Effects of carbon source and light intensity on the growth and total lipid production of three microalgae under different culture conditions

We attempted to enhance the growth and total lipid production of three microalgal species, Isochrysis galbana LB987, Nannochloropsis oculata CCAP849/1, and Dunaliella salina, which are capable of accumulating high content of lipid in cells. Low nitrogen concentration under photoautotrophic conditions stimulated total lipid production, but a decreasing total lipid content and an increasing biomass were observed with increasing nitrogen concentration. Among the different carbon sources tested for heterotrophic cultivation, glucose improved the growth of all three strains. The optimal glucose concentration for growth of I. galbana LB987 and N. oculata CCAP849/1 was 0.02 M, and that of D. salina was 0.05 M. Enhanced growth occurred when they were cultivated under heterotrophic or mixotrophic conditions compared with photoautotrophic conditions. Meanwhile, high total lipid accumulation in cells occurred when they were cultivated under photoautotrophic or mixotrophic conditions. During mixotrophic cultivation, biomass production was not affected significantly by light intensity; however, both chlorophyll concentration and total lipid content increased dramatically with increasing light intensity up to 150 µmol/m²/s. The amount and composition ratio of saturated and unsaturated fatty acids in cells were different from each other depending on both species and light intensity. The highest accumulation of total fatty acid (C16–C18) among the three strains was found from cells of N. oculata CCAP849/1, which indicates that this species can be used as a source for production of biodiesel.     

Effects of nitrogen source and irradiance on <Emphasis Type="Italic">Porphyridium cruentum</Emphasis>

We determined the effects of two nitrogen sources (ammonium and nitrate) and two irradiance levels (50 and 200 μmol photons m^?2 s^?1) on the growth rate, cell size, proximate composition, pigment content, and photosynthesis of the unicellular red alga, Porphyridium cruentum. Irradiance significantly affects growth rate, as well as carbohydrate, protein, and phycoerythrin content. Nitrogen form significantly affects cell size, total dry weight, organic dry weight, ash content, carotene content, phycocyanin content, allophycocyanin content, maximum relative electron transport rate (rETRm), and photosynthetic efficiency (α). However, the irradiance and nitrogen source had significantly interaction with the content of lipids and chlorophyll a content, relative electron transport rate (rETR), and irradiance of saturation (I_k). These findings demonstrate that irradiance and nitrogen source influence the metabolism of P. cruentum and that the combination of these two variables induces the production of chemical products for biotechnological, aquaculture, and nutraceutical industry.     

Mixotrophic cultivation of <Emphasis Type="Italic">Chlorococcum</Emphasis> sp. under non-controlled conditions using a digestate from pig manure within a biorefinery

A number of business opportunities may arise from microalgae and wastewater treatment becoming an integrated system, as biofuels and high-added value products could be obtained simultaneously. This study, performed under controlled and non-controlled conditions, aimed at cultivating Chlorococcum sp. using a digestate from pig manure as culture medium and assessing its growth and biochemical composition for further applications. Under controlled conditions, cultures of Chlorococcum sp. were established testing various digestate dilutions (v/v). It was found that all tested dilutions (up to 8% v/v) promoted a higher biomass density, compared to the control culture in modified Bold’s Basal Medium (modified BBM). Under non-controlled conditions, it was found that the biomass productivity using the digestate diluted 5.6% v/v (23.4 mg L^?1 day^?1) was statistically similar to the one obtained using modified BBM (26.4 mg L^?1 day^?1). The volatile fatty acids contained in the digestate might have allowed mixotrophic growth for Chlorococcum sp. The intracellular lipid content in Chlorococcum sp. remained constant throughout the experiments in both, treatment and control cultures, while carbohydrates increased from 20 to 45% of the cell dry weight in the treatment and from 20 to 42% in the control one. It was concluded that conditions of nitrogen starvation and fluctuating irradiance and temperature benefit carbohydrate accumulation in this strain, since intracellular carbohydrate content increased nearly two-fold during this period. Additionally, the obtained biomass has the potential to be used as feedstock for bioethanol production. This system can meet the concept of a microalgae-based biorefinery, in which biofuels and high-added value products are produced from microalgae and wastewater.     

