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Chlorella sorokiniana green microalga offers many environmentally friendly applications, including wastewater treatment, biofertilizers, animal feed, and biofuel production. Different strains of C. sorokiniana have unique properties that may suit one application but not another. There is a need to distinguish between the many available strains of C. sorokiniana to choose the one that best fits the application. Consequently, our research goal was to develop strain-specific simple sequence repeat (SSR) markers to differentiate between the different strains. Seventeen markers spanning ten out of the twelve chromosomes of the C. sorokiniana genome were developed and validated on eight different strains from culture collections and our lab, and were then analyzed by fragment analysis. The results demonstrate the potential of these polymorphic markers to detect the genetic differences between the strains of C. sorokiniana , and to serve as useful tools for the intra-species population genetic analysis and conservation genetics studies of C. sorokiniana .

Chloroviruses (family Phycodnaviridae ) are large double-stranded DNA (dsDNA) viruses that infect unicellular green algae present in inland waters. These viruses have been isolated using three main chlorella-like green algal host cells, traditionally called NC64A, SAG, and Pbi, revealing extensive genetic diversity. In this study, we performed a functional genomic analysis on 36 chloroviruses that infected the three different hosts. Phylogenetic reconstruction based on the DNA polymerase B family gene clustered the chloroviruses into three distinct clades. The viral pan-genome consists of 1,345 clusters of orthologous groups of genes (COGs), with 126 COGs conserved in all viruses. Totals of 368, 268, and 265 COGs are found exclusively in viruses that infect NC64A, SAG, and Pbi algal hosts, respectively. Two-thirds of the COGs have no known function, constituting the "dark pan-genome" of chloroviruses, and further studies focusing on these genes may identify important novelties. The proportions of functionally characterized COGs composing the pan-genome and the core-genome are similar, but those related to transcription and RNA processing, protein metabolism, and virion morphogenesis are at least 4-fold more represented in the core genome. Bipartite network construction evidencing the COG sharing among host-specific viruses identified 270 COGs shared by at least one virus from each of the different host groups. Finally, our results reveal an open pan-genome for chloroviruses and a well-established core genome, indicating that the isolation of new chloroviruses can be a valuable source of genetic discovery. IMPORTANCE Chloroviruses are large dsDNA viruses that infect unicellular green algae distributed worldwide in freshwater environments. They comprise a genetically diverse group of viruses; however, a comprehensive investigation of the genomic evolution of these viruses is still missing. Here, we performed a functional pan-genome analysis comprising 36 chloroviruses associated with three different algal hosts in the family Chlorellaceae , referred to as zoochlorellae because of their endosymbiotic lifestyle. We identified a set of 126 highly conserved genes, most of which are related to essential functions in the viral replicative cycle. Several genes are unique to distinct isolates, resulting in an open pan-genome for chloroviruses. This profile is associated with generalist organisms, and new insights into the evolution and ecology of chloroviruses are presented. Ultimately, our results highlight the potential for genetic diversity in new isolates.

The green alga Chlorella ohadii was isolated from a desert biological soil crust, one of the harshest environments on Earth. When grown under optimal laboratory settings it shows the fastest growth rate ever reported for a photosynthetic eukaryote and a complete resistance to photodamage even under unnaturally high light intensities. Here we examined the energy distribution along the photosynthetic pathway under four light and carbon regimes. This was performed using various methodologies such as membrane inlet mass spectrometer with stable O 2 isotopes, variable fluorescence, electrochromic shift and fluorescence assessment of NADPH level, as well as the use of specific inhibitors. We show that the preceding illumination and CO 2 level during growth strongly affect the energy dissipation strategies employed by the cell. For example, plastid terminal oxidase (PTOX) plays an important role in energy dissipation, particularly in high light- and low-CO 2 -grown cells. Of particular note is the reliance on PSII cyclic electron flow as an effective and flexible dissipation mechanism in all conditions tested. The energy management observed here may be unique to C. ohadii, as it is the only known organism to cope with such conditions. However, the strategies demonstrated may provide an insight into the processes necessary for photosynthesis under high-light conditions.

Transcription factor engineering to regulate multiple genes has shown promise in the field of microalgae genetic engineering. Here, we report the first use of transcription factor engineering in Chlorella sp. HS2, thought to have potential for producing biofuels and bioproducts. We identified seven endogenous bZIP transcription factors in Chlorella sp. HS2 and named them HSbZIP1 through HSbZIP7. We overexpressed HSbZIP1, a C-type bZIP transcription factor, in Chlorella sp. HS2 with the goal of enhancing lipid production. Phenotype screening under heterotrophic conditions showed that all transformants exhibited increased fatty acid production. In particular, HSbZIP1 37 and 58 showed fatty acid methyl ester (FAME) yields of 859 and 1,052 mg/l, respectively, at day 10 of growth under heterotrophic conditions, and these yields were 74% and 113% higher, respectively, than that of WT. To elucidate the mechanism underlying the improved phenotypes, we identified candidate HSbZIP1-regulated genes via transcription factor binding site analysis. We then selected three genes involved in fatty acid synthesis and investigated mRNA expression levels of the genes by qRTPCR. The result revealed that the possible HSbZIP1-regulated genes involved in fatty acid synthesis were upregulated in the HSbZIP1 transformants. Taken together, our results demonstrate that HSbZIP1 can be utilized to improve lipid production in Chlorella sp. HS2 under heterotrophic conditions.

Chlorella microalgae are increasingly used for various purposes such as fatty acid production, wastewater processing, or as health-promoting food supplements. A mass spectrometry-based survey of N-glycan structures of strain collection specimens and 80 commercial Chlorella products revealed a hitherto unseen intragenus diversity of N-glycan structures. Differing numbers of methyl groups, pentoses, deoxyhexoses, and N-acetylglucosamine culminated in c. 100 different glycan masses. Thirteen clearly discernible glycan-type groups were identified. Unexpected features included the occurrence of arabinose, of different and rare types of monosaccharide methylation (e.g. 4-O-methyl-N-acetylglucosamine), and substitution of the second N-acetylglucosamine. Analysis of barcode ITS1-5.8S-ITS2 rDNA sequences established a phylogenetic tree that essentially went hand in hand with the grouping obtained by glycan patterns. This brief prelude to microalgal N-glycans revealed a fabulous wealth of undescribed structural features that finely differentiated Chlorella-like microalgae, which are notoriously poor in morphological attributes. In light of the almost identical N-glycan structural features that exist within vertebrates or land plants, the herein discovered diversity is astonishing and argues for a selection pressure only explicable by a fundamental functional role of these glycans., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Microalgae accumulate bioavailable selenium-containing amino acids (Se-AAs), and these are useful as a food supplement. While this accumulation has been studied in phototrophic algal cultures, little data exists for heterotrophic cultures. We have determined the Se-AAs content, selenium/sulfur (Se/S) substitution rates, and overall Se accumulation balance in photo- and heterotrophic Chlorella cultures. Laboratory trials revealed that heterotrophic cultures tolerate Se doses ∼8-fold higher compared to phototrophic cultures, resulting in a ∼2-3-fold higher Se-AAs content. In large-scale experiments, both cultivation regimes provided comparable Se-AAs content. Outdoor phototrophic cultures accumulated up to 400 μg g -1 of total Se-AAs and exhibited a high level of Se/S substitution (5-10%) with 30-60% organic/total Se embedded in the biomass. A slightly higher content of Se-AAs and ratio of Se/S substitution was obtained for a heterotrophic culture in pilot-scale fermentors. The data presented here shows that heterotrophic Chlorella cultures provide an alternative for Se-enriched biomass production and provides information on Se-AAs content and speciation in different cultivation regimes.

