Inhibition of abscisic acid biosynthesis inCercospora rosicola by triarimol

The fungicide triarimol was tested for its effect on abscisic acid (ABA) accumulation in growing culturesof Cercospora rosicola. ABA accumulation was reduced by approximately 50% with 10^?8 M triarimol. Growth ofC. rosicola, as measured by dry weight accumulation, was inhibited by triarimol concentrations at or greater than 10^?7 M. These results are compared with those obtained with clomazone, ancymidol, and paclobutrazol, which inhibit ABA accumulation by 50% at concentrations of 5 × 10^?5, 5 × 10^?6, and 5 × 10^?7 M, respectively. Triarimol, therefore, is among the most potent inhibitors of ABA biosynthesis reported to date. Feeding studies with [¹⁴C]mevalonic acid confirmed the inhibition of ABA biosynthesis by 5 × 10^?8 M triarimol. These results support previous suggestions that one or more of the steps in the ABA biosynthetic pathway from mevalonic acid is catalyzed by cytochrome P-450. Feeding studies with 1′-deoxy-[²H]-ABA in resuspended cultures ofC. rosicola show that the conversion of this substrate is not inhibited by triarimol.     

Optimization of pressurized liquid extraction of zeaxanthin from Chlorella ellipsoidea

The total zeaxanthin level in Chlorella ellipsoidea, a green microalga, was more than nine times that of red pepper, a plant source of zeaxanthin. Additionally, the zeaxanthin in C. ellipsoidea consisted of the free form, while those in other plants exist as zeaxanthin mono- and diesters. Pressurized liquid extraction (PLE) was used to extract zeaxanthin from C. ellipsoidea. Both the extraction temperature and extraction time, the two main factors in PLE, were optimized with a central composite design to obtain the highest extraction efficiency of zeaxanthin. Hexane, ethanol, and isopropanol were used as PLE extraction solvents. Ethanol extracted zeaxanthin most efficiently from C. ellipsoidea. Temperature was the parameter with the strongest influence on the extraction of zeaxanthin. The optimum extraction temperature and time for zeaxanthin were 115.4°C and 23.3?min, respectively. The maximum predicted value of 4.28?mg?g^?1 agreed with the experimental value of 4.26?mg?g^?1, supporting the quality of the fitted model. These results indicate that PLE using ethanol may be a useful method for extracting zeaxanthin from C. ellipsoidea.     

Efficiency of using green algae as biological controllers against toxic algal taxa in cultured Nile tilapia Oreochromis niloticus,based on histopathological examinations

The incidence of harmful cyanobacterial blooms in surface waters has increased in frequency and outbreaks have become more severe. This research aimed at studying the effect of a culture of two green algal species as biological control of the growth of toxic blue-green algae. Nile tilapia of an initial mean weight of 55 g fish^?1 (SE 5) were used for each of four treatments in triplicate. All algal seedings were done at 4 × 10³ cells ml^?1. Treatment I (untreated) served as a control, Treatment II was seeded with Microcystis aeruginosa, Treatment III was seeded with green algae Chlorella ellipsoidea and Scenedesmus bijuga, and Treatment IV was seeded with a mixture of M. aeruginosa and C. ellipsoidea and S. bijuga. After 10 days, Treatment IV showed 3.4% viable cell survival, compared to 35% and 55% in Treatments II and III, respectively. Histopathological examination revealed mild degenerative changes and focal necrosis, as well as a depletion of haematopoietic tissues in Treatment IV compared to Treatment II. These findings suggest the efficacy of C. ellipsoidea and S. bijuga in controlling the growth of M. aeruginosa and minimising its side effects on cultured Nile tilapia.     

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.     

Identification and localization of carbonic anhydrase in two chlorella species

Carbonic anhydrase (CA) activity and localization have been examined in two species of the eukaryotic green alga Chlorella. Mass spectrometric and potentiometric assays of CA activity indicate that C. ellipsoidea contains very little extracellular CA activity whereas C. saccharophila exhibits significant extracellular activity when grown at alkaline pH values. Extracellular CA activity appears to be correlated with the presence of a 36 kilodalton polypeptide that was detected immunologically using a polyclonal antibody directed against the 37 kilodalton Chlamydomonas CA monomer. Both Chlorella species and enzymatically isolated C. ellipsoidea chloroplasts also contain an immunologically similar 38 kilodalton polypeptide that may be a cytosolic or chloroplastic form of CA.     

