Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

A method is described for growing cell suspension cultures of Chenopodium rubrum photoautotrophically for prolonged periods of time. By using a two-tier culture vessel the growth medium with the cells was separated from the CO₂ reservoir. Definite CO₂ concentrations were established by a K₂CO₃/KHCO₃ buffer. Photoautotrophic growth in C. rubrum cell suspension cultures was correlated with the CO₂ level. At 0.5% CO₂ the cell cultures contained 68 μg chlorophyll/g fresh weight and showed an increase in fresh weight of about 80% in 18 days. At 1% CO₂ an increase in fresh weight of 165% in 18 days was observed. The chlorophyll content rose up to 84 μg/g fresh weight. The photoautotrophic growth was also greatly influenced by the 2,4-D content of the medium. Cell growth was enhanced by lowering the auxin concentration. Best growth was attained (210% increase in fresh weight) at 10^?8M 2,4-D. The photosynthetic activity of the cells was measured by the light dependent ¹⁴CO₂ incorporation. At 0.5% CO₂ the cell suspensions assimilated about 100 μmol CO₂/mg chlorophyll × h. In the presence of 1% CO₂ the light driven assimilation was raised up to 185 μmol CO₂/mg chlorophyll × h. In both cases, the dark incorporation of CO₂ was merely 1.8% of the values obtained in light.     

Inorganic Carbon Limitation and Chemical Composition of Two Freshwater Green Microalgae

Two freshwater chlorophytes, Chlorella vulgaris and Scenedesmus obliquus, were grown in inorganic carbon-limited continuous cultures in which HCO₃⁻ was the sole source of inorganic carbon. The response of the steady-state growth rate to the external total inorganic carbon concentration was reasonably well described by the Monod equation; however, the response to the internal nutrient concentration was only moderately well represented by the Droop equation when the internal carbon concentration was defined on a cellular basis. The Droop equation was totally inapplicable when total biomass (dry weight) was used to define internal carbon because the ratio of carbon to dry weight did not vary over the entire growth rate spectrum. In batch cultures, maximum growth rates were achieved at the CO₂ levels present in atmospheric air and at HCO₃⁻ concentrations of 3 mM. No growth was observed at 100% CO₂. Both nitrogen uptake and chlorophyll synthesis were tightly coupled to carbon assimilation, as indicated by the constant C/N and C/chlorophyll ratios found at all growth rates. The main influence of inorganic carbon limitation appears to be not on the chemical structure of the biomass, but rather on cell size; higher steady-state growth rates lead to bigger cells.     

Growth and respiratory activity of mold fungus (Trichoderma lignorum)

The growth ofTrichoderma lignorum was studied in relation to the carbon balance of the culture system and the respiratory activity at different growth phases. Conidia, after inoculation into the medium, swelled and germinated rapidly. The growth rate of the hyphae at the exponential phase was 0.46 hr⁻¹ (2.2 hr for mass doubling) at 25 C. The yield efficiency of hyphal biomass-C at the cost of glucose-C was 67%, while those of the waste products-C excreted and of CO₂-C evolved were, 6.5% and 26.5%, respectively. The yield efficiency of conidia-C to the decrease of hyphae-C was 34%. The germination, growth rate and carbon balance were not affected by different concentrations of glucose from 10 to 2×10³ mg glucose-C/l. Carbon dioxide was needed as the growth factor for the initiation of the germination of conidia, but there was no increase in yield efficiency as a result of CO₂ fixation. The respiratory rate of the fungus changed drastically as the growth proceeded. The rate of endogenous respiration of conidia was less than 0.2 mg CO₂-C/g conidia-C/hr which increased immediately after inoculation into the medium. The highest respiratory rate of hyphae (100–110 mg CO₂-C/g hyphae-C/hr) was obtained throughout the exponential phase. Thereafter, decreasing rapidly, the respiratory rate of submerged hyphae of 1-week-old showed only 1.8 mg CO₂-C/g biomass-C/hr, whereas the rate of aerial hyphae forming conidia increased again, but did not exceed 10 mg CO₂-C/g biomass-C/hr.     