Microalgal Chlorella has been demonstrated to process wastewater efficiently from piggery industry, yet optimization through genetic engineering of such a bio-treatment is currently challenging, largely due to the limited data and knowledge in genomics. In this study, we first investigated the differential growth rates among three wastewater-processing Chlorella strains: Chlorella sorokiniana BD09, Chlorella sorokiniana BD08 and Chlorella sp. Dachan, and the previously published Chlorella sorokiniana UTEX 1602, showing us that BD09 maintains the best tolerance in synthetic wastewater. We then performed genome sequencing and analysis, resulting in a high-quality assembly for each genome with scaffold N50 > 2 Mb and genomic completeness ≥91%, as well as genome annotation with 9,668, 10,240, 9,821 high-confidence gene models predicted for BD09, BD08, and Dachan, respectively. Comparative genomics study unravels that metabolic pathways, which are involved in nitrogen and phosphorus assimilation, were enriched in the faster-growing strains. We found that gene structural variation and genomic rearrangement might contribute to differential capabilities in wastewater tolerance among the strains, as indicated by gene copy number variation, domain reshuffling of orthologs involved, as well as a ~1 Mb-length chromosomal inversion we observed in BD08 and Dachan. In addition, we speculated that an associated bacterium, Microbacterium chocolatum, which was identified within Dachan, play a possible role in synergizing nutrient removal. Our three newly sequenced Chlorella genomes provide a fundamental foundation to understand the molecular basis of abiotic stress tolerance in wastewater treatment, which is essential for future genetic engineering and strain improvement.

Objective: Microalgae gained interest for potential use as biodiesel producers, since they synthesize and accumulate significant quantities of lipids. The aim of this work was to isolate indigenous microalgae strains from Greek habitats, study their physicochemical growth conditions and finally select the best ones with respect to overall lipid production and profile., Results: Two sampling sites of marine aquatic ecosystems were selected in Attica prefecture, Greece in order to screen for novel wild type strains with lipid production capacity. Microalgae isolates (59) were obtained from the selected areas and were morphologically and molecularly characterized. Fatty acids were estimated through Flow Cytometry combined with BODIPY staining method. Four isolates were selected for their lipid production properties and were cultivated in 15 L tank cultures. The four isolates were also identified by 18S rDNA gene sequencing. Two of them, Chlorella sp. ΑCΑ9 and ACA17, exhibited both maximum biomass and lipid productivity. Optimization of growth conditions with respect to pH and initial NaNO 3 concentration was performed for the two microalgae in 15 L cultures. Finally, 20 L fed batch cultures were set up using the optimum culture conditions. Lipid profiles were stabilized for both strains at dry biomass levels over 1 g L -1 and lipid content of 25% (w/w)., Conclusions: Two Chlorella strains (ACA9 and ACA17) were promising candidates for biodiesel production as they were easily grown in sea water in fed batch systems and produce lipids suitable for biodiesel-especially Chlorella sp. ACA9.

The ciliate Spirostomum semivirescens is a large freshwater protist densely packed with endosymbiotic algae and capable of building a protective coating from surrounding particles. The species has been rarely recorded and it lacks any molecular investigations. We obtained such data from S. semivirescens isolated in the UK and Sweden. Using single-cell RNA sequencing of isolates from both countries, the transcriptome of S. semivirescens was generated. A phylogenetic analysis identified S. semivirescens as a close relative to S. minus. Additionally, rRNA sequence analysis of the green algal endosymbiont revealed that it is closely related to Chlorella vulgaris. Along with the molecular species identification, an analysis of the ciliates' stop codons was carried out, which revealed a relationship where TGA stop codon frequency decreased with increasing gene expression levels. The observed codon bias suggests that S. semivirescens could be in an early stage of reassigning the TGA stop codon. Analysis of the transcriptome indicates that S. semivirescens potentially uses rhodoquinol-dependent fumarate reduction to respire in the oxygen-depleted habitats where it lives. The data also shows that despite large geographical distances (over 1,600km) between the sampling sites investigated, a morphologically-identical species can share an exact molecular signature, suggesting that some ciliate species, even those over 1mm in size, could have a global biogeographical distribution., (Copyright © 2018 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Ciliated protists often form symbioses with many diverse microorganisms. In particular, symbiotic associations between ciliates and green algae, as well as between ciliates and intracellular bacteria, are rather wide-spread in nature. In this study, we describe the complex symbiotic system between a very rare ciliate, Paramecium chlorelligerum, unicellular algae inhabiting its cytoplasm, and novel bacteria colonizing the host macronucleus. Paramecium chlorelligerum, previously found only twice in Germany, was retrieved from a novel location in vicinity of St. Petersburg in Russia. Species identification was based on both classical morphological methods and analysis of the small subunit rDNA. Numerous algae occupying the cytoplasm of this ciliate were identified with ultrastructural and molecular methods as representatives of the Meyerella genus, which before was not considered among symbiotic algae. In the same locality at least fifteen other species of "green" ciliates were found, thus it is indeed a biodiversity hot-spot for such protists. A novel species of bacterial symbionts living in the macronucleus of Paramecium chlorelligerum cells was morphologically and ultrastructurally investigated in detail with the description of its life cycle and infection capabilities. The new endosymbiont was molecularly characterized following the full-cycle rRNA approach. Furthermore, phylogenetic analysis confirmed that the novel bacterium is a member of Holospora genus branching basally but sharing all characteristics of the genus except inducing connecting piece formation during the infected host nucleus division. We propose the name "Candidatus Holospora parva" for this newly described species. The described complex system raises new questions on how these microorganisms evolve and interact in symbiosis., Competing Interests: The authors have declared that no competing interests exist.

The complete nucleotide sequences of the mitochondrial (mtDNA) and chloroplast (cpDNA) genomes of Chlorella variabilis NC64A (Trebouxiophyceae) have been determined in this study (GenBank accession no. KP271968 and KP271969, respectively). The mt genome assembles as a circle of 78,500 bp and contains 62 genes, including 32 protein-coding, 27 tRNA and 3 rRNA genes. The overall GC content is 28.2%, while the coding sequence is 34%. The cp genome forms a circle of 124,793 bp, containing 114 genes, including 79 protein-coding, 32 tRNA and 3 rRNA genes. The overall GC content is 33,9%, while the coding sequence is 50%.

