Relationship between the algal partners and the growth of lichen-forming fungus Porpidia crustulata

In recent years, examinations based on morphological characters and phylogenetic analyses have begun to reveal the diversity of photobionts in lichen symbioses. However, still little is known regarding genetic diversity and ecological adaptation of algal partners in lichen symbioses. In this study, we investigated the photobiont Chlorella “sp. GC” (Trebouxiophyceae), a partner of Porpidia crustulata from the Guancen Mountains, China. We examined the relationship between photobiont layer thickness and Porpidia crustulata growth over a 6-year period (2007–2012). Although Porpidia crustulata exhibited moderate growth rates (0.4–0.62 mm year^?1), photobiont layer became increasingly thinner over the six-year period. We speculate that prolonged exposure to sunlight and desiccation may deleteriously alter photobiont morphology and physiology. Porpidia crustulata may be forced into a state of physiological dormancy.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

The genetic relationships of nine strains of Chlorella saccharophila were determined by DNA hybridization techniques. Four strains are closely related to the type strain 211-9a and one strain seems to be moderately related, whereas the taxonomic position of the remaining three strains is not clear. C. saccharophila, like C. sorokiniana, is another species of Chlorella containing strains which are heterogeneous in their overall DNA base sequence and partly also in morphological, biochemical and physiological characters.     

Effect of triarimol on sterol and fatty acid composition of three species of Chlorella

The concentration of triarimol giving ca 50% inhibition of growth was different for each of 3 species of Chlorella [C. emersonii, 1 mg/l. (1.5 × 10^?6 M), C. ellipsoidea 10 mg/l. (3 × 10^?5 M), C. sorokiniana, 2 mg/l. (6 × 10^?6 M)]. The total lipid of 3 species of Chlorella grown in a culture medium containing triarimol were analysed for chlorophyll, fatty acids and sterol composition. Growth rates were studied in the presence of different concentrations of triarimol. The growth rates of the 3 species were differentially inhibited by triarimol. The growth of Chlorella sorokiniana was 50% inhibited by 2 mg/l. triarimol but 20 mg/l. did not produce a cessation of growth. The greatest inhibition of growth rates and chlorophyll content was observed in Chlorella emersonii. The quantity of unsaturated fatty acids was increased by triarimol treatment in all 3 species of Chlorella. Triarimol strongly inhibited 14α-demethylation in Chlorella emersonii, and C. ellipsoidea and less in C. sorokiniana, resulting in accumulation of 14α-methyl sterols. Triarimol also inhibited the second alkylation of the side chain in C. ellipsoidea and C. emersonii. The introduction of the 22-double bond was inhibited in all 3 species of Chlorella studied. Although some differences were apparent, the effect of triarimol was quite similar to that of triparanol and AY-9944 in these 3 species of Chlorella.     

Upper limits of temperature for growth inChlorella (Chlorophyceae)

The upper limit of temperature for growth is a species-specific character in the genusChlorella. The limits of 14Chlorella species range from 26–30°C (C. saccharophila) to 38–42°C (C. sorokiniana), withC. fusca var.vacuolata (34°C) andC. kessleri (34–36°C) assuming an intermediate position. Thus, there is no wide gap in the temperature limits between the normal (“low-temperature”) species ofChlorella and the “high-temperature” species,C. sorokiniana.     

Nitrogen Starvation Induced Oxidative Stress in an Oil-Producing Green Alga Chlorella sorokiniana C3

Microalgal lipid is one of the most promising feedstocks for biodiesel production. Chlorella appears to be a particularly good option, and nitrogen (N) starvation is an efficient environmental pressure used to increase lipid accumulation in Chlorella cells. The effects of N starvation of an oil-producing wild microalga, Chlorella sorokiniana C3, on lipid accumulation were investigated using thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). The results showed that N starvation resulted in lipid accumulation in C. sorokiniana C3 cells, oil droplet (OD) formation and significant lipid accumulation in cells were detected after 2 d and 8 d of N starvation, respectively. During OD formation, reduced photosynthetic rate, respiration rate and photochemistry efficiency accompanied by increased damage to PSII were observed, demonstrated by chlorophyll (Chl) fluorescence, 77K fluorescence and oxygen evolution tests. In the mean time the rate of cyclic electron transportation increased correspondingly to produce more ATP for triacylglycerols (TAGs) synthesis. And 0.5 d was found to be the turning point for the early stress response and acclimation of cells to N starvation. Increased level of membrane peroxidation was also observed during OD formation, and superoxide dismutase (SOD), peroxide dismutase (POD) and catalase (CAT) enzyme activity assays suggested impaired reactive oxygen species (ROS) scavenging ability. Significant neutral lipid accumulation was also observed by artificial oxidative stress induced by H₂O₂ treatment. These results suggested coupled neutral lipid accumulation and oxidative stress during N starvation in C. sorokiniana C3.     

