Distribution of the Water-Soluble Astaxanthin Binding Carotenoprotein (AstaP) in Scenedesmaceae.

Photooxidative stress-inducible water-soluble astaxanthin-binding proteins, designated as AstaP, were identified in two Scenedesmaceae strains, Coelastrella astaxanthina Ki-4 and Scenedesmus obtusus Oki-4N; both strains were isolated under high light conditions. These AstaPs are classified as a novel family of carotenoprotein and are useful for providing valuable astaxanthin in water-soluble form; however, the distribution of AstaP orthologs in other microalgae remains unknown. Here, we examined the distribution of AstaP orthologs in the family Scenedesmaceae with two model microalgae, Chlamydomonas reinhardtii and Chlorella variabilis. The expression of AstaP orthologs under photooxidative stress conditions was detected in cell extracts of Scenedesmaceae strains, but not in model algal strains. Aqueous orange proteins produced by Scenedesmaceae strains were shown to bind astaxanthin. The protein from Scenedesmus costatus SAG 46.88 was purified. It was named ScosAstaP and found to bind astaxanthin. The deduced amino acid sequence from a gene encoding ScosAstaP showed 62% identity to Ki-4 AstaP. The expression of the genes encoding AstaP orthologs was shown to be inducible under photooxidative stress conditions; however, the production amounts of AstaP orthologs were estimated to be approximately 5 to 10 times lower than that of Ki-4 and Oki-4N.

Morphological characterization, growth appraisal, and probing biofuels potential of newly isolated Scenedesmus sp. from desert Cholistan.

Microalgae have an excellent potential for producing valuable natural products, including biofuels. Therefore, it is imperative to explore and document the existing microalgal flora and utilize their potentials to cope the increasing human needs. The present work aims at exploring and characterizing newly isolated microalgae from desert Cholistan, a habitat with myriad algal diversity. Light microscopy, scanning electron microscopy, and molecular phylogenetic approaches were used for species-level identification. Characterization and growth optimization of Scendesmus sp. were analyzed under three different growth modes to determine the most favorable conditions for increasing biomass, growth rate, and lipid content. The results revealed that mixotrophic (MT) mode significantly increases photosynthetic activity, growth rate, and lipid content with glycerol as supplement carbon source. The investigated Scenedesmus dimorphous produced a maximum dry weight of 1.73 g L-1 , improved fatty acid methyl esters profile and yield lipid up to 40% of DCW (68 g L-1 ) under MT mode, which is almost double to that of photoautotrophic cultivation. The glycerol availability in medium has been identified as the critical element for boosting growth and lipid content. Thus, it can reduce the cost of biofuel production.

How DNA barcoding can be more effective in microalgae identification: a case of cryptic diversity revelation in Scenedesmus (Chlorophyceae).

Microalgae identification is extremely difficult. The efficiency of DNA barcoding in microalgae identification involves ideal gene markers and approaches employed, which however, is still under the way. Although Scenedesmus has obtained much research in producing lipids its identification is difficult. Here we present a comprehensive coalescent, distance and character-based DNA barcoding for 118 Scenedesmus strains based on rbcL, tufA, ITS and 16S. The four genes, and their combined data rbcL + tufA + ITS + 16S, rbcL + tufA and ITS + 16S were analyzed by all of GMYC, P ID, PTP, ABGD, and character-based barcoding respectively. It was apparent that the three combined gene data showed a higher proportion of resolution success than the single gene. In comparison, the GMYC and PTP analysis produced more taxonomic lineages. The ABGD generated various resolution in discrimination among the single and combined data. The character-based barcoding was proved to be the most effective approach for species discrimination in both single and combined data which produced consistent species identification. All the integrated results recovered 11 species, five out of which were revealed as potential cryptic species. We suggest that the character-based DNA barcoding together with other approaches based on multiple genes and their combined data could be more effective in microalgae diversity revelation.

Effects of simulated flue gas on components of Scenedesmus raciborskii WZKMT.