Several different barcoding methods of distinguishing species have been advanced, but which method is the best is still controversial. Chlorella is becoming particularly promising in the development of second-generation biofuels. However, the taxonomy of Chlorella-like organisms is easily confused. Here we report a comprehensive barcoding analysis of Chlorella-like species from Chlorella, Chloroidium, Dictyosphaerium and Actinastrum based on rbcL, ITS, tufA and 16S sequences to test the efficiency of traditional barcoding, GMYC, ABGD, PTP, P ID and character-based barcoding methods. First of all, the barcoding results gave new insights into the taxonomic assessment of Chlorella-like organisms studied, including the clear species discrimination and resolution of potentially cryptic species complexes in C. sorokiniana, D. ehrenbergianum and C. Vulgaris. The tufA proved to be the most efficient barcoding locus, which thus could be as potential "specific barcode" for Chlorella-like species. The 16S failed in discriminating most closely related species. The resolution of GMYC, PTP, P ID, ABGD and character-based barcoding methods were variable among rbcL, ITS and tufA genes. The best resolution for species differentiation appeared in tufA analysis where GMYC, PTP, ABGD and character-based approaches produced consistent groups while the PTP method over-split the taxa. The character analysis of rbcL, ITS and tufA sequences could clearly distinguish all taxonomic groups respectively, including the potentially cryptic lineages, with many character attributes. Thus, the character-based barcoding provides an attractive complement to coalescent and distance-based barcoding. Our study represents the test that proves the efficiency of multiple DNA barcoding in species discrimination of microalgaes.

Marine chlorophytes of the genus Chlorella are unicellular algae capable of accumulating a high proportion of cellular lipids that can be used for biodiesel production. In this study, we examined the broad physiological capabilities of a subtropical strain (C596) of Chlorella sp. "SAG-211-18" including its heterotrophic growth and tolerance to low salt. We found that the alga replicates more slowly at diluted salt concentrations and can grow on a wide range of carbon substrates in the dark. We then sequenced the RNA of Chlorella strain C596 to elucidate key metabolic genes and investigate the transcriptomic response of the organism when transitioning from a nutrient-replete to a nutrient-deficient condition when neutral lipids accumulate. Specific transcripts encoding for enzymes involved in both starch and lipid biosynthesis, among others, were up-regulated as the cultures transitioned into a lipid-accumulating state whereas photosynthesis-related genes were down-regulated. Transcripts encoding for two of the up-regulated enzymes-a galactoglycerolipid lipase and a diacylglyceride acyltransferase-were also monitored by reverse transcription quantitative polymerase chain reaction assays. The results of these assays confirmed the transcriptome-sequencing data. The present transcriptomic study will assist in the greater understanding, more effective application, and efficient design of Chlorella-based biofuel production systems.

We previously showed that both the linear photosynthetic electron transportation rate and the respiration rate dropped significantly during N starvation-induced neutral lipid accumulation in an oil-producing microalga, Chlorella sorokiniana, and proposed a possible role for cyclic electron flow (CEF) in ATP supply. In this study, we further exploited this hypothesis in both Chlorella sorokiniana C3 and the model green alga Chlamydomonas. We found that both the rate of CEF around photosystem I and the activity of thylakoid membrane-located ATP synthetase increased significantly during N starvation to drive ATP production. Furthermore, we demonstrated that the Chlamydomonas mutant pgrl1, which is deficient in PGRL1-mediated CEF, accumulated less neutral lipids and had reduced rates of CEF under N starvation. Further analysis revealed that Ca(2+) signaling regulates N starvation-induced neutral lipid biosynthesis in Chlamydomonas by increasing calmodulin activity and boosting the expression of the calcium sensor protein that regulates Pgrl1-mediated CEF. Thus, Ca(2+)-regulated CEF supplies ATP for N starvation-induced lipid biosynthesis in green alga. The increased CEF may re-equilibrate the ATP/NADPH balance and recycle excess light energy in photosystems to prevent photooxidative damage, suggesting Ca(2+)-regulated CEF also played a key role in protecting and sustaining photosystems.

Chloroplasts are essential organelles, in which genes have widely been used in the phylogenetic analysis of green plants. Here, we took advantage of the breadth of plastid genomes (cpDNAs) sequenced species to investigate their dynamic changes. Our study showed that gene rearrangements occurred more frequently in the cpDNAs of green algae than in land plants. Phylogenetic trees were generated using 55 conserved protein-coding genes including 33 genes for photosynthesis, 16 ribosomal protein genes and 6 other genes, which supported the monophyletic evolution of vascular plants, land plants, seed plants, and angiosperms. Moreover, we could show that seed plants were more closely related to bryophytes rather than pteridophytes. Furthermore, the substitution rate for cpDNA genes was calculated to be 3.3×10(-10), which was almost 10 times lower than genes of nuclear genomes, probably because of the plastid homologous recombination machinery., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Global warming and ozone depletion, and the resulting increase of ultraviolet radiation (UVR), have far-reaching impacts on biota, especially affecting the algae that form the basis of the food webs in aquatic ecosystems. The aim of the present study was to investigate the interactive effects of temperature and UVR by comparing the photosynthetic responses of similar taxa of Chlorella from Antarctic (Chlorella UMACC 237), temperate (Chlorella vulgaris UMACC 248) and tropical (Chlorella vulgaris UMACC 001) environments. The cultures were exposed to three different treatments: photosynthetically active radiation (PAR; 400-700 nm), PAR plus ultraviolet-A (320-400 nm) radiation (PAR + UV-A) and PAR plus UV-A and ultraviolet-B (280-320 nm) radiation (PAR + UV-A + UV-B) for one hour in incubators set at different temperatures. The Antarctic Chlorella was exposed to 4, 14 and 20°C. The temperate Chlorella was exposed to 11, 18 and 25°C while the tropical Chlorella was exposed to 24, 28 and 30°C. A pulse-amplitude modulated (PAM) fluorometer was used to assess the photosynthetic response of microalgae. Parameters such as the photoadaptive index (Ek) and light harvesting efficiency (α) were determined from rapid light curves. The damage (k) and repair (r) rates were calculated from the decrease in ΦPSIIeff over time during exposure response curves where cells were exposed to the various combinations of PAR and UVR, and fitting the data to the Kok model. The results showed that UV-A caused much lower inhibition than UV-B in photosynthesis in all Chlorella isolates. The three isolates of Chlorella from different regions showed different trends in their photosynthesis responses under the combined effects of UVR (PAR + UV-A + UV-B) and temperature. In accordance with the noted strain-specific characteristics, we can conclude that the repair (r) mechanisms at higher temperatures were not sufficient to overcome damage caused by UVR in the Antarctic Chlorella strain, suggesting negative effects of global climate change on microalgae inhabiting (circum-) polar regions. For temperate and tropical strains of Chlorella, damage from UVR was independent of temperature but the repair constant increased with increasing temperature, implying an improved ability of these strains to recover from UVR stress under global warming.

