Effects of Different Nitrogen Sources and Concentrations on CAM Photosynthesis in Kalanchoe blossfeldiana

When Kalanchoë blossfeldiana Poelln. cv. Hikan plants werecultured in solutions containing 0.2, 1.0, 5.0 or 10 mM of nitrateor ammonium under a long-day photoperiod, some criteria of CAM(Crassulacean acid metabolism) photosynthesis (diurnal changesof CO₂ uptake, titratable acidity and malate content in leaves)were examined. The plants absorbed 90 to 100% of CO₂ duringthe light phase regardless of the supplied nitrogen. Nitrate-grownplants absorbed about 10% of CO₂ during the dark phase regardlessof the supplied concentration, whereas in ammonium-grown plantsthe nocturnal CO₂ uptake occurred at 0.2 mM, at which the plantsdepleted nitrogen and no uptake was observed at the higher concentrations.Changes of nocturnal increase in titratable acidity and malatecontent almost corresponded with the changes in the amount ofnocturnal CO₂ uptake. Also K. daigremontiana plants suppliedwith 10 mM of ammonium had a less CAM-like pattern of diurnalCO₂ uptake than the plants supplied with 10 mM of nitrate. Theseresults suggest that a sufficient supply of ammonium depressesCAM photosynthesis.     

Coupling of malate decarboxylation to CO2 fixation and the reduction of 3-phosphoglyceric acid in corn bundle sheath cells,2

1. In the presence of NADP⁺ and Mg⁺⁺, the bundle sheath strandsisolated from corn (Zea mays) leaves by cellulase treatmentsdecarboxylated malate in the light at an initial rate (200 µmoles/mgchl.hr), which was sufficient to account for photosyntheticCO₂ fixation in intact leaves. This rate gradually slowed downand then stopped. The final level of the malate decarboxylatedwas approximately equal to the amount of NADP⁺ added.
2. Rapidand continued decarboxylation of malate was observed whenNADP⁺,3-phosphoglyceric acid and ATP (and Mg⁺⁺) were addedtogether.The addition of ADP instead of ATP showed a similareffect.Light did not show any effect on the malate decarboxylationin the presence of ATP or ADP.
3. When malate was added to thebundle sheath strands in the presenceof exogenous NADP⁺ NADP⁺was rapidly reduced. The reductionstopped after 2 min when,73% of the added NADP⁺ was reduced.The further addition of3-phosphoglyceric acid and ATP broughtabout a decrease in theNADPH-level, which rose again to attaina new steady level.
4. The transfer of radioactivity from (1-¹⁴C-3-phosphoglycericacid to dihydroxyacetone phosphate in the bundle sheath strandsin the presence of ATP and NADP⁺ was greatly enhanced by theaddition of malate.
5. In the presence of ribose 5-phosphateand ATP, the rate of ¹⁴C-transferfrom (4-¹⁴C)-malate to theintermediates of the reductive pentosephosphate cycle was equalto that of ¹⁴CO₂ fixation in the light.

All these results support the current view that in the bundlesheath cells of C₄ plants belonging to the NADP-malic enzyme-group,the decarboxylation of malate is coupled to the fixation ofthe released CO₂ and the reduction of 3-phosphoglyceric acidformed as a result of CO₂ fixation. ¹ Part of this research was reported at the 40th Annual Meetingof the Botanical Society of Japan Osaka, December, 1975. ³ Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, 359 Otsuka, Hachioji-City, Tokyo 173, Japan. (Received April 30, 1977; )     

Accumulation of tartaric acid in the ripening process of grapes

1. 1. Tartaric acid content in grapes gradually increased withripening and reached a plateau about 50 days after flowering.
2. 2. Tartaric acid synthesis from ^14C0₂ was predominant in anearly ripening stage. When the berries were exposed to ^14CO₂8 days after flowering and examined two days later, 30% of thetotal ¹⁴C fixed was found in tartaric acid. Subsequently, apart of the tartaric acid decomposed, but the greater part remainedin the berries in a salt form. At the last stage of the ripeningprocess (82-100 days after flowering), some of the tartaratewas again converted to free acid. No ^14CO₂ was incorporatedinto tartaric acid when berries were exposed 61 days after flowering.
3. 3. L(+)-Tartaric acid-l,4-¹⁴C fed to the berries was catabolizedto ¹⁴CO₂. The ratio of radio activity recovered as ^{14 CO₂} tothat fed was nearly constant throughout the ripening process.
4. The cause of tartaric acid accumulation in grape berries isnot thought to be due to a lack of catabolizable enzymes, butto formation of an insoluble salt which is scarcely effectedby such enzymes.