Wachstum und photosyntheseleistung der halophyten Mesembryanthemum nodiflorum L. und Suaeda maritima (L.) Dum. bei variierter NaCl-Salinität des anzuchtmediums

Summary The effect of various concentrations of NaCl in the culture solution (up to 400 mM) on growth (fresh weight, dry weight, chlorophyll content) and net CO₂ gas exchange was studied in two halophytes, Mesembryanthemum nodiflorum L. and Suaeda maritima (L.) Dum. Mesembryanthemum nodiflorum reaches optimal growth at concentrations of 100 to 200 mM NaCl in the culture medium, whereas growth of Suaeda maritima is stimulated up to concentrations of 400 mM NaCl. Independent of this stimulated plant growth, net CO₂ uptake in the light appears to be decreased with increased salinity when net CO₂ assimilation is calculated on a fresh weight or dry weight basis. However, expressed per mg chlorophyll, net CO₂ uptake is clearly correlated with plant productivity at different levels of salinity.     

Biochemical responses and photosynthetic performance of Gracilaria sp. (Rhodophyta) from Cádiz,Spain, cultured under different inorganic carbon and nitrogen levels

Photosynthetic acclimation and the interactions between carbon (C) and nitrogen (N) metabolism have been studied in the red macroalga Gracilaria sp. from Cádiz, Spain, cultured under different inorganic C and N levels. The use of chemostats and buffered medium allowed continuous restoration of the alkaline reserve and constancy of pH during the experiments. The N:C ratios and phycobiliprotein, chlorophyll a and soluble protein contents decreased when Gracilaria sp. was grown at low N levels. Algae grown in a high inorganic C concentration (5% CO₂) displayed a higher soluble carbohydrate concentration and maximum photosynthesis rates but a lower photosynthesic affinity for inorganic C, and lower phycobiliprotein and Rubisco contents, than those cultured at low inorganic C levels (air CO₂). The inorganic C enrichment also affected the N uptake and assimilation in Gracilaria sp., causing a decrease in the N uptake rate even under conditions of N sufficiency. These results reflect the significant influence of the inorganic C growth regime on N assimilation in Gracilaria sp.     

Metabolism of Acetivibrio cellulolyticus during optimized growth on glucose,cellobiose and cellulose

Summary The growth of Acetivibrio cellulolyticus in 2.5 l batch cultures was optimized by controlling the growth pH at 6.7, the dissolved inorganic sulphide concentration at 0.4–0.6 mM, and by constant removal of hydrogen from the cultures by sparging with N₂/CO₂ or N₂ gas. An initial ethanol concentration of 0.15% (w/v) in cellobiose media resulted in specific growth rates which were reduced by about 75% compared to growth rates of 0.17 h^–1 in control cultures. Acetivibrio cellulolyticus had to be adapted for growth on glucose and ¹⁴C-radiotracer studies indicated that glucose was metabolized by the Embden-Meyerhof pathway. The specific growth rate (=0.03h^–1) and molar growth yield (Y_glucose=21.5) were considerably lower than those obtained (=0.17 h^–1, Y_cellobiose=68.9) in cellobiose media. A Y_ATP of 12.8 was obtained during growth on cellobiose. The mol product formed per mol Avicel cellulose fermented (on anhydroglucose equivalent basis) were 3.70 H₂, 2.64 CO₂, 0.73 acetate, 0.39 ethanol and 0.03 total soluble sugars on glucose basis. Maximum cellulase activity was observed in cellulose-grown cultures.National Research council of Canada No. 20826     

Growth and tissue senescence inPrunus avium shoots grownin vitro at different CO3/O2 ratios