Proteomics in conjunction with biochemical strategy was employed to unravel regulation of lipid metabolism in the green microalga Chlorella protothecoides by heterotrophy-photoinduction cultivation regime (HPC). Interestingly, HPC triggered transiently synthesis of starch followed by substantial lipid accumulation. And a marked decrease in intracellular protein and chlorophyll contents was also observed after 12h of photo-induction. The highest lipid content of 50.5% was achieved upon the photo-induction stage, which represented 69.3% higher than that of the end of heterotrophic cultivation. Results suggested that turnover of carbon-nitrogen-rich compounds such as starch, protein, and chlorophyll might provide carbon or energy for lipid accumulation. The proteomics analysis indicated that several pathways including glycolysis, TCA cycle, β-oxidation of fatty acids, Calvin cycle, photosynthesis, energy and transport, protein biosynthesis, regulate and defense were involved in the lipid biosynthesis. Malate dehydrogenase and acyl-CoA dehydrogenase were suggested as key regulatory factors in enhancing lipid accumulation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this study, flocculation of Chlorella sorokiniana cultivated in swine manure wastewater, BG-11 medium and BG-11 medium supplemented with different organic matters (glucose, urea and tryptone) was investigated. The results demonstrated that the minimum amount of Al(3+) required for complete flocculation in wastewater would increase substantially, and flocculation efficiency became highly sensitive to pH. Tryptone could cause similar extent of inhibition on flocculation as in wastewater. Meanwhile, glucose could increase concentrations of Algogenic Organic Matter (AOM), inhibiting flocculation strongly at higher pH, including flocculation induced by Al(3+) and autoflocculation. However, urea had little effect on flocculation of C. sorokiniana. Moreover, the major factors: dilution times, pH and flocculants dosage, which had significant impact on flocculation efficiency of C. sorokiniana in piggery wastewater, were optimized using response surface methodology (RSM). The optimal flocculation efficiency (100%) was achieved at pH 8.5, 7-folds of dilution and 52.14 mg L(-1) of Al(3+)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Reserve lipids of microalgae are promising for biodiesel production. However, economically feasible and sustainable energy production from microalgae requires optimization of cultivation conditions for both biomass yield and lipid production of microalgae. Biomass yield and lipid production in microalgae are a contradictory problem because required conditions for both targets are different. Simultaneously, the mass cultivation of microalgae for biofuel production also depends extremely on the performance of the microalgae strains used. In this study a green unicellular microalgae Chlorella sorokiniana (DS6) isolated from the holding tanks of farm wastewater treatment plant using multi-step screening and acclimation procedures was found high-lipid producing facultative heterotrophic microalgae strain capable of growing on dairy farm effluent (DFE) for biodiesel feedstock and wastewater treatment. Morphological features and the phylogenetic analysis for the 18S rRNA identified the isolated strains. A novel three stage cultivation process of facultative strain of C. sorokiniana was examined for lipid production., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A Chlorella strain tolerant to high-strength anaerobic digestion effluent was isolated from the anaerobic digestion effluent with a long-term exposure to air. The strain was identified as a Chlorella by morphological and molecular biological methods, and named Chlorella sp. BWY-1, The anaerobic digestion effluent used in this study was from a biogas plant with the raw materials of swine wastewater after solid-liquid separation. The Chlorella regularis (FACHB-729) was used as the control strain. The comparative study showed that Chlorella sp, BWY-Ihad relatively higher growth rate, biomass accumulation capacity and pollutants removal rate in BG11. and different concentrations of anaerobic digestion effluent. Chlorella sp. BWY-1 had the highest growth rate and biomass productivity (324.40 mg.L-1) in BG11, but its lipid productivity and lipid content increased with the increase of anaerobic digestion effluent concentration, In undiluted anaerobic digestion effluent, the lipid productivity and lipid content of Chlorella sp. BWY-1 were up to 44. 43% and 108. 70 mg.L-1, respectively. Those results showed that the isolated algal strain bad some potential applications in livestock wastewater treatment and bioenergy production, it could be combined with a solid-liquid separation, anaerobic fermentation and other techniques for processing livestock wastewater and producing biodiesel.

In the experiment, Chlorella pyrenoidosa and Microcystis aeruginosa were chosen to test the individual, combined and second exposure effect of Cu(2+) and chlortetracycline (CTC). The 96 h EC50s of each test were calculated, with the ranges of 0.972-15.6 μmol/L (Cu(2+)), 29.5-102.5 μmol/L (CTC), 14.4-78.9 μmol/L (mixture). The combined toxicities were evaluated with toxicity units (TU) method. The toxicity of complex of Cu(2+) and chlortetracycline was analyzed using concentration addition (CA) model. In the initial test, the combined effect of the two substances was partly additive to C. pyrenoidosa and antagonistic to M. aeruginosa, while in the second exposure test, the combined effect was synergistic to both algae. The biochemical indicators measured in the experiment included chlorophyll fluorescence (Fv/Fm), MDA content, SOD activity and content of soluble proteins. When under combined stress, the biochemical features of both algae were significantly different between the initial test and the second exposure test., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chlorella, the archetype of unicellular green algae, is a high-performance primary producer in aquatic and terrestrial ecosystems. Under the simple spherical morphology of Chlorella, many other 'green balls' unfolded as independent phylogenetic lineages as a result of convergent evolution. By contrast, green algae with strikingly different phenotypes were unmasked as close relatives of Chlorella by modern molecular techniques. Here, we point to the increasing impact of these diverse protists on ecology, evolution, and biotechnology in the light of integrative taxonomy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Microalgae-derived biodiesel is a promising substitute for conventional fossil fuels. In particular, the green alga Chlorella protothecoides sp. 0710 is regarded as one of the best candidates for commercial manufacture of microalgae-derived biofuel. This is due not only to its ability to live autotrophically through photosynthesis, but also to its capacity to produce a large amount of biomass and lipid through fermentation of glucose. However, until the present study, neither its genome sequence nor the platform required for molecular manipulations were available., Results: We generated a draft genome for C. protothecoides, and compared its genome size and gene content with that of Chlorella variabilis NC64A and Coccomyxa subellipsoidea C-169. This comparison revealed that C. protothecoides has a reduced genome size of 22.9 Mbp, about half that of its close relatives. The C. protothecoides genome encodes a smaller number of genes, fewer multi-copy genes, fewer unique genes, and fewer genome rearrangements compared with its close relatives. In addition, three Chlorella-specific hexose-proton symporter (HUP)-like genes were identified that enable the consumption of glucose and, consequently, heterotrophic growth. Furthermore, through comparative transcriptomic and proteomic studies, we generated a global perspective regarding the changes in metabolic pathways under autotrophic and heterotrophic growth conditions. Under heterotrophic conditions, enzymes involved in photosynthesis and CO2 fixation were almost completely degraded, either as mRNAs or as proteins. Meanwhile, the cells were not only capable of quickly assimilating glucose but also showed accelerated glucose catabolism through the upregulation of glycolysis and the tricarboxylic acid (TCA) cycle. Moreover, the rapid synthesis of pyruvate, upregulation of most enzymes involved in fatty acid synthesis, and downregulation of enzymes involved in fatty acid degradation favor the synthesis of fatty acids within the cell., Conclusions: Despite similarities to other Chlorella, C. protothecoides has a smaller genome than its close relatives. Genes involved in glucose utilization were identified, and these genes explained its ability to grow heterotrophically. Transcriptomic and proteomic results provided insight into its extraordinary ability to accumulate large amounts of lipid. The C. protothecoides draft genome will promote the use of this species as a research model.

