination and nitrogen starvation on accumulation of arachidonic acid by the microalga Parietochloris incisa

Parietochloris incisa is a unicellular freshwater green alga capable of accumulating high amounts of the valuable long-chain polyunsaturated arachidonic acid (AA) in triacylglycerols (TAG) of cytoplasmic oil bodies. To find the cultivation conditions providing maximum AA yield, the effects of illumination and N-availability on the dry weight (DW), chlorophyll, carotenoid, and AA content were studied. Under nitrogen starvation, TAG accounted for over 30% of dry weight (DW) and the AA content became as high as about 55% of total fatty acids. For biomass accumulation, light intensity of ca 400 μE m^?2 s^?1 was found to be optimal for growing P. incisa on a complete medium. Lower light intensities (or a higher cell density of inoculum) resulted in a higher AA yield when the alga was cultivated on nitrogen-free media. In the absence of nitrogen, algal cells were unable to cope with high illumination and suffered from photooxidative damage, whereas the nutrientsufficient culture survived under such illumination conditions, probably due to accumulation of carotenoids. Nitrogen-deprived P. incisa cells displayed elevated sensitivity to light.     

The effect of nitrogen starvation on the ultrastructure and pigment composition of chloroplasts in the acidothermophilic microalga Galdieria sulphuraria

We studied the effect of nitrogen starvation on growth indices, vitality, ultrastructure, and the photosynthetic apparatus of unique acidothermophilic microalga Galdieria sulphuraria (Galdieri) Merola. Long-term nitrogen starvation ceased G. sulphuraria growth and cell division. During the first days of starvation, phycobiliproteins degraded first, then the content of chlorophyll and carotenoids decreased to trace amounts, chloroplast reduced, cell wall became thinner, and storage compounds accumulated. However, the cells were alive. A comparison with the effects of nitrogen starvation on other photosynthesizing organisms showed that suppression of cell division, reduction of the photosynthetic apparatus to some minimum, and accumulation of storage compounds are a universal response to this stress.     

Bioremediation of wastewater from edible oil refinery factory using oleaginous microalga Desmodesmus sp. S1

Edible oil industry produced massive wastewater, which requires extensive treatment to remove pungent smell, high phosphate, carbon oxygen demand (COD), and metal ions prior to discharge. Traditional anaerobic and aerobic digestion could mainly reduce COD of the wastewater from oil refinery factories (WEORF). In this study, a robust oleaginous microalga Desmodesmus sp. S1 was adapted to grow in WEORF. The biomass and lipid content of Desmodesmus sp. S1 cultivated in the WEORF supplemented with sodium nitrate were 5.62 g·L^?1 and 14.49%, whereas those in the WEORF without adding nitrate were 2.98 g·L^?1 and 21.95%. More than 82% of the COD and 53% of total phosphorous were removed by Desmodesmus sp. S1. In addition, metal ions, including ferric, aluminum, manganese and zinc were also diminished significantly in the WEORF after microalgal growth, and pungent smell vanished as well. In comparison with the cells grown in BG-11 medium, the cilia-like bulges and wrinkles on the cell surface of Desmodesmus sp. S1 grown in WEORF became out of order, and more polyunsaturated fatty acids were detected due to stress derived from the wastewater. The study suggests that growing microalgae in WEORF can be applied for the dual roles of nutrient removal and biofuel feedstock production.     

Effects of light and nitrogen starvation on the content and composition of carotenoids of the green microalga <Emphasis Type="Italic">Parietochloris incisa</Emphasis>