Unveiling antimicrobial activity of microalgae Chlorella sorokiniana (UKM2), Chlorella sp. (UKM8) and Scenedesmus sp. (UKM9)

Microalgae represent promising sources of bioactive compounds for pharmaceutical and industrial applications. The emergence of antibiotic resistant bacteria leads to the need to explore new cost-effective, safe, and potent bioactive compounds from the microalgae. This study aimed to investigate the potential of local microalgae for their antimicrobial properties and bioactive compounds. Three local microalgae namely Chlorella sorokiniana (UKM2), Chlorella sp. UKM8, and Scenedesmus sp. UKM9 biomass methanol extracts (ME) were prepared and tested against Gram-positive and Gram-negative bacteria. Chlorella sp. UKM8-ME showed the highest antibacterial activity. UKM8-ME minimum inhibitory concentrations were in the range of 0.312 to 6.25 mg/mL. Cytotoxicity evaluation using MTT assay showed that the microalgae methanolic extracts did not exhibit cytotoxicity against Vero-cells. The UKM8-ME was mainly containing 28 compounds from the Gas Chromatography-Mass Spectrometry (GC–MS) analysis. Major compounds of UKM8-ME included phenol (18.5%), hexadecanoic acid (18.25%), phytol (14.43%), 9,12-octadecadienoic acid (13.69%), and bicyclo[3.1.1]heptane (7.23%), which have been previously described to possess antimicrobial activity. Hence, Chlorella sp. (UKM8) methanol extracts showed promising antibacterial activity. More comprehensive studies are required to purify these antimicrobial compounds and develop our understanding on their mechanism in UKM8-ME to unleash their specific potential.     

Molecular characterization of Chlorella cultures of the National Institute for Environmental Studies culture collection with description of Micractinium inermum sp. nov., Didymogenes sphaerica sp. nov., and Didymogenes soliella sp. nov. (Chlorellaceae,Trebouxiophyceae)

Chlorella Beijerinck (Chlorellaceae, Trebouxiophyceae) strains from the collection of the National Institute for Environmental Studies (NIES) were characterized using gene sequence data. The misidentification of a number of strains was rectified. Chlorella vulgaris Beijerinck NIES‐2173 was reclassified as C. sorokiniana Shihira et Krauss. Chlorella sp. NIES‐2171 was described as a new species in the genus Micractinium Fresinius, M. inermum Hoshina et Fujiwara. Chlorella sorokiniana NIES‐2167 and Chlorella sp. NIES‐2330 were found to be phylogenetically related to Didymogenes Schmidle. We propose these two strains be transferred to the genus Didymogenes and given new names: D. sphaerica Hoshina et Fujiwara and D. soliella Hoshina et Fujiwara. Taxonomic decisions were primarily based on small subunit‐internal transcribed spacer ribosomal DNA phylogeny for genus assignment and ITS2 sequence‐structure to determine species autonomy. Our findings suggest that this strategy is the most effective way to use the species concept among autosporic coccoids.     

