Acetate metabolism in the process of "acetate-bleaching" of Chlorella protothecoides

Green cells of Chlorella protothecoides when incubated in amedium containing acetate but no nitrogen source, have beenshown to be bleached as strongly as in glucose-induced bleaching.Using U-¹⁴C-acetate as tracer, the acetate metabolism of algalcells during the process of acetate-induced bleaching was investigated.Changes in algal cell activities for respiration and assimilationof added ¹⁴C-acetate were followed during bleaching processesin "acetate-adapted" and "non-adapted" green cells. As in glucose-inducedbleaching of algal cells, algal cell activity for incorporating¹⁴C into lipids showed the most characteristic change, suggestingthat lipogenesis is causally related to the occurrence of bleachingin algal cells. (Received March 5, 1969; )     

Studies on red pigments excreted by cells of Chlorella protothecoides during the process of bleaching induced by glucose or acetate I. Chemical properties of the red pigments

During the process of bleaching of Chlorella protothecoidescells induced by the addition of glucose or acetate in a nitrogen-deficientmineral nutrient medium, at least four red pigments were foundto be excreted into the incubation medium. These pigments wereseparated by thin-layer chromatography on silica gel, and theirabsorption spectra in visible and infrared regions as well astheir NMR spectra were measured. Elementary composition andthe Gmelin colour reaction of the pigments were also examined.The results obtained suggest that the four pigments are certainacidic substances—containing a structure similar to thatof a bile pigment—which are most probably derived fromchlorophyll through oxidative rupture of the tetrapyrrole ringof the latter. (Received July 23, 1968; )     

Application of Flow Microflorometry to the Study of Algal Cells and Isolated Chloroplasts

The applicability of flow microfluorometry (FMF) to the studyof chlorophyll-containing cells was investigated through theuse of the blue-green alga Agmenellum quadruplicatum, the greenalgae Trebouxia, Chlorella, and Euglena spp., and isolated spinachchloroplasts. When excited by laser radiation (488 nm), algalcells emitted fluorescence with intensity positively relatedto the chloro-phyll content. The chlorophyll fluorescent signalswere used further as a differential criterion in determiningrelative size based on light scattering logic and to sort mixturesof algal cells having different chlorophyll content The FMFalso was useful in estimating nucleic acid and protein contentin completely dechlorophyUized algal cells with the use of ethidiumbromide (EB) and fluoresceinisothiocyanate (FITC), respectively.     

Digestion of chlorophylls and carotenoids by the marine protozoan Oxyrrhis marina studied by h.p.l.c. analysis of algal pigments

Algal pigments were measured, by reverse-phase h.p.l.c., duringgrazing experiments with the protozoan Oxyrrhis marina Dujardinon Rhodomonas sp. Chlorophylls and carotenoids were degradedto colourless residues; the rate of degradation of pigmentswas highest in the light. Very little chlorophyll a (5%) wasdegraded to phaeophytin and none to phaeophorbide during grazing.This suggests that phaeopigment concentrations cannot be usedas a measure of algal mortality due to grazing when heterotrophicProtozoa are a component of the grazer community.     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: V. DEGENERATION OF CHLOROPLASTS INDUCED BY DIFFERENT CARBON SOURCES, AND EFFECTS OF SOME ANTIMETABOLITES UPON THE PROCESS INDUCED BY GLUCOSE

1. The green cells of Chlorella protothecoides were bleached todifferent extents when incubated (in the dark) in the nitrogen-freemedia containing, besides basal mineral nutrients, glucose,fructose, galactose, glycerol or acetate. Glucose and fructosewere found to have the strongest bleaching effect. Additionof a nitrogen source (urea) caused a considerable reductionof the bleaching. It was assumed that from the different carbonsources a certain common intermediate(s) causing the bleachingis formed, and that in the presence of the nitrogen source thesubstance is removed by reacting with it.
2. Using glucose asbleach-inducing agent, the effects of someantimetabolites uponthe processes of bleaching, division andgrowth of green algalcells were investigated, and it was demonstratedthat the processof bleaching occurs without being accompaniedby growth anddivision of the algal cells.
3. It was found that during theprocess of bleaching no net increasesin RNA and protein tookplace.