The changes in pigment content and composition of the unicellular alga Parietochloris incisa comb. nov (Trebouxiophyceae, Chlorophyta) were studied. This alga is unique in its ability to accumulate high amounts of arachidonic acid in the cell during cultivation under different irradiances and nitrogen availability in the medium. Under low irradiance of 35 μE/(m₂ s) photosynthetically active radiation the P. incisa cultures possessed slow growth and a relatively low carotenoid-to-chlorophyll ratio. At higher irradiances (200 and 400 μE/(m₂ s)) on complete medium, the alga displayed higher growth rate and an increase in the carotenoid content, especially that of β-carotene and lutein. Both on nitrogen-free (regardless of illumination intensity) and nitrogen-replete medium (under high light), a considerable increase in the ratio of carotenoid and chlorophyll contents was recorded. Predominant accumulation of xanthophylls took place in thylakoid membranes, whereas β-carotene deposition occurred mainly in the cytoplasmic lipid globules (oil bodies); lower amounts of carotenoids were accumulated in the absence of nitrogen. Under high light and nitrogen-deficiency conditions, an increase in violaxanthin de-epoxidation and nonphotochemical quenching was recorded together with a decline in variable chlorophyll fluorescence (F _v/F _m) level. A possible photoprotective role of carotenoids in adaptation of P. incisa to high light under nitrogen starvation conditions is discussed.     

Optimization of cultivation conditions for fatty acid composition and EPA production in the eustigmatophycean microalga Trachydiscus minutus

We determined the effects of cultivation conditions (nitrogen source, salinity, light intensity, temperature) on the composition of polyunsaturated fatty acids (PUFAs) and the production of eicosapentaenoic acid (EPA) in the laboratory cultured eustigmatophycean microalga, Trachydiscus minutus. T. minutus was capable of utilizing all nitrogen compounds tested (potassium nitrate, urea, ammonium nitrate, ammonium carbonate) with no differences in growth and only minor differences in fatty acid (FA) compositions. Ammonium carbonate was the least appropriate for lipid content and EPA production, while urea was as suitable as nitrates. Salinity (0.2 % NaCl) slightly stimulated EPA content and inhibited growth. Increasing salinity had a marked inhibitory effect on growth and PUFA composition; salinity at or above 0.8 % NaCl was lethal. Both light intensity and temperature had a distinct effect on growth and FA composition. The microalga grew best at light intensities of 470–1,070 μmol photons m^?2 s^?1 compared to 100 μmol photons m^?2 s^?1, and at 28 °C; sub-optimal temperatures (20, 33 °C) strongly inhibited growth. Saturated fatty acids increased with light intensity and temperature, whereas the reverse trend was found for PUFAs. Although the highest level of EPA (as a proportion of total FAs) was achieved at a light intensity of 100 μmol photons m^?2 s^?1 (51.1?± 2.8 %) and a temperature of 20 °C (50.9?±?0.8 %), the highest EPA productivity of about 30 mg L^?1?day^?1 was found in microalgae grown at higher light intensities, at 28 °C. Overall, for overproduction of EPA in microalgae, we propose that outdoor cultivation be used under conditions of a temperate climatic zone in summer, using urea as a nitrogen source.     

Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under nitrogen-limited conditions

Photosynthetic carbon partitioning into starch and neutral lipid was investigated in the oleaginous green microalga Pseudochlorococcum sp. When grown under low light and nitrogen-replete conditions, the algal cells possessed a basal level of starch. When grown under high light and nitrogen-limited conditions, starch synthesis was transiently up-regulated. After nitrogen depletion, starch content decreased while neutral lipid rapidly increased to 52.1% of cell dry weight, with a maximum neutral lipid productivity of 0.35 g L⁻¹ D⁻¹. These results suggest that Pseudochlorococcum used starch as a primary carbon and energy storage product. As nitrogen was depleted for an extended period of time, cells shift the carbon partitioning into neutral lipid as a secondary storage product. Partial inhibition of starch synthesis and degradation enzymes resulted in a decrease in neutral lipid content, indicating that conversion of starch to neutral lipid may contribute to overall neutral lipid accumulation. Biotechnological application of Pseudochlorococcum sp. as a production strain for biofuel was assessed.     

