Photosynthetic characteristics of photomixotrophic and photoautotrophic cell suspension cultures ofChenopodium rubrum

Summary Cell suspension cultures ofChenopodium rubrum have been grown for more than 2 years photoautotrophically with CO₂ as sole carbon source. Average increase in fresh weight is appr. 600% within 14 days. The chlorophyll content of photoautotrophic cells (200 g/g fresh weight) is much higher than of photomixotrophic cells (50 g/g fresh weight). The photosynthetic activity of the cells (190 moles CO₂×mg^–1 chlorophyllXh^–1) is comparable to the values found with intact leaves. As shown by short-term¹⁴CO₂ photosynthesis, both, the photomixotrophic and the photoautotrophic cell suspension cultures assimilate CO₂ predominantly via the Calvin pathway.Major differences were found with cells from either exponential or stationary phase of growth with regard to differential labelling of 3-phosphoglyceric acid, malate, sucrose and glucose/fructose.In vitro measurements of carboxylation reactions only partially corroborate our findings with¹⁴CO₂ incorporation. The ratio of ribulosebisphosphate to phosphoenolpyruvate carboxylase activity is 4.7 for leaves of C.rubrum, 1.2 for photoautotrophic cells during stationary growth and 0.5 for cells during exponential growth phase, however, 0.18 was found for photomixotrophic cells. Though the¹⁴CO₂ incorporation into 3-phosphoglyceric acid is clearly higher than into malate, thein vitro activity of phosphoenolpyruvatecarboxylase is 2–6 fold higher than that of ribulosebisphosphate carboxylase. We postulate that anaplerotic reactions of the tricarboxylic acid cycle are involved in the regulation of phosphoenolpyruvate carboxylase.Abbreviations 2,4-D didilorophenoxyacetic acid - EDTA ethylene-diamine-tetraacetic acid - fr. w. fresh weight - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PGA 3-phosphoglyceric acid - PPO 2,5-diphenyloxazole - PEP phosphoenolpyruvate - RuBP nbulosebisphosphate     

Photoautotrophic growth of cell suspension cultures fromChenopodium rubrum in an airlift fermenter

Summary This communication describes the construction and operation of an airlift fermenter for the photoautotrophic growth of cell suspension cultures fromChenopodium rubrum. The basic batch culture unit provides a culture of 1.51 volume, sufficient to permit frequent aseptic sampling. It can be maintained at any desired temperature and aerated to different extents. Using an initial cell density of about 400,000 cells per ml suspension, the increase in cell number is 270% after a 14 days' growth period, although the stationary phase of growth is not yet reached. The transfer of photoautotrophic cell suspensions fromChenopodium rubrum from stationary growth into the large volume of fresh culture medium in the airlift fermenter results in an immediate protein formation, followed by an exponential phase of cell division, whereas rapid chlorophyll accumulation is delayed by 2 days.The growth capacities of photoautotrophic fermenter cultures including protein and chlorophyll formation as well asin vitro activities of the ribulosebisphosphate carboxylase and the phosphoenolpyruvate carboxylase are greatly lower as compared to photoautotrophic cells propagated in standard two-tier culture vessels using 30 ml culture medium. However the pattern of change in the activities of carboxylation enzymes is quite similar in both culture systems.Photoautotrophic cell suspensions fromChenopodium rubrum grown in an airlift fermenter assimilate about 90 mol CO₂/mg chlorophyll × hour. Dark CO₂ fixation is about 1.5% of the light values.Abbreviations PEF phosphoenolpyruvate - RuDP ribulosebisphosPhate - NS ground glass joints of standardized size made from Duran glass, Schott, Germany     

Physical properties of the cell wall of photoautotrophic suspension cells fromChenopodium rubrum L.