Scenedesmus raciborskii WZKMT cultured with simulated flue gas was investigated. Cellular components, including total sugar, starch, chlorophyll, protein and lipid, were compared between simulated flue gas and 7% (v/v) CO2. Dissolution of SO2 and NO in simulated flue gas led to pH decrease and toxicity to microalgae cells. Furthermore, the death or aging of microalgae cells reduced the buffer capacity and caused decrease of simulated flue gas absorption. With 7% CO2, the highest total sugar and starch content could attain to 66.76% and 53.16%, respectively, which indicated S. raciborskii WZKMT is a desired feedstock candidate for bioethanol production. Microalgae growth and starch accumulation was inhibited, while cells produced more chlorophyll, protein and lipid when simulated flue gas was the carbon source. Fatty acids composition analysis indicated that there was no significant distinction on fatty acids relative content (fatty acid/TFA) between cells aerated using simulated flue gas and 7% CO2.

Using carbon dioxide to maintain an elevated oleaginous microalga concentration in mixed-culture photo-bioreactors.

Microbial contamination of growth reactors is a major concern for microalgal biofuel production. In this study, the oleaginous, CO2-tolerant microalga Scenedesmus dimorphus was combined with a wastewater-derived microbial community and grown in replicated sequencing batch photobioreactors. The reactors were sparged with either ambient air or 20% v/v CO2. In the initial growth cycles, air and the 20% CO2 reactors were similar in terms of growth and microbial community structure. Beyond the fourth growth cycle, however, the ambient air reactors had larger decreases in cell density and growth rate, and increases in species richness and non-algal microorganisms compared to the 20% CO2 reactors. Both qPCR and rDNA sequence analyses demonstrated a greater loss in S. dimorphus enrichment in the ambient-air reactors compared to the 20% CO2 reactors. These results demonstrate that environmental parameters can be used to delay the adverse impacts of microbial contamination in open, mixed-culture microalgae bioreactors.

Agrobacterium-mediated transformation of three freshwater microalgal strains.

Microalgal transformation has gained interest in recent years. Agrobacterium-mediated transformation remains as the most efficient method for the development of transgenic plants and microalgae due to its wide host range, inexpensive procedure and transfer of large segments of DNA. In the present study, three different microalgal species were isolated from freshwater environment and identified based on the morphological characteristics and ITS-2 region amplification. Agrobacterium-mediated transformation was successful for the isolates Chlorella sp., Ankistrodesmus sp and Scenedesmus bajacalifornicus. Gene integration and expression was confirmed by PCR amplification of hptII and GUS histochemical assay. A. tumifaciens contamination was checked by amplification of npt II gene (kanamycin resistant) which lies outside the T-border. Based on GUS assay, transformation efficiencies were found to be 12.25% for Chlorella sp. 2.96% for Scenedesmus bajacalifornicus and 3.5% for Ankistrodesmus sp.

Enhanced CO2 sequestration by a novel microalga: Scenedesmus obliquus SA1 isolated from bio-diversity hotspot region of Assam, India.

The present study aimed to isolate a high CO2 and temperature tolerant microalga capable of sequestering CO2 from flue gas. Microalga strain SA1 was isolated from a freshwater body of Assam and identified as Scenedesmus obliquus (KC733762). At 13.8±1.5% CO2 and 25 °C, maximum biomass (4.975±0.003 g L(-1)) and maximum CO2 fixation rate (252.883±0.361 mg L(-1) d(-1)) were obtained which were higher than most of the relevant studies. At elevated temperature (40 °C) and 13.8±1.5% CO2 maximum biomass (0.883±0.001 g L(-1)) was obtained. The carbohydrate, protein, lipid, and chlorophyll content of the CO2 treated SA1 were 30.87±0.64%, 9.48±1.65%, 33.04±0.46% and 6.03±0.19% respectively, which were higher than previous reports. Thus, SA1 could prove to be a potential candidate for CO2 sequestration from flue gas as well as for the production of value added substances.

