Enhanced Chlorella vulgaris Buitenzorg growth by photon flux density alteration in serial photobioreactors

Microalgae perform oxygenic photosynthesis and are capable of taking up a large amount of CO₂, using an inducible CO₂ concentrating mechanism (CCM), and fixing CO₂ into higher compounds. These characteristics make the microalgae potentially useful for removal and utilization of CO₂ emitted from industrial plants and, generally, the usage of photosynthetic microorganisms has increased and significantly improved as a solution for CO₂ emissions. In this light and based on previous research using Anabaena cylindrica IAM M1 and Spirulina platensis IAM M 135, enhancement was sought for CO₂ fixation and biomass production by Chlorella vulgaris Buitenzorg by increasing the photon flux density concurrent with increases in culture biomass during the cellular growth phase and was compared to cultures of Chlorella grown at optimal constant illumination, with all cultures grown using Bennick basal medium, 29°C, and a flow of 1.0 atm. 10% CO₂ enriched air delivered to three in serial photobioreactors of 0.200 dm³ capacity each. The results showed that increasing illumination during culture increased biomass production of Chlorella by ∼60% as well as increased CO₂ fixation ability by ∼7.0%. It was also demonstrated that the non-competitive inhibition of [HCO₃ ⁻] as a carbon source significantly affected the cultivation in both the increasing and constant photon flux density regimes.     

TES and HEPES buffers in mammalian cell cultures and viral studies: Problem of carbon dioxide requirement

In order to evaluate TES and HEPES as a buffer system for cell culture, the proliferative capacities of cells of several mammalian cell lines in the medium buffered with either of these compounds were examined in cultures in stoppered and open flasks at high and low cell densities. When cultivated in stoppered flasks, cells grew equally well or even better in TES- and HEPES-buffered medium than in NaHCO₃-buffered medium irrespective of cell culture density. In open flasks or Petri dishes in TES- or HEPES-buffered medium, however, the proliferative capacity of cells in low density cultures was limited. The inhibition of cell growth in the latter condition was restored (1) as the cell density of the cultures increased; (2) by feeding continuously the cultures with the gas produced by high density cultures; (3) by introducing a small amount of CO₂ to the environment.These and other evidences presented suggest that, in agreement with the prevailing notions, CO₂ is required by cells as an essential nutrient for growth, and that the desired level of CO₂ in culture can be maintained efficiently by its production by even a small number of cells in culture as long as the culture flasks are stoppered. If flasks are not stoppered, however, the level of CO₂ tension is determined by an equilibrium between the rate of its production by the cells and that of escape from culture to air, resulting in the observed failure in growth of cells in TES- and HEPES-buffered medium at low cell densities unless cultures were further supplemented with added CO₂.     

CO2 accumulation in bioreactor suspension cultures of Cyclamen persicum Mill. and its effect on cell growth and regeneration of somatic embryos

CO₂ accumulation in different culture systems containing embryogenic cell suspension cultures of cyclamen (Cyclamen persicum Mill.) was analyzed. In bioreactors equipped with a bubble-free or a bubble aeration system, CO₂ mole fractions in the gas phase of more than 10% were determined whereas in Erlenmeyer flasks, CO₂ mole fractions were below 2%. CO₂ accumulation in bioreactors was severely growth inhibiting in comparison to the flasks. By removing CO₂ in the aeration gas of a bubble-free aerated bioreactor, cell growth comparable to that in flasks was achieved. The regeneration ability of cell suspensions after being cultured in bioreactors with CO₂ accumulation was better than those after culture in bioreactors without CO₂ accumulation or in flasks. Received: 16 June 1998 / Revision received: 13 August 1998 / Accepted: 1 December 1998     

Simultaneous measurement of ethylene and CO2 production and of O2 consumption in soil and pure microbial cultures