The Chlorella variabilis NC64A Genome Reveals Adaptation to Photosymbiosis,Coevolution with Viruses,and Cryptic Sex

Chlorella variabilis NC64A, a unicellular photosynthetic green alga (Trebouxiophyceae), is an intracellular photobiont of Paramecium bursaria and a model system for studying virus/algal interactions. We sequenced its 46-Mb nuclear genome, revealing an expansion of protein families that could have participated in adaptation to symbiosis. NC64A exhibits variations in GC content across its genome that correlate with global expression level, average intron size, and codon usage bias. Although Chlorella species have been assumed to be asexual and nonmotile, the NC64A genome encodes all the known meiosis-specific proteins and a subset of proteins found in flagella. We hypothesize that Chlorella might have retained a flagella-derived structure that could be involved in sexual reproduction. Furthermore, a survey of phytohormone pathways in chlorophyte algae identified algal orthologs of Arabidopsis thaliana genes involved in hormone biosynthesis and signaling, suggesting that these functions were established prior to the evolution of land plants. We show that the ability of Chlorella to produce chitinous cell walls likely resulted from the capture of metabolic genes by horizontal gene transfer from algal viruses, prokaryotes, or fungi. Analysis of the NC64A genome substantially advances our understanding of the green lineage evolution, including the genomic interplay with viruses and symbiosis between eukaryotes.     

Deep genomic analysis of the Chlorella sorokiniana SAG 211-8k chloroplast

The chloroplast genome contains information that is applicable in many scientific fields, such as plant systematics, phylogenetic reconstruction and biotechnology, because its features are highly conserved among species. To date, several complete green algal chloroplast genomes have been sequenced and assembled. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Chlorella sorokiniana SAG 211-8k is reported and compared for the first time to the chloroplast genomes of 10 Chlorellaceae. The recently updated Chlorella sorokiniana cpDNA sequence, assembled as a circular map of 109?811 bp, encodes 113 genes. Similar to other Chlorella strains, this chloroplast genome does not show a quadripartite structure and lacks the large rRNA operon-encoding Inverted Repeat (IR). The Chlorella sorokiniana plastid encodes the tRNA(Ile)-lysidine synthetase (tilS), which is responsible for modifying the CAU anticodon of a unique tRNA. Gene ordering and clustering highlight the close relationships among Chlorella clade members and the preservation of crucial gene clusters in photosynthetic strains. The features of Chlorella sorokiniana presented here reinforce the monophyletic character of Chlorellaceae and provide important information that sheds light on chloroplast genome evolution among species of Chlorella.     

On the characteristics and taxonomic position of symbiotic Chlorella

Three strains of symbiotic Chlorella (NC64A, CE/76, UTEX-130) could be assigned to C. vulgaris by physiological and DNA-hybridization studies and a fourth symbiotic strain, 3N8/13-1, exhibited characteristics intermediate between C. vulgaris and C. sorokiniana. None of the strains contained detectable sporopollenin or was nutritionally fastidious but the capacity for sugar release (usually maltose) may be a characteristic of all symbiotic Chlorella.     

Recognition mechanisms during the pre-contact state of lichens: I. Mycobiont-photobiont interactions of the mycobiont of Fulgensia bracteata

Lichens are an association of a photoautotrophic alga/cyanobacteria (photobiont) and a heterotrophic fungus (mycobiont) constituting the lichen thallus as a complex phenotype. Many mycobionts reproduce sexually and the ascospores are dispersed without the photobiont. For successful re-lichenization the specific photobiont must be recognized, contacted, and incorporated by the mycobiont. A so-called pre-contact stage has been postulated as the initial step of a gradual recognition process. In the present study, the effect of the specific Trebouxia photobiont, an unspecific Asterochloris photobiont and the non-lichenizing green alga Myrmecia bisecta on the development of the mycobiont Fulgensia bracteata was assessed by pre-contact assays. Three hypotheses were confirmed: (i) the pre-contact stage exists, (ii) it is characterized by morphological reactions in the development of the mycobiont, and (iii) the reactions depend on the interacting alga. Control conditions revealed a mycelial growth arrest but this effect was not observed in the presence of any of the three algae. Different algae induce distinct growth patterns with respect to hyphal length, morphological characteristics, and formation of mucilage. The specific Trebouxia photobiont had a positive impact on hyphal growth, branching frequency, and mucilage formation. These effects were less explicit with the non-specific Asterochloris photobiont. Myrmecia bisecta induced uncharacteristic growth patterns with pronounced hyphal growth and high numbers of aerial hyphae but less formation of mucilage. These results indicate that symbiont recognition mechanisms are established before physical contact. Pre-contact reactions may be an evolutionary advantage that supports the persistence of the mycobiont on newly colonized sites and improves the probability of re-lichenization.     