Influence of nitrogen sources on biomass productivity of microalgae Scenedesmus bijugatus.

The influence of different nitrogen source of varying concentrations on biomass production of green algae Scenedesmus was investigated. The result revealed that there was a significant difference among nitrogen sources in promoting algal biomass growth at lower concentrations of 5 and 10 mM. Nitrate was found to be a preferred form of nitrogen source and potassium (0.32 g/L) and sodium nitrates (0.28 g/L) performed better for biomass growth of Scenedesmus. Among the ammonical forms, urea (0.25 g/L) resulted in almost equal biomass as nitrates, making it an economical substitute for nitrogen source in large scale culturing of algae being commercially available.

Scenedesmus sp. NJ-1 isolated from Antarctica: a suitable renewable lipid source for biodiesel production.

Microalgal lipids are promising alternative feedstocks for biodiesel production. Scenedesmus sp. NJ-1, an oil-rich freshwater microalga isolated from Antarctica, was identified to be a suitable candidate to produce biodiesel in this study. This strain could grow at temperatures ranging from 4 to 35 °C. With regular decrease in nitrate concentration in the medium, large quantities of triacylglycerols accumulated under batch culture conditions detected by thin layer chromatography and BODIPY 505/515 fluorescent staining. Scenedesmus sp. NJ-1 achieved the average biomass productivity of 0.105 g l⁻¹ d⁻¹ (dry weight) and nearly the highest lipid content (35 % of dry cell weight) was reached at day 28 in the batch culture. Neutral lipids accounted for 78 % of total lipids, and C18:1 (n-9), C16:0 were the major fatty acids in total lipids, composing 37 and 20 % of total fatty acids of Scenedesmus sp. NJ-1 grown for 36 days, respectively. These results suggested that Scenedesmus sp. NJ-1 was a good source of microalgal oils for biodiesel production.

'Y'Scenedesmus (Chlorophyta, Chlorophyceae): the internal transcribed spacer 2 rRNA secondary structure re-revisited.

Including RNA secondary structures improves accuracy and robustness in reconstruction of phylogenetic trees. It is possible to simultaneously infer alignments and phylogenies on the primary sequence and the secondary structure information. For the internal transcribed spacer 2 (ITS2), a phylogenetic RNA transcript marker, two different structure conformations (I or Y shape for helix I) were published for Scenedesmaceae, and a third appeared in the ITS2 database. We contrast the effects on phylogenetic tree reconstruction of different structure sets for a small scenedesmacean subset, using neighbour-joining, maximum parsimony and, for the first time, maximum likelihood, on sequence-structure alignments. Generally our study supports inclusion of secondary structure information. However, we found that any of the three structure conformations is equally fit for phylogenetic studies, but prefer the I shape for helix I. Moreover, our results enable us to give general recommendations on how to build a phylogenetic tree using ITS2 sequence-structure alignments, including different methods to obtain the secondary structures. Thus, we hope to provide a valuable contribution not only for scenedesmacean ITS2 phylogeny, but also for other approaches using RNA transcript markers.

The effect of different trophic modes on lipid accumulation of Scenedesmus quadricauda.

In this study, the effects of different carbon sources on cell growth and lipid accumulation of Scenedesmus quadricauda were investigated. Results showed that S. quadricauda could grow on photoautotrophic, heterotrophic and mixotrophic modes. The lipid yield of S. quadricauda was much lower in the culture containing NaHCO(3) as only carbon source, while CO(2) and glucose concentration significantly influenced cell yield and lipid accumulation in photoautotrophic and heterotrophic culture, respectively. Furthermore, lipid content of S. quadricauda in mixotrophic culture (33.1% of cell dry weight) was much higher than that in photoautotrophic and heterotrophic cultivation (14-28%). Therefore, upon comparing these three trophic modes, present results revealed mixotrophy was the optimal culture method for S. quadricauda to produce lipid. Besides, it was a feasible and promising strategy to culture S. quadricauda using starch wastewater as raw material, which could reduce chemical oxygen demand (COD) of wastewater and the cost of biodiesel production.