Summary The method described here makes it possible to determine gaseous and volatile compounds produced by stationary and shaken cultures of microorganisms, and by soil samples. In a closed system oxygen consumed by a biological sample is electrolytically replenished and CO₂ produced is trapped in a KOH solution. Oxygen from the electrolyzer was replenished through a distribution bottle with the aid of a system of polyethylene tubings equipped with injection needles at both ends. The amount of the oxygen consumed is permanently reflected by volume of the electrolytically released hydrogen. The reverse flow of atmosphere from the cultivation flasks was prevented by a liquid seal formed by a KOH solution or the cultivation medium itself. Suba-seal type flasks (125 ml) with rubber caps served for the cultivation. Glass cylinders placed inside cultivation flasks or penicillin flasks (20 ml) connected with cultivation flasks by polyethylene tubings served as adsorption vessels. By exchanging the KOH solution it is possible to follow CO₂ production even during individual cultivation phases. Volatile acids adsorbed in the KOH solution could be determined after their release by mineral acid. Samples of atmosphere were analyzed by gas chromatography. The whole system had to be temperature equilibrated.     

The Influence of Precipitation Regimes and Elevated CO2 on Photosynthesis and Biomass Accumulation and Partitioning in Seedlings of the Rhizomatous Perennial Grass Leymus chinensis

Leymus chinensis is a dominant, rhizomatous perennial C₃ species in the grasslands of Songnen Plain of Northern China, and its productivity has decreased year by year. To determine how productivity of this species responds to different precipitation regimes, elevated CO₂ and their interaction in future, we measured photosynthetic parameters, along with the accumulation and partitioning of biomass. Plants were subjected to combinations of three precipitation gradients (normal precipitation, versus normal ± 40%) and two CO₂ levels (380±20 µmol mol^-1,760±20 µmol mol^-1) in controlled-environment chambers. The net photosynthetic rate, and above-ground and total biomass increased due to both elevated CO₂ and increasing precipitation, but not significantly so when precipitation increased from the normal to high level under CO₂ enrichment. Water use efficiency and the ratio of root: total biomass increased significantly when precipitation was low, but decreased when it was high under CO₂ enrichment. Moreover, high precipitation at the elevated level of CO₂ increased the ratio between stem biomass and total biomass. The effect of elevated CO₂ on photosynthesis and biomass accumulation was higher at the low level of precipitation than with normal or high precipitation. The results suggest that at ambient CO₂ levels, the net photosynthetic rate and biomass of L. chinensis increase with precipitation, but those measures are not further affected by additional precipitation when CO₂ is elevated. Furthermore, CO₂ may partly compensate for the negative effect of low precipitation on the growth and development of L. chinensis.     

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.     

Organic acid production from CO2/H2 and CO/H2 by mixed-culture anaerobes

The gases CO, CO₂, and H₂ were used as substrates in anaerobic fermentations producing organic acids. Various mixed bacterial sources were used, including sewage sludge digester effluent, rabbit feces, and soil. Nonsterile microorganism selection was carried out using CO₂/H₂ and CO/H₂ as the primary carbon and energy sources. Cultures were grown in specially designed, high-pressure (to 70 psig) flasks. Methanogenic bacteria were eliminated from the cultures. Liquid products of the fermentations were acetic through caproic acids, with the even-numbered acids predominating. Carbon balances showed conclusively that acetic acid was formed from carbon contained in the CO or CO₂ feed gas. Measurements made included rates of acid product formation, cell density, and degree of gas utilization. Limited characterization of the microorganisms was also performed. Production of organic acids by mixed culture inocula from CO₂/H₂ or CO/H₂ had not been reported previously. Application of this work is to the production of organic chemicals from synthesis gas (SNG), produced by the gasification of fossil fuels (peat, lignite, and various ranks of coals), biomass (agricultural and forest residues, and various biomass crops grown expressly for energy recovery), and municipal solid waste.     