Photoautotrophic suspension cells ofChenopodium rubrum were used to determine Donnan potential, charge density and pore-radius distribution in the cell wall. Experiments were done either with turgescent cells or with isolated cell walls. Titration of a cell-wall-generated 9-aminoacridine fluorescence quench with salts of mono- and divalent cations was used to determine Donnan potential and charge density. The experiments and theory were adapted from measurements of membrane surface charges. A tenfold increase in ionic strength, which decreases the repellant forces between charges of the same sign, led to an approximately threefold increase in the measured charge density, thus resulting in a much smaller decrease of the Donnan potential than would be expected if the charge density remained fixed. This decreased influence of ionic strength on the Donnan potential, resulting from the elasticity of the cell wall, was also measurable but less pronounced when the wall of intact cells was stretched by turgor. The porosity of the cell wall was determined by longterm uptake of polyethylene glycols of different molecular weights, and by gel filtration of polyethylene glycols and dextrans as well as mono- and disaccharides using intact suspension cells as matrix. Both methods gave a mean pore diameter of about 4.5 nm and a maximum pore size of 5.5 nm. The resulting pores-size distribution was slightly broader with the latter method.Abbreviations 9-AA 9-aminoacridine - DMBr₂ decamethoniumbromide=N,N,N,N,N,N hexamethyldecane-1,10-diaminebromide - DW dry weight after lyophilization - EDTA ethylene diaminetetra acetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FW fresh weight - Mops 3-(N-morpholino)propanesulfonic acid - MW molecular weight - PEG polyethylene glycol     

The lipids in photoautotrophic and heterotrophic cell suspension cultures of Chenopodium rubrum

Resembling the lipids in the leaves and other green organs of intact plants, the lipids in photoautotrophic cell cultures of Chenopodium rubrum were found to contain high proportions of monogalactosyldiacylglycerols and digalactosyldiacylglycerols, as well as fair amounts of sulfoquinovosyldiacylglycerols and diacylglycerophosphoglycerols. Conversely, the heterotrophic cell cultures, from which the photoautotrophic cultures had been derived, contained only traces of these compounds. The heterotrophic cultures were rich in sterols, sterol esters, sterol glycosides, and esterified sterol glycosides. The lipids of photoautotrophic cell cultures contained higher proportions of constituent linolenic acid, but lower concentrations of linoleic acid than those of heterotrophic cultures. In the photoautotrophic cultures, as in green leaves, linolenic acid was predominantly estrified in monogalactosyldiacylglycerols and digalactosyldiacylglycerols. This investigation shows that it is possible to select strains of cell cultures, which are capable of grosing photoautotrophically, with the aim of activating the biosynthesis of specific metabolites.     

Embryogenesis of photoautotrophic cell cultures of Daucus carota L.

In this paper photoautotrophic carrot (Daucus carota L.) suspension cultures are described which are able to produce somatic embryos. The development of somatic embryos, however, requires a sucrose supplement. Although an elevation of the CO₂ concentration up to 2.3% results in the same level of dry weight production as with sucrose in the medium, somatic embryos could not be observed.Results on the influence of sucrose on some aspects of the photosynthetic apparatus of cultured cells are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DW dry weight - ELISA enzyme-linked-immunosorbent-assay - FW fresh weight - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - PEPCase phosphoenol-pyruvate carboxylase - Rubisco Ribulose- 1,5-bisphosphate carboxylase/oxygenase - se somatic embryogenesis     

Growth characteristics of Glycyrrhiza glabra cell suspension cultures

Sweet potato (Ipomoea batatas (L.) Lam.) breeding has been hampered by self-and cross-incompatibilities that are frequently encountered among the plants in the section Batatas. Ovule culture techniques were developed to assist in overcoming some of these incompatibilities. Ovules that contain embryos at the late globular to heart shaped stage of development were cultured on MS medium containing full strength or one-half strength salts with 3%, 8% or 12% sucrose. Ovules were cultured either intact or after slicing. Ovules of I. triloba and I. trifida were successfully cultured as early as 3 and 4 days after pollination while sweet potato ovules were successfully cultured 5 and 6 days after pollination. The percentage of ovules with developing embryos on the media tested ranged from 27.8% to 50.2%. The highest percentage of embryos developed when the ovules were sliced and cultured on medium containing one-half MS salts and 8% sucrose. Three plants were recovered from cultured ovules of incompatible interspecific crosses.Abbreviations DAP days after pollination - MS medium Murashige and Skoog (1962) medium     

Metabolism and compartmentation of dihydrozeatin exogenously supplied to photoautotrophic suspension cultures of Chenopodium rubrum

