Far‐red dependent changes in the chemical composition of Spirulina platensis

The influence of far‐red light (FRL) was studied on the chemical composition of Spirulina platensis biomass. The following light compositions were used during the culture white light, blue‐red LED light (BRL) and BRL supplemented with FRL (BRFRL). Chlorophyll and phenol contents were measured by spectrophotometric methods, whereas presence of carotenoids, lipids, and phycobiliproteins were estimated by Fourier‐transform Raman spectrometry. Additionally, phenol content was investigated by fluorescence intensity of algae culture in the range of 430–650 nm. The content of chlorophyll and phenols in algae cells depended on the spectral composition of light and was the highest under BRL (16.7 ± 0.5 and 9.1 ± 0.6, respectively). It was shown that there is a positive linear correlation (R = 0.902 at p < 0.0000001) between the ratio of relative fluorescence intensity of S. platensis suspensions at 450 nm to the suspensions at 540 nm (F450/F540) and the content of phenolic compounds in the biomass. Changes in the F450/F540 ratio can explain approximately 80% changes of phenol contents in algae cells. Spirulina platensis Raman spectra demonstrated that the biomass of algae growing under white light and BRL had a significantly higher intensity of phycobiliprotein bands than the algae growing under BRFRL.     

Lipid analysis of Neochloris oleoabundans by liquid state NMR

This study is an evaluation of liquid state NMR as a tool for analyzing the lipid composition of algal cultures used for biodiesel production. To demonstrate the viability of this approach, ¹³C NMR was used to analyze the lipid composition of intact cells of the algal species, Neochloris oleoabundans (UTEX #1185). Two cultures were used in this study. One culture was “healthy” and grown in conventional media, whereas the other culture was “nitrogen‐starved” and grown in media that lacked nitrate. Triglyceride was determined to be present in both cultures by comparing the algal NMR spectra with published chemical shifts for a wide range of lipids and with a spectrum obtained from a triglyceride standard (glyceryl trioleate). In addition, it is shown that (1) the signal‐to‐noise ratio of the ～29.5 ppm methylene peak is indicative of the lipid content and (2) the nitrogen‐starved culture contained a greater lipid content than the healthy culture, as expected. Furthermore, the nitrogen‐starved culture produced spectra that primarily contained the characteristic peaks of triglyceride (at ～61.8 and ～68.9 ppm), whereas the healthy culture produced spectra that contained several additional peaks in the glycerol region, likely resulting from the presence of monoglyceride and diglyceride. Finally, potential interferences are evaluated (including the analysis of phospholipids via ³¹P NMR) to assess the specificity of the acquired spectra to triglyceride. These results indicate that NMR is a useful diagnostic tool for selectively identifying lipids in algae, with particular relevance to biodiesel production. Biotechnol. Bioeng. 2010;106: 573–583. © 2010 Wiley Periodicals, Inc.     

Comparative Analyses of Three Chlorella Species in Response to Light and Sugar Reveal Distinctive Lipid Accumulation Patterns in the Microalga C. sorokiniana