(Received May 2, 1968; )     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM II. THE SYSTEM OF CO2-FIXATION IN THIOBAC1LLUS THIOOXIDANS

1. In the early stage of CO₂-fixation by Thiobacillus thiooxidans,which was incubated aerobically in the presence of sulfur, mostpart of the fixed carbon was found in the phosphate ester fraction.
2. The fixation was inhibited by NaF, picolinic acid, PCMB, azide,dipyridyl, o-phenanthroline, monoiodoacetic acid, and arsenite,each in the concentration range where the sulfur oxidation wasnot affected strongly.
3. The crude extract of this organismcould fix CO₂ in the presenceof ATP, R-5-P and Mg⁺⁺. Most partof the fixed carbon was foundin PGA.
4. The crude extract showedthe CO₂-fixation coupled with the H₂S-oxidationin the presenceof ADP.
5. An appreciable reduction of PGA could not be detectedin thepresence of reducing systems, involving TPNH and DPNH.

(Received March 6, 1962; )     

STUDIES ON THE PATHWAY OF SULFIDE PRODUCTION IN A COPPER-ADAPTED YEAST

Metabolism of some sulfur-containing substances was studiedin a copper-resistant strain of yeast (R), its parent strain(P) and respiratory-deficient(RD) mutants from them. The resultsobtained are as follows:

1. Using sulfate, sulfite and thiosulfate as sulfur sources, Rproducedmore H₂S than P, and both of these had the activityhigher than their RD mutants. All of them produced a large amountof H₂S from cysteine, but only little from methionine, cysteinesulfinic acid and S-sulfocysteine.
2. From sulfite and thiosulfate,P and R produced more H₂S inaerobicthan in anaerobic condition.With sulfate and cysteine, however,H₂S production did not differunder those conditions.
3. In both P and R, the sulfate-to-sulfiteand sulfite-to-sulfidereactions were remarkably lowered byiron and zinc deficiencies.But the cysteine-to-sulfide reactionwas not affected by themetal-deficiencies.
4. H₂S productionfrom sulfate was remarkably depressed by highconcentrationsof pantothenate.
5. Rates of reaction steps on a plausible pathway from sulfatetosulfide and to organic sulfur compounds areestimated forthe strainsused. R is characterized by its largecapacity ofthe reaction step from sulfate to sulfite, and excessivesulfitethus formed is liberatedas sulfide not by the way ofcysteine.

¹Present address: Research Reactor Institute, Kyoto University,Kumatori-cho, Sennan-gun, Osaka     

Oxygen enhancement of photosynthesis in Anacystis nidulans cells1

Oxygen enhanced photosynthetic ¹⁴CO₂ fixation in Anacystis nidulanscells. Results obtained under different conditions revealedthe following properties of the oxygen enhancement:

1. The enhancement was most significant at ca. 10% O₂. Furtherincrease in oxygen concentration decreased the enhancing effect.The rate under 100% O₂ was equivalent to or a little higherthan that under N₂ gas.
2. b) With the increase in CO₂ concentration,the magnitude ofthe enhancing effect decreased. No oxygen enhancementwas observedwhen the CO₂ concentration. was raised to 9,000ppm.
3. c) The enhancement was observed only at high light intensities.No enhancement was observed when the rate of photosynthesiswas limited by light intensity.
4. Ribulose 1,5-diphosphate (RuDP)carboxylase activity was demonstratedin the extract obtainedfrom A. nidulans cells. We also foundthat the RuDP carboxylaseactivity in this extract was competitivelyinhibited by oxygen.
5. Based on the above-mentioned results, the possible mechanismunderlying the observed enhancing effect of oxygen was discussed.

(Received May 10, 1976; )     

ORGANIC ACID SYNTHESIS IN RESPONSE TO EXCESS CATION ABSORPTION IN VACUOLATE AND NON-VACUOLATE SECTIONS OF CORN AND BARLEY ROOTS

1. ¹⁴CO₂ fixation into organic acids in tips and proximal sectionsof both corn and barley roots was studied as a function of thenature and concentration of the salt in the external solution.
2. In comparison with the level of ¹⁴CO₂ fixation by vacuolateproximal sections in KCI, incorporation was markedly enhancedin K₂SO₄ and diminished in CaCk. By contrast, non-vacuolateroot tips were indifferent to the type of external salt withrespect to UCO₂ incorporation into organic acids.
3. The effectof salt type on organic acid formation from ¹⁴CO₂was most pronouncedat relatively high concentrations.
4. The conclusion was reachedthat organic acid synthesis in responsetoexcess cation uptakeis the result of cation movement into thevacuole, and thattransport into the vacuole is mediated bythe low-affinity componentof the dual mechanisms involved inion absorption.