Summary The rate of metabolism and biosynthetic processes makein vitro cultures very sensitive to environmental changes, and therefore subject to physiological and morphological alterations leading to senescence in the short term. The effect of three different calibrated atmospheric compositions were studied duringin vitro culture ofPrunus avium shoots. At 0.034% CO₂-21% O₂ (vol/vol), which stimulate the natural atmosphere, the highest growth rate and chlorophyll content were recorded. When grown at 0.09% CO₂-8% O₂ (vol/vol), a favorable condition for photosynthesis and growth, cultures showed a higher percentage of dry matter and elevated ethylene production, but total chlorophyll was lower. These shoots were also highly lignified and fibrous with red pigmentation along the leaves and stems. At 0% CO₂-21% O₂ (vol/vol), in contrast, growth and ethylene formation were inhibited; chlorophyll content was lowest in comparison with the other two environmental conditions, but regreening of tissues was observed after the first half of the culture period. Senescence symptoms, as indicated by decreased chlorophyll, appeared after about 18 d of culture for tissues grown in CO₂-containing atmospheres. These experiments provided evidence that in CO₂-enriched cultures biomass production steadily increased even when chlorophyll decreased. A possible role of CO₂ in promoting tissue-senescence through activation of photooxidative events and ethylene synthesis is discussed.     

Vertical thin-layer photoreactor for controlled cultivation of cyanobacteria

Nostoc sp. was cultivated in an air-lift reactor with continuous recirculation of the head gas phase that aerated and agitated the cyanobacterial suspension at regulated flow rates. The supply of inorganic carbon for growth was coupled with pH control, in the range of 7.7 to 8.1, by intermittent sparging of CO₂-head gas mixtures. The formation of irregular bubbles with swirling motion at the photostage of the reactor promoted efficient CO₂ transference in dense populations of Nostoc sp. (1.1 g/l) when bubbling at flow rates of 10 l/min. Biomass productivity was almost six-fold higher in the photoreactor (16.4 mg/l.h) than in a conventional system (2.8 mg/l.h). The exponential growth phase of cultures in the photoreactor amounted to 60% of the total growth period.The authors are with the Laboratorio de Alimentos, Area Microbiologia, Facultad de Quimica Bioquimica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina     

A culture method for microalgal forms using two-tier vessel providing carbon-dioxide environment: studies on growth and carotenoid production

A culture method was developed for photoautotrophic culture of Haematococcus pluvialis, Chlorella vulgaris, Scenedesmus obliquus, Spirulina platensis, Nostoc and Stigonema in a two-tier flask consisting of nutrient media in the upper chamber and CO₂ generating buffer mixture (KHCO₃/K₂CO₃) in the lower chamber. The concentration of buffer mixture was varied to obtain desired levels of CO₂. CO₂ at 2.0% (v/v) level enhanced growth and chlorophyll content over control cultures (without CO₂ supplementation) in all microalgal species. Haematococcus pluvialis culture in BBM and KM1 media showed 6.71- and 2.07-fold increase in biomass yields with astaxanthin productivity at 7.26 and 7.48 mg l^–1 level respectively. CO₂ supplementation to C. vulgaris and S. obliquus cultures resulted in 5.97- and 7.30-folds increase in biomass with 2–3 fold increase in chlorophyll and carotenoid contents over their respective controls. Similarly 2–3 fold increase in chlorophyll and carotenoid contents were observed in Sp. platensis, Nostoc and Stigonema spp. This culture methodology will provide information on CO₂ requirement for growth of algae and metabolite production and also facilitates studies on the influence of light and temperature conditions.     