While photosynthetic microalgae, such as Chlorella, serve as feedstocks for nutritional oils and biofuels, heterotrophic cultivation can augment growth rates, support high cell densities, and increase triacylglycerol (TAG) lipid content. However, these species differ significantly in their photoautotrophic and heterotrophic characteristics. In this study, the phylogeny of thirty Chlorella strains was determined in order to inform bioprospecting efforts and detailed physiological assessment of three species. The growth kinetics and lipid biochemistry of C. protothecoides UTEX 411, C. vulgaris UTEX 265, and C. sorokiniana UTEX 1230 were quantified during photoautotrophy in Bold''s basal medium (BBM) and heterotrophy in BBM supplemented with glucose (10 g L⁻¹). Heterotrophic growth rates of UTEX 411, 265, and 1230 were found to be 1.5-, 3.7-, and 5-fold higher than their respective autotrophic rates. With a rapid nine-hour heterotrophic doubling time, Chlorella sorokiniana UTEX 1230 maximally accumulated 39% total lipids by dry weight during heterotrophy compared to 18% autotrophically. Furthermore, the discrete fatty acid composition of each strain was examined in order to elucidate lipid accumulation patterns under the two trophic conditions. In both modes of growth, UTEX 411 and 265 produced 18∶1 as the principal fatty acid while UTEX 1230 exhibited a 2.5-fold enrichment in 18∶2 relative to 18∶1. Although the total lipid content was highest in UTEX 411 during heterotrophy, UTEX 1230 demonstrated a two-fold increase in its heterotrophic TAG fraction at a rate of 28.9 mg L⁻¹ d⁻¹ to reach 22% of the biomass, corresponding to as much as 90% of its total lipids. Interestingly, UTEX 1230 growth was restricted during mixotrophy and its TAG production rate was suppressed to 18.2 mg L⁻¹ d⁻¹. This constraint on carbon flow raises intriguing questions about the impact of sugar and light on the metabolic regulation of microalgal lipid biosynthesis.     

New lipid‐producing,cold‐tolerant yellow‐green alga isolated from the rocky mountains of colorado

A new strain of yellow‐green algae (Xanthophyceae, Heterokonta), tentatively named Heterococcus sp. DN1 (UTEX accession number UTEX ZZ885), was discovered among snow fields in the Rocky Mountains. Axenic cultures of H. sp. DN1 were isolated and their cellular morphology, growth, and composition of lipids were characterized. H. sp. DN1 was found to grow at temperatures approaching freezing to accumulate large intracellular stores of lipids. H. sp. DN1 produces the highest quantity of lipids when grown undisturbed with high light in low temperatures_. Of particular interest was the accumulation of eicosapentaenoic acid, known to be important for human nutrition, and palmitoleic acid, known to improve biodiesel feedstock properties. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:853–861, 2013     

Biochemical composition of three algal species proposed as food for captive freshwater mussels

To identify potential diets for rearing captive freshwater mussels, the protein, carbohydrate (CHO), and lipid contents of two green algae, Neochloris oleoabundans, Bracteacoccus grandis, and one diatom, Phaeodactylum tricornutum, were compared at different growth stages. The fatty acid and sterol composition were also identified. Protein was greatest (55–70%) for all species at late log growth stage (LL), and declined in late stationary (LS) growth. CHO was greatest at LS stage for all species (33.9–56.4% dry wt). No significant change in lipid levels occurred with growth stage, but tended to increase in N. oleoabundans. Mean lipid content differed significantly in the order: N. oleoabundans > P. tricornutum > B. grandis. Total fatty acids (TFA) were higher at LS stage compared to other stages in the two green algae, and stationary stage in the diatom. Mean unsaturated fatty acids (UFA) as %TFA was significantly higher in N. oleoabundans than the other species. The green algae contained high percentages of C-18 polyunsaturated fatty acids (PUFAs), while the diatom was abundant in C-16 saturated and mono-unsaturated fatty acids and C-20 PUFA fatty acids. Growth stage had no effect on sterol concentration of any species. B. grandis showed significantly higher sterol levels than the other species except P. tricornutum at S stage. B. grandis was characterized by predominantly ⁵, C-29 sterols, while N. oleoabundans synthesized ^5,7, ^5,7,22 , and ⁷, C-28 sterols. P. tricornutum produced primarily a ^5,22, C-28 sterol, and a small amount of a ^7,22, C-28 sterol.     