¹Present address: Government Forst Experiment Station, Meguro,Tokyo.     

EFFECT OF LIGHT ON THE CHLOROPHYLL FORMATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. Previous studies have shown that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), apparently chlorophyll-less cells with profoundly degeneratedplastids—referred to as "glucose-bleached cells—areproduced either in the light or in darkness. When the glucose-bleachedcells are incubated in a medium enriched with the nitrogen sourcebut without added glucose, an active formation of chlorophylloccurs after a certain lag period under illumination, whilein darkness a very small amount of chlorophyll is formed atabout the same time as in the light. The stimulating effectof light on the chlorophyll formation is not appreciably affectedwhen the photosynthetic CO₂-fixation of greening algal cellsis blocked by the addition of CMU. In the present study, itwas further found that the light-enhanced chlorophyll formationproceeds, although at a somewhat lower rate, under aerationof CO₂-free air. All the experiments in this work were doneunder these non-photosynthetic conditions to exclude any influenceof photosynthates.
2. The effect of light (from daylight fluorescentlamps) on thechlorophyll formation in the glucose-bleachedalgal cells wassaturating at about 1,000 lux. Blue light wasfound to be mosteffective; yellow, green and red light followingin the orderof decreasing effectiveness.
3. When the bleachedalgal cells were illuminated for a short periodin the lag phaseof chlorophyll formation and subsequently incubatedin darkness,there occurred an appreciable enhancement of chlorophyllformationin the dark. When the short illumination was appliedat differenttimes of the lag phase, the enhancement was inducedto almostthe same extent. But the longer the duration of theilluminationduring the lag phase, the greater was the enhancementof chlorophyllformation in the subsequent dark incubation.In such experimentsblue light was most effective and red lightleast, as it wasthe case in the experiments of continuous illumination.An intervenientillumination of the bleached cells at lowertemperatures orunder the atmosphere of N2 produced little orno enhancementof the chlorophyll formation in the subsequentdark incubation.
4. Based on these results, it was concluded that the light enhancementof chlorophyll formation in the glucose-bleached algal cellsis mediated by a non-chlorophyllous photoreceptor(s), absorbingmaximally blue and yellow light, and that a light-induced changeof the photoreceptor is immediately followed by a certain dark(temperaturedependent and aerobic) process(es) which is connected,directly or indirectly, to the chlorophyll synthesis.

(Received August 10, 1967; )     

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; )     

EFFECT OF SOME SULFHYDRYL REAGENTS ON LIGHT-INDUCED CO2-FIXATION IN CHLORELLA

1. Using intact cells of Chlorella ellipsoidea, investigationswere made on the effects of some SH-reagents (IAA, CMB and arsenite)upon various reactions pertaining to the primary photogenicagent (designated by R) formed in the mechanism of photosynthesis.
2. The pre-illumination experiments using ¹⁴CO₂ as a tracer haveled us to the inference that (i) the process of photochemicalformation of R is inhibited by IAA, that (ii) the spontaneousdecay of R in the dark is markedly accelerated by CMB and arsenite,but not at all affected by IAA, and that (iii) the participationof R in the cyclic path of carbon leading to the fixation ofCO₂ is not affected by IAA and CMB.
3. Based on the assumptionthat R is a reducing agent, it was discussedthat the fact mentionedunder (iii) is incompatible with theidea that the reductionof PGA to triose phosphate be the soleor rate-determining reductivestep in the cyclic path of carbonin photosynthesis.
4. The possibilitythat R may have a functional SH-group (s) wasinvoked to accountfor the observation that the decay of R inthe dark was markedlyaccelerated by CMB and arsenite.