Innovative development of membrane sparger for carbon dioxide supply in microalgae cultures

The present study was aimed to develop a membrane sparger (MS) integrated into a tubular photobioreactor to promote the increase of the carbon dioxide (CO₂) fixation by Spirulina sp. LEB 18 cultures. The use of MS for the CO₂ supply in Spirulina cultures resulted not only in the increase of DIC concentrations but also in the highest accumulated DIC concentration in the liquid medium (127.4 mg L⁻¹ d⁻¹). The highest values of biomass concentration (1.98 g L⁻¹), biomass productivity (131.8 mg L⁻¹ d⁻¹), carbon in biomass (47.9% w w⁻¹), CO₂ fixation rate (231.6 mg L⁻¹ d⁻¹), and CO₂ use efficiency (80.5% w w⁻¹) by Spirulina were verified with MS, compared to the culture with conventional sparger for CO₂ supply. Spirulina biomass in both culture conditions had high protein contents varying from 64.9 to 69% (w w⁻¹). MS can be considered an innovative system for the supply of carbon for the microalgae cultivation and biomass production. Moreover, the use of membrane system might contribute to increased process efficiency with a reduced cost of biomass production.     

Seasonal physiological responses and biomass growth in a bioenergy crop (<Emphasis Type="Italic">Phalaris arundinacea</Emphasis> L.) under elevated temperature and CO<Subscript>2</Subscript>, subjected to different water regimes in boreal conditions

We investigated the seasonal variability of effects of elevated temperature (+3.5°C), CO₂ elevation (700 μmol mol⁻¹) and varying water regimes (high to low water content) on physiological responses and biomass growth of reed canary grass (Phalaris arundinacea L., local field-grown cultivar) grown in a boreal environment. In controlled environment greenhouses, various physiological and growth parameters of grass, i.e., light-saturated net photosynthetic rates (P _nmax), water use efficiency (WUE) and optimal photochemical efficiency of photosystem II (F _v/F _m), and leaf area development and biomass of plant organs (leaf, stem, coarse, and fine root) were measured. During the early measurement periods, elevated temperature enhanced leaf photosynthesis and above-ground biomass of reed canary grass; however, this resulted in earlier senescence and lower biomass at the end of measurement period, compared to ambient temperature. This effect was more pronounced under water limitation. Elevated CO₂ enhanced P _nmax, WUE, and leaf area and total plant biomass (above- and below-ground) over growing season. The explanation for imbalance between stimulated photosynthesis and increase in above-ground biomass was that CO₂ enrichment causes a greater increase in the plant’s root system. The combination of elevated temperature and CO₂ slightly increases the growth of plant. Adequate water availability favored photosynthesis and biomass growth of reed canary grass. The temperature- and drought-induced stresses were partially mitigated by elevated CO₂. Other cultivars should be tested in order to identify those that are better adapted to elevated temperatures and CO₂ and variable water levels.     

Photosynthetic efficiency and rate of CO2 assimilation by Arthrospira (Spirulina) platensis continuously cultivated in a tubular photobioreactor

Similar to other photosynthetic microorganisms, the cyanobacterium Arthrospira platensis can be used to produce pigments, single cell proteins, fatty acids (which can be used for bioenergy), food and feed supplements, and biofixation of CO₂. Cultivation in a specifically designed tubular photobioreactor is suitable for photosynthetic biomass production, because the cultivation area can be reduced by distributing the microbial cells vertically, thus avoiding loss of ammonia and CO₂. The aim of this study was to investigate the influence of light intensity and dilution rate on the photosynthetic efficiency and CO₂ assimilation efficiency of A. platensis cultured in a tubular photobioreactor in a continuous process. Urea was used as a nitrogen source and CO₂ as carbon source and for pH control. Steady‐state conditions were achieved in most of the runs, indicating that continuous cultivation of this cyanobacterium in a tubular photobioreactor could be an interesting alternative for the large‐scale fixation of CO₂ to mitigate the greenhouse effect while producing high protein content biomass.     

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.     