A Constitutive Enzyme System for Glucose Transport by Chlorella sorokiniana

It was found that the transport system for glucose (as measured by deoxyglucose uptake) in the high temperature strain of Chlorella (strain 07-11-05 or C. sorokiniana) was constitutive and the rate of uptake did not increase upon incubation of autotrophically grown cells with either deoxyglucose or glucose. The uptake obeyed Michaelis-Menten type kinetics with a concentration of 200 micromolar for half-saturation. The maximum rate of uptake was nearly 10 times faster per cell (at 38 C) than that reported for any other Chlorella. This rapid accumulation of deoxyglucose causes the passive efflux to become significant compared to the pump-driven influx and nonlinear uptake appears even after only 3 to 4 minutes.     

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.     

Optimization of Chlorella biomass harvesting by flocculation and its potential for biofuel production

Optimization of microalgal biomass harvesting is essential to produce effective and optimum outcomes that can contribute towards a feasible and economical harvesting technique. Two Chlorella species were used, namely, C. vulgaris and C. sorokiniana UKM3. Two essential factors affecting microalgal biomass harvesting via flocculation, namely, the initial pH of the microalgal broth and flocculant (chitosan) concentration were studied. The optimization process was conducted by using a response surface methodology (RSM) based on the model of face-centered-central composite design (FC-CCD). The potential for biofuel application of the harvested biomass was evaluated based on the production of fatty acid methyl esters (FAMEs) by transesterification. The quadratic models obtained from the RSM significantly fitted the experiment data as the p-values were less than 0.05. The initial pH of the microalgal suspension was found to have a more significant effect on the flocculation process than flocculant concentration. For C. vulgaris, the highest flocculant efficiency of 98.7% was obtained at a chitosan concentration of 0.2 g L^?1 and an initial pH of 12.0, whereas for C. sorokiniana UKM3, at 0.15 g L^?1 of chitosan and initial pH of 12.0 produced the highest efficiency of 97.1%. The harvested biomass of both species exhibited a high content of palmitic acid (C16:0) with 29.74 wt% and 11.81 wt% of dry biomass for C. vulgaris and C. sorokiniana UKM3, respectively. This study showed that Chlorella species can be harvested efficiently using the flocculation process and manifested an excellent potential for biodiesel production where palmitic acid (C16:0) is one of the main compounds for high-acid oil-biodiesel.

     

Infectivity of Chlorella species for the ciliate Paramecium bursaria is not based on sugar residues of their cell wall components, but on their ability to localize beneath the host cell membrane after escaping from the host digestive vacuole in the early infection process

Summary. Paramecium bursaria cells harbor several hundred symbiotic algae in their cytoplasm. Algae-free cells can be reinfected with algae isolated from algae-bearing cells or cultivated Chlorella species through the digestive vacuoles. To determine the relationship between the infectivity of various Chlorella species and the nature of their cell wall components, algae-free P. bursaria cells were mixed with 15 strains of cultivated Chlorella species and observed for the establishment of endosymbiosis at 1 h and 3 weeks after mixing. Only 2 free-living algal strains, C. sorokiniana C-212 and C. kessleri C-531, were maintained in the host cells, whereas free-living C. sorokiniana C-43, C. kessleri C-208, C. vulgaris C-27, C. ellipsoidea C-87 and C-542, C. saccharophila C-183 and C-169, C. fusca var. vacuolata C-104 and C-28, C. zofingiensis C-111, and C. protothecoides C-150 and C-206 and the cultivated symbiotic Chlorella sp. strain C-201 derived from Spongilla fluviatilis could not be maintained. These infection-incapable strains could escape from the host digestive vacuole but failed to localize beneath the host cell membrane and were eventually digested. Labeling of their cell walls with Alexa Fluor 488-conjugated wheat germ agglutinin, GS-II, or concanavalin A, with or without pretreatment with 0.4 N NaOH, showed no relationship between their infectivity and the stainability with these lectins. Our results indicate that the infectivity of Chlorella species for P. bursaria is not based on the sugar residues on their cell wall and on the alkali-insoluble part of the cell wall components, but on their ability to localize just beneath the host cell membrane after escaping from the host digestive vacuole. Correspondence and reprints: Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Cytological,genetic, and biochemical characteristics of an unusual non‐Chlorella photobiont of Stentor polymorphus collected from an artificial pond close to the shore of Lake Biwa,Japan

