Chlorophyll breakdown in Chlorella protothecoides: characterization of degreening and cloning of degreening-related genes

Chlorella protothecoides cultures grown in a nitrogen-free bleaching medium (BM–N) in the dark rapidly degraded chlorophyll (Chl) to red catabolites. This degreening process was investigated under different growth conditions. Supply of nitrogen to the culture medium (BM+N) inhibited bleaching and the synthesis of catabolites as did the addition to BM–N of cycloheximide or a chelator, 2,2-bipyridyl. In contrast, chloramphenicol or the protease inhibitor E64 had no effect. During bleaching, Chl breakdown was accompanied by the degradation of cellular proteins such as light-harvesting complex II, cytochrome f and protochlorophyllide oxido-reductase. During growth in BM–N, protease activity increased and proteins immunologically detectable with an antibody against a senescence-enhanced cysteine protease accumulated. cDNAs from BM–N and BM+N cells were used for differential and subtractive screening to isolate cDNAs representing genes with degreening-enhanced expression (dee) in C. protothecoides. Several different dees were identified with different patterns of expression during Chlorella growth but which were all expressed at higher levels during bleaching. Among these, dee4 was most abundant and its expression was exclusive in BM–N cultures. Analysis of the dee sequences showed that they encode different proteins including a novel amino acid carrier (dee4), ferritin, ATP-dependent citrate lyase, a Ca²⁺-binding protein, MO25, ubiquinone-cytochrome c-reductase and several new proteins.     

Pyrolytic characteristics of heterotrophic Chlorella protothecoides for renewable bio-fuel production

Heterotrophic Chlorella protothecoides cells were pyrolyzed in athermogravimetric analyzer to investigate the pyrolytic characteristics anddetermine the kinetic parameters. Heating rates of 15, 40, 60 and 80 ^°C^-1 up to a final temperature of 800 ^°C wereused. The pyrolysis reactions mainly took place between 160–520^°C with a volatile yield of about 80%. The devolatilization stageconsisted of two main temperature zones (I and II) with a transition at300–320 ^°C. Crude lipid in cells decomposed at Zone II whileother main components at Zone I. The increase of heating rate caused alateral shift to higher temperatures in the thermograms, a decrease ofactivation energies for the devolatilization stage and an increase of both theinstantaneous maximum and average reaction rates. The difference ofactivation energies between two zones implied that more energy input forlipid pyrolysis seems needed in comparison with other main components.These data are useful for the design, operation, and modeling of thepyrolysis systems for microalgae.     

Mercury Inhibits the Activity of the NADPH:Protochlorophyllide Oxidoreductase (POR)

The effect of Hg⁺⁺ was studied on the arrangement and photoactivity of NADPH:protochlorophyllide oxidoreductase (POR) in homogenates of dark-grown wheat (Triticum aestivum L.) leaves. 77 K fluorescence emission spectra of the homogenates were recorded before and after the irradiation of the homogenates and the spectra were deconvoluted into Gaussian components. The mercury treatment caused a precipitation of the membrane particles, which was followed by a remarkable decrease of the fluorescence yield. 10^-3 M Hg⁺⁺ decreased the ratio of the 655 nm-emitting protochlorophyllide (Pchlide) form to the 633 nm-emitting form. 10^-2 M Hg⁺⁺ shifted the short wavelength band to 629–630 nm and a 655 nm form was observed which was inactive on irradiation. This inhibition may be caused by serious alteration of the enzyme structure resulting in the trans-localisation of NADPH within the active site of POR.     

Effects of temperature and holding time on production of renewable fuels from pyrolysis of Chlorella protothecoides

To investigate the influence of temperature andholding time on the pyrolyzate yields of Chlorella protothecoides, the microalgal cells weresubjected to pyrolysis at 200, 300, 400, 500 and 600 ^°Cfor 5, 20, 60 and 120 min, separately. High oil yields above 40% dry weight cells wereobtained both at relatively low temperature (300 ^°C)with relatively long holding times (20–120min) and relatively high temperatures (400–500 ^°C)with relatively short holding times (5–20min). The maximum oil yield of 52.0% was achieved at500 ^°C for 5 min. The gas yield was generallyincreased with the increasing temperature and holdingtime. It could reach 63.3–76.0% at 600 ^°C.High pyrolytic rates of 72–87% were obtained at allexperiments except at 200 ^°C for 5–20 min or300 ^°C for 5 min. Thermogravimetric analysisindicated that the main thermal degradation of thismicroalga occurred at 200–520 ^°C. The resultsimply that C. protothecoides is a good candidatefor the production of renewable fuels by pyrolysis.     