Combined Effects of CO2 Enrichment and Drought Stress on Growth and Energetic Properties in the Seedlings of a Potential Bioenergy Crop Jatropha curcas

The rapid growth of worldwide energy demands has led to mounting concerns about energy shortages and has promoted the development of biofuels, which are susceptible to climate change. To evaluate the effects of future environmental changes such as CO₂ enrichment and water stress on the growth and biodiesel production of bioenergy plants, we exposed Jatropha curcas to two levels of CO₂ concentration (ambient and elevated) and three watering regimes (well-watered, moderate drought, and severe drought) to study its biomass accumulation and allocation, energy cost-gain properties, and photosynthetic response. Elevated CO₂ enhanced biomass accumulation of J. curcas by 31.5, 25.9, and 14.4 % under well-watered, moderate drought, and severe drought treatments, respectively, indicating that the stimulating effect was greater under optimum water conditions than in water-deficit conditions. Drought stress significantly increased the biomass allocation to roots, especially the fine roots. CO₂ enrichment also increased the root mass fraction, though not significantly. CO₂ enrichment significantly enhanced the photosynthetic rate measured under growth CO₂ concentration (A _growth) and decreased foliar N content and therefore construction cost irrespective of watering conditions. Under elevated CO₂, J. curcas employed a quicker return energy use strategy indicated by the higher photosynthetic energy use efficiency and lower payback time. There was a pronounced downregulation in the light-saturated photosynthetic rate under the common CO₂ concentration (P _max) under long-term CO₂ exposure, due to a decrease in the initial and total ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities and partially lower foliar N content. The significant interaction of CO₂ enrichment and watering regimes implied that the stimulation of plant growth by CO₂ enrichment may be negated by soil drought in the future. Long-term field experiments manipulating multiple factors simultaneously are needed to explore how the ecophysiological traits measured for J. curcas translate into bioenergy production.     

On-line estimation of O<Subscript>2</Subscript> production,CO<Subscript>2</Subscript> uptake,and growth kinetics of microalgal cultures in a gas-tight photobioreactor

Growth of the green algae Chlamydomonas reinhardtii and Chlorella sp. in batch cultures was investigated in a novel gas-tight photobioreactor, in which CO₂, H₂, and N₂ were titrated into the gas phase to control medium pH, dissolved oxygen partial pressure, and headspace pressure, respectively. The exit gas from the reactor was circulated through a loop of tubing and re-introduced into the culture. CO₂ uptake was estimated from the addition of CO₂ as acidic titrant and O₂ evolution was estimated from titration by H₂, which was used to reduce O₂ over a Pd catalyst. The photosynthetic quotient, PQ, was estimated as the ratio between O₂ evolution and CO₂ up-take rates. NH₄ ⁺, NO₂ ⁻, or NO₃ ⁻ was the final cell density limiting nutrient. Cultures of both algae were, in general, characterised by a nitrogen sufficient growth phase followed by a nitrogen depleted phase in which starch was the major product. The estimated PQ values were dependent on the level of oxidation of the nitrogen source. The PQ was 1 with NH₄ ⁺ as the nitrogen source and 1.3 when NO₃ ⁻ was the nitrogen source. In cultures grown on all nitrogen sources, the PQ value approached 1 when the nitrogen source was depleted and starch synthesis became dominant, to further increase towards 1.3 over a period of 3–4 days. This latter increase in PQ, which was indicative of production of reduced compounds like lipids, correlated with a simultaneous increase in the degree of reduction of the biomass. When using the titrations of CO₂ and H₂ into the reactor headspace to estimate the up-take of CO₂, the production of O₂, and the PQ, the rate of biomass production could be followed, the stoichiometrical composition of the produced algal biomass could be estimated, and different growth phases could be identified.     