Pre-treatment optimization of Scenedesmus obliquus microalga for bioethanol production.

The present work deals with the optimization of cellular disruption and sugar extraction from the microalgae Scenedesmus obliquus (Sc) for bioethanol production. Among the physical and physicochemical methods tested, the best results were obtained with acid hydrolysis by H(2)SO(4) (2N), at 120 °C for 30 min and using dried biomass. The sugar extraction efficiency level reached was 95.6% when compared to the harsh quantitative acid hydrolysis. The influence of other parameters such as biomass loading and number of extraction cycles were also evaluated. The results obtained in the latter case showed that a unique hydrolysis step is sufficient.

Scenedesmus obliquus CNW-N as a potential candidate for CO(2) mitigation and biodiesel production.

This study aimed to achieve higher CO(2) consumption ability and lipid productivity of an indigenous microalgal isolate Scenedesmus obliquus CNW-N by a two-stage cultivation strategy. The microalga strain was first cultivated with 10% CO(2) using a nutrient-rich medium to promote cell growth, which was followed by a nutrient-deficient condition to trigger lipid accumulation. The optimal biomass productivity, lipid productivity, and CO(2) consumption rate were 292.50mgL(-1)d(-1), 78.73mgL(-1)d(-1) (38.9% lipid content per dry weight of biomass), and 549.90mgL(-1)d(-1), respectively. This performance is superior to the results from most of the related studies. Under the nutrient-deficient condition, the microalgal lipid was mainly composed of C16/C18 fatty acids (accounting for 89% of total fatty acids), which is suitable for biodiesel synthesis.

ITS2 data corroborate a monophyletic chlorophycean DO-group (Sphaeropleales).

BACKGROUND: Within Chlorophyceae the ITS2 secondary structure shows an unbranched helix I, except for the 'Hydrodictyon' and the 'Scenedesmus' clade having a ramified first helix. The latter two are classified within the Sphaeropleales, characterised by directly opposed basal bodies in their flagellar apparatuses (DO-group). Previous studies could not resolve the taxonomic position of the 'Sphaeroplea' clade within the Chlorophyceae without ambiguity and two pivotal questions remain open: (1) Is the DO-group monophyletic and (2) is a branched helix I an apomorphic feature of the DO-group? In the present study we analysed the secondary structure of three newly obtained ITS2 sequences classified within the 'Sphaeroplea' clade and resolved sphaeroplealean relationships by applying different phylogenetic approaches based on a combined sequence-structure alignment. RESULTS: The newly obtained ITS2 sequences of Ankyra judayi, Atractomorpha porcata and Sphaeroplea annulina of the 'Sphaeroplea' clade do not show any branching in the secondary structure of their helix I. All applied phylogenetic methods highly support the 'Sphaeroplea' clade as a sister group to the 'core Sphaeropleales'. Thus, the DO-group is monophyletic. Furthermore, based on characteristics in the sequence-structure alignment one is able to distinguish distinct lineages within the green algae. CONCLUSION: In green algae, a branched helix I in the secondary structure of the ITS2 evolves past the 'Sphaeroplea' clade. A branched helix I is an apomorph characteristic within the monophyletic DO-group. Our results corroborate the fundamental relevance of including the secondary structure in sequence analysis and phylogenetics.

Comparative study of zooid and non-zooid forming strains of Scenedesmus obliquus. Physiology and cytomorphology.

Two zooid forming strains and four non-zooid strains of the green chlorococcal alga Scenedesmus obliquus were compared in terms of growth, morphological and physiological characteristics. Large differences were observed among the strains grown under various growth conditions (light and temperature). The assumption that the zooid forming strains may be similar was not confirmed. Since they considerably differed in daughter cells morphology, photosynthesis, growth rate in batch culture or commitment to cellular division. Molecular-genetic comparison of 18S RNA/DNA might distinguish zooid forming strains from non-zooid ones.