(Received February 11, 1960; )     

EFFECT OF CHROMATIC LIGHTS ON PHYCOBILIN FORMATION IN A BLUE-GREEN ALGA, TOLYPOTHRIX TENUIS

1. The formation of phycobilin pigments in a blue-green alga Tolypothrixtenuis was investigated with special reference to the effectsof preillumination with colored lights.
2. It was discoveredthat the algal cells are capable of formingphycobilin pigmentsin the dark, if they have been previouslyilluminated for severalhours in the presence of CO₂.
3. The color of light applied inthe later period of preillumination(chromatic illumination)was found to affect the ratio of phycoerythrinto phycocyaninformed in the subsequent dark period. A greenlight acceleratesthe dark-formation of phycoerythrin, a redlight that of phycocyanin,and the two lights counteractingwith each other in their effects.
4. These directive effects of the "chromatic illumination" canbe accomplished within a very short period, for instance, in3 minutes if it is preceded by sufficient "preillumination"with an incandescent or day light fluorescent light. The reactionsoccurring during the period of chromatic illumination does notrequire the presence of CO₂ and the aerobic condition.
5. Thealga can be grown heterotrophically when supplied with casaminoacids and glucose. Under such a condition the alga forms phycocyanintogether with chlorophyll and carotenoids, but not phycoerythrin.
6. On the basis of the results obtained, a tentative scheme forthe biosynthesis of phycobilin pigments in the alga was proposed,assuming the light-induced formation of unknown precursors whichare converted into phycocyanin and phycoerythrin in the subsequentdark period.

(Received July 4, 1960; )     

DEVELOPMENT OF PHOTOSYNTHETIC ACTIVITIES DURING THE PROCESS OF CHLOROPLAST FORMATION IN CHLORELLA PROTOTHECOIDES

1. By growing Chlorella protothecoides in a medium rich in glucoseand poor in nitrogen source (urea), entirely chlorophyll-lesscells, called "glucose-bleached’ cells, were obtained.These cells were found to have neither discernible plastid structuresnor photosynthetic activities. When these cells were incubatedin a nitrogenenriched mineral medium without added glucose,a remarkable formation of fully organized chloroplasts occurredin the light and only partially organized chloroplasts weredeveloped in darkness.
2. In the dark-incubated algal cells asmall but appreciable amountof chlorophyll was formed, beingaccompanied by developmentof significant activities for thePMS- and FMN-catalyzed photophosphorylationsand the HILL reaction.The development of the capacity for performingphotosyntheticCO₂-fixation, however, was negligible.
3. During the processof "re-generation" of chloroplasts in thelight, there occurredactive formation of chlorophyll followedby development of allthe photic activities mentioned above.Chlorophyll formationas well as development of the photic activitiesproceeded firstin a manner of autocatalytic reaction and laterin the formof the first-order reaction. It was inferred thatthe light-absorbingagent which mediates the chlorophyll synthesisis chlorophyllitself.
4. The activities for the PMS- and FMN-photophosphorylations,theHILL reaction and photosynthetic CO₂-fixation were recognizedalready in the algal cells at an early stage of greening inthe light, in which the "discs" were developed but no completelamellar structure was observed. Further processes of increaseof these photosynthetic and related activities—as measuredat a high and a lower light intensities—were studied inrelation to the chlorophyll formation under continuous illuminationand under light-dark conditions. It was found that the PMS-photophosphorylationactivity was developed always in parallel with the chlorophyllformation under these different light conditions. Developmentof the activities for the other photic reactions, however, lagged,to different extents, behind the formation of chlorophyll inthe later phase of greening of algal cells under these conditions.
5. Based on these results the modes of formation of the componentsinvolved in these photic reactions were surmised.

(Received September 15, 1965; )     

Organic Acid Metabolism of Sedum praealtum

1. The organic acids present are citric, isocitric, and l-malic,with a small residue of unidentified acids.
2. The diurnal variationin acidity is due chiefly to changes,in malic acid, with aparallel fluctuation shown by citric acid.Under these conditionsisocitric acid shows little change.
3. The importance of carbondioxide during acidification is confirmed,and it is shown thatat room temperatures or higher the CO₂produced in respirationis sufficient to produce maximum acidification.At lower temperaturesthe supply of CO₂ limits acid production.
4. In the absence ofoxygen no acidification occurs, but even smallquantities (approx.1 per cent.) are sufficient to cause someacid production.
5. Completebalance-sheets are presented for acids, carbohydrates,CO₂ andoxygen for leaves maintained in the dark at high andlow temperatures.As acids are produced there is a correspondingloss of carbohydrate(chiefly starch). A scheme of reactionsis suggested to explainthe experimental results.