Effects of Carbon Dioxide on Coccidian Oocysts from 6 Host Species*

SYNOPSIS Activation of sporozoites in oocysts of Eimeria acervulina (chicken), E. intricata (sheep), and E. scabra (swine) occurred after pretreatment in aqueous 0.02 M cysteine hydrochloride under an atmosphere of CO₂, followed by incubation in a trypsin-bile mixture. Sporozoites of E. stiedae (rabbit), E. bilamellata (squirrel), and Isospora canis (dog) became activated when incubated in trypsin and bile with or without prior CO₂-pretreatment of oocysts; however, when CO₂-pretreatment was used, activation of these species in trypsin and bile was greatly enhanced. For E. acervulina, 12% of the oocysts were activated after 4 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 43 C; higher temperatures or longer pretreatment times did not cause greater activation. Eimeria intricata oocysts became activated after 1 hr pretreatment and 10 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively. The highest activation (31%) occurred after 20 hr pretreatment and 10 hr incubation in trypsin and bile at 41 C. Ninety percent of E. scabra oocysts contained active sporozoites after 1 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 37 C. At 39 or 41 C, 100% activation occurred with this species after similar pretreatment and treatment periods. With E. bilamellata, 64% activation occurred in nonpretreated oocysts incubated 10 hr in trypsin and bile at 41 C, whereas 100% activation occurred if oocysts were pretreated with CO₂ for 1 hr before treatment with trypsin and bile. Thirty-one, 35, and 36% of CO₂-pretreated E. stiedae oocysts were activated after 1 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively, whereas 1, 2, and 20% activation occurred in nonpretreated oocysts incubated at the same temperatures. Sporozoites in 99-100% of I. canis oocysts were activated after 10 hr treatment in trypsin and bile with or without 1 hr CO₂-pretreatment at 23, 37, 39 or 41 C.     

Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

Effect of dissolved inorganic carbon on the expression of carboxysomes,localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO₂+HCO _inf3 ^sup- ) in the growth medium. In cells grown under 5% CO₂ and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section^-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO₂ · 1^-1 possessed many carboxysomes (>8 · longitudinal section^-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO _inf3 ^sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon (CO₂+HCO _inf3 ^sup- +CO _inf3 ^sup2- ) - PRK phosphoribulokinase - RuBP ribulose 1,5-bisphosphate - Rubisco LS large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase     

Methanobacterium arbophilicum sp. nov. An obligate anaerobe isolated from wetwood of living trees

The isolation and characterization of a new methanogenic bacterium,Methanobacterium arbophilicum, is described. Isolation from wetwood enrichment cultures, that were obtained from methane-positive trees, required a medium containing inorganic salts, vitamins, and an atmosphere consisting of an 80∶20 mixture of hydrogen-carbon dioxide. Isolates ofM. arbophilicum were gram-positive, non-motile short rods that occurred singly, in pairs, or chains. The organism was found to be an autotroph and a strict anaerobe, and to have a pH optimum of 7.5–8.0. The optimal temperature for growth was 30 to 37C, the maximum being 45C and the minimum about 10C. The organism had obligate growth requirements for H₂ and CO₂, and organic compounds greatly stimulated growth. The generation time in shake flask culture was about 17 hr in mineral salts medium and about 13 hr in complex medium. The DNA base composition was 27.5 mol % GC.     

Photorespiratory toxicity in autotrophic cell cultures of a mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity

Procedures were devised for heterotrophic culture and autotrophic establishment of protoplast-derived cell cultures from the sat mutant of Nicotiana sylvestris Speg. et Comes lacking serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) activity. Increasing photon flux rates (dark, 40, 80 mol quanta·m^-2·s^-1) enhanced the growth rate of autotrophic (no sucrose) wild-type (WT) cultures in air and 1% CO₂. Mutant cultures showed a similar response to light under conditions suppressing photorespiration (1% CO₂), and maintained 65% of WT chlorophyll levels. In normal air, however, sat cultures developed severe photorespiratory toxicity, displaying a negligible rate of growth and rapid loss of chlorophyll to levels below 1% of WT. Low levels of sucrose (0.3%) completely reversed photorespiratory toxicity of the mutant cells in air. Mutant cultures maintained 75% of WT chlorophyll levels in air, displayed light stimulation of growth, and fixed ¹⁴CO₂ at rates identical to WT. Autotrophic sat cultures accumulated serine to levels nearly nine-fold above that of WT cultures in air. Serine accumulated to similar levels in mixotrophic (0.3% sucrose) sat cultures in air, but had no deleterious effect on fixation of ¹⁴CO₂ or growth, indicating that high levels of serine are not toxic, and that toxicity of the sat mutation probably stems from depletion of intermediates of the Calvin cycle. Autotrophic sat cultures were employed in selection experiments designed to identify spontaneous reversions restoring the capacity for growth in air. From a population of 678 000 sat colonies, 23 plantlets were recovered in which sustained growth in air resulted from reacquisition of SGAT activity. Twenty-two had SGAT levels between 25 and 50% of WT, but one had less than 10% of WT SGAT activity, and eventually developed symptoms typical of the sat mutant. The utility of autotrophic sat cultures for selection of chloroplast mutations diminishing the oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is discussed.Abbreviations Chl chlorophyll - DW dry weight - FW fresh weight - SGAT Serine:glyoxylate aminotransferase - WT wild-type     