Ammonium,nitrate, and urea play different roles for lipid accumulation in the nervonic acid—producing microalgae <Emphasis Type="Italic">Mychonastes afer</Emphasis> HSO-3-1

Producing valuable coproducts from oleaginous microalgae is an option to reduce the total cost of biofuel production. Here, the influence of nitrogen sources on biomass yield and lipid accumulation of a newly identified oleaginous green microalgal strain, Mychonastes afer HSO-3-1, was evaluated. Carbon assimilation and the following lipid biosynthesis of M. afer were inhibited to some extent under weak acidic conditions (6 < pH < 7) and any of the tested nitrogen source. The highest lipid productivity of 50.7 mg L^?1 day^?1 was achieved with a 17.6 mM nitrogen supplement in the form of urea. The cell polar lipid content was significantly higher than triacylglycerol (TAG), and saturated palmitic acid (C16:0) occupied a dominant position in the fatty acid profiles while culturing M. afer in acidic medium with NH₄ ⁺ as the nitrogen source. Under neutral conditions, the lipid productivities of M. afer cultivated in media containing 17.6 mM of NaNO₃, NH₄Cl, and NH₄NO₃ were 76.2, 77.5, and 79.0 mg L^?1 day^?1, respectively. The greatest TAG content (58.56%) of total lipids was obtained when NaNO₃ was used as the nitrogen source. There was no significant difference in the fatty acid composition of M. afer cells when they were cultivated in neutral media supplemented with NaNO₃, urea, NH₄Cl, and NH₄NO₃. Therefore, NH₄ ⁺ was not a suitable nitrogen source for M. afer cultivation due to the additional labor, working procedures, and alkali required to adjust the medium pH. Considering that using urea as nitrogen source could reduce the cost of nutrient salts substantially and urea can be taken up and utilized by most microalgae, it is a preferred nitrogen source. The major properties of biodiesel derived from M. afer HSO-3-1 met biodiesel quality, and nervonic acid concentrations remained at approximately 3.0% of total fatty acids.     

Enhancement of thiamine release during synthetic mutualism between <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> and <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> growing under stress conditions

Thiamine release during synthetic mutualism between Chlorella sorokiniana co-immobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense was measured under stress conditions of pH, light intensity, and nitrogen starvation in short-term experiments. Thiamine release in the co-immobilized treatment was significantly higher at acidic pH compared to thiamine released by either microorganism alone. Under slightly alkaline pH, C. sorokiniana released the highest amount of thiamine. At stressful pH 6, the co-immobilized treatment released a higher quantity of thiamine than the sum of thiamine released by either microorganisms when immobilized separately. Release of thiamine by C. sorokiniana alone or co-immobilized was light intensity dependent; with higher the light intensity, more thiamine was released. Extreme light intensity negatively affected growth of the microalgae and release of thiamine. Nitrogen starvation during the first 24 h of culturing negatively affected release of thiamine by both microorganisms, where C. sorokiniana was more severely affected. Partial or continuous nitrogen starvation had similar negative effects on C. sorokiniana, but co-immobilization improved thiamine release. These results indicate that thiamine is released during synthetic mutualism between C. sorokiniana and A. brasilense, and this happens specifically during the alleviation of pH stress in the microalgae.     

Assessment and comparison of the properties of biodiesel synthesized from three different types of wet microalgal biomass

In recent years, microalgae-based carbon-neutral biofuels (i.e., biodiesel) have gained considerable interest due to high growth rate and higher lipid productivity of microalgae during the whole year, delivering continuous biomass production as compared to vegetable-based feedstocks. Therefore, biodiesel was synthesized from three different microalgal species, namely Tetraselmis sp. (Chlorophyta) and Nannochloropsis oculata and Phaeodactylum tricornutum (Heterokontophyta), and the fuel properties of the biodiesel were analytically determined, unlike most studies which rely on estimates based on the lipid profile of the microalgae. These include density, kinematic viscosity, total and free glycerol, and high heating value (HHV), while cetane number (CN) and cold filter plugging point (CFPP) were estimated based on the fatty acid methyl ester profile of the biodiesel samples instead of the lipid profile of the microalgae. Most biodiesel properties abide by the ASTM D6751 and the EN 14214 specifications, although none of the biodiesel samples met the minimum CN or the maximum content of polyunsaturated fatty acids with ≥4 double bonds as required by the EN 14214 reference value. On the other hand, bomb calorimetric experiments revealed that the heat of combustion of all samples was on the upper limit expected for biodiesel fuels, actually being close to that of petrodiesel. Post-production processing may overcome the aforementioned limitations, enabling the production of biodiesel with high HHV obtained from lipids present in these microalgae.     