[³H]Dihydrozeatin supplied to photoautotrophically growing cell suspension cultures of Chenopodium rubrum was rapidly taken up and metabolized by the cells. The predominant metabolites in extracts of the cells were [³H]dihydrozeatin-O-glucoside and [³H]dihydrozeatin riboside-O-glucoside. Both these compounds could be shown to be compartmented within the vacuole, whereas [³H]dihydrozeatin and [³H]dihydrozeatin riboside, which were both present to a minor extent in cell extracts, were both present to a minor extent in cell extracts, were localized predominantly outside the vacuole. Analysis of the culture medium at the end of the 36-h incubation period showed that there had been an efflux of [³H]dihydrozeatin metabolites out of the cells. Whereas [³H]dihydrozeatin riboside was found to be the major extracellular [³H]dihydrozeatin metabolite, the O-glucosides of neither this compound nor [³H]dihydrozeatin could be detected in the medium. The differential compartmentation of [³H]dihydrozeatin metabolites found with the C. rubrum suspension-culture system is discussed with respect to possible mechanisms governing the metabolism of cytokinins in plants cells.Abbreviations (diH)Z dihydrozeatin - (diH) [9R]Z 9--D-ribofuranosyl dihydrozeatin - HPLC high-performance liquid chromatography - ODS octododecyl silica - PEP phosphoenolyruvate     

CO2-Fixation and ATP synthesis in continuous cultures of Chlorella fusca

In continuous cultures of Chlorella fusca under steady state conditions, the CO₂-fixation rate, the ATP-level, the apparent rate of photophosphorylation as calculated from the changes in the ATP-level during light to dark or dark to light transients and the energy charge were measured at various environmental conditions. During growth the energy charge was around 0.64. CO₂-assimilation and the apparent ATP-synthesis were strongly dependant on light intensity, however the ATP-level was independant on it. Since the rates of apparent ATP-synthesis and of the CO₂-fixation do not seem to be strictly correlated in a logic way when environmental factors are changed and furthermore the stoichiometry of 3 ATP necessary per CO₂ fixed was never achieved, the described method frequently used for procaryotes to determine the in vivo rate of phosphorylation does not give valid results in highly compartimented eukaryotic cells.     

Alternative formation of anthraquinones and lipoquinones in heterotrophic and photoautotrophic cell suspension cultures of Morinda lucida Benth.

Photoheterotrophic and photoautotrophic cell suspension cultures were raised from a callus tissue derived from a Morinda lucida Benth. plant (Rubiaceae). The cultures were characterized with regard to fresh weight, dry weight, cell number, pH, chlorophyll and quinoid natural products. The amount of lipoquinones (phylloquinone, -tocopherol, plastoquinone, ubiquinone) isolated from the photoautotrophic cultures matched the amount detected in an intact leaf. Anthraquinone glycosides which are found in the roots of Morinda plants were not present in the photoautotrophic culture. The photoheterotrophic culture contained only trace amounts of these pigments. Abundant anthraquinone synthesis was observed when photoautotrophic and photoheterotrophic suspension cultures were transferred into darkness, provided sucrose was present in the medium. Induction of synthesis of anthraquinone pigments coincided with a rapid disappearance of lipoquinones from the culture. Thus, in the suspension culture, photoautotrophy correlates with lipoquinone synthesis and heterotrophy correlates with anthraquinone synthesis. This reflects the situation in the intact plants where lipoquinones are chloroplast-associated whereas anthraquinones occur in the roots.Abbreviation HPLC high-performance liquid chromatography     

Semicontinuous cultivation of photoautotrophic cell suspension cultures in a 20 l airlift-reactor