Screening of the culture media with different concentrations of nutrients for cultivation of the microalgae associated with the invertebrates of the White Sea

The growth and biomass accumulation of three microalgal strains of Desmodesmus (Scenedesmaceae, Chlorophyceae), 1Рm66В, 2Cl66E, and 3Dp86Е-1, isolated from the White Sea benthic invertebrates were studied under conditions of batch culture in different standard media (BG-11, Prat, Goldberg, Gromov, Tamiya, artificial seawater) and modified media. The culture condition, biomass accumulation, and uptake of nitrate and phosphate were recorded. A significant alkalization of the culture medium up to pH 10 has been observed during a vigorous growth of the microalgae. The most significant biomass accumulation has been recorded in BG-11 (in complete or modified medium with addition of artificial seawater), Tamiya, and Prat media. Addition of seawater did not affect the growth of Desmodesmus sp. in the nitrate-containing media, although that maintained growth of the microalgae in the nitrogen-lacking media without cell aggregation. The BG-11 medium appears suitable for isolation and cultivation of both symbiotic and free-living microalgae by all the tested features. The Prat medium is the best for maintaining the microalgal strains in living collection.     

Rapid estimation of lipid content in an Antarctic ice alga (Chlamydomonas sp.) using the lipophilic fluorescent dye BODIPY505/515

Chlamydomonas sp. ICE-L, isolated from Antarctic coastal marine environments, was selected as a high lipid producer, which may be useful for biodiesel production. The lipophilic fluorescent dye BODIPY505/515 was used to determine the algal lipid content. Lipid bodies stained with BODIPY505/515 have a characteristic green fluorescence, and their volumes were determined using the sphere volume formula. In this study, lipid accumulation by Chlamydomonas ICE-L was analyzed under different cultivation conditions (nitrogen deficiency and UV-B radiation). The results demonstrated that nitrogen deficiency and UV-B radiation could significantly promote the accumulation of lipid content per cell. The highest yields of total lipid content (reaching 84?μL?L^?1) were obtained in full Provasoli medium after 12?days of cultivation, but not in the nitrogen-deficient medium. The inoculum used in this experiment was obtained from the late-exponential growth phase. The main reason was that the cell numbers in nitrogen-deficient medium had not increased and total lipid contents were offset by the lower growth rate. Considering the high lipid content in Chlamydomonas sp. ICE-L, this alga might be a promising alternative species for production of microalgal oil for the production of renewable biodiesel in the future.     

Expression of fatty acid synthesis genes and fatty acid accumulation in haematococcus pluvialis under different stressors

Background

Biofuel has been the focus of intensive global research over the past few years. The development of 4^th generation biofuel production (algae-to-biofuels) based on metabolic engineering of algae is still in its infancy, one of the main barriers is our lacking of understanding of microalgal growth, metabolism and biofuel production. Although fatty acid (FA) biosynthesis pathway genes have been all cloned and biosynthesis pathway was built up in some higher plants, the molecular mechanism for its regulation in microalgae is far away from elucidation.

Results

We cloned main key genes for FA biosynthesis in Haematococcus pluvialis, a green microalga as a potential biodiesel feedstock, and investigated the correlations between their expression alternation and FA composition and content detected by GC-MS under different stress treatments, such as nitrogen depletion, salinity, high or low temperature. Our results showed that high temperature, high salinity, and nitrogen depletion treatments played significant roles in promoting microalgal FA synthesis, while FA qualities were not changed much. Correlation analysis showed that acyl carrier protein (ACP), 3-ketoacyl-ACP-synthase (KAS), and acyl-ACP thioesterase (FATA) gene expression had significant correlations with monounsaturated FA (MUFA) synthesis and polyunsaturated FA (PUFA) synthesis.

Conclusions

We proposed that ACP, KAS, and FATA in H. pluvialis may play an important role in FA synthesis and may be rate limiting genes, which probably could be modified for the further study of metabolic engineering to improve microalgal biofuel quality and production.     