(Received March 11, 1965; )     

Changes of ribulose 1,5-diphosphate carboxylase level during the processes of degeneration and regeneration of chloroplasts in Chlorella protothecoides

RuDP carboxylase was active mainly in chloroplasts and PEP carboxylaseactive principally outside of chloroplasts in Chlorella protothecoides. During the process of chloroplast degeneration in algal cellsinduced by addition of glucose, the activity of RuDP carboxylasesignificantly decreased, whereas the activities of PEP-carboxylaseand -carboxykinase markedly increased. During the process of chloroplast regeneration in "glucose-bleached"algal cells, which contained no detectable amounts of FractionI protein and showed only traces of RuDP carboxylase activity,a light-dependent development of RuDP carboxylase proceededalmost in parallel with the light-induced formation of chlorophyll.The activities of PEP-carboxylase and -carboxykinase, whichwere negligibly low in glucose-bleached cells, developed independentlyof light. Both chloramphenicol and cycloheximide severely inhibited thedevelopment of RuDP carboxylase activity. A relatively low concentrationof glucose also caused a significant suppression. Under theseconditions, chlorophyll formation was inhibited only slightlyby chloramphenicol and very strongly by cycloheximide and glucose. ¹ Deceased, 11 June, 1972. (Received April 25, 1972; )     

Studies on chlorophyll formation in Chlorella protothecoides I. Enhancing effects of light and added {delta}-aminoIevulinic acid, and suppressive effect of glucose on chlorophyll formation

Light-induced formation of chlorophyll in "etiolated" cellsof Chlorella protothecoides was studied under various experimentalconditions, (i) Two different types of enhancing effect of lightwere demonstrated: a "long-term" effect lasting for many hoursafter a relatively short illumination of etiolated cells anda "short-term" effect disappearing in a few hours after illumination,(ii) Addition of ALA caused enhancement of chlorophyll synthesisin etiolated cells in darkness as well as in light; the ALA-enhancedrate of dark chlorophyll synthesis, however, was much lowerthan the rate in light without added ALA. ALA was replaceablewith succinic acid plus glycine in light, but not in the dark,for enhancement of chlorophyll formation, (iii) Adding glucose,fructose, galactose, glycerol or acetate—at concentrationsmuch lower than those previously shown to induce "bleaching"of green algal cells-caused a more or less marked suppressionof light-induced greening in etiolated cells, (iv) Added glucosealmost instantaneously and completely stopped chlorophyll synthesisin light as well as in darkness either with or without addedALA. On the basis of these and other results, a tentative schemeis presented for the enhancing effects of light and the suppressiveeffects of glucose on chlorophyll formation in algal cells. (Received April 1, 1970; )     

Studies on ribonucleic acids from Chlorella protothecoides with special reference to the degradation of chloroplast RNA during the process of "glucose-bleaching"

By growing Chlorella protothecoides under certain nutritionaland light conditions the following three different types ofalgal cells were obtained: (i) normal "green" cells grown ina medium rich in a nitrogen source (urea) and poor in glucoseunder illumination, (ii) "etiolated" cells cultivated in thesame medium in darkness, and (iii) "glucose-bleached" cellsgrown, in the light or in darkness, in a medium rich in glucoseand poor in the nitrogen source. The "glucose-bleached" cellscontain profoundly degenerated plastids, and the "etiolated"cells have only partially organized plastids. From these algalcells RNA was extracted by the cold phenol method, and fractionatedby MAK column chromatography and sucrose density gradient centrifugation,making use of ³²P-labelled E. coli RNA as the internal marker.It was found that in comparison with the green cells that arerich in chloroplast ribosomal RNA as well as in nonchloroplastic("cytoplasmic") ribosomal RNA, the etiolated cells possess acomparable amount of "cytoplasmic" rRNA but a significantlylesser amount of chloroplast rRNA. Both types of rRNA existat extremely low levels in the glucose-bleached cells. During the process of bleaching (chloroplast degeneration) ofthe green cells induced by the addition of a high concentrationof glucose, marked changes were observed in the patterns offractionation of RNA as followed by the above procedures. Itwas disclosed that the chloroplast rRNA is rapidly degradedduring an early phase of the bleaching process, while the quantityof "cytoplasmic" rRNA remained almost unaltered. ¹Part of this work was reported at the Symposium on Cell Differentiationsponsored by the Institute of Applied Microbiology, Universityof Tokyo, in November 1965, and at the Symposium on Biogenesisof Subcellular Particles, the 7th Internatl. Congress of Biochemistry,Tokyo, 1967. ²Present address: Faculty of Pharmaceutical Sciences, Universityof Hokkaido, Sapporo.     