An airlift-bioreactor system was established for semicontinuous growth of photosynthetically active plant cell suspension cultures in a controlled environment. The bioreactor unit was constructed as a conventional, internal draught tube airlift-reactor, which is characterized by a H D^-1 ratio of 2.9, a ratio of the cross-sectional area of the riser to the cross-sectional area of the downcomer of 0.25 and a surface area of 0.435 m² for illumination. Cultivation experiments could be scaled up to working volumes of maximal 20 1. Sixteen fluorescent tubes were fixed around the outer glass cylinder to provide cells continuously with light. An external cooling device was used to keep the temperature constantly at 27°C. Agitation as well as supply with CO₂ was performed by injecting air enriched with CO₂ through a ring-shaped sparger at the bottom of the vessel. A first set of experiments was carried out with a photoautotrophic culture of Chenopodium rubrum L. Cell material adapted to large scale culture conditions was used to inoculate a modified MS medium (Murashige & Skoog 1962) without any organic constituents. Under these conditions a biomass increase of 1870% was achieved in 18 days. Several physiological parameters (e.g. pigmentation, photosynthetic O₂ evolution, carbohydrate content) were measured routinely to elucidate the growth characteristics of large-scale grown Chenopodium cells. Electron microscopic photographs from different phases of culture growth clearly demonstrate the pattern of cellular development. Special emphasis was placed upon the differentiation of chloroplast ultrastructure. The presented data confirm the feasibility of large-scale culture techniques with photosynthetic active plant cell cultures.Abbreviations D diameter - DW dry weight - FW fresh weight - H height - K_La volumetric oxygen transfer coefficient (h^-1) - MES 2-(N-morpholino)-ethanesulfonic acid - specific growth rate (d^-1) - PAR photosynthetically active radiation (400–700 nm) - Pepcase phosphoenolpyruvate carboxylase - Rubisco ribulose-1,5-biphosphate carboxylase/oxygenase - t_d doubling time (d) - vvm (aeration volume) (medium volume)^-1 min^-1 Dedicated to Prof. F.-C. Czygan on the Occasion of his 60th Birthday     

Analysis of morphological characteristics of Solanum chrysotrichum cell suspension cultures

Cell size distribution of Solanum chrysotrichum cell suspension cultures was determined using mechanical sieving and an image analysis system. The results were compared using the sieve size (<0.25, 0.25–0.50, 0.5–1.0 and >1.00 mm) as the criterion. Mechanical sieving as well as image analysis showed that S. chrysotrichum cultures developed in shake flasks present a similar tendency to increase in aggregate size as growth persists. However, there are considerable differences in the values of each fraction. Fractions obtained by mechanical sieving were characterized by image analysis demonstrating that an inefficient separation of the cell population exists. The results demonstrate that digital image analysis was more precise than mechanical sieving to describe the cell size distribution changes occurring during cell growth. It was also possible to achieve a total characterization of S. chrysotrichum morphology.     

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

The UFL-AG-286 cell line, established from embryonic tissue of the lepidopteran insect Anticarsia gemmatalis, has been identified as a good candidate to be used as a cellular substrate in the development of a process for in vitro production of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, a baculovirus widely used as bioinsecticide. In order to characterize the technological properties of this cell line and evaluate its feasibility to use it for the large-scale production of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, UFL-AG-286 cells were adapted to grow as agitated suspension cultures in spinner-flasks. Batch suspension cultures of adapted cells in serum-supplemented TC-100 medium grew with a doubling time of about 29 h and reached a maximum cell density higher than 3.5 × 10⁶ viable cells ml⁻¹. At the end of the growth period glucose was completely depleted from the culture medium, but l-lactate was not produced. Amino acids, with the exception of glutamine, were only negligibly consumed or produced. In contrast to other insect cell lines, UFL-AG-286 cells appeared to be unable to synthesize alanine as a metabolic way to dispose the by-product ammonia. The synchronous infection of suspension cultures with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus in the early to medium exponential growth phase yielded high amounts of both viral progenies per cell and reduced the specific demands of UFL-AG-286 cells for the main nutrients.     

Ammonium and nitrate uptake and nitrate reductase activity of photoautotrophic callus cultures of the fernPlatycerium coronarium (Koenig) DESV

Summary The uptake of nitrate and ammonium by callus ofPlatycerium coronarium from the culture medium was examined. Nitrate reductase activity of photoautotrophic callus cultures under CO₂ enrichment was significantly lower compared to the cultures without CO₂ enrichment, but higher than that of heterotrophic callus cultured on medium with 2% (wt/vol) sucrose. When sucrose concentration of the heterotrophic culture was lowered to 0.2%, nitrate reductase activity increased. The level of nitrate reductase activity increased by about 25% in the heterotrophic callus with an increase in 2,4-D from 2 μM to 10 μM, despite a decline in fresh weight gain. However, photoautotrophic cultures with 1% CO₂ enrichment showed 20% decline in nitrate reductase activity and 45% decline in fresh weight gain with a similar increase in 2,4-D level. The rate of uptake of nitrate from the culture medium was unrelated to the level of nitrate reductase activity in the callus. For photoautotrophic callus under CO₂ enrichment, the presence of 1% (vol/vol) CO₂ generally resulted in the highest rate of nitrate uptake. The rate of uptake of ammonium was higher for callus cultured on 2 μM 2,4-D compared to that on 10 μM 2,4-D.     