The air‐lift photobioreactors with flow patterning for high‐density cultures of microalgae and carbon dioxide removal

A photobioreactor containing microalgae is a highly efficient system for converting carbon dioxide (CO₂) into biomass. Using a microalgal photobioreactor as a CO₂ mitigation system is a practical approach to the problem of CO₂ emission from waste gas. In this study, a marine microalga, Chlorella sp. NCTU‐2, was applied to assess biomass production and CO₂ removal. Three types of photobioreactors were designed and used: (i) without inner column (i.e. a bubble column), (ii) with a centric‐tube column and (iii) with a porous centric‐tube column. The specific growth rates (μ) of the batch cultures in the bubble column, the centric‐tube and the porous centric‐tube photobioreactor were 0.180, 0.226 and 0.252 day^?1, respectively. The porous centric‐tube photobioreactor, operated in semicontinuous culture mode with 10% CO₂ aeration, was evaluated. The results show that the maximum biomass productivity was 0.61 g/L when one fourth of the culture broth was recovered every 2 days. The CO₂ removal efficiency was also determined by measuring the influent and effluent loads at different aeration rates and cell densities of Chlorella sp. NCTU‐2. The results show that the CO₂ removal efficiency was related to biomass concentration and aeration rate. The maximum CO₂ removal efficiency of the Chlorella sp. NCTU‐2 culture was 63% when the biomass was maintained at 5.15 g/L concentration and 0.125 vvm aeration (volume gas per volume broth per min; 10% CO₂ in the aeration gas) in the porous centric‐tube photobioreactor.     

Effect of carbon dioxide and ethylene on berberine production and cell browning in Thalictrum minus cell cultures

Cultured cells of Thalictrum minus L. (Ranunculaceae), transferred from culture flasks to a bubble column bioreactor, produced little berberine and turned dark brown, even when supplied with sufficient oxygen. This phenomenon was ascribed to the removal of CO₂ from the culture medium by bubbling air, and could be reproduced in flask cultures artificially deprived of CO₂. The induction of cell browning by exogenously administered ethylene suggested that CO₂ probably acts antagonistically against endogenously generated C₂H₄. The physiological damage caused by forced aeration could be prevented by adding 2 % CO₂ to the air in the bioreactor.     

<Emphasis Type="Italic">In situ</Emphasis> carbon supplementation in large-scale cultivations of <Emphasis Type="Italic">Spirulina platensis</Emphasis> in open raceway pond

The objective of this study was to estimate the CO₂ absorptivity provided by an in situ carbon supply system using a photosynthetic culture of the cyanobacterium Spirulina platensis in an open raceway pond. The effects of initial total carbon concentrations (ranging from 0 to 0.1 mol/L), suspension depths (ranging from 5 to 20 cm) and pH values (ranging from 8.9 to 11.0) on the CO₂ absorptivity were studied. The results indicated that CO₂ absorptivity was positively correlated with pH value, negatively correlated with total carbon concentration, and only negligibly affected by the suspension depth. The optimum total carbon concentration range and pH range were 0.03 ∼ 0.09 mol/L and 9.7 ∼ 10.0, respectively. An average CO₂ absorptivity of 86.16% and average CO₂ utilization efficiency of 79.18% were achieved using this in situ carbon-supply system in large-scale cultivation of Spirulina platensis, with an initial total carbon concentration of 0.06 mol/L and pH value of 9.8. Our results demonstrated that this system could obtain a favorable CO₂ utilization efficiency in outdoor, large-scale cultivation of Spirulina platensis in open raceway ponds.     