Biomass-pigment relationships in potamoplankton

During most of the growing season of 1994, pigment content,as determined by HPLC analysis of algal sample extracts, wasfollowed in the River Meuse (Belgium) potamoplankton. The concentrationof some algal pigments (chlorophylls a and b, fucoxanthin, lutein,echinenone and alloxanthin) was related to biomass estimatesof total phytoplankton and of major taxonomic components (diatoms,green algae, cyanobacteria and cryptomonads). Highly significantlinear regressions were obtained for chlorophyll a-total biomass,fucoxanthin-diatoms, lutein-green algae, chlorophyll b-greenalgae. However, no relationship was found for cyanobacteriaor cryptomonads and their specific pigments, which may be attributedto poor accuracy of biomass estimates for these non-dominantalgae. In conclusion, the good relationship found for dominantalgae and their specific pigments confirms the value of pigmentsas quantitative markers of phytoplankton, as detected in othermarine and freshwater environments.     

Further studies on the metabolism of glucose in the process of "glucose-bleaching" of Chlorella protothecoides

Previous studies have demonstrated that when cells of Chlorellaprotothecoides are incubated in a medium containing glucosebut no nitrogen source, they are profoundly bleached with degenerationof chloroplast structure and photosynthetic activity. When anitrogen source (urea) is added to the glucose medium, bleachingof algal cells is greatly suppressed. In this work the metabolismof glucose in the process of glucose-induced bleaching was studiedusing ¹⁴C-glucose as tracer. Changes in algal cell activityfor ¹⁴CO₂-evolution and ¹⁴C-incorporation into various cellularsubstances from ¹⁴C-glucose were followed. Most conspicuouswere increases in cellular activities for assimilating ¹⁴C-glucoseinto lipids (fatty acids) and glucose polymer. When urea wasadded to the glucose medium, the incorporation of ¹⁴C by algalcells into fatty acids was greatly reduced, while the assimilationof ¹⁴C into glucose polymer was increased. These and previous observations suggest that the formation oflarge amounts of lipids (fatty acids) probably is causally relatedto the induction of algal cell bleaching. (Received March 5, 1969; )     

Effect of cycloheximide on the process of "glucose-bleaching" in Chlorella protothecoides

The process of bleaching of Chlorella protothecoides inducedby the addition of glucose was strongly inhibited by cycloheximide,an inhibitor of protein synthesis, whereas it was suppressedonly weakly by chloramphenicol, puromycin and ethionine. Whencycloheximide was added simultaneously with glucose at the beginningof die bleaching experiment, no bleaching of algal cells occurredduring the subsequent incubation. When it was added after glucose,the bleaching of algal cells proceeded for a period of timeas actively as in the control, then gradually ceased. Cycloheximidewas found to suppress the uptake of glucose by algal cells,and to severely inhibit the assimilation of glucose into lipidswhen added at the beginning of the bleaching experiment. Theseinhibitory effects of cycloheximide are discussed in relationto the induction of "glucose-bleaching" in algal cells. (Received December 16, 1968; )     

DEGRADATION AND FORMATION OF SULFOLIPID OCCURRING CONCURRENTLY WITH DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES

1. It has been demonstrated that when the cells of Chlorella protothecoidesare grown mixotrophically under illumination in a medium richin nitrogen source (urea) and poor in glucose, the normal greencells are obtained, while in a medium rich in glucose and poorin the nitrogen source, entirely chlorophyll-less cells withprofoundly degenerated plastids ("glucose-bleached" cells) areproduced, irrespective of whether in the light or in darkness.The "glucose-bleached" cells turn green with regeneration offully organized chloroplasts when incubated in a nitrogen-enrichedmedium in the light ("light-greening"), while in the dark theybecome pale green with formation of only partially organizedchloroplasts ("dark-greening"). When, on the other hand, thegreen cells are transferred into a medium enriched with glucose,they are bleached fairly rapidly with degeneration of chloro-plastsin the light as well as in darkness ("bleaching"). Using ³⁵Sas a tracer, investigations were made on the changes of contentsof the algal cells in sulfolipid and other sulfur compoundsduring the processes of the greening and bleaching.
2. By determiningthe radioactivities of chromatographically separatedsulfur-containingcompounds of the uniformly ³⁵S-labeled green("G") and "glucose-bleached"("W") cells, it was found thatthe concentration of a speciesof sulfolipid (discovered byBENSON et al.) as well as thoseof glutathione, sulfotriosesand most of the other sulfur-containingcompounds were at least5 times higher in the "G" cells thanin the "W" cells, whilesulfoquinovosyl glycerol was presentin approximately equalamounts in the two types of cells.
3. Phospholipidcontents and compositions in the two types of algalcells werefound to be practically identical.
4. The sulfolipid contentof algal cells increased and decreasedalmost in parallel withthe processes of greening and bleaching,respectively.
5. Studyingthe mode of incorporation of radiosulfate into varioussulfurcompounds of algal cells during the processes of "light-anddark-greening" and "bleaching" (lasting about 70 hr), itwasfound that active ³⁵S-incorporation into sulfolipid occurredthroughout the process of "light-greening," while in the "dark-greening"and "bleaching" the active incorporation abruptly ceased afterthe initial 24 hr period of experiments. It was suggested thatthe biosynthesis of the sulfolipid is closely related to theformation of photosynthetic apparatus in chloroplast.
6. Whenthe ³⁵S-labeled green cells were bleached in a medium containingno radiosulfate, the ³⁵S-sulfolipid and most of other ³⁵S-sulfurcompounds decreased markedly but the ³⁵S-sulfoquinovosyl glycerolincreased considerably. It was inferred that the deacylationof the sulfolipid, a surfactant lipid, with formation of watersoluble sulfoquinovosyl glycerol may be a cardinal event ofbleaching process, causing a disintegration of the intact architechtureof photosynthetic apparatus.
7. Based on these observations itwas concluded that the sulfolipidis an integral component ofphotosynthetic structure.

¹This work was partly reported at the Symposium on Biochemistryof Lipids, sponsored by the Agricultural Chemical Society ofJapan, Sapporo, July, 1964.     

Formation of water-solubleMonascus red pigments by biological and semi-synthetic processes

Summary New water-soluble red pigments were produced byMonascus sp. in a chemically defined fermentation medium containing glutamate as nitrogen source. They were isolated and characterized as glutamate derivatives of the well-known orangeMonascus pigments (monascorubrin and rubropunctatin). The new pigments have several advantages over the known redMonascus pigments (rubropunctamine and monascorubramine) including very high water-solubility, higher absorption coefficient, and greater resistance to decoloration by light. Adding glutamate, glycine or leucine to a resting-cell system led to the formation of specific water-soluble red pigments corresponding to the exogenous amino acid. The water-soluble red pigments produced by resting-cells have retention times identical to those of the corresponding red derivatives made chemically from the orange pigments in methanol-phosphate buffer at pH 7. The hydrophobicities of the amino acid sources correspond to the HPLC retention times of the red pigments derived from them.     

HPLC analysis of phytoplankton pigments from Lake Kinneret with special reference to the bloom-forming dinoflagellate Peridinium gatunense (Dinophyceae) and chlorophyll degradatio products

Photosynthetic pigments extracted from the paniculate materialof the water column of Lake Kinneret were studied throughoutthe periods of May 1988-June 1989, and November 1993-November1994, by means of HPLC. The temporal and vertical variationof the pigment suite found agreed with the microscopically determinedphytoplankton record. The regression calculations of taxon-specificbiomass with the corresponding signature pigments suggest thatpigment analysis may be a useful tool for the monitoring ofbloom-forming species, e.g. the dinoflagellate Peridinium gatunenseNygaard. The HPLC pigment analysis permitted the identificationand quantification of chlorophyll degradation products, providingfor the first time information about their composition in LakeKinneret. Chlorophyllide a was the major detectable degradationproduct of chlorophyll a, varying between 1 and 9% of the chlorophylla concentration. Other chlorophyll a derivatives appeared mostlyin minor quantities. Pheophytin a was virtually lacking in allthe samples. Removal rates of pigments, measured by sedimentationtraps, indicated that the degradation of chlorophyll a via chlorophyllidea is a dynamic process that continues during the sedimentationof the phytoplankton particles.     