Separation ofChlorella ellipsoidea fromC. saccharophila (Chlorophyceae): No growth on mannitol and cadmium sensitivity

This paper presents physiological evidence for the existence as separate taxonomic entities ofChlorella saccharophila andC. ellipsoidea. C. saccharophila is able to utilize mannitol for growth in the dark, whereasC. ellipsoidea is unable to grow on mannitol. In addition,C. ellipsoidea is characterized by an extreme sensitivity towards cadmium.—Among the otherChlorella species, onlyC. luteoviridis is capable of growing on mannitol.     

Microalgal growth enhancement by levoglucosan isolated from the green seaweed <Emphasis Type="Italic">Monostroma nitidum</Emphasis>

Microalgal growth was enhanced by the addition of levoglucosan to the culture medium. The growth-enhancing compound levoglucosan was isolated from the green seaweed Monostroma nitidum using water extraction, molecular fractionation, DEAE-cellulose column chromatography, and high-performance liquid chromatography. Yield of the compound from seaweed powder was 5 × 10⁻³% (w/w). At 10 mM concentration, levoglucosan enhanced cell growth and the specific growth rate of all feed microalgal species tested (Chaetoceros gracilis, Chlorella ellipsoidea, Dunaliella salina, Isochrysis galbana, Nannochloris oculata, Navicula incerta, Pavlova lutheri, Tetraselmis suecica) in most culture media by approximately 150%. Cellular fatty acid profiles and cell size differed marginally between cultures with and without levoglucosan.     

Microalgae growth on concentrated human urine

In this study, for the first time, a microalga was grown on non-diluted human urine. The essential growth requirements for the species Chlorella sorokiniana were determined for different types of human urine (fresh, hydrolysed, male and female). Batch experimental results using microtiter plates showed that both fresh and synthetic urine supported rapid growth of this species, provided additional trace elements (Cu, Fe, Mn, and Zn) were added. When using hydrolysed urine instead of fresh urine, additional magnesium had to be added as it precipitates during hydrolysis of urea. C. sorokiniana was able to grow on non-diluted urine with a specific growth rate as high as 0.104 h^?1 under light-limited conditions (105 μmol photons m^?2 s^?1), and the growth was not inhibited by ammonium up to a concentration of 1,400 mg NH₄ ⁺-N L^?1. The highest growth rate on human urine was as high as 0.158 h^?1. Because it was demonstrated that concentrated urine is a rich and good nutrient source for the production of microalgae, its application for a large-scale economical and sustainable microalgae production for biochemicals, biofuels and biofertilizers becomes feasible.     

Inhibition of abscisic acid biosynthesis inCercospora rosicola by triarimol

The fungicide triarimol was tested for its effect on abscisic acid (ABA) accumulation in growing culturesof Cercospora rosicola. ABA accumulation was reduced by approximately 50% with 10^–8 M triarimol. Growth ofC. rosicola, as measured by dry weight accumulation, was inhibited by triarimol concentrations at or greater than 10^–7 M. These results are compared with those obtained with clomazone, ancymidol, and paclobutrazol, which inhibit ABA accumulation by 50% at concentrations of 5 × 10^–5, 5 × 10^–6, and 5 × 10^–7 M, respectively. Triarimol, therefore, is among the most potent inhibitors of ABA biosynthesis reported to date. Feeding studies with [¹⁴C]mevalonic acid confirmed the inhibition of ABA biosynthesis by 5 × 10^–8 M triarimol. These results support previous suggestions that one or more of the steps in the ABA biosynthetic pathway from mevalonic acid is catalyzed by cytochrome P-450. Feeding studies with 1-deoxy-[²H]-ABA in resuspended cultures ofC. rosicola show that the conversion of this substrate is not inhibited by triarimol.     

Effect of cement industry flue gas simulation on the physiology and photosynthetic performance of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Cement plants account for significant emissions of CO₂ and other pollutants into the atmosphere. As a means for its mitigation, we tested the effect of a cement industry-based flue gas simulation (FGS — 18% CO₂, 9% O₂, 300 ppm NO₂, 140 ppm SO₂) on the green alga, Chlorella sorokiniana. Culture pH, cell density, cell viability and productivity, specific growth rates, photosynthetic performance, and biochemical composition were monitored. The treatments consisted of different FGS volumes (0.1, 0.3, 0.8, 1.5, 6, and 48 L day^?1) that were applied in a series of laboratory-scale semi-continuous batch cultures under controlled conditions. Controls were exposed to 18% CO₂ enriched air. Cell density showed that C. sorokiniana was able to grow in all treatments, but compared to the controls, low pH (~ 5.0) caused by 48 L FGS day^?1 led to 27% decrease in specific growth rate. Increasing FGS exposure decreased maximum and operational quantum yields obtained by pulse amplitude modulated fluorometry, while photochemical quenching remained constant (~ 0.93). The α and rETR _max parameters calculated from rapid light curves decreased with increasing FGS exposure. Total proteins and carbohydrates (per cell basis) increased after 6 and 48 L FGS day^?1, which can be advantageous for biotechnological applications, but cell productivity (cells L^?1 day^?1) decreased. Despite the effects in physiology, C. sorokiniana could withstand a pH range of 6.0–5.0 imposed by 48 L FGS day^?1. Overall, C. sorokiniana can be considered a robust species in flue gas bioremediation.     