Background: Chorella is the representative taxon of Chlorellales in Trebouxiophyceae, and its chloroplast (cp) genomic information has been thought to depend only on studies concerning Chlorella vulgaris and GenBank information of C. variablis. Mitochondrial (mt) genomic information regarding Chlorella is currently unavailable. To elucidate the evolution of organelle genomes and genetic information of Chlorella, we have sequenced and characterized the cp and mt genomes of Arctic Chlorella sp. ArM0029B., Results: The 119,989-bp cp genome lacking inverted repeats and 65,049-bp mt genome were sequenced. The ArM0029B cp genome contains 114 conserved genes, including 32 tRNA genes, 3 rRNA genes, and 79 genes encoding proteins. Chlorella cp genomes are highly rearranged except for a Chlorella-specific six-gene cluster, and the ArM0029B plastid resembles that of Chlorella variabilis except for a 15-kb gene cluster inversion. In the mt genome, 62 conserved genes, including 27 tRNA genes, 3 rRNA genes, and 32 genes encoding proteins were determined. The mt genome of ArM0029B is similar to that of the non-photosynthetic species Prototheca and Heicosporidium. The ArM0029B mt genome contains a group I intron, with an ORF containing two LAGLIDADG motifs, in cox1. The intronic ORF is shared by C. vulgaris and Prototheca. The phylogeny of the plastid genome reveals that ArM0029B showed a close relationship of Chlorella to Parachlorella and Oocystis within Chlorellales. The distribution of the cox1 intron at 721 support membership in the order Chlorellales. Mitochondrial phylogenomic analyses, however, indicated that ArM0029B shows a greater affinity to MX-AZ01 and Coccomyxa than to the Helicosporidium-Prototheca clade, although the detailed phylogenetic relationships among the three taxa remain to be resolved., Conclusions: The plastid genome of ArM0029B is similar to that of C. variabilis. The mt sequence of ArM0029B is the first genome to be reported for Chlorella. Chloroplast genome phylogeny supports monophyly of the seven investigated members of Chlorellales. The presence of the cox1 intron at 721 in all four investigated Chlorellales taxa indicates that the cox1 intron had been introduced in early Chorellales as a cis-splice form and that the cis-splicing intron was inherited to recent Chlorellales and was recently trans-spliced in Helicosporidium.

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.

The microalgae family Chlorella species are known to accumulate starch and lipids. Although nitrogen or phosphorous deficiencies promote starch and lipids formation in many microalgae, these deficiencies also limit their growth and productivity. Therefore, the Chlorellaceae strains were attempted to increase starch and lipids productivity under high-light-intensity conditions (600-μmol photons m(-2)s(-1)). The 12:12-h light-dark (LD) cycle conditions elicited more stable growth than the continuous light (LL) conditions, whereas the starch and lipids yields increased in LL conditions. The amount of starch and lipids per cell increased in Chlorella viscosa and Chlorella vulgaris in sulfur-deficient medium, and long-chain fatty acids with 20 or more carbon atoms accumulated in cells grown in sulfur-deficient medium. Accumulation of starch and lipids was investigated in eight strains. The accumulation was strain-dependent, and varied according to the medium and light conditions. Five of the eight Chlorella strains exhibited similar accumulation patterns., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

We recently isolated a small green alga from a biological sand crust (BSC) in the NW Negev, Israel. Based on its 18S rRNA and rbcL genes, it is a close relative of Chlorella sorokiniana and of certain strains of C. vulgaris and C. variabilis, but differs substantially in many aspects from C. sorokiniana. Because the classification of Chlorellales is still not resolved, we designated this species as C. ohadii (Trebouxiophyceae) in honor of Professor Itzhak Ohad. Under controlled laboratory conditions, C. ohadii showed marked structural and photosynthetic performance changes, depending on the carbon source used during growth, as well as remarkable resistance to photoinhibition. CO2 -dependent O2 evolution was not affected even when exposed to a light intensity of 3500 μmole photons m(-2)  s(-1) , over 1.5 times the maximal intensity reached at the BSC surface, whereas the variable fluorescence declined sharply. We briefly discuss the use of fluorescence to assess photosynthetic rate and the implications of this finding for the assessment of global BSCs activity., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

One of the main limitations of productivity in photobioreactor is the inefficient conversion of the available light into biomass. Photoautotrophic cells such as microalgae only absorb a small fraction of supplied illumination due to limitation of its photosystem's (PS) absorbing rate. However, phenomenon of Flashing Light Effect (FLE) allows microalgae to utilize strong light exceptionally through intermittent exposure. Exposure of strong light at correct frequency of light and dark photoperiod would allow two pigment-protein complexes, PSI and PSII to be at the equilibrium mid-point potential to allow efficient light conversion. Narrow range of optimum frequency is crucial since overexposure to strong light would injured photosynthetic apparatus whereas longer dark period would contributed to loss of biomass due to triacylglycerol metabolism. The behaviour of microalgae towards various illumination conditions of FLE was determined at batch Photobioreactor (PBR) by varying the aeration flow rate: 16.94, 33.14 and 49.28 mL sec(-1) which yield, respectively the light exposure time of 3.99, 1.71 and 1.1 seconds per cycle. Maximum cell density in FLE-PBR was significantly higher at the exponential phase as compared to the continuously illuminated culture (p = 5.62 x 10(-5), a = 0.05) under the flow rate of 25.07 mL sec(-1). Maximum cell density yield of FLE-PBR and continuously illuminated PBR was, respectively 3.1125 x 10(7) and 2.947 x 10(7) cells mL(-1). Utilization of FLE as an innovative solution to increase the efficiency of microalgae to convert light into chemical energy would revolutionize the microalgae culture, reduce the time for cultivation and produce higher maximum biomass density.

Algal lipids are ideal biofuel sources. Our objective was to determine the contributors to triacylglycerol (TAG) accumulation and lipid body formation in Chlorella UTEX29 under nitrogen (N) deprivation. A fivefold increase in intracellular lipids following N starvation for 24 h confirmed the oleaginous characteristics of UTEX29. Ultrastructural studies revealed increased number of lipid bodies and decreased starch granules in N-starved cells compared to N-replete cells. Lipid bodies were observed as early as 3 h after N removal and plastids collapsed after 48 h of stress. Moreover, the identification of intracellular pyrenoids and differences in the expected nutritional requirements for Chlorella protothecoides (as UTEX29 is currently classified) led us to conduct a phylogenetic study using 18S and actin cDNA sequences. This indicated UTEX29 to be more phylogenetically related to Chlorella vulgaris. To investigate the fate of different lipids after N starvation, radiolabeling using ¹⁴C-acetate was used. A significant decrease in ¹⁴C-galactolipids and phospholipids matched the increase in ¹⁴C-TAG starting at 3 h of N starvation, consistent with acyl groups from structural lipids as sources for TAG under N starvation. These results have important implications for the identification of key steps controlling oil accumulation in N-starved biofuel algae and demonstrate membrane recycling during lipid body formation.