Presence of the CO2-concentrating mechanism in some species of the pyrenoid-less free-living algal genus Chloromonas (Volvocales, Chlorophyta)

Physiological and morphological characteristics related to the CO₂-concentrating mechanism (CCM) were examined in several species of the free-living, unicellular volvocalean genus Chloromonas (Chlorophyta), which differs morphologically from the genus Chlamydomonas only by lacking pyrenoids. The absence of pyrenoids in the chloroplasts of Chloromonas (Cr.) rosae UTEX 1337, Cr. serbinowii UTEX 492, Cr.␣clatharata UTEX 1970, Cr. rosae SAG 26.90, and Cr. palmelloides SAG 32.86 was confirmed by light and electron microscopy. In addition, immunogold electron microscopy demonstrated that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were distributed almost evenly throughout the chloroplasts in all five Chloromonas strains. However, Chloromonas exhibited two types of physiological characteristics related to the CCM depending on the species or strains examined. Chloromonas rosae UTEX 1337 and Cr. serbinowii had high photosynthetic affinities for CO₂ in cells grown in culture medium bubbled with air (low-CO₂ cells), compared with those grown in medium bubbled with 5% CO₂ (high-CO₂ cells), indicating the presence of the low-CO₂-inducible CCM. In addition, these two Chloromonas strains exhibited low-CO₂-inducible carbonic anhydrase (CA; EC 4.2.1.1) activity and seemed to have small intracellular inorganic carbon pools. Therefore, it appears that Cr. rosae UTEX 1337 and Cr. serbinowii possess the CCM as in pyrenoid-containing microalgae such as Chlamydomonas reinhardtii. By contrast, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides showed low photosynthetic affinities for CO₂ when grown under both CO₂ conditions. Moreover, these three strains exhibited an apparent absence of intracellular inorganic carbon pools and lacked low-CO₂-inducible CA activity. Thus, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides, like other pyrenoid-less algae (lichen photobionts) reported previously, seem to lack the CCM. The present study is the first demonstration of the CCM in pyrenoid-less algae, indicating that pyrenoids or accumulation of Rubisco in the chloroplasts are not always essential for the CCM in algae. Focusing on this type of CCM in pyrenoid-less algae, the physiological and evolutionary significance of pyrenoid absence is discussed. Received: 1 May 1997 / Accepted: 11 September 1997     

乙酸钠调控小球藻生长及代谢产物

【背景】小球藻是一种单细胞绿藻,在不同培养条件下可积累高附加值的代谢产物,这些产物可用于生产生物燃料、食品、保健品、药品等。然而这些代谢产物在藻细胞中的生产率较低且很难通过经济可行的方法将其分离,这使其工业化规模生产受到限制。【目的】研究乙酸钠对小球藻生物量的影响,并分析其对小球藻代谢产物的调控作用。【方法】通过在小球藻培养液中添加不同浓度的乙酸钠(1.0、2.0、3.0、4.0、5.0 g/L),研究其调控小球藻生长和代谢的作用机理。【结果】在添加3.0 g/L乙酸钠的培养液中,小球藻的生物量是对照组的5.2倍,尽管藻细胞中蛋白质含量无明显变化,但油脂和类胡萝卜素含量是对照组的2.4倍和1.2倍,多糖和叶绿素a含量却仅为对照组的54.6%和54.4%。【结论】乙酸钠不仅会影响藻细胞的生长,还会调控其代谢过程,这为深入探索乙酸钠在调控小球藻生长及代谢过程的作用机制提供了理论基础和技术资料。     

The costs and benefits of multicellular group formation in algae*

The first step in the evolution of complex multicellular organisms involves single cells forming a cooperative group. Consequently, to understand multicellularity, we need to understand the costs and benefits associated with multicellular group formation. We found that in the facultatively multicellular algae Chlorella sorokiniana: (1) the presence of the flagellate Ochromonas danica or the crustacean Daphnia magna leads to the formation of multicellular groups; (2) the formation of multicellular groups reduces predation by O. danica, but not by the larger predator D. magna; (3) under conditions of relatively low light intensity, where competition for light is greater, multicellular groups grow slower than single cells; (4) in the absence of live predators, the proportion of cells in multicellular groups decreases at a rate that does not vary with light intensity. These results can explain why, in cases such as this algae species, multicellular group formation is facultative, in response to the presence of predators.     