Development of a New Method for Genetic Transformation of the Green Alga Chlorella ellipsoidea

Chlorella ellipsoidea is a single-celled eukaryotic green microalgae with high nutritional value. Its value may be further increased if a simple, reliable and cost-effective transformation method for C. ellipsoidea can be developed. In this paper, we describe a novel transformation method for C. ellipsoidea . This system is based on treatment of C. ellipsoidea cells with cellulolytic enzymes to weaken their cell walls, making them become competent to take up foreign DNA. To demonstrate the usefulness and effectiveness of this method, we treated C. ellipsoidea cells with a cell wall-degrading enzyme, cellulase, followed by transformation with plasmid pSP-Ubi-GUS harbouring both the zeocin resistance gene and the beta-glucuronidase (GUS) reporter gene that serve as selective makers for transformation. Transformants were readily obtained on zeocin selection medium, reaching transformation efficiency of 2.25 × 10³ transformants/μg of plasmid DNA. PCR analysis has also demonstrated the presence of the GUS reporter gene in the zeocin-resistant transformants. Histochemical assays further showed the expression of the GUS activity in both primary transformants and transformants after long-term growth (10 months) with antibiotic selection on and off. Availability of a simple and efficient transformation system for C. ellipsoidea will accelerate the exploration of this microalga for a broader range of biotechnological applications, including its use as a biologic factory for the production of high-value human therapeutic proteins.     

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.     

Morphological and phylogenetic study of algal partners associated with the lichen-forming fungus Porpidia crustulata from the Guancen Mountains, northern China

The algal photobiont of the lichen Porpidia crustulata from the Guancen Mountains was successfully cultivated under axenic conditions and tentatively designated as Chlorella sp. GC. The phylogenetic analysis of ribulose bisphosphate carboxylase (rbcL) and internal transcribed spacer (ITS) nucleotide sequence data revealed that this alga is closely related to C. sorokiniana. There are clear morphological differences between Chlorella sp. GC associated with Porpidia crustulata and the free-living C. sorokiniana, the former having a larger central pyrenoid. Based on our genetic and morphological characterization, the Chlorella photobiont of Porpidia crustulata is distinct from closely related Chlorella species.     

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.     

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.

     

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.     

Characterization of Nitrate Reductase Deficient Mutants of Chlorella sorokiniana

After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO₃⁻ as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH₄⁺-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein.     

Active uptake of inorganic carbon by Chlorella saccharophila is not repressed by growth in high CO2

Regulation of transport of dissolved inorganic carbon (DIC)in response to CO₂ concentration in the external medium hasbeen compared in two closely-related green algae, Chlorellaellipsoidea and Chlorella saccharophila. C. ellipsoidea, whengrown in high CO₂, had reduced activities of both CO₂ and transport and DIC transport activitieswere increased after the cells had acclimated to air. However,high CO₂-grown C. saccharophila had a comparable level of photosyntheticaffinity for DIC to that of air-grown C. ellipsoidea and thiswas accompanied by a capacity to accumulate high internal concentrationsof DIC. The high photosynthetic affinity and the high intracellularDIC accumulation did not change in cells grown in air exceptthat the occurrence of external carbonic anhydrase (CA) in air-grownC. saccharophila stimulated the intracellular DIC accumulationin the absence of added CA. These data indicate that activeDIC transport is constitutively expressed in C. saccharophila,presumably because this alga is insensitive to the repressiveeffect of high CO₂ on DIC transport. This strongly supportsthe existence of a direct sensing mechanism for external CO₂in Chlorella species, but also indicates that external CA isregulated independently of DIC transport in Chlorella species. Key words: Carbonic anhydrase, Chlorella, CO₂-insensitive, DIC transport, wild type