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PubMed1977

Studies on the purification of wastes from the nitrogen fertilizer industry by intensive algal cultures. IV. growth of Chlorella vulgaris in wastes with high nitrogen content in continuous and intermittent light.

The growth and utilization of nitrogen by intensive Chlorella vulgaris in wastes from production of urea, containing 1300 mg NH4+-N and 4000 mg urea-N/1, was investigated. In these conditions only Chlorella vulgaris AA strain, adapted to high concentrations of ammonia nitrogen, was able to grow. The elimination of nitrogen by continuous cultures was 750 mg urea-N/1 with 5-day flow rate. A considerable part of the urea was hydrolized by urease bacteria and removed in the form of NH3. The effect of intermittent light on the growth of algae was also studied. The better growth than in continuous light, was obtained with alternate one hour periods of light and darkness. Good results were also obtained with the use of 12 hour light and 12 hour darkness.

Ammonia↗
PubMed1981

Purification and characterization of demolybdo nitrate reductase (NADH-cytochrome c oxidoreductase) of Chlorella vulgaris.

Chlorella vulgaris was cultured on an ammonia-mineral salts medium until the nitrate reductase content reached a minimal level. These ammonia-grown cells were then induced by nitrate in the absence of molybdenum and of tungsten. A demolybdo nitrate reductase developed and reached high levels. This protein contained very little nitrate-reducing capacity, but had the full cytochrome c-reducing capacity of normal nitrate reductase. It was purified to homogeneity by the same procedures previously developed for the purification of nitrate reductase. The purified enzyme contained 1 molecule of heme and 1 molecule of FAD/subunit, but no detectable molybdenum or tungsten. This cytochrome c reductase was completely inhibited by antibodies raised against purified nitrate reductase of Chlorella. Mixtures prepared from normal nitrate reductase and the demolybdoenzyme could not be resolved by disc gel electrophoresis or by centrifugation in a density gradient. By a two-step enzyme induction (1, incubation with nitrate in absence of Mo; 2, incubation with Mo in absence of nitrate) the process of nitrate reductase synthesis could be cleanly separated from growth into two steps: Step 1, induction of cytochrome c reductase, was completely inhibited by cycloheximide. Step 2 was unaffected by cycloheximide, and most of the nitrate reductase synthesized accumulated in the form of the reversibly inactivated HCN complex of the enzyme.

Chlorella↗
PubMed1982

Quaternary structure of assimilatory NADH:nitrate reductase from Chlorella.

Gel chromatography experiments over a wide range of protein concentrations showed that Chlorella nitrate reductase is a nonassociating protein with a Stokes radius of 81 A. Sedimentation equilibrium of nitrate reductase in H2O-D2O solvents yielded a partial specific volume of 0.800 +/- 0.014 (n = 12) and a Mr = 360,000 +/- 25,000. No lipid was found associated with nitrate reductase. Cross-linking with the bifunctional reagent, dimethyl suberimidate, and subsequent separation of products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded four protein-staining bands in which the molecular weights of the cross-linked products were integral multiples of the monomeric molecular weight (90,000). Extensive cross-linking of the enzyme resulted in one principal protein-staining band of 360,000, corresponding to a tetramer. The cross-linked tetramer of nitrate reductase appeared to have identical physical properties as the native enzyme. The cross-linking pattern produced by reaction with dimethyl suberimidate suggested that nitrate reductase is an isologous tetramer which has at least two different types of bonding domains. Gel filtration, sedimentation equilibrium, and density gradient experiments at very low enzyme concentrations indicated that nitrate reductase dissociates to a species with a Stokes radius of 54 A, s20.w of 7.1, and Mr = approximately 200,000 at these low enzyme concentrations. No change in specific activity of the enzyme was observed over this concentration range. Treatment of nitrate reductase with trypsin or with cyanogen bromide yielded the number of peptides expected for identical subunits. From these results, it is concluded that Chlorella nitrate reductase is a homotetramer with dihedral symmetry ("dimer of dimers").

Chlorella↗
PubMed1978

[Ferredoxin-dependent glutamate synthase of Chlorella].

Ferredoxin-dependent glutamate synthase has been found in cells of thermophylic Chlorella strain Ch. pyrenoidosa 82T. The enzyme is active with its own ferredoxin and that of Spirulina. Glutamate synthase activity increases during nitrogen starvation and than decreases in the course of successive ammonium assimilation. The scheme of ammonium assimilation in Chlorella pyrenoidosa 82T cells is proposed.

Chlorella↗
PubMed1980

Toxic effects of cobalt and zinc to Chlorella pyrenoidosa (26) in soft and hard water.