Mutagenesis and Triazine Herbicide Effects in Strawberry Shoot Cultures

The effects of simazine on shoot culture of two varieties ofstrawberry were investigated with a view to establishing a systemfor in vitro selection of resistant mutants. In single shootssome chlorophyll loss occurred at herbicide levels producinglittle growth inhibition, but efficient bleaching was associatedwith severe suppression of growth. Varying simazine and sucroselevels, in both single and multiple shoot cultures did not revealany conditions where chlorophyll destruction and growth inhibitioncould be separated. A protective effect, against bleaching,of high sucrose levels was not linked to suppression of photosynthesis,as assessed by CO₂-fixation measurements. Mutagenesis with nitrosomethylurea resulted in chlorophyll deficiencies in about 10% of shootsarising from axillary buds, but no resistant sectors emergedfrom 8000 and 400 mutagenized explants placed on medium containinginhibitory levels of simazine and streptomycin sulphate, respectively. Key words: Strawberry, simazine, triazine, mutagenesis, shoot cultures     

Effect of inorganic carbon on photoautotrophic growth of microalga Chlorococcum littorale

The growth rate of a highly CO₂‐tolerant green alga, Chlorococcum littorale, was investigated in semi‐batch cultures at a temperature of 22°C, a light intensity of 170 μmol‐photon m^?2 s^?1 and CO₂ concentrations ranging from 1 to 50% (v/v) at atmospheric pressure. In the experiments, solutions were bubbled with CO₂ and N₂ gas mixtures to adjust CO₂ concentrations to minimize the influence of O₂. Growth rate, which was defined in terms of a specific growth rate μ, decreased with increasing CO₂ concentration at the conditions studied. The inhibition of growth by CO₂ gas could be attributed to the concentration of inorganic carbon in the culture medium. A growth model is proposed where key assumptions are the formation of bicarbonate ion HCO as substrate for algal growth and equilibrium between CO₂ inhibitor. The proposed growth model based on the Monod equation agreed with the experimental data to within 5% and provides better correlation than the conventional inhibition model, especially in the high CO₂ concentration region. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Culturing of stoneworts and submersed angiosperms with phosphate uptake exclusively from an artificial sediment

1. We aimed to demonstrate reproducible nutrition and growth of macrophytes in non‐axenic laboratory cultures preventing growth of phytoplankton and epiphytes. 2. Macrophyte shoot segments were planted in a mixture of commercial acid‐washed silica sand with crystalline tricalcium phosphate, and this artificial sediment was covered with a layer of pure silica sand. The liquid mineral media used did not contain phosphorus but were rich in all other nutrient elements. A CO₂ reservoir provided sustainable CO₂ supply to macrophyte cultures by gas diffusion through a polyethylene membrane. 3. Chara hispida, Chara tomentosa, Chara baltica, Elodea canadensis, Potamogeton pectinatus and Zanichellia palustris could be cultivated for long term without medium exchange and aeration. Microalgae growth was prevented by the absence of phosphate in the water column. Mobilisation of tricalcium phosphate and phosphate uptake by the rhizoids of C. hispida enabled sustainable rapid shoot growth and increased the concentration of inorganic phosphate in the shoot dry weight by five to six times in comparison with plants cultivated on pure silica sand. A significant growth support from tricalcium phosphate was also observed for E. canadensis, but the rate of phosphate uptake by the roots was not sufficient to maintain a storage pool of inorganic phosphate (P_i) in the growing shoots of this plant. 4. Membrane‐controlled CO₂ supply from a reservoir and artificial sediments like the one described provide attractive options for the laboratory culture of macrophytes.     