Regeneration of photosystem II and phycobilin pigments in photoorganotrophically grown Anabaena variabilis

Regeneration of photosynthetic activity and phycobilin pigmentswas studied with cells of Anabaena variabilis lacking photosystemII activity and phycobilin pigments. Regeneration was achievedonly when the cells were incubated in the presence of nitrateor nitrite. The addition of ammonium salts or urea was far lesseffective. Nitrate-directed regeneration was independent oflight and inhibited by chlorate. Dark-regenerated cells, however,differed from light-regenerated ones in that the former wereincapable of excitation transfer from phycocyanin to pigmentsystemII chlorophyll a, although they emitted fluorescence of pigmentsystem II chlorophyll a origin, if illuminated by the lightabsorbed by chlorophyll. The regeneration process inAnabaenacells is assumed to consist of two steps: [1] light-independent,nitratesupported synthesis of phycobilin pigments and photosystemII integrity, followed by [2] light-directed formation of excitationtransfer from phycocyanin to pigment system II chlorophyll a.An antibiotic study revealed that the former is associated withprotein synthesis, while the latter isnot. ¹ Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo 164, Japan. (Received November 19, 1975; )     

Production and release of pigments by culture of transformed hairy root of red beet

Adventitious roots were induced from red beet (Beta vulgaris L. cv. Detroit dark red) by infecting the plant with a soil bacterium, Agrobacterium rhizogenes. Based on analysis of opines which are uniquely produced in transformed hairy roots, the established clone was proved to be a transformed hairy root. In a shake culture of the beet hairy root clone with a liquid medium, it was found that significant amounts of pigments, mainly betanin and vulgaxanthin-I, were released into the medium by the cessation of culture shaking (temporary limitation of oxygen supply). The hairy root cells were capable of propagation even after the cells were subjected to shaking cessation. Repeated-batch culture of the beet hairy root was performed with the cell growth phases for 9 or 10 d and with pigment leakage phases during shaking cessation for 2 d, and more than 20% of the total intracellular pigments were recovered from the culture broth at a culture time of 35 d. The released pigments were confirmed to be substantially identical to those extracted from the hairy root and original plant cells of red beet.     

METABOLISM OF GLUCOSE IN THE PROCESS OF "GLUCOSE-BLEACHING" OF CHLORELLA PROTOTHECOIDES

1. As previously demonstrated, normal cells of Chlorella protothecoidesare bleached with degeneration of chloroplasts when they areincubated, under aerobic conditions—either in the lightor in darkness—, in a glucose-containing medium withoutadded nitrogen source ("glucose-bleaching"). It was found inthe present study that under the atmosphere of N₂, neither bleachingnor growth of algal cells occurs in the dark, while in the lighta significant growth of cells takes place with formation ofa certain amount of chlorophyll.
2. Studies on the effects ofvarious inhibitors (ammonium ion,DNP, CMU, -hydroxysulphonates,arsenate, cyanide, azide, andantimycin A) under different conditionsshowed that oxidativephosphorylation is a necessary processfor the occurrence ofthe glucosebleaching as well as the assimilationof glucose(cellular growth). Under light-anaerobic conditionsin the presenceof glucose, assimilation of glucose (cellulargrowth) takesplace being supported by photophosphorylation,but no bleachingoccurs.
3. When the algal cells in the courseof bleaching were transferredto the glucose-free mineral medium,the cell growth ceased immediatelybut the cell bleaching proceededfor several hours before itscessation. The respiratory activity,which was high in the glucose-containingmedium, became loweron transferring the algal cells into theglucose-free medium.The lowered level of respiration was maintained,for more than8 hr after the transfer of cells to the glucose-freemedium.
4. When the cells in the course of bleaching were placed underthe atmosphere of N₂, the cell bleaching ceased almost instantaneously.
5. Based on these observations and other inhibition experiments,it was inferred that a certain intermediate(s) produced by theaerobic respiration of glucose is closely associated with theoccurrence of cell bleaching, and that an O₂-requiring stepmay be involved in the process of chlorophyll degradation.