To augment biomass and lipid productivities of heterotrophic cultured microalgae Chlorella sorokiniana, the influence of environmental temperature and medium factors, such as carbon source, nitrogen source, and their initial concentrations was investigated in this study. The microalga C. sorokiniana could tolerate up to 42°C and showed the highest growth rate of 1.60d(-1) at 37°C. The maximum dry cell weight (DCW) and corresponding lipid concentration was obtained with 80gL(-1) of initial glucose and 4gL(-1) of initial KNO3 at 37°C. In 5-L batch fermentation, the DCW increased dramatically from 0.9gL(-1) to 37.6gL(-1) in the first 72h cultivation, with the DCW productivity of 12.2gL(-1)d(-1). The maximum lipid content of 31.5% was achieved in 96h and the lipid productivity was 2.9gL(-1)d(-1). The results showed C. sorokiniana could be a promising strain for biofuel production., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The influence of sulfur deficiency on biomass production was analyzed in the four Chlorellaceae species, Chlorella vulgaris, Chlorella sorokiniana, Chlorella lobophora, and Parachlorella kessleri. Culturing under sulfur-deficient conditions promoted transient accumulation of starch followed by a steady increase in lipid storage. Transmission electron microscopy indicated an increase and decrease in starch granules and subsequent enlargement of lipid droplets under sulfur-deficient conditions. Chlorellaceae spp. accumulated 1.5-2.7-fold higher amounts of starch and 1.5-2.4-fold higher amounts of lipid under sulfur-deficient conditions than under sulfur-sufficient conditions. More than 75% of the fatty acids that accumulated in Chlorellaceae spp. under the sulfur-sufficient condition were unsaturated and culturing under sulfur-deficient conditions increased the saturated fatty acid content from 24.3% to 59.7% only in P. kessleri. These results indicate that the sequential accumulation of starch and lipid is a response to the sulfur depletion that commonly occurs in Chlorellaceae spp., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The concentration of NaNO(3), MgSO(4) · 7H(2)O and proteose, in Chlorella protothecoides medium were optimized for algal biomass and lipid production by using response surface methodology with Box-Behnken design. The optimal concentrations were 0.45 g/L of NaNO(3), 6 mg/L of MgSO(4) · 7H(2)O, and 0.25 g/L of proteose for maximum biomass production and 2 mg/L of MgSO(4) · 7H(2)O and no addition of NaNO(3) and proteose for lipid accumulation. In optimized biomass production medium, a final biomass concentration of 1.19 g/L was obtained, which was 1.8 times higher than that in the original medium. For lipid accumulation, a 12.9% lipid content was obtained from the biomass in the lipid production medium, which was three times higher than that from the original medium. The fatty acid profile of algae grown in the optimized medium demonstrated a higher unsaturated fatty acid content (i.e. methyl linoleate (C18:2) and methyl linolenate (C18:3)) than that of the algae grown in the original medium. The results provide a strategy for limiting the amount of nutrients required in large scale outdoor cultivation systems of C. protothecoides to make the production of algal biomass more economically attractive., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Microalgae are a potential resource for biodiesel production. A green alga, Chlorella sp., was isolated from Arctic sea ice, which was named ArM0029B. These algae displayed faster growth at a wide temperature range of 4-32°C compared to Chlorella vulgaris. ArM0029B also accumulated high levels of total fatty acids under nitrogen starvation conditions, reaching 39% of dry cell weight, with the proportion of oleic acid (18:1) and linoleic acid (18:2) reaching 54% of total fatty acids. Taken together, these results indicate that the newly identified Chlorella species, ArM0029B, is a promising candidate for biodiesel production., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A novel green unicellular microalgal isolate from the freshwater of the Inner Mongolia Province of China and named as CCTCC M209220, grows between pH 6 and 11 and temperatures of 20-35°C with optimal conditions at pH 9 and 30°C. Morphological features and the phylogenetic analysis for the 18S rRNA gene reveal that the isolate is a Chlorella sorokiniana strain. A nitrogen source test reveals that this strain can grow well with nitrate and urea, but not ammonium. The strain can grow heterotrophically with glucose as the carbon source and accumulates lipid content as high as 56% (w/w) dry weight after 7 days in high glucose concentrations compared to 19% lipids achieved in 30 days of photoautotrophic culture. The relative neutral lipid content as a fraction of the total lipid is also much higher in heterotrophic culture as compared to photoautotrophic culture., (Copyright © 2012 Wiley Periodicals, Inc.)

High costs and issues such as a high cell concentrations in effluents are encountered when utilizing microalgae for wastewater treatment. The present study analyzed nitrogen and phosphate removal under autotrophic, heterotrophic, mixotrophic and micro-aerobic conditions by Chlorella sorokiniana GXNN 01 immobilized in calcium alginate. The immobilized cells grew as well as free-living cells under micro-aerobic conditions and better than free-living cells under the other conditions. The immobilized cells had a higher ammonium removal rate (21.84%, 43.59% and 41.46%) than free living cells (14.35%, 38.57% and 40.59%) under autotrophic, heterotrophic, and micro-aerobic conditions, and higher phosphate removal rate (87.49%, 88.65% and 84.84%) than free living cells (20.21%, 42.27% and 53.52%) under heterotrophic, mixotrophic and micro-aerobic conditions, respectively. The data indicate that immobilized Chlorella sorokiniana GXNN 01 is a suitable species for use in wastewater treatment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lipids obtained from Chlorella protothecoides in heterotrophic cultivation are considered a suitable feedstock for biodiesel production. In this study, glucose fed-batch fermentation was performed to increase final biomass and lipid production. The biomass productivity and lipid productivity were 6.28 and 2.06 g/L day, respectively. Biomass/glucose conversion and the lipid/glucose conversion were 43.3% and 14.2%, respectively. Extraction of lipids from algae has been identified as a key bottleneck in bioprocessing operations. Supercritical carbon dioxide (SC-CO(2)) was applied for neutral lipids extraction and the SC-CO(2) kinetics was investigated by the Goto et al. model. The modeling showed a good fit with experimental data. Additionally, neutral lipids extracted by SC-CO(2) displayed a suitable fatty acid profile for biodiesel [mainly C18:1 (60.0%), C18:2 (18.7%) and C16:0 (11.5%)]. Our study demonstrated the ability to produce high levels of neutral lipids through heterotrophic algal culture and subsequent extraction of lipids with SC-CO(2) method developed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Piggery wastewater that is used for microalgae cultivation has usually been treated by secondary treatment processes. The present study investigates mixotrophic cultivation of the green microalgae Chlorella pyrenoidosa with primary piggery wastewater that has merely been diluted before use. The biomass productivity of the microalgae exhibited a positive linear correlation with the initial COD values, which ranged from 250 to 1000 mg/L. Nutrients in the piggery wastewater were removed efficiently; for example, the removal rate of ammonium was over 90% in all diluted samples. Profiles of fatty acids in the algal lipids were different after cultivation in piggery wastewater medium compared to Bristol's solution. The maximum lipid productivity of 6.3 mg L(-1) day(-1), which was achieved when the initial COD was 1000 mg/L, is not superior to other reports, but it suggests a convenient way to reduce the high organic content of piggery waste with the production of algal lipids., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A colony PCR technique was applied for both genomic and chloroplast DNA in the green microalgae Chlorella. Of five different lysis buffers, Chelex-100 was superior for DNA extraction, PCR and DNA storage. It also was insensitive to variations in cell density. The conditions established for an improved PCR formulation are applicable for screening of genetically-engineered transformants as well as bioprospecting of natural microalgal isolates. Besides multiple Chlorella species, we also demonstrate the efficacy of Chelex-100 for colony PCR with a number of other microalgal strains, including Chlamydomonas reinhardtii, Dunaliella salina, Nannochloropsis sp., Coccomyxa sp., and Thalassiosira pseudonana.