The combined effects of blue light and dilution rate on lipid class and fatty acid composition of <Emphasis Type="Italic">Tisochrysis lutea</Emphasis>

The lipid class and the fatty acid compositions of microalgae highly influence bivalve larval and post-larval development. Light is an essential environmental factor for microalgal culture, and quantity and quality of light may induce changes in the biochemical composition of the algae. The objective of this study was to investigate the effect of light spectrum (blue vs. white light) on lipid class and fatty acid compositions of Tisochrysis lutea cultured in a chemostat. Two different dilution rates (D) were assayed for each light spectrum: 0.2 and 0.7 day^?1. Triacylglycerol (TAG), sterol, and hydrocarbon (HC) content increased sharply at low D. The proportion of alkenones was significantly reduced under blue light. Polyunsaturated fatty acids (PUFA), and particularly n-3 PUFA, content in phospholipids (PL) increased under blue light compared to white light at low D. Thus, blue light raised 22:6(n-3) levels in total lipids of T. lutea at low D. The cultivation of T. lutea in a chemostat at low D under blue light may improve nutritional value as feed for bivalve larvae by modifying the PUFA profile, especially increasing 22:6(n-3).     

Different effects of the combined stress of nitrogen depletion and high temperature than an individual stress on the synthesis of biochemical compounds in <Emphasis Type="Italic">Arthrospira platensis</Emphasis> C1 (PCC 9438)

In nature, several abiotic stresses occur simultaneously, leading to retarded growth and biochemical changes in microalgae, including the commercial cyanobacterium, Arthrospira platensis. To gain more understanding of stress response, we investigated the integrative effects of nitrogen depletion and high temperature stress on physiological changes of A. platensis C1. The results revealed that photosynthetic activities of the stressed cells were markedly reduced by more than a half in comparison to the non-stressed cells. Moreover, a reduction of biomass was observed within 24 h after prolonged exposure to combined stress of nitrogen depletion and high temperature. The total protein contents, including phycocyanin (PC), in the stressed cells, decreased rapidly within 8 h of incubation. This finding was concomitant with the increase in carbohydrate content. However, the accumulation of carbohydrates in the nitrogen depletion-treated cells was greater than that in the cells under the combined stress. Furthermore, the levels of polysaccharides increased only under long-term incubation under nitrogen depletion but not under the combined stress. In addition, the combination of nitrogen depletion and high temperature stress resulted in an increase in the proportion of linoleic acid but a decrease in γ-linolenic acid within 24 h. These results suggest that the response of A. platensis to the combined stress was different from the responses of cells to individual stress. The PC degradation, the increased carbohydrates, and the alteration in fatty acids profiles were required for physiological response to combined nitrogen depletion and high temperature stress of A. platensis C1.     

Regulation of carbon metabolic fluxes in response to CO<Subscript>2</Subscript> supplementation in phototrophic <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>: a cytomic and biochemical study

As one of the promising species of microalgae for biofuel production, Chlorella vulgaris CS-42 was cultivated phototrophically in two cylindrical photobioreactors with aeration of 5 % (v/v) CO₂ or air for 13 days to evaluate the effects of CO₂ supplementation on biomass, CO₂ fixation performance, and biochemical content. Significant increases of specific growth rate and total carbon content in biomass resulting in a higher CO₂ fixation rate were found with 5 % CO₂. The maximum biomass concentration, carbohydrate and fatty acid contents with 5 % CO₂ were significantly higher than those with air, while carbohydrate biosynthesis was most affected as compared to other biochemical components. Cytomic analysis revealed a rapid accumulation of neutral lipid in the late growth phase with more lipid bodies visualized by confocal laser scanning microscopy (CLSM), when nitrate consumption was accelerated with CO₂ supplementation. Gas chromatography mass spectrometry (GC-MS) analysis indicated that 5 % CO₂ favored the formation of C18:2, which led to a decrease in the degree of lipid unsaturation (DLU). These results proved that CO₂ supplementation was one of the most efficient methods to significantly prompt the growth of microalgae and increase the C/N ratio in the medium, which in turn regulated the carbon metabolic flux to enhance neutral lipid and fatty acid production in C. vulgaris.     

Exploiting Phosphate-Starved cells of <Emphasis Type="Italic">Scenedesmus</Emphasis> sp. for the Treatment of Raw Sewage

Phosphate depletion is one of the favorable ways to enhance the sewage water treatment with the algae, however, detailed information is essential with respect to internal phosphate concentration and physiology of the algae. The growth rate of the phosphate-starved Scenedesmus cells was reduced drastically after 48 h. Indicating cells entered in the stationary phase of the growth cycle. Fourier Transform Infrared analysis of phosphate-starved Scenedesmus cells showed the reduction in internal phosphate concentration and an increase in carbohydrate/phosphate and carbohydrate/lipid ratio. The phosphate-starved Scenedesmus cells, with an initial cell density of, 1 × 10⁶ cells mL^?1 shows 87% phosphate and 100 % nitrogen removal in 24 h. The normal Scenedesmus cells need approximately 48 h to trim down the nutrients from wastewater up to this extent. Other microalgae, Ankistrodesmus, growth pattern was not affected due to phosphate starvation. The cells of Ankistrodesmus was able to reduce 71% phosphate and 73% nitrogen within 24 h, with an initial cell density of, 1 × 10⁶ cells mL^?1.     