A microtest system for the serial assay of phytotoxic compounds using photoautotrophic cell suspension cultures of Chenopodium rubrum

Chenopodium rubrum photoautotrophic cell suspensions were grown in plastic tissue culture dishes under photoautotrophic conditions. Growth was monitored by measuring cell number, packed cell volume, chlorophyll content and oxygen production. Such microtiter dishes are suitable systems for the serial assay of growth inhibition and various physiological effects (i.e. chlorophyll fluorescence, cell viability, oxygen production) of photoautotrophic cells as caused by herbicides and fungal phytotoxins. The applicability of the test system is discussed.Abbreviations pcv packed cell volume - fr.w. fresh weight - rpm revol. per minute - DMSO dimethyl sulfoxide - PMS phenazine methosulfate - NBT nitro-blue tetrazolium chloride     

Fluorescence characteristics of photoautotrophic soybean cells

We report here the first measurements on chlorophyll (Chl) a fluorescence characteristics of photoautotrophic soybean cells (cell lines SB-P and SBI-P). The cell fluorescence is free from severe distortion problems encountered in higher plant leaves. Chl a fluorescence spectra at 77 K show, after correction for the spectral sensitivity of the photomultiplier and the emission monochromator, peaks at 688, 696 and 745 nm, representing antenna systems of photosystem II-CP43 and CP47, and photosystem I, respectively. Calculations, based on the complementary area over the Chl a fluorescence induction curve, indicated a ratio of 6 of the mobile plastoquinone (including Q_B) to the primary stable electron acceptor, the bound plastoquinone Q_A. A ratio of one between the secondary stable electron acceptor, bound plastoquinone Q_B, and its reduced form Q_B ^- was obtained by using a double flash technique. Owing to this ratio, the flash number dependence of the Chl a fluorescence showed a distinct period of four, implying a close relationship to the S state of the oxygen evolution mechanism. Analysis of the Q_A ^- reoxidation kinetics showed (1) the halftime of each of the major decay components ( 300 s fast and 30 ms slow) increases with the increase of diuron and atrazine concentrations; and (2) the amplitudes of the fast and the slow components change in a complementary fashion, the fast component disappearing at high concentrations of the inhibitors. This implies that the inhibitors used are able to totally displace Q_B. In intact soybean cells, the relative amplitude of the 30 ms to 300 s component is higher (40:60) than that in spinach chloroplasts (30:70), implying a larger contribution of the centers with unbound Q_B. SB-P and SBI-P soybean cells display a slightly different sensitivity of Q_A ^- decay to inhibitors.Abbreviations CA complementary area over fluorescence induction curve - Chl chlorophyll, diuron - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F _m maximum chlorophyll a fluorescence - F ₀ minimum chlorophyll a fluorescence - F _v = F _t-F₀ - where F _v = variable chlorophyll a fluorescence - and F_t = chlorophyll a fluorescence at time t - PS II photosystem II - Q _a primary (plastoquinone) electron acceptor of PS II - Q _b secondary (plastoquinone) electron acceptor of PS II - t₅₀ the time at which the concentration of reduced Q _a is 50% of that at its maximum value     

Dynamics of limiting cell wall porosity in plant suspension cultures

Changes in the limiting porosity of cell walls, i.e. the size limit for permeation of neutral molecules through the wall, were studied in several higher-plant cell-suspension cultures. For this purpose, samples of biomass fixed at different cultivation times were investigated using a method based on size-exclusion chromatography of polydisperse dextrans before and after equilibration with the extracted cell clusters. In suspension cultures of Chenopodium album L., Dioscorea deltoidea Wall. and Medicago sativa L., the mean size limit (MSL; critical Stokes' radius for exclusion of neutral polymers from half of the intracellular space) was found to vary between 2.4 and 3.8 nm. It decreased significantly during transition from the growth phase to the stationary phase. In the case of the C. album culture this change was found to be irrespective of whether sucrose in the medium was completely depleted at the end of the growth phase or not. The MSL was kept constant for long periods of the stationary phase if cell viability was maintained by repeated sucrose supplement. In a suspension strain of Triticum aestivum L., the MSL of cell wall permeation was comparatively small (1.75 nm) and remained constant during all cultivation phases. Relations between limiting porosity and cell wall growth, loss of pectic compounds to the medium, cross-linking activities and cell wall stiffening are discussed. Received: 19 December 1996 / Accepted: 23 April 1997     