Tolerance of the photosynthetic apparatus to acidification of the growth medium as a possible determinant of CO<Subscript>2</Subscript>-tolerance of the symbiotic microalga <Emphasis Type="Italic">Desmodesmus</Emphasis> sp. IPPAS-2014

The symbiotic unicellular chlorophyte Desmodesmus sp. IPPAS-2014 capable of growth at extremely high CO₂ levels prohibitive for most other microalgae is an interesting model for studies of CO₂ tolerance mechanisms and a promising organism for CO₂ biocapture. We studied the initial (0-60 min) phase of acclimation of this microalga to an abrupt decrease in pH of the medium sparged with air/20% CO₂ mixture. Acclimation of the culture to these conditions was accompanied by a sharp decrease in photochemical activity of the chloroplast followed by its recovery with a characteristic time of 10-50 min. We hypothesize that acidification of the cultivation medium by dissolving CO₂ plays a key role in the observed decrease in the photochemical activity. The possible role of photosynthetic apparatus tolerance to abrupt acidification in overall high tolerance of symbiotic microalgae to extremely high CO₂ levels is discussed.     

Cultivation of <Emphasis Type="Italic">Chlorella emersonii</Emphasis> with flue gas derived from a cement plant

The present study reviews the options of cultivating the green alga, Chlorella emersonii, under photoautotrophic conditions with flue gas derived from a cement plant. It was conducted in the Lafarge Perlmooser plant in Retznei, Austria, where stone coal and various surrogate fuels such as used tyres, plastics and meat-and-bone meal are incinerated for heating limestone. During 30 days of cultivation, flue gas had no visible adverse effects compared to the controls grown with pure CO₂. The semi-continuous cultivation with media recycling was performed in 5.5-L pH-stat photobioreactors. The essay using CO₂ from flue gas yielded a total of 2.00 g L⁻¹ microalgal dry mass and a CO₂ fixation of 3.25 g L⁻¹. In the control, a total of 2.06 g L⁻¹ dry mass was produced and 3.38 g L⁻¹ CO₂ was fixed. Mean growth rates were between 0.10 day⁻¹ (control) and 0.13 day⁻¹ (flue gas). No accumulation of flue gas residues was detected in the culture medium. At the end of the experiment, however, the concentration of lead was three times higher in algal biomass compared to the control, indicating that cultures aerated with this type of flue gas should not be used as food supplements or animal feed.     

Anaerobic digestates are useful nutrient sources for microalgae cultivation: functional coupling of energy and biomass production

We investigated the extent to which nitrogenous and phosphorus nutrients from liquid anaerobic digestates could be recycled for photosynthetic growth of a microalga, Scenedesmus sp. AMDD. Digestates recovered from the anaerobic digestion of cow manure and swine manure and a co-digestion of swine manure and algal biomass were diluted in distilled water and used for algal growth with and without supplemental CO₂ addition. Nutrient assimilation and final biomass yield were retarded in all but the swine manure/algae co-digestate cultures supplemented with high CO₂. Swine manure digestate cultures supplemented with the typical complement of micronutrients normally added with a commonly used growth medium or with Fe/EDTA failed to grow any better than unamended controls. When the culture medium was prepared by blending swine manure digestate with 25 or 50 % algal biomass digestate, diluting it with lake water or by supplementing with magnesium, nutrient assimilation and final algal biomass yields were maximized, indicating that magnesium was critically limiting for algal growth in swine manure digestates. Magnesium amendment thus appears to be essential if nutrients from swine manure digestates are recycled for algal growth. No such requirement is necessary for recycling nutrients from digestates generated wholly or in part from algal biomass.     

Ultrastructural and biochemical development of the photosynthetic apparatus during callus induction in carrot root explants

During the first 8–12 days of cultivation in a nutrient solution containing IAA, inositol and kinetin freshly isolated carrot root explants develop into a chlorophyllous and photosynthetically active tissue culture. Electron microscopy, low temperature pigment absorption spectra and fluorescence induction profile recording as well as the determination of the activity of some enzymes (ribulosebisphosphate carboxylase, phosphoenolpyruvate carboxylase) and CO₂-fixation experiments were carried out. Based on the results, a sequence of developmental stages of the photosynthetic system will be proposed. During the autotropic period from about the 20th day of culture onward the light reaction system of these tissue cultures is quite comparable to that of carrot leaves; however, some differences in the CO₂-fixation mechanism were observed.