Different concentrations (0.83, 3.33, 10 ppm) of cobalt (cobalt sulphate) and zinc (zinc chloride) were added to bristol media prepared using soft water (addition of 20 mg/l calcium carbonate) and hard water (addition of 400 mg/l calcium carbonate). A known amount of Chlorella pyrenoidosa (26) was inoculated into various media, and the number of Chlorella cells was counted every other day for a period of 8 days. Cobalt and zinc were more toxic in media prepared using soft water than hard water as revealed by the growth rate of C. pyrenoidosa. These results agree with studies elsewhere on other aquatic organisms.

Calcium Carbonate↗
PubMed1993

Expression and characterization of the heme-binding domain of Chlorella nitrate reductase.

A recombinant protein corresponding to the putative heme-binding domain of assimilatory NADH:nitrate reductase from Chlorella vulgaris has been expressed and purified from transformed Escherichia coli BL21 cells. The recombinant protein, exhibited a subunit molecular mass of approximately 10 kDa with a N-terminal sequence beginning with the residues PAGA in agreement with that predicted by cDNA analysis. The UV-visible spectrum of the protein confirmed the incorporation of heme with maxima at 413 nm and 423, 528, and 557 nm for the oxidized and reduced forms, respectively. Circular dichroism spectra indicated the environment of the heme chromophore was very similar to that of the native enzyme. Potentiometric titrations of the recombinant heme domain yielded a midpoint potential of +16 mV (n = 1, pH 7), substantially higher than the values of -160 mV obtained for the native enzyme and -28 mV obtained for a previously expressed recombinant heme domain that contained part of the Mo-pterin domain. These results indicate that portions of the amino acid sequence that are involved in the formation of the Mo-pterin domain of Chlorella nitrate reductase influence the redox potential of the heme prosthetic group.

Base Sequence↗
PubMed1995

[Warfarin therapy and chlorella].

Warfarin potassium (warfarin) is one of anticoagulants. Its anticoagulant effect is induced by inhibiting vitamin K in a complex manner. It is used effectively and safely in preventing cerebral embolism of cardiac origin, provided that thrombotest (TT) values is maintained from 10 to 20%. A 75-year-old man with atrial fibrillation and cardiomyopathy had cerebral embolism in the territory of the right middle cerebral artery. Warfarin therapy was started to prevent the recurrence of embolic stroke. TT values had been well controlled until intake of chlorella, but they rose above the therapeutic limit after its intake. There was no evidence of discontinuing warfarin, or taking drugs containing vitamin K or Natto. Chlorella is one of vitamin K-rich foods. Thus, it may inhibit the anticoagulant effect of warfarin.

Aged↗
PubMed1995

Telomere and LINE-like elements at the termini of the Chlorella chromosome I.

The telomeres of Chlorella chromosomes consisted of 5'-TTTAGGG repeats, which are exactly the same as those of higher plants. This sequence was reiterated approximately 70 times at both termini of chromosome I. Subtelomeric sequences next to the telomeres were totally different between the right and left arms. On the left-side subtelomeric region, polyA associated LINE (long interspersed element)-like elements were found tandemly repeated just next to the telomeric repeats. It is very interesting to compare this unique structure with telomeres of Drosophila, where a transposable element play a major role in forming telomerase-generated repeats. We propose a mechanism of transposon-mediated healing of a broken chromosomal end that would operate in the unicellular green alga Chlorella.

Animals↗
PubMed1995

Molecular characterization of Chlorella chromosomes: screening of bent DNAs.

As a model for plant-type chromosomes, we have been characterizing molecular organization of Chlorella vulgaris (C-169) chromosome I. To locate structurally important chromosome regions, bent DNA fragments of this chromosome were systematically obtained and analyzed. By two-dimensional polyacrylamide gel electrophoresis, 150 clones containing bent DNA fragments were screened from C. vulgaris genomic libraries. These clones were classified into 3 groups by Southern hybridization. A group that hybridized with all Chlorella chromosomes contained fragments carrying a retroposon-like element. A clone (p2DS-66A) derived from chromosome VI strongly hybridized with chromosome I as well as the others. The corresponding chromosome I sequences (pI-21 and pI-31) were analyzed in detail.

Base Sequence↗
PubMed1976

[Effect of univalent cations on the glutamate dehydrogenase of chlorella].