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [³5S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [³⁵S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

DNA homologies of 14 strains of Chlorella protothecoides were determined. All strains are related by a high degree of DNA similarity (96–102% D) with the exception of strain 211-11 a which proved to belong to C. kessleri. There is, however, no detectable DNA homology with strains of the genus Prototheca which is supposed to have evolved from C. protothecoides by loss of photosynthetic pigments. Even within Prototheca the low degree of DNA similarity indicates a heterogeneity similar to that observed in the genus Chlorella.     

Production of Deuterated Lutein by Chlorella protothecoides and its Detection by Mass Spectrometric Methods

Chlorella protothecoides, a lutein-producing microalga, was grown aerobically in a mineral medium prepared with 70% (v/v) deuterated water. HPLC/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MS) analysis revealed 58% replacement of hydrogen by deuterium atoms as indicated by the molecular mass cluster at around m/z 599. The rapidly growing microalga had much higher levels (58%) of deuterium substitution relative to previously reported (9–15%) natural sources of lutein.     

Novel Insights into the Enzymology,Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms

The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently, angiosperms become etiolated in the absence of light, since the reduction of Pchlide in angiosperms is solely dependent on POR. In eukaryotic phototrophs, POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids. POR possesses unique features, its light-dependent catalytic activity, accumulation in plastids of dark-grown angiosperms (etioplasts) via binding to its substrate, Pchlide, and cofactor, NADPH, resulting in the formation of prolamellar bodies (PLBs), and rapid degradation after catalysis under subsequent illumination. During the last decade, considerable progress has been made in the study of the gene organization, catalytic mechanism, membrane association, regulation of the gene expression, and physiological function of POR. In this review, we provide a brief overview of DPOR and then summarize the current state of knowledge on the biochemistry and molecular biology of POR mainly in angiosperms. The physiological and evolutional implications of POR are also discussed.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

Four species of the unicellular green alga Chlorella, C. vulgaris, C. luteoviridis, C. minutissima, and C. zofingiensis, were characterized with respect to DNA similarities as determined by quantitative DNA hybridization procedures. In contrast to previous DNA hybridization procedures. In contrast to previous results, C. vulgaris turned out to be a homogeneous species with the exception of strain 211-11c of the Göttingen collection, which was shown to belong to C. kessleri. Similary, C. luteoviridis and C. minutissima represent well defined species in terms of phenotypic and genotypic features. Whitin C. zofingiensis on strain is clearly different with respect to DNA base composition and DNA hybridization data even though it shares phenotypic characteristics with the other strains of C. zofingiensis.     

Cultivation of Chlorella pyrenoidosa in soybean processing wastewater

Chlorella pyrenoidosa was cultivated in soybean processing wastewater (SPW) in batch and fed-batch cultures without a supply of additional nutrients. The alga was able to remove 77.8 ± 5.7%, 88.8 ± 1.0%, 89.1 ± 0.6% and 70.3 ± 11.4% of soluble chemical oxygen demand (SCOD_Cr), total nitrogen (TN), NH₄⁺-N and total phosphate (TP), respectively, after 120 h in fed-batch culture. C. pyrenoidosa attained an average biomass productivity of 0.64 g L⁻¹ d⁻¹, an average lipid content of 37.00 ± 9.34%, and a high lipid productivity of 0.40 g L⁻¹ d⁻¹. Therefore, cultivation of C. pyrenoidosa in SPW could yield cleaner water and useful biomass.     

Molecular Characterization of Puroindolines and their Encoding Genes in Aegilops Ventricosa

Puroindolines, the tryptophan-rich proteins controlling grain hardness in wheat, appeared as two pairs of 13 kDa polypeptides in the Acid-PAGE (A-PAGE) and two-dimensional A-PAGE×SDS-PAGE patterns of starch-granule proteins from wild allotetraploid wheat Aegilops ventricosa Tausch. (2n = 4x = 28, genomes D^vD^vN^vN^v). Puroindoline pair a1 + a2 reacted strongly with an antiserum specific for puroindoline-a from common wheat (Triticum aestivum L.), whereas puroindoline pair b1 + b2 exhibited A-PAGE relative mobilities similar to that of puroindoline-b in Aegilops tauschii (Coss.), the D-genome donor to both common wheat and Ae. ventricosa. Puroindolines a2 and b1 were found to be encoded by alleles Pina-D1a and Pinb-D1h on chromosome 5D^v, respectively, whereas puroindolines a1 and b2 were assumed to be under the genetic control of chromosome 5N^v. Puroindoline a1 encoded by the novel Pina-N1a allele exhibited a high level of amino acid variation with respect to puroindoline-a. On the other hand, the tryptophan-rich region of puroindoline b2 encoded by allele Pinb-N1a showed a sequence change from lysine-42 to arginine, with no effect on the amount of protein b2 accumulated on the starch granules. A partial duplication of the pin-B gene (Pinb-relic) was identified about 1100 bp downstream from Pinb-D1 on chromosome 5D^v. The present findings are the first evidence of a tetraploid wheat species in which four puroindoline genes are expressed. The potential of Ae. ventricosa as a source of genes that may be used to modulate endosperm texture and other valuable traits in cultivated wheat species is discussed.     