Toxicity, transformation and accumulation of inorganic arsenic species in a microalga Scenedesmus sp. isolated from soil

Arsenic speciation and cycling in the natural environment are highly impacted via biological processes. Since arsenic is ubiquitous in the environment, microorganisms have developed resistance mechanisms and detoxification pathways to overcome the arsenic toxicity. This study has evaluated the toxicity, transformation and accumulation of arsenic in a soil microalga Scenedesmus sp. The alga showed high tolerance to arsenite. The 72-h 50 % growth inhibitory concentrations (IC₅₀ values) of the alga exposed to arsenite and arsenate in low-phosphate growth medium were 196.5 and 20.6 mg? L^?1, respectively. When treated with up to 7.5 mg? L^?1 arsenite, Scenedesmus sp. oxidised all arsenite to arsenate in solution. However, only 50 % of the total arsenic remained in the solution while the rest was accumulated in the cells. Thus, this alga has accumulated arsenic as much as 606 and 761 μg? g^?1 dry weight when exposed to 750 μg? L^?1 arsenite and arsenate, respectively, for 8 days. To our knowledge, this is the first report of biotransformation of arsenic by a soil alga. The ability of this alga to oxidise arsenite and accumulate arsenic could be used in bioremediation of arsenic from contaminated water and soil.     

Culture of the green microalga Botryococcus braunii Showa with LED irradiation eliminating violet light enhances hydrocarbon production and recovery

The green microalga Botryococcus braunii (B. braunii), race B, was cultured under light-emitting diode (LED) irradiation with and without violet light. This study examined the effect of violet light on hydrocarbon recovery and production in B. braunii. C34 botryococcene hydrocarbons were efficiently extracted by thermal pretreatments at lower temperatures when the alga was cultured without violet light. The hydrocarbon content was also higher (approximately 3%) in samples cultured without violet light. To elucidate the mechanism of effective hydrocarbon recovery and production, we examined structural components of the extracellular matrix (ECM). The amounts of extracellular carotenoids and water-soluble polymers extracted by thermal pretreatment from the ECM were decreased when the alga was cultured without violet light. These results indicate that LED irradiation without violet light is more effective for hydrocarbon recovery and production in B. braunii. Furthermore, structural ECM components are closely involved in hydrocarbon recovery and production in B. braunii.     

Evaluation of closed photobioreactor types and operation variables for enhancing lipid productivity of <Emphasis Type="Italic">Nannochloropsis sp.</Emphasis> KMMCC 290 for biodiesel production

Previously, we have successfully produced biodiesel using the marine microalga Nannochloropsis sp. KMMCC 290 cultivated in a raceway open pond. Here, we investigated the effects of closed photobioreactors and operating variables on cell concentration and lipid content of the microalga to increase its lipid productivity. The flatplate photobioreactor (FPP) showed higher performance than bubble column and air-lift photobioreactors. Among the variables evaluated, light intensity, aeration rate, and carbon dioxide feeding significantly influenced cell concentration, whereas a simultaneous increase in light intensity and aeration rate, as well as carbon dioxide feeding noticeably increased the lipid content. The lipid productivity in the FPP was 26.7 × 10^-3 g/L/day, which was 16.6 times higher than that produced by the microalga cultivated in the raceway pond, 4.8 times higher than that from the simple flask-grown control culture, and 2.1 times higher than that from the FPP under initial conditions.     

Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta)

In order to study the effects of different nitrogen source and concentrationon the growth rate and fatty acid composition, a marine microalga Ellipsoidion sp. with a high content of eicosapentaenoic acid (EPA) wascultured in media with different nitrogen sources and concentrations.During the pre-logarithmic phase, the alga grew faster with ammoniumas N source than with nitrate, but the reverse applied during thepost-logarithmic phase. The alga grew poorly in N-free mediumor medium with urea as the sole N source. In the same growth phase,ammonium medium resulted in higher yield of total lipid, but the EPA yielddid not differ significantly different from that using nitrate medium. Themaximum growth rate occurred in medium containing 1.28 mmolL^-1 sodium nitrate, while maximum EPA and total lipid contents werereached at 1.92 mmol L^-1, when EPA accounted for 27.9% totalfatty acids. The growth rate kept stable when NH₄Cl ranged from0.64 to 2.56 mmol L^-1, and the maximum content of total lipidand EPA occurred in the medium with 2.56 mmol L^-1NH₄Cl. The EPA content was higher in the pre- thanpost-logarithmic phase, though the total lipid content was lower. Thehighest EPA content expressed as percent total fatty acid was 27.9% innitrate medium and and 39.0% in ammonium medium.     

Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution

There is a particularly high interest to derive carotenoids such as β-carotene and lutein from higher plants and algae for the global market. It is well known that β-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased β-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.     

Metabolite Profiling and Integrative Modeling Reveal Metabolic Constraints for Carbon Partitioning under Nitrogen Starvation in the Green Algae Haematococcus pluvialis

The green alga Hematococcus pluvialis accumulates large amounts of the antioxidant astaxanthin under inductive stress conditions, such as nitrogen starvation. The response to nitrogen starvation and high light leads to the accumulation of carbohydrates and fatty acids as well as increased activity of the tricarboxylic acid cycle. Although the behavior of individual pathways has been well investigated, little is known about the systemic effects of the stress response mechanism. Here we present time-resolved metabolite, enzyme activity, and physiological data that capture the metabolic response of H. pluvialis under nitrogen starvation and high light. The data were integrated into a putative genome-scale model of the green alga to in silico test hypotheses of underlying carbon partitioning. The model-based hypothesis testing reinforces the involvement of starch degradation to support fatty acid synthesis in the later stages of the stress response. In addition, our findings support a possible mechanism for the involvement of the increased activity of the tricarboxylic acid cycle in carbon repartitioning. Finally, the in vitro experiments and the in silico modeling presented here emphasize the predictive power of large scale integrative approaches to pinpoint metabolic adjustment to changing environments.     

Changes in pigments profile in the green alga Haeamtococcus pluvialis exposed to environmental stresses

Haematococcus green culture starved for either nitrogen or phosphate accumulated astaxanthin up to 4% cell dry wt (2.6 g l^–1). While under nitrogen starvation astaxanthin accumulation was faster (maximum achieved after 8 days in comparison to 14 days in the phosphate-starved culture) and accompanied by a drop in the chlorophyll content per cell down to 50% of its original value (30 pg cell^–1); in the phosphate-starved culture this parameter did not change. HPLC profiles of carotenoids monitored along the starvation process revealed that astaxanthin esters accounted for more than 99% of total carotenoids at the end of the exposure period at both starvations.     

Microbial Formation of Manganese Oxides

Microbial manganese oxidation was demonstrated at high Mn²⁺ concentrations (5 g/liter) in bacterial cultures in the presence of a microalga. The structure of the oxide produced varied depending on the bacterial strain and mode of culture. A nonaxenic, acid-tolerant microalga, a Chlamydomonas sp., was found to mediate formation of manganite (γ-MnOOH). Bacteria isolated from associations with crude cultures of this alga grown in aerated bioreactors formed disordered γ-MnO₂ from Mn²⁺ at concentrations of 5 g/liter over 1 month, yielding 3.3 g of a semipure oxide per liter. All algal-bacterial cultures removed Mn²⁺ from solution, but only those with the highest removal rates formed an insoluble oxide. While the alga was an essential component of the reaction, a Pseudomonas sp. was found to be primarily responsible for the formation of a manganese precipitate. Medium components—algal biomass and urea—showed optima at 5.7 and 10 g/liters, respectively. The scaled-up culture (50 times) gave a yield of 22.3 g (53 mg/liter/day from a 15-liter culture) of semipure disordered γ-MnO₂, identified by X-ray diffraction and Fourier transform infrared (FTIR) spectroscopy, and had a manganese oxide O/Mn ratio of 1.92. The Mn(IV) content in the oxide was low (30.5%) compared with that of mined or chemically formed γ-MnO₂ (ca. 50%). The shortfall in the bacterial oxide manganese content was due to biological and inorganic contaminants. FTIR spectroscopy, transmission electron microscopy, and electron diffraction studies have identified manganite as a likely intermediate product in the formation of disordered γ-MnO₂.