Effect of univalent cations (Li+, K+, Na+ and Cs+) on the activity and some kinetic properties of the constitutive and the inducible glutamate dehydrogenases (GDH) of Chlorella pyrenoidosa Pringsheim 82T has been studied. All the cations used activate the inducible GDH and produced no such effect on the constitutive GDH. From the analysis of the kinetic behaviour in the presence of K+ the conclusion was made that K+ promotes and stabilyzes a catalitically advantagenous conformation of the inducible GDH. This phenomenon appears to have a physiological meaning, because of a higher K+ concentration in Chlorella cells (about 0.1 M) and its important role in metabolism.

Cesium↗
PubMed1995

Large deletions in antigenic variants of the chlorella virus PBCV-1.

Four spontaneously derived, antigenic variants of chlorella virus PBCV-1 contained 27- to 37-kb deletions in the left end of the 330-kb genome. Two of the mutants, which were serologically identical, had deletions that began from map position 4.9 or 16 and ended at position 42.2 kb. In total, the two deleted regions encoded 28 putative functional open reading frames (ORFs); these deletions probably arose from homologous recombination. The other two mutants, which were serologically identical but distinct from the first two mutants, lacked the entire left terminal 37 kb of the PBCV-1 genome, including an identical 2.2-kb inverted terminal repeat region present at both ends of the wild-type genome. The deleted left end region was replaced by the transposition of an inverted 7.7- or 18.5-kb copy of the right end of the PBCV-1 genome. The region deleted in these two viruses encoded 26 single-copy ORFs, of which 23 were common to those deleted in the first two mutant viruses. The junctions of the deletions/transpositions probably arose from nonhomologous recombination. Taken together, the results indicate that 40.1 kb of single-copy DNA encoding 31 ORFs at the left end of the genome are unnecessary for PBCV-1 replication in Chlorella strain NC64A in the laboratory. The results also indicate that the size of the inverted terminal repeat region in this virus can be highly variable and that the PBCV-1 DNA packaging process tolerates large changes in genome size.

Antigenic Variation↗
PubMed2000

Intron conservation in a UV-specific DNA repair gene encoded by chlorella viruses.

Large dsDNA-containing chlorella viruses encode a pyrimidine dimer-specific glycosylase (PDG) that initiates repair of UV-induced pyrimidine dimers. The PDG enzyme is a homologue of the bacteriophage T4-encoded endonuclease V. The pdg gene was cloned and sequenced from 42 chlorella viruses isolated over a 12-year period from diverse geographic regions. Surprisingly, the pdg gene from 15 of these 42 viruses contain a 98-nucleotide intron that is 100% conserved among the viruses and another 4 viruses contain an 81-nucleotide intron, in the same position, that is nearly 100% identical (one virus differed by one base). In contrast, the nucleotides in the pdg coding regions (exons) from the intron-containing viruses are 84 to 100% identical. The introns in the pdg gene have 5'-AG/GTATGT and 3'-TTGCAG/AA splice site sequences which are characteristic of nuclear-located, spliceosomal processed pre-mRNA introns. The 100% identity of the 98-nucleotide intron sequence in the 15 viruses and the near-perfect identity of an 81-nucleotide intron sequence in another 4 viruses imply strong selective pressure to maintain the DNA sequence of the intron when it is in the pdg gene. However, the ability of intron-plus and intron-minus viruses to repair UV-damaged DNA in the dark was nearly identical. These findings contradict the widely accepted dogma that intron sequences are more variable than exon sequences.

Amino Acid Sequence↗
PubMed2002

Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea.

Chlorella is an attractive organism for complex recombinant protein production because of its eukaryotic characteristics and low cost for large-scale culture. Protoplasts of C. ellipsoidea were transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the cauliflower mosaic virus 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. The presence of introduced DNA was first determined by PCR amplification of both the fGH and Sh ble genes from genomic DNA isolated from transformants and fGH protein expression was detected by immunoblot analysis. Over 400 microg of fGH protein expression per one liter culture containing 1 x 10(8) cells/ml was estimated by ELISA. Stable integration of introduced DNA was confirmed by Southern blot analysis of genomic DNA digested with restriction enzymes. The introduced DNA and fGH expression were detected after seven successive transfers in media devoid of phleomycin, but stably remained in the presence of the antibiotic. Flounder fry fed on the transformed Chlorella revealed a 25% growth increase after 30 days of feeding.

Journal Article↗
PubMed1986

Characterization of viruses infecting a eukaryotic Chlorella-like green alga.