     

The Influence of Nitrogen, Light and Water Stress on CO2 Exchange and Organic Acid Accumulation in the Tropical C3-CAM Tree, Clusia minor

The carbon balance and changes in leaf structure in Clusia minorL., were investigated in controlled conditions with regardto nitrogen supply and responses to low and high photosyntheticallyactive radiation (PAR). Nitrogen deficiency and high PAR ledto the production of smaller leaves with higher specific leafdry weight (SLDW) and higher leaf water content, but with lowerchlorophyll content. Nitrogen and PAR levels at growth alsoaffected CO₂ exchange and leaf area. In – N conditions,total daily net CO₂ uptake and leaf area accumulation were slightlyless for high-PAR-grown plants. In contrast, high-PAR-grownplants supplied with nitrogen showed about a 4-fold higher totaldaily CO₂ uptake and about twice the total leaf area of low-PAR-grownplants. Although total daily net CO₂ uptake of +N plants wasonly slightly higher than –N plants under the low PARlevel, –N plants produced almost three times more leafarea but with lower SLDW. Under well-watered conditions, low-PAR-grownplants showed only CO₂ evolution during the night and malicacid levels decreased. However, there was considerable night-timeaccumulation of titratable protons due to day/night changesin citric acid levels. High-PAR-grown plants showed net CO₂uptake, malate and citrate accumulation during the dark period.However, most of the CO₂ fixed at night probably came from respiratoryCO₂. Positive night-time CO₂ exchange was readily observed forlow-PAR-grown plants when they were transferred to high PARconditions or when they were submitted to water stress. In plantsgrown in high and low PAR, CAM leads to a substantial increasein daily water use efficiency for water-stressed plants, althoughtotal net CO₂ uptake decreased.     

The Organic Acid Metabolism of Cox's Orange Pippin Apples: I. SOME EFFECTS OF THE ADDITION OF ORGANIC ACIDS TO THE PEEL OF THE FRUIT

1. The basic respirations (CO₂-output and O₂-uptake) of Cox'sOrangePippin apples and of the peel tissue prepared from themwerecompared in fruit in various stages of development, bothinitiallyand after storage at 12°C. Both show the samegeneral trend,although as the apples become mature the peakvalue of the respirationclimacteric tends to rise in the wholefruit and fall in thepeel.
2. The effect of adding malate orcitrate on the respiration ofthe same samples of peel was studied.
3. Three broad stages of development were observed. During thefirst stage (petal fall to 60 days after) the metabolic patternappears to be different from the two later stages. Here O₂-uptakeas well as C₂-output are influenced by the addition of bothmalate and, to a considerably less extent, citrate. In stage2 (60–125 days from petal fall), the malate effect (CO₂-output)is small until after detachment from the tree, when it risessharply. In stage 3 (125 days to full maturity) the malate effectfollows the course expected for earlier work, namely, it developsat the same time as the climacteric rise in respiration. Thepossible reasons for the different behaviour of the peel atthe three stages is discussed.
4. Results were similar in generaltrend for Cox's Orange Pippinapples grown on different rootstocksand under different culturalconditions.
5. It is suggested thatthe malate effect is most active in theepidermal and hypodermaltissues of the fruit.

     

The Effect of Aeration on the Metabolism of Acetate by Aspergillus niger

1. The effect of passing different rates of air or graded concentrationsof CO₂, over cultures of A. niger growing on an acetate mediumhas been investigated. 2. There is a rate of aeration or concentration of CO₂ whichis optimal for most rapid utilization of acetate. High ratesof air-flow severely depress consumption of acetate, whereasvery slow rates only slightly increase the metabolic period. 3. Production of oxalate is not greatly affected by aerationor by different concentrations of CO₂ except where consumptionof acetate is reduced. 4. Citrate formation, is markedly affected by the experimentalconditions. Under optimal treatment citric acid yield is increasedby amounts up to 64 per cent. with controlled aeration and byamounts up to a further i80 per cent. with regulated concentrationof CO₂     

CHANGES IN LEVELS OF PYRIDINE NUCLEOTIDES IN CHLORELLA CELLS DURING THE COURSE OF PHOTOSYNTHESIS

1. Using intact cells of Chlorella, the effects of CO₂ on thelevelsof oxidized and reduced forms of DPN and TPN in the lightandin the dark were investigated.
2. It was found that the light-inducedchanges of the DPNH-levelwere not affected by the presenceor absence of CO₂. On theother hand, the light-induced increaseof TPNH was suppressedin the presence of CO₂ and the levelof TPNH which was raisedon illumination in the absence of CO₂was lowered by the provisionof CO₂.
3. On the basis of thesefindings, it was concluded that TPNH,but not DPNH, is participating,in some way, in the mechanismof photosynthesis.
4. Discussionswere made on the difference in the sites of participationofTPNH and of the photogenic reducing agent (R) in the pathofcarbon in photosynthesis.