(Received September 9, 1965; )     

CHANGES IN CONTENTS OF CARBOHYDRATE AND FATTY ACID IN THE CELLS OF CHLORELLA PROTOTHECOIDES DURING THE PROCESSES OF DE- AND RE-GENERATION OF CHLOROPLASTS

1. Previous work has demonstrated that when cells of Chlorellaprotothecoides are grown mixotrophically under illuminationin a medium rich in nitrogen source (urea) and poor in glucose,normal green cells are obtained, while in a medium rich in glucoseand poor in the nitrogen source, strongly bleached cells containingapparently no discernible chloroplast structures — called"glucose-bleached" cells — are produced either in thelight or in darkness. When the green cells are incubated ina glucose-enriched mineral medium without added nitrogen source,they are fairly rapidly bleached with concomitant degenerationof chloroplast structures (" bleaching "). When, on the otherhand, the "glucose-bleached" cells are transferred in a nitrogen-enrichedmedium without added glucose under illumination, they turn greenwith regeneration of chloroplasts (" greening "). In the presentstudy changes in contents of carbohydrate and fatty acid inalgal cells were followed during these processes of "bleaching"and "greening.".
2. During the process of "bleaching", the quantityof glucose existingin the insoluble carbohydrate fraction ofalgal cells increasedrapidly and markedly. A considerable increasewas also observedin the contents of cells in oleic, linoleicand palmitic acids.It was noted, however, that linolenic aciddecreased in quantityduring the most active phase of cell bleaching.
3. During the process of "greening", the glucose in the insolublecarbohydrate fraction rapidly decreased, suggesting that itis utilized, as carbon and energy sources, for the chloroplastregeneration. Linolenic acid was found to be synthesized inparallel with formation of chlorophyll. A peculiar pattern ofchange in contents was observed with oleic and palmitic acids,which was interpreted as being related with the process of cellulardivision occurring incidentally during the process of greening.

(Received September 24, 1966; )     

Photooxidative damage to the cyanobacterium Spirulina platensis mediated by singlet oxygen

Experiments on the specific growth rate, bleaching of pigments, O₂ evolution, lipid peroxidation, and loss of sulfhydryl (-SH) content in response to the varying light intensities (2–28 W/m²) suggested that photodamage to the Spirulina cells was maximum at or beyond the photosynthesis saturating light intensity (12 W/m²). However, photobleaching of the chlorophyll a was relatively higher than carotenoid. The results on the N,N-dimethyl-p-nitrosoaniline (RNO) bleaching in the presence of oxygen radical quenchers exhibited maximum effect of sodium azide and indicated about the generation of singlet oxygen. The chlorophyll a-sensitized production of singlet oxygen by a type II reaction cannot be ruled out because of maximum oxidative damage to the cells at or beyond the photosynthesis saturating light intensity, i.e., 12 W/m², when the availability of triplet chlorophyll is maximum.     

Negative effect of ammonium nitrate as nitrogen source on the production of water-soluble red pigments by Monascus sp.

Ammonium salts, especially ammonium nitrate, have been used as nitrogen sources for production of traditional water-insoluble Monascus pigments. However, we noted that defined media employing NH₄NO₃ as the sole nitrogen source in fermentations supported only poor pigment production by Monascus sp., and the pigments produced were mainly cell-bound. NH₄NO₃ was found not to (a) repress pigment synthase formation, (b) enhance synthase decay, or (c) serve as a nitrogen source for pigment production by resting cells; it had a weak inhibitory effect on the action of pigment synthase(s). The high level of cell-bound did not exert a feedback effect on the further synthesis of pigments. These observations indicate that the reason why NH₄NO₃ supports only low pigment production during fermentations is the poor ability of NH₄NO₃ to donate nitrogen in the Schiff-base reaction converting orange pigments to red ones.