Nineteen plaque-forming viruses of the unicellular, eukaryotic Chlorella-like green alga, strain NC64A, were isolated from various geographic regions in the United States and characterized. Like the previously described virus, PBCV-1, all of the new viruses were large polyhedrons, sensitive to chloroform, and contained large dsDNA genomes of ca. 300 kbp. All of the viral DNAs contained 5-methyldeoxycytidine which varied from 0.1 to 47% of the deoxycytidine. In addition, 10 of the viral DNAs contained N6-methyldeoxyadenosine which varied from 8.1 to 37% of the deoxyadenosine. These viruses, along with 11 previously described viruses which replicate in the same Chlorella host, were grouped into 11 classes based on at least one of the following properties: plaque size, reaction with PBCV-1 antiserum, or the nature and abundance of methylated bases in their genomic DNA.

Base Composition↗
PubMed1994

Effects of two herbicidal wastewaters on Chlorella sp. and Phaeodactylum tricornutum.

The effects on Chlorella sp. and Phaeodactylum tricornutum of two industrial wastewaters known to contain the herbicide residues of Trifluralin and Propanil have been determined by monitoring the number of cells, the chlorophyll fluorescence and the carbon dioxide assimilation simultaneously for a period of 14 days. The growth of the diatom, Phaeodactylum tricornutum, was inhibited by concentrations of herbicidal waste of the order of 0.1-0.5%, apparently because the rate of reproduction was reduced. Chlorella sp. cells, on the other hand, whilst dramatically inhibited by 1% concentrations of herbicidal waste, were able to recover over a period of 14 days. If discharged at concentrations below 0.01%, the industrial wastes appeared not to affect phytoplankton.

Journal Article↗
PubMed2003

Possible function for virus encoded K+ channel Kcv in the replication of chlorella virus PBCV-1.

The K+ channel Kcv is encoded by the chlorella virus PBCV-1. There is evidence that this channel plays an essential role in the replication of the virus, because both PBCV-1 plaque formation and Kcv channel activity in Xenopus oocytes have similar sensitivities to inhibitors. Here we report circumstantial evidence that the Kcv channel is important during virus infection. Recordings of membrane voltage in the host cells Chlorella NC64A reveal a membrane depolarization within the first few minutes of infection. This depolarization displays the same sensitivity to cations as Kcv conductance; depolarization also requires the intact membrane of the virion. Together these data are consistent with the idea that the virus carries functional K+ channels in the virion and inserts them into the host cell plasma membrane during infection.

Animals↗
PubMed2000

Increase in Chlorella strains calorific values when grown in low nitrogen medium.

The calorific value of five strains of Chlorella grown in Watanabe and low-nitrogen medium was determined. The algae were grown in small (2L) stirred tank bioreactors and the best growth was obtained with Chlorella vulgaris with a growth rate of 0.99 d(-1) and the highest calorific value (29 KJ/g) was obtained with C. emersonii. The cellular components were assayed at the end of the growth period and the calorific value appears to be linked to the lipid content rather than any other component.

Journal Article↗
PubMed2000

pH-dependent toxicity of copper and uranium to a tropical freshwater alga (Chlorella sp.).

Copper (Cu) and uranium (U) are of potential ecotoxicological concern to tropical freshwater organisms in northern Australia as a result of mining activity. No local data on the toxicity of these metals to tropical freshwater algae are currently available. The aim of this study was to investigate the effect of pH (5.7 and 6.5) on the toxicity of Cu and U to the green alga Chlorella sp. in a synthetic softwater representative of fresh surface waters in sandy-streams of tropical northern Australia. The effects of Cu and U on algal growth (cell division) rate after a 72-h exposure were determined. Intracellular and extracellular (membrane-bound) metal concentrations at the two selected pH values were also compared. Based on the 72-h minimum detectable effect concentrations (MDEC), Chlorella sp. was approximately 20-fold more sensitive to Cu (0.7 and 1.4 µg l(-1) at pH 6.5 and 5.7, respectively) than U (13 and 34 µg l(-1) at pH 6.5 and 5.7, respectively), and more sensitive than other Australian tropical freshwater organisms. The toxicity of Cu and U was highly pH-dependent. Copper concentrations required to inhibit growth (cell division) rate by 50% (72-h EC(50)) increased from 1.5 to 35 µg l(-1) as the pH decreased from 6.5 to 5.7. Similarly, the 72-h EC(50) values for U increased from 44 to 78 µg l(-1) over the same pH range. Calculation of Cu and U speciation using the geochemical model HARPHRQ, showed that differences in the concentrations of the free metal ions (Cu(2+) and UO(2)(2+)) were only minimal (<10%) between pH 5.7 and 6.5. The decreased toxicity at pH 5.7 was due to lower concentrations of cell-bound and intracellular Cu and U compared to those at pH 6.5. These results are explained in terms of the possible mechanism of competition between H(+) and the metal ion at the cell surface.

Journal Article↗