(Received February 28, 1960; )     

COMPONENTS OF THE ELECTRON-TRANSFERRING SYSTEM IN ACETOBACTER SUBOXYDANS AND RECONSTRUCTION OF THE LACTATE OXIDATION SYSTEM

1. Cytochromes a₁⁵⁹⁰, b⁵⁶⁰, c₁⁵⁵⁴ and c₁⁵⁵² were isolated andpurifiedfrom a strain of Acetobacter suboxydans. The proceduresusedwere described in detail.
2. The main cytochrome band at550-560 mµ in intact cellssplitted at liquid air temperatureinto two bands, 551 mµ(strong) and 559 mµ (weak).
3. Optical and physiological properties of the four cytochromeswere investigated. Lactic dehydrogenase activity was found tobe associated with cytochrome c₁⁵⁵⁴. The two c₁-type cytochromes,especially cytochrome c₁⁵⁵⁴, persisted in their reduced formafter the purification through many steps.
4. By some combinationsof isolated components reconstruction ofthe oxygen uptake systemcould be realized.
5. The oxygen-consuming activity of purifiedoxidase preparationswas accelerated by a-tocopherol but notby Emasoll 4130 andTween 80.
6. Some discussions were made onthe nature of terminal oxidase,the role of cytochrome c₁⁵⁵²in the electron-transport system,and persistence of reducedstate of c₁-type cytochromes.
7. A possible scheme of the electron-transferringsystem of Acetobactersuboxydans was presented.

(Received May 16, 1960; )     

The Aeration of Catharanthus roseus L. G. Don Suspension Cultures in Airlift Bioreactors: The Inhibitory Effect at High Aeration Rates on Culture Growth

An investigation was carried out to examine the effect of aerationon the growth of Catharanthus roseus suspension cultures inairlift bioreactors. A high aeration rate (0·86 v.v.m.)was found to inhibit the growth of cultures. Venting culturesat a high rate with low oxygen content gas mixtures was equallyinhibitory to culture growth, showing that high aeration wasnot inhibitory as a result of oxygen toxicity. The dissolvedcarbon dioxide tension was found to be lower in cultures operatedat high aeration than those operated at low aeration. Supplyingexogenous CO₂ to cultures at high aeration restored the CO₂tension to values normally encountered at a low aeration rate,and was found to alleviate the inhibitory effects at high aeration.However, further increasing the CO₂ supply to cultures was foundto be severely inhibitory to growth. Therefore, the growth ofC. roseus cultures is very sensitive to dissolved CO₂ concentration,growth being inhibited at values either higher or lower thanan optimum. Key words: Aeration, carbon dioxide, Catharanthus roseus suspension culture     

LIGHT-INDUCED REDUCTION OF NITRATE, NITRITE AND HYDROXYLAMINE IN A BLUE-GREEN ALGA, ANABAENA CYLINDRICA

1. Reduction of nitrate, nitrite and hydroxylamine by intact cellsof Anabaena cylindrica was investigated with special referenceto the stimulating effect of light on these processes.
2. Itwas found that in light and under anaerobic condition thesecompounds are reduced to ammonia, with the production of extraoxygen. The stoichiometry of the reactions under these conditionscan be represented as follows: HNO₂+H₂O=NH₂+2O₂ HNO₂+H₂O=NH₂+1O₂ NH₂OH+H₂O=NH₂+O₂+H₂O
3. Reduction of nitrite and hydroxylaminewas markedly suppressedby CMU in the light but not in the dark.KCN inhibited reductionto the same extent both in the lightand in the dark. Reductionin the light was much less sensitiveto the uncoupling agent,DNP, than was that in the dark.
4. Atlow light intensities, CO_2– was suppressed by 20–30per cent by the simultaneous provision of nitrite, but the nitritereduction was not affected at all by CO₂. At high light intensities,reduction of nitrate and nitrite was considerably acceleratedby CO₂
5. On the basis of these findings, a possible mechanismfor thelight stimulation of the reactions in question was brieflydiscussed.

(Received August 22, 1962; )