Chlorosis during nitrogen starvation is altered by carbon dioxide and temperature status and is mediated by the ClpP1 protease in <Emphasis Type="Italic">Synechococcus elongatus</Emphasis>

The interactive effects of inorganic carbon status, temperature and light on chlorosis induced by nitrogen deficiency, and the roles of Clp proteases in this process were investigated. In wild-type cultures grown in high or ambient CO₂, following transfer to media lacking combined nitrogen, phycocyanin per cell dropped primarily through dilution of the pigment through cell division, and also suffered variable degrees of net degradation. When grown at high CO₂ (5%), chlorophyll (Chl) suffered net degradation to a greater extent than phycocyanin. In marked contrast, growth at ambient CO₂ resulted in Chl per cell dropping through dilution. Conditions that drove net Chl degradation in the wild-type resulted in little or no net Chl degradation in a clpPI inactivation mutant, with Chl content dropping largely through growth dilution in the mutant. The chlorotic response of a clpPII inactivation strain was nearly the same as that of wild-type, although phycocyanin degradation may have been slightly accelerated in the former.     

Glucose fermentation byClostridium butyricum grown under a self generated gas atmosphere in chemostat culture

Summary Clostridium butyricum was grown anae-robically under glucose-limited conditions in che-mostat cultures under self generated gas atmo-sphere. It is shown that the quantitative composi-tion of the fermentation products is dependent on the pH value, the growth rate, the concentration of glucose in the growth medium and the compo-sition of the gas atmosphere developed in the reactor. The ratio q_acetate/q_butyrate increases from 0.06 to 0.66 in parallel with an increase in growth rate from 0.02 h⁻¹ to 0.29 h⁻¹ (at pH = 6.0). De-creasing the partial pressure of H₂ results in an in-crease of the q_acetate/q_butyrate ratio. The partial pressure of CO₂ in the reactor does not influence the fermentation products whatsoever. Increasing pH values (>6.8) and concentrations of glucose in the growth medium also result in increasing q_acetate/q_butyrate ratios. The maximal Y_ATP is con-stant from pH 4.8–6.0. The functioning of NADH₂-ferredoxin oxi-doreductase is discussed.     

Effect of carbon dioxide on pigment and membrane content in Synechococcus lividus

The effect of carbon dioxide on pigment and membrane content in Synechococcus lividus was studied by depriving cells of CO₂ and examining cell populations biochemically and by electron microscopy. After 120 h of CO₂ deprivation, S. lividus lost all detectable chlorophyll a and C-phycocyanin. Such bleached cultures were mustard yellow, the result of approximately 1.8 times more carotenoid per cell than green control cultures.Although cells from beached cultures appeared morphologically identical to control green cells when examined by light microscopy, electron microscopic examination revealed them to be devoid of detectable thylakoid membrane. Thylakoid membrane could not be recovered by physical isolation or revealed by freeze etching of bleached S. lividus. In addition, inclusion bodies characteristically found in S. lividus were also absent.Reintroduction of CO₂ into bleached cultures resulted in a rapid resynthesis of both chlorophyll a and C-phycocyanin. Electron microscopic examination of these regreening cultures revealed that thylakoid membrane was also rapidly resynthesized. Growth of regreened cultures did not occur until there was the synthesis of a full complement of chlorophyll a, C-phycocyanin, and thylakoid membrane.A time course study of the cytological events occurring during bleaching and regreening is presented.