The isolation and characterization of the phytoene synthase gene from the green microalga Chlorella zofingiensis (CzPSY), involved in the first step of the carotenoids biosynthetic pathway, have been performed. CzPSY gene encodes a polypeptide of 420 amino acids. A single copy of CzPSY has been found in C. zofingiensis by Southern blot analysis. Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to catalyze the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to form phytoene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the phytoene synthases (PSY) of the chlorophycean microalgae studied, being very closely related to PSY of plants. This new isolated gene has been adequately inserted in a vector and expressed in Chlamydomonas reinhardtii. The overexpression of CzPSY in C. reinhardtii, by nuclear transformation, has led to an increase in the corresponding CzPSY transcript level as well as in the content of the carotenoids violaxanthin and lutein which were 2.0- and 2.2-fold higher than in untransformed cells. This is an example of manipulation of the carotenogenic pathway in eukaryotic microalgae, which can open up the possibility of enhancing the productivity of commercial carotenoids by molecular engineering.

Symbiosis of green algae with protozoa and invertebrates has been studied for more than 100 years. Endosymbiotic green algae are widely distributed in ciliates (e.g. Paramecium, Stentor, Climacostomum, Coleps, Euplotes), heliozoa (e.g. Acanthocystis) and invertebrates (e.g. Hydra, Spongilla), and have traditionally been identified as named or unnamed species of Chlorella Beij. or Zoochlorella K. Brandt or referred to as Chlorella-like algae or zoochlorellae. We studied 17 strains of endosymbionts isolated from various hosts and geographical localities using an integrative approach (nuclear encoded small subunit and internal transcribed spacer regions of rRNA gene sequences including their secondary structures, morphology, physiology and virus sensitivity). Phylogenetic analyses have revealed them to be polyphyletic. The strains examined belong to five independent clades within the Trebouxiophyceae (Choricystis-, Elliptochloris-, Auxenochlorella- and Chlorella-clades) and Chlorophyceae (Scenedesmus-clade). The most studied host organism, Paramecium bursaria, harbours endosymbionts representing at least five different species. On the basis of our results, we propose a taxonomic revision of endosymbiotic 'Chlorella'-like green algae. Zoochlorella conductrix K. Brandt is transferred to Micractinium Fresen. and Zoochlorella parasitica K. Brandt to Choricystis (Skuja) Fott. It was shown that Choricystis minor (Skuja) Fott, the generitype, is a later heterotypic synonym of Choricystis parasitica (K. Brandt) comb. nov. A new species, Chlorella heliozoae, is proposed to accommodate the endosymbiont of Acanthocystis turfacea.

All four Chlorella species, including one commercially available species, Chlorella vulgaris and three local isolates, Chlorella sp. (1 uoai), Chlorella sp. (2f5aia) and Chlorellaminiata (WW1), had a rapid and high ability to remove nonylphenol (NP). Among these species, C. vulgaris had the highest NP removal (nearly all NP was removed from the medium) and degradation abilities (more than 80% of NP was degraded) after 168 h, followed by WW1 and 1 uoai; 2f5aia had the lowest NP degradation ability. The NP removal by C. vulgaris was less affected by growth conditions, but its biodegradation efficiency was significantly increased by temperature and light intensity, suggesting that the biodegradation ability was positively related to photosynthetic and metabolic activities. These results indicated that C. vulgaris was the most suitable species for effective removal and biodegradation of NP, especially under 25 °C with light illumination and initial biomass between 0.5 and 1.0 mg chlorophyll l(-1)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The lipid contents and fatty acid profiles of Chlorella zofingiensis cultured in the dark with various carbon sources were investigated. Glucose was found to be the best carbon source for the growth and lipid production. When cultivated with 50gL(-1) glucose, C. zofingiensis accumulated lipids up to 52% of the dry biomass, with triacylglycerols (TAGs) accounting for 72.1% of the total lipids. Fatty acid profiles revealed that glucose contributed to the highest yield of total fatty acids (TFAs) and proportion of oleic acid (35.7% of TFAs), which corresponded to the strongest up-regulation of biotin carboxylase (BC) and stearoyl ACP desaturase (SAD) genes. In fed-batch cultivation based on glucose, the lipid yield and productivity of C. zofingiensis were further increased to 20.7gL(-1) and 1.38gd(-1)L(-1) respectively, representing 3.9-fold of those achieved in batch culture. We conclude that C. zofingiensis has great potential for biodiesel production.

Using a combined set of sequences of SSU and ITS regions of nuclear-encoded ribosomal DNA, the concept of the experimental algal genus Chlorella was evaluated. Conventionally in the genus Chlorella, only coccoid, solitary algae with spherical morphology that do not possess any mucilaginous envelope were included. All Chlorella species reproduce asexually by autospores. However, phylogenetic analyses showed that within the clade of 'true'Chlorella species (Chlorella vulgaris, C. lobophora, and C. sorokiniana), taxa with a mucilaginous envelope and colonial lifeform have also evolved. These algae, formerly designated as Dictyosphaerium, are considered as members of the genus Chlorella. In close relationship to Chlorella, five different genera were supported by the phylogenetic analyses: Micractinium (spherical cells, colonial, with bristles), Didymogenes (ellipsoidal cells, two-celled coenobia, with or without two spines per cell), Actinastrum (ellipsoidal cells within star-shaped coenobia), Meyerella (spherical cells, solitary, without pyrenoids), and Hegewaldia (spherical cells, colonial, with or without bristles, oogamous propagation). Based on the secondary structures of SSU and ITS rDNA sequences, molecular signatures are provided for each genus of the Chlorella clade.