Light acclimation potential and xanthophyll cycle pigments in photoautotrophic suspension cells of Chenopodium rubrum

The present study investigates the light acclimation potential of photoautotrophic suspension culture cells of Chenopodium rubrum L. grown in 16 h light/8 h dark cycles. Typical features of sun/shade acclimation could be demonstrated in cultures grown at photon flux densities of 30 and 150 μmol m⁻² s⁻¹. Low light grown cells had lower chlorophyll a/b ratios, lower respiration rates and lower light compensation points than high light grown cells. Maximum photosynthetic rate per cell dry weight was highest in low light conditions, indicating that the cells did not enlarge their photosynthetic machinery upon exposure to high light. Transfer of cultures to 800 μmol m⁻² s⁻¹ caused photoinhibition as indicated by a decrease in photosynthetic efficiency and by the occurrence of a slowly reversible quenching of variable chlorophyll fluorescence. Extension of the photoinhibitory treatment over six light dark cycles did not result in further dramatic changes of these parameters, whereas the chlorophyll content per dry weight and the chlorophyll a/b ratio decreased. Measurements of photochemical quenching showed that the capability of the cells to dissipate excessive energy had increased during the acclimation process. The presence of the xanthophyll cycle pigments and the operation of the cycle could be demonstrated. In agreement with the putative photoprotective function of antheraxanthin and zeaxanthin these pigments could only be detected under photoinhibitory conditions. Prolonged photoinhibitory treatment resulted in increases in the xanthophyll pigment concentration but not of the potential to deepoxidate violaxanthin. The limited potential of the cells to accumulate zeaxanthin and antheraxanthin might indicate that the xanthophyll cycle is not the main factor determining their resistance to high light stress.     

Expression of a sugar-transporter gene family in a photoautotrophic suspension culture of Chenopodium rubrum L.

Photoautotrophic suspension-culture cells of Chenopodium rubrum L. were shifted to mixotrophic growth by adding glucose to investigate whether the activities of plant sugar transporters, as well as the expression of the corresponding genes, are regulated in response to sugars. The rate of d-glucose uptake was shown not to be affected by mixotrophic growth in the presence of d-glucose. The polymerase chain reaction (PCR) technique was applied to amplify cDNA and genomic fragments from monosaccharide-carrier genes. Seven members of a monosaccharide-carrier family were identified of which three were found to be expressed in the suspension-culture cells. The expression of the monosaccharide-carrier genes was independent of the presence of d-glucose.Abbreviation PCR polymerase chain reaction We would like to thank Michaela Bittner, Rainer Ehneß and Monika Kammerer for skillful technical assistance and S. Buchhauser and H. Hallmer for photographic work. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 43) and by Fonds der Chemischen Industrie.     

Absolute measurement of cell expansion in plant cell suspension cultures

A method is described for the measurement of intracellular volume (V_i) in cell cultures. In principle, any stable compound that neither penetrates the plasma membrane nor binds to the cells can be used to trace the total extracellular (apoplastic) volume and hence to estimate the intracellular volume. No suitable coloured or UV-absorbing compound could be found among those tested; the main problems were binding to the cell surface and/or instability in the medium. However, [¹⁴C]mannitol was an acceptable apoplastic marker, by use of which we showed that 21–47% of total packed cell volume (PCV) was intracellular, and 14–33% of total settled cell volume (SCV) was intracellular. Therefore, measurements of PCV and SCV misrepresent cell expansion to a variable extent. Cultures of Acer, Rosa, Spinacia and Zea achieved final symplastic volumes of only 9, 14, 6 and 6%, respectively, of the total suspension culture volume.     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.