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.     

The effect of dynamic light regimes on Chlorella

The patterns of diurnal variations in pigmentation and optical cross-section were compared for two cyclostat cultures of Chlorella pyrenoidosa, where the dynamics of the photoperiod differed. Populations were light-limited, nutrient rich and growing on an 8:16 light-dark (LD) cycle. One light regime was an 8 h sine function of the light period (sinusoidal culture), while the second had an 1 h sine function super-imposed on the 8 hour sine function (oscillating sinusoidal culture). Hourly samples were taken throughout a 12 h period including the light period. Determinations were made of chlorophyll (Chl) a and b abundance, in vivo absorption spectra, cell number and volume and used to derive both cell-specific (cell) and optical chlorophyll specific (chl) cross sections, as well as the absorption efficiency, Q, of the cells. The results indicate that C. pyrenoidosa is capable of adapting to dynamics in light intensity within an 8 h photoperiod. The sinusoidal culture showed a constant decrease in the Chl a/b ratio of 28% while the total Chl content per cell increased slightly and chl and Q remained constant, suggesting coordinated changes in reaction centers and light harvesting complexes. Over the oscillating photoperiod, however, the second culture displayed a diurnal variation in Chl a/b ratio, a 20% increase in chl and an apparent oscillation in Q. These observations suggest that an oscillating photoperiod promoted the capability of Chl molecules to collect light and that the fractional area of all Chl molecules exposed to the photon flux is inversely related to the photon flux.     

Cycloheximide induces synchronous swelling of perialgal vacuoles enclosing symbiotic Chlorella vulgaris and digestion of the algae in the ciliate Paramecium bursaria

Cycloheximide is known to inhibit preferentially protein synthesis of symbiotic Chlorella of the ciliate Paramecium bursaria, but to hardly host protein synthesis. Treatment of algae-bearing Paramecium cells with cycloheximide induces synchronous swelling of all perialgal vacuoles that are localized immediately beneath the host's cell membrane. In this study, the space between the symbiotic algal cell wall and the perialgal vacuole membrane widened to about 25 times its normal width 24 h after treatment with cycloheximide. Then, the vacuoles detached from beneath the host's cell membrane, were condensed and stained with Gomori's solution, and the algae in the vacuoles were digested. Although this phenomenon is induced only under a fluorescent light condition, and not under a constant dark condition, this phenomenon was not induced in paramecia treated with cycloheximide in the light in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that algal proteins synthesized in the presence of algal photosynthesis serve some important function to prevent expansion of the perialgal vacuole and to maintain the ability of the perialgal vacuole membrane to protect itself from host lysosomal fusion.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

DNA hybridization techniques showed Chlorella fusca var. vacuolata and C. kessleri to be homogeneous species with DNA homologies of 90–100% C. fusca var. fusca and var. rubescens, however, have only about 15% DNA homology with C. fusca var. vacuolata and should no longer be regarded as varieties. A good correlation was found so far between biochemical and physiological characters used in the taxonomy of Chlorella and DNA relatedness. Mutant strains of Chlorella were tested for DNA homologies to prove the reliability of the taxonomical interpretation.     

Nicotinamide is a specific inhibitor of dark-operative protochlorophyllide oxidoreductase,a nitrogenase-like enzyme,from Rhodobacter capsulatus

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme consisting of two components, L-protein as a reductase component and NB-protein as a catalytic component. Elucidation of the crystal structures of NB-protein (Muraki et al., Nature 2010, 465: 110–114) has enabled us to study its reaction mechanism in combination with biochemical analysis. Here we demonstrate that nicotinamide (NA) inhibits DPOR activity by blocking the electron transfer from L-protein to NB-protein. A reaction scheme of DPOR, in which the binding of protochlorophyllide (Pchlide) to the NB-protein precedes the electron transfer from the L-protein, is proposed based on the NA effects.