Single nucleotide polymorphism-mismatch primer development for rapid molecular identification of selected microalgal species

Microalgae have been a great source for food, cosmetic, pharmacological, and biofuel production. The adoption of effective diagnostic assays for monitoring all stages of algal cultivation has become essential. In addition to microscopy identification, molecular assays can aid greatly in the identification and monitoring of algal species of interest. In this study the 18S ribosomal RNA (rRNA) sequences of 12 microalgal species and/or strains were used to design algal identification primers. Sequence alignment revealed five highly variable regions and multiple unique single nucleotide polymorphisms (SNPs). To design target algae specific primers, a SNP identified as unique to each microalgal species was incorporated into the 3’-terminus of forward and reverse primer pairs, respectively. To further enhance primer specificity, transverse mutation was introduced into each primer at the third base upstream of the respective SNP. The SNP-mismatch primer pairs yield size-specific amplicons, enabling the rapid molecular detection of 12 microalgae by circumventing cloning and sequencing. To verify the primer specificity, two SNP-mismatch primer pairs designed for Chlorella sorokiniana DOE1412 and wildtype species of Scenedesmus were tested in the outdoor reactor run inoculated with C. sorokiniana DOE1412. The primer pairs were able to identify C. sorokiniana DOE1412 as well as the environmental invader Scenedesmus sp. Furthermore, the “relative concentration” of two microalgae was accessed throughout the entire cultivation run. The use of SNPs primers designed in this study offers a cost-effective, easy to use alternative for routine monitoring of microalgal cultures in laboratories, in scale-ups, and in cultivation reactors, independent of the production platform.

     

Two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production

A two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production was studied, wherein high density heterotrophic cultures of Chlorellasorokiniana serve as seed for subsequent phototrophic growth. The data showed growth rate, cell density and productivity of heterotrophic C.sorokiniana were 3.0, 3.3 and 7.4 times higher than phototrophic counterpart, respectively. Hetero- and phototrophic algal seeds had similar biomass/lipid production and fatty acid profile when inoculated into phototrophic culture system. To expand the application, food waste and wastewater were tested as feedstock for heterotrophic growth, and supported cell growth successfully. These results demonstrated the advantages of using heterotrophic algae cells as seeds for open algae culture system. Additionally, high inoculation rate of heterotrophic algal seed can be utilized as an effective method for contamination control. This two-stage heterotrophic phototrophic process is promising to provide a more efficient way for large scale production of algal biomass and biofuels.     

In Vivo Blood Glucose Quantification Using Raman Spectroscopy

We here propose a novel Raman spectroscopy method that permits the noninvasive measurement of blood glucose concentration. To reduce the effects of the strong background signals produced by surrounding tissue and to obtain the fingerprint Raman lines formed by blood analytes, a laser was focused on the blood in vessels in the skin. The Raman spectra were collected transcutaneously. Characteristic peaks of glucose (1125 cm^-1) and hemoglobin (1549 cm^-1) were observed. Hemoglobin concentration served as an internal standard, and the ratio of the peaks that appeared at 1125 cm^-1 and 1549 cm^-1 peaks was used to calculate the concentration of blood glucose. We studied three mouse subjects whose blood glucose levels became elevated over a period of 2 hours using a glucose test assay. During the test, 25 Raman spectra were collected transcutaneously and glucose reference values were provided by a blood glucose meter. Results clearly showed the relationship between Raman intensity and concentration. The release curves were approximately linear with a correlation coefficient of 0.91. This noninvasive methodology may be useful for the study of blood glucose in vivo.     

At high temperature lipid production in Ettlia oleoabundans occurs before nitrate depletion