Characterization of cell structural change,growth, lipid accumulation,and pigment profile of a novel oleaginous microalga, <Emphasis Type="Italic">Vischeria stellata</Emphasis> (Eustigmatophyceae), cultured with different initial nitrate supplies

The appropriate microalgal species and the optimal nitrogen supply in culture medium are vital factors in maximizing biomass and metabolite productivities in microalgae. Vischeria stellata is an edaphic unicellular eustigmatophycean microalga. Cytological and ultrastructural characteristics and the effects of different initial nitrate-nitrogen concentrations on growth, lipid accumulation, fatty acid profile, and pigment composition were investigated in the present study. The cell structures of V. stellata changed with the degree of nutrient utilization and growth phase. The initial nitrate concentration for the optimal growth of V. stellata ranged from 6.0 to 9.0 mM. The maximum total lipid (TLs), neutral lipid (NLs), and total fatty acid (TFAs) contents were 55.9, 51.9, and 44.7 % of dry biomass, respectively. The highest volumetric productivity of TLs, NLs, and TFAs reached 0.28, 0.25, and 0.21 g L^?1 day^?1, respectively. V. stellata had a suitable fatty acid profile for biodiesel production, as well as containing eicosapentaenoic acid (EPA) for nutraceutical applications. In addition, the content β-carotene, increased gradually as culture time was prolonged, resulting in its exclusive production at the end of cultivation. V. stellata is a promising microalgal strain for the production of biofuels and bioproducts.     

Requirement of the isocitrate lyase gene <Emphasis Type="Italic">ICL1</Emphasis> for <Emphasis Type="Italic">VPS41</Emphasis>-mediated starvation response in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans is a major cause of fungal meningitis in individuals with impaired immunity. Our previous studies have shown that the VPS41 gene plays a critical role in the survival of Cryptococcus neoformans under nitrogen starvation; however, the molecular mechanisms underlying VPS41-mediated starvation response remain to be elucidated. In the present study, we show that, under nitrogen starvation, VPS41 strongly enhanced ICL1 expression in C. neoformans and that overexpression of ICL1 in the vps41 mutant dramatically suppressed its defects in starvation response due to the loss of VPS41 function. Moreover, targeted deletion of ICL1 resulted in a dramatic decline in viability of C. neoformans cells under nitrogen deprivation. Taken together, our data suggest a model in which VPS41 up-regulates ICL1 expression, directly or indirectly, to promote survival of C. neoformans under nitrogen starvation.     

Effects of iron sources on the growth and lipid/carbohydrate production of marine microalga <Emphasis Type="Italic">Dunaliella tertiolecta</Emphasis>

The effects of iron sources with different speciation and anionic moieties (ferric chloride, ferrous chloride, ferric EDTA, ferrous EDTA, ferric ammonium sulfate, and ferrous ammonium sulfate) on the cell growth and the production of energy storage (lipid and carbohydrate) from Dunaliella tertiolecta were investigated. The influence of iron dosage was also compared in the range from 0.65 mg/L (1X) to 6.5 mg/L (10X) as Fe concentration. Best cell growth rate was achieved when ferrous ammonium sulfate was used. Ferric EDTA resulted in higher lipid content than other iron sources, while ferrous ammonium sulfate favored the accumulation of carbohydrate among six iron sources. The accumulations of lipid and carbohydrate as energy storage competed each other and thus both contents did not increase together. In the presence of ferric EDTA, lipid content is increasing, while carbohydrate content is decreasing. On the contrary, lipid content is decreasing while carbohydrate is increasing in the presence of ferric ammonium sulfate. Because the overall carbohydrate content was larger than that of lipid, bioethanol production would be more advantageous than biodiesel production with the present D. tertiolecta strain if the carbohydrate in D. tertiolecta contains a high fraction of glucose with a good saccharification yield.     