Stentor polymorphus (Müller) Ehrenberg (Heterotrichea, Ciliophora) is a trumpet‐form ciliate with endosymbiotic green algae (photobionts). Previous reports have indicated that this photobiont has a form typical of Chlorella Beijerinck (Trebouxiophyceae), with one chloroplast and a distinct pyrenoid. We collected S. polymorphus from an artificial pond on the shore of Lake Biwa in Japan. Microscopic examination demonstrated that there was no pyrenoid in the photobiont, and thus we analyzed the cytological, genetic, and biochemical characteristics of these algal cells. Cultured photobionts were smaller than those in hospite. Phylogenetic analyses placed the photobiont within the genus Mychonastes Simpson and Van Valkenburg, a chlorophycean alga occurring in fresh/brackish waters that lacks pyrenoids. Mychonastes provided little photosynthate to its host; the quantity of carbohydrate amounted to only 2% of that provided to Paramecium bursaria (Ehrenberg) Focker by its photobiont.     

Symbiosis as a successful strategy in continental Antarctica: performance and protection of Trebouxia photosystem II in relation to lichen pigmentation

Lichens as symbiotic associations consisting of a fungus (the mycobiont) and a photosynthetic partner (the photobiont) dominate the terrestrial vegetation of continental Antarctica. The photobiont provides carbon nutrition for the fungus. Therefore, performance and protection of photosystem II is a key factor of lichen survival. Potentials and limitations of photobiont physiology require intense investigation to extend the knowledge on adaptation mechanisms in the lichen symbiosis and to clarify to which extent photobionts benefit from symbiosis. Isolated photobionts and entire lichen thalli have been examined. The contribution of the photobiont concerning adaptation mechanisms to the light regime and temperature conditions was examined by chlorophyll a fluorescence and pigment analysis focusing on the foliose lichen Umbilicaria decussata from North Victoria Land, continental Antarctica. No photoinhibition has been observed in the entire lichen thallus. In the isolated photobionts, photoinhibition was clearly temperature dependent. For the first time, melanin in U. decussata thalli has been proved. Though the isolated photobiont is capable of excess light protection, the results clearly show that photoprotection is significantly increased in the symbiotic state. The closely related photobiont of Pleopsidium chlorophanum, a lichen lacking melanin, showed a higher potential of carotenoid-based excess light tolerance. This fact discriminates the two photobionts of the same Trebouxia clade. Based on the results, it can be concluded that the successful adaptation of lichens to continental Antarctic conditions is in part based on the physiological potential of the photobionts. The findings provide information on the success of symbiotic life in extreme environments.     

Phylogenetic relationships ofSphaerophoraceae (Ascomycetes) inferred from SSU rDNA sequences

SSU rDNA was sequenced from the lichenized fungiBunodophoron scrobiculatum andLeifidium tenerum (Sphaerophoraceae), andStereocaulon ramulosum andPilophorus acicularis (Stereocaulaceae) and analysed by maximum parsimony with 44 homologous ascomycete sequences in a cladistic study. A small insertion (c. 60 nt.) was found in the sequence ofLeifidium tenerum. Sphaerophoraceae constitutes a strongly supported monophyletic group which groups together withLecanora dispersa and theStereocaulaceae. Together withPorpidia crustulata, this larger group is a sistergroup to thePeltigerineae. This analysis thus supports theLecanorales as monophyletic, includingSphaerophoraceae and thePeltigerineae, but does not provide strong support for this monophyly. The analysis also suggests that the prototunicate ascus in theSphaerophoraceae is a reversion to the plesiomorphic state. Based on morphological, anatomical and chemical reasons,Sphaerophoraceae is proposed to belong to one of the groups presently included in the paraphyletic suborderCladoniineae within theLecanorales.     

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.