Temperature and light intensity effects on biomass and lipid production were investigated in Ettlia oleoabundans to better understand some fundamental properties of this potentially useful but poorly studied microalgal species. E. oleoabundans entered dormant state at 5 °C, showed growth at 10 °C, and when exposed to light at 70 μmol photons per square meter per second at 10 °C, cells reached a biomass concentration of >2.0 g?L^?1 with fatty acid methyl esters of 11.5 mg?L^?1. Highest biomass productivity was at 15 °C and 25 °C regardless of light intensity, and accumulation of intracellular lipids was stimulated by nitrate depletion under these conditions. Although growth was inhibited at 35 °C, at 130 μmol photons per square meter per second lipid content reached 10.37 mg?L^?1 with fatty acid content more favorable to biodiesel dominating; this occurred without nitrate depletion. In a two-phase temperature shift experiment at two nitrate levels, cells were shifted after 21 days at 15 °C to 35 °C for 8 days. Although after the shift growth continued, lipid productivity per cell was less than that in the 35 °C cultures, again without nitrate depletion. This study showed that E. oleoabundans grows well at low temperature and light intensity, and high temperature can be a useful trigger for lipid accumulation independent of nitrate depletion. This will prove useful for improving our knowledge about lipid production in this and other oleaginous algae for modifying yield and quality of algal lipids being considered for biodiesel production.     

Cultivation of the microalga <Emphasis Type="Italic">Neochloris oleoabundans</Emphasis> for biofuels production and other industrial applications (a review)

Microalgae cultivation for biofuels production and other applications has gained considerable interest recently. Despite their simple structures, microalgae can accumulate significant amounts of neutral lipids per dry cell weight compared to other energy crops. Neochloris oleoabundans is a promising microalga known for its high lipid content and biomass growth rate compared to other species cultivated for biofuels synthesis; therefore, it is considered as a suitable candidate for biodiesel synthesis. This review paper covers several key aspects associated with the cultivation and applications of the microalga N. oleoabundans. Biomass composition, factors affecting the growth, and biomass and lipid productivities of this species were addressed. In addition, different growth conditions as well as alternative readily available nutrient media to support the growth of N. oleoabundans were presented in this review.     

ISOLATION AND CHARACTERIZATION OF NATIVE PIGMENT-PROTEIN COMPLEXES FROM TWO EUSTIGMATOPHYCEAE1

Pigment-protein complexes were isolated from two species of Eustigmatophyceae, Monodus subterraneus Peterson and Vischeria punctata Vischer, by digitonin treatment followed by density gradient centrifugation. Absorption and fluorescence spectra of the samples were monitored at various steps of preparation, and pigment composition was analyzed by reverse phase HPLC. Although the fluorescence emission spectra were very different in the two species, the absorption spectra were similar, and each exhibited an absorption band with a maximum at 487 nm attributable to violaxanthin and vaucheriaxanthin ester (the molar concentration of these pigments in Monodus was, respectively, 28 and 10 per 100 Chl a). The light-harvesting role of these xanthophylls was ascertained by fluorescence excitation spectra. The light-harvesting fractions (LH) collected in the upper part of the gradient were depleted in β-carotene, whereas their xanthophyll/chlorophyll ratio was almost the same as in whole cells. This is consistent with the presence in these algae of large LH antennae and relatively small core antennae in the photosystems. In Monodus, a polypeptide of 23 kDa, immunologically related to the major LH polypeptide of brown algae, constituted the majority of the LH protein moiety.     

Morphology,Physiology, and Virulence of <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> Isolates

Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases in cereal crops. The high morphological, physiological, and genetic variability makes the control of this fungus a difficult task. The aim of this work was to study the virulence, morphological, and physiological variability of B. sorokiniana isolates. For this, 35 B. sorokiniana isolates from different geographic regions in Brazil and other countries were used. The isolates were evaluated for their morphological variability, considering mycelium color, sector formation, and growth rate. Based on these morphological characteristics, the isolates were grouped in five different morphological groups. Extracellular enzymes activity in solid medium, virulence in wheat seeds and seedlings, and analysis of total proteins by SDS-PAGE were evaluated for all isolates. Variations among the isolates were found for enzymatic activity, and esterase was the enzyme that showed the highest activity indices. The results obtained from infection of seeds and seedlings showed that isolates from the same geographical region and morphological group had different degrees of virulence. The total protein profile shown by the isolates varied in the number of bands and intensity, where some of them may be used to characterize the specie.     