Microalgae from the Selenastraceae as emerging candidates for biodiesel production: a mini review

Over the years, microalgae have been identified to be a potential source of commercially important products such as pigments, polysaccharides, polyunsaturated fatty acids and in particular, biofuels. Current demands for sustainable fuel sources and bioproducts has led to an extensive search for promising strains of microalgae for large scale cultivation. Prospective strains identified for these purposes were among others, mainly from the genera Hematococcus, Dunaliella, Botryococcus, Chlorella, Scenedesmus and Nannochloropsis. Recently, microalgae from the Selenastraceae emerged as potential candidates for biodiesel production. Strains from the Selenastraceae such as Monoraphidium sp. FXY-10, M. contortum SAG 47.80, Ankistrodesmus sp. SP2-15 and M. minutum were high biomass and lipid producers when cultivated under optimal conditions. A number of Selenastraceae strains were also reported to be suitable for cultivation in wastewater. This review highlights recent reports on potential strains from the Selenastraceae for biodiesel production and contrasts their biomass productivity, lipid productivity as well as fatty acid profile. Cultivation strategies employed to enhance their biomass and lipid productivity as well as to reduce feedstock cost are also discussed in this paper.     

Metabolic engineering of lipid pathways in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and staged bioprocess for enhanced lipid production and cellular physiology

Microbially produced lipids have attracted attention for their environmental benefits and commercial value. We have combined lipid pathway engineering in Saccharomyces cerevisiae yeast with bioprocess design to improve productivity and explore barriers to enhanced lipid production. Initially, individual gene expression was tested for impact on yeast growth and lipid production. Then, two base strains were prepared for enhanced lipid accumulation and stabilization steps by combining DGAT1, ΔTgl3 with or without Atclo1, which increased lipid content ~?1.8-fold but reduced cell viability. Next, fatty acid (FA) biosynthesis genes Ald6-SEACS^L641P alone or with ACC1** were co-expressed in base strains, which significantly improved lipid content (8.0% DCW, 2.6-fold than control), but severely reduced yeast growth and cell viability. Finally, a designed two-stage process convincingly ameliorated the negative effects, resulting in normal cell growth, very high lipid productivity (307 mg/L, 4.6-fold above control) and improved cell viability.     

The effect of temperature on growth and lipid and fatty acid composition on marine microalgae used for biodiesel production

Cultivation temperature is one of the major factors affecting the growth and lipid accumulation of microalgae. In this study, the effects of temperature on the growth, lipid content, fatty acid composition and biodiesel properties of the marine microalgae Chaetoceros sp. FIKU035, Tetraselmis suecica FIKU032 and Nannochloropsis sp. FIKU036 were investigated. These species were cultured at different temperatures (25, 30, 35 and 40 °C). The results showed that the specific growth rate, biomass and lipid content of all microalgae decreased with increasing temperature. With regards to fatty acids, the presence of saturated fatty acids (SFAs) in T. suecica FIKU032 and Nannochloropsis sp. FIKU036 decreased with increasing temperature, in contrast with polyunsaturated fatty acids (PUFAs). Moreover, Chaetoceros sp. FIKU035 was the only species that could grow at 40 °C. The highest lipid productivity was observed in Chaetoceros sp. FIKU035 when cultivated at 25 °C (66.73 ± 1.34 mg L^?1 day^?1) and 30 °C (61.35 ± 2.89 mg L^?1 day^?1). Moreover, the biodiesel properties (cetane number, cold filter plugging point, kinematic viscosity and density) of the lipids obtained from this species were in accordance with biodiesel standards. This study indicated that Chaetoceros sp. FIKU035 can be considered as a suitable species for biodiesel production in outdoor cultivation.     

Investigation of lipid production by nitrogen-starved <Emphasis Type="Italic">Parachlorella kessleri</Emphasis> under continuous illumination and day/night cycles for biodiesel application

This study aims to investigate the triacylglycerol (TAG) productivity of Parachlorella kessleri grown under continuous illumination and to investigate its metabolism in simulated day/night cycles in order to estimate the feasibility of a large-scale production in outdoor solar photobioreactors. The strain was chosen for its ability to accumulate large amounts of triacylglycerol during nitrogen starvation. Several protocols of nitrogen starvation were tested in continuous illumination as well as in simulated day/night cycles. Sudden and progressive nitrogen starvation conditions have enhanced the TAG concentration and productivity of P. kessleri reaching up to 48 dry wt% and 4.4 × 10^?3 kg m^?2 day^?1, respectively. Microalgal cell metabolism was significantly affected by the day/night illumination cycles. The energy-rich compounds (TAGs and carbohydrates) were accumulated by P. kessleri during the photoperiods and partly consumed during the dark to sustain the microalgae vitality. This TAG oxidation ultimately led to a 26% decrease in TAG productivity in cultures exposed to day/night cycles compared to ones exposed to continuous illumination of equal 24-h average photon flux density. The results can dictate the optimal time for harvesting cells for recovering the largest amount of TAGs.