Objective: Lycopene epsilon cyclase (LCYE) is the key enzyme in the lutein synthesis pathway and catalyses linear lycopene to form cyclic epsilon-carotene, a presucursor of lutein. We aimed to clone the full-length cDNA of LCYE gene from Chlorella protothecoides CS-41, to predict the functional sites and the three-dimensional structure of LCYE through bioinformatics analysis and to confirm its activities and functions., Method: We used RACE (rapid-amplification of cDNA ends) essay and RT-PCR for the cloning of the full-length cDNA of LCYE from C. protothecoides CS-41. The online software such as PredictProtein, Pfam HMMs and Swiss-Model were used in bioinformatics analysis of the amino acid sequence of LCYE protein. We constructed the expression vector for LCYE gene with pET-28a(+) and transformed into Escherichia coli BL21 (DE3). Furthermore, the E. coli strain containing the pAC-LYC plasmid which could accumulate lycopene was used for the functional confirmation of LCYE from C. protothecoides CS-41., Results: A 2107 bp cDNA (GenBank Accession No. FJ752528) sequence was cloned with 1731 bp open reading frame, encoding a putative LCYE, from C. protothecoides CS-41. Homology studies showed that the deduced amino acid sequence of LCYE gene had a significant similarity with the corresponding sequences of other green algae and higher plants. It shared the highest sequence identity, up to 67%, with the LCYE gene from Chlamydomonas reinhardtii. One typical lycopene cyclase protein domain (Pfam05834) was predicted between the 48th-459th amino acid. In addition, the sequence between 261th -284th was one typical conserved lycopene cyclase protein motif. The SDS-PAGE result showed that the LCYE gene was overexpressed in Escherichia coli BL21 (DE3) after the addition of IPTG. The prokaryotically expressed LCYE protein was able to transfer the color of the E. coli strain containing the pAC-LYC plasmid from pink to yellow., Conclusion: The full-length cDNA sequence of LCYE gene was successfully cloned with the size of 2107 bp. Several typical motifs were found and the three-dimensional structure of LCYE was constructed from Bioinformatics analysis. The generated phylogenetic tree showed the closest relationship between C. protothecoides CS-41 and C. reinhardtii among the listed organisms. Finally, the expression product of the LCYE gene cloned in the study was confirmed to hold the function and activity of lycopene epsilon cyclase.

Permanently low temperature environments are one of the most abundant microbial habitats on earth. As in most ecosystems, photosynthetic organisms drive primary production in low temperature food webs. Many of these phototrophic microorganisms are psychrophilic; however, functioning of the photosynthetic processes of these enigmatic psychrophiles (the "photopsychrophiles") in cold environments is not well understood. Here we describe a new chlorophyte isolated from a low temperature pond, on the Ross Ice Shelf near Bratina Island, Antarctica. Phylogenetic and morphological analyses place this strain in the Chlorella clade, and we have named this new chlorophyte Chlorella BI. Chlorella BI is a psychrophilic species, exhibiting optimum temperature for growth at around 10 degrees C. However, psychrophily in the Antarctic Chlorella was not linked to high levels of membrane-associated poly-unsaturated fatty acids. Unlike the model Antarctic lake alga, Chlamydomonas raudensis UWO241, Chlorella BI has retained the ability for dynamic short term adjustment of light energy distribution between photosystem II (PS II) and photosystem I (PS I). In addition, Chlorella BI can grow under a variety of trophic modes, including heterotrophic growth in the dark. Thus, this newly isolated photopsychrophile has retained a higher versatility in response to environmental change than other well studied cold-adapted chlorophytes.

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.

The most recent revision of the genus Chlorella, based on biochemical and SSU rDNA analyses, suggested a reduction to a set of four "true" spherical Chlorella species, while a growing number of morphologically different species such as Micractinium (formerly Micractiniaceae) were found to cluster within the clade of "true"Chlorella. In this study, the generic concept in Chlorellaceae to Chlorella and Micractinium was evaluated by means of combined SSU and ITS-2 rDNA sequence analyses and biotests to induce development of bristles on the cell wall. Molecular phylogenetic analyses of Chlorella and Micractinium strains confirmed their separation into two different genera. In addition, non-homoplasious synapomorphies (NHS) and compensatory base changes (CBC) in the secondary structures of SSU and ITS-2 rDNA sequences were found for both genera using this approach. The Micractinium clade can be differentiated into three different genotypes. Using culture medium of the rotifer Brachionus calyciflorus, phenotypic plasticity in Chlorella and Micractinium was studied. Non-bristled Micractinium cells developed bristles during incubation with Brachionus culture medium, whereas Chlorella did not produce bristles. Grazing experiments with Brachionus showed the rotifer preferred to feed on non-bristled cells. The dominance of colonies versus solitary cells in the Micractinium culture was not correlated with the "Brachionus factor". These results suggest that morphological characteristics like formation of bristles represent phenotypic adaptations to the conditions in the ecosystem.

Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to < 1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. A Cyanidioschyzon merolae sequence was predominant at temperatures of > or = 49 degrees C. A Chlorella protothecoides var. acidicola sequence and a Paradoxia multisita-like sequence were predominant at temperatures of < or = 39 degrees C.

Chlorella sorokiniana IAM C-212 has long been maintained in slant culture as a mixed strain, representing an associated natural microbial consortium. In this study, the consortium was separated and five nonalgal constituents, a fungal strain (CSSF-1), and four bacterial strains (CSSB-1, CSSB-2, CSSB-3, and CSSB-4) were isolated and identified. 16S rDNA sequence analysis revealed that strains CSSB-1, CSSB-2, CSSB-3, and CSSB-4 were close to Ralstonia pickettii (99.8% identity), Sphingomonas sp. DD38 (99.4% identity), Microbacterium trichotecenolyticum (98.6% identity), and Micrococcus luteus (98.6% identity) respectively. 18S rDNA sequence analysis revealed that strain CSSF-1 resembled Acremonium-like hyphomycete KR21-2 (98.8%). The fungal strain CSSF-1 and one of the bacterial strains, CSSB-3, were found to promote the growth of Chlorella while the presence of bacterial strains CSSB-1 and CSSB-2 had no effect. Strain CSSB-4 could not be subcultured so its role was not elucidated. These results show that the interaction between Chlorella and its symbionts under photoautotrophic conditions involved both mutualism and commensalisms. The chlorophyll content of mixed strain was stable in long-term cultivation (7 months) while the chlorophyll content of a pure culture showed a marked decline. Electron microscopic analysis showed the two bacterial strains CSSB-2 and CSSB-3 were harbored on the sheath excreted by Chlorella, while the fungal strain CSSF-1 and the bacterial strain CSSB-1 directly adhered to the Chlorella cell surface. This report is the first observation of a symbiotic relationship among fungus, bacteria, and Chlorella, and the first observation of direct adhesion of fungus and bacteria to Chlorella in a consortium.