Toxic and non-toxic Nodularia strains can be distinguished from each other and from eukaryotic algae with chlorophyll fluorescence fingerprinting

Chlorophyll fluorescence induction curves of toxic and non-toxic strains of the cyanobacterium Nodularia were measured and compared with fluorescence curves measured from four species of eukaryotic algae. Both cyanobacteria and algae were isolated from the Baltic Sea. The results show that Nodularia strains can be distinguished from the eukaryotes by applying a pattern recognition procedure to the fluorescence induction curves, suggesting that the fluorescence fingerprinting technique might be useful in environmental monitoring of marine algae. The six studied Nodularia strains could not be distinguished from each other from their fluorescence induction kinetics. However, their fluorescence curves fell into two clear categories, the toxic and the non-toxic Nodularia. Emission spectroscopy and differences in the fluorescence induction curves showed that the ratio of the intensity of the Photosystem I emission peak to the Photosystem II peak is higher in non-toxic Nodularia than in the toxic strains, suggesting that the toxicity affects the structure of the photosynthesis machinery. The effect on photosynthesis may be related to the ability of the microcystins to chelate iron.     

STEROL,FATTY ACID,AND PIGMENT CHARACTERISTICS OF UTEX 2341, A MARINE EUSTIGMATOPHYTE IDENTIFIED PREVIOUSLY AS CHLORELLA MINUTISSIMA (CHLOROPHYCEAE)1

An examination of the sterols of UTEX 2341, a small (ca. 2μm), nonmotile unicellular marine alga identified as Chlorella minutissima Fott et Novakova, yielded results inconsistent with any of 35 Chlorella strains analyzed previously. UTEX 2341 contained cholesterol as the principal sterol, with 24-methylenecholesterol, fucosterol, and isofucosterol also present; these are not dominant sterols in any other Chlorella species. Presence of eicosapentaenoic acid in UTEX 2341 also contrasted with fatty acids of Chlorella strains analyzed previously. Pigment analysis of UTEX 2341 revealed that it contained chlorophyll a, but not chlorophylls b or c; violaxanthin was the only major xanthophyll pigment. Both lipid and pigment compositions suggest that UTEX 2341 is not a member of the genus Chlorella but, rather, belongs in the Eustigmatophyceae; it may be Nannochloropsis sp. Cells with possible extracellular structures were present at an appreciable percentage of the stationary-phase population studied; centrifuging removed or collapsed these structures. The high cholesterol and polyunsaturated fatty acid contents of UTEX 2341 make it attractive as a potential aquaculturefeed, provided it is, or can be made, digestible.     

AN UNUSUAL PHYCOCYANOBILIN-CONTAINING PHYCOERYTHRIN OF SEVERAL BLUISH-COLORED,ACROCHAETIOID, FRESHWATER RED ALGAL SPECIES1

The reproductive biology and phycobiliproteins of four different culture isolates of the freshwater algae Audouinella and‘Chantransia’were investigated.‘Chantransia’sp. (3585/UTEX 2623) and Audouinella macrospora (Wood) Sheath et Burkholder (3394,3395) from California and Minnesota reproduced only by monospores. However, A. macrospora (3603/Necchi 1) reproduced by monosporangia that formed successive generations of the Audouinella phase, and Batrachospermum shoots developed from the basal and erect systems. The major light-harvesting phycobiliprotein in all of these isolates was a phycocyanobilin-containing phycoerythrin not previously detected in red algae or cyanobacteria. As in the commonly found R- and B-phycoerythrins, Audouinella phycoerythrin had a native molecular mass of ～ 240,000 and was made up of α, β, and γ subunits. Audouinella phycoerythrin carried two phycoerythrobilins on the α subunit; one phycourobilin, one phycoerythrobilin, and one phycocyanobilin on the β subunit; and one phycourobilin and two phycoerythrobilins on the γ subunit. With excitation at 495, 563, or 603 nm, the fluorescence emission peak of Audouinella phycoerythrin was at 626 nm, showing that phycocyanobilin was the terminal energy acceptor.