Photoautotrophic culture conditions and photosynthetic photon flux influence growth of <Emphasis Type="Italic">Lilium</Emphasis> bulblets <Emphasis Type="Italic">in vitro</Emphasis>

Summary Lilium Asiatic hybrid ‘Mona’ bulblets were cultured in vitro for 100 d under photoautotrophic (CO₂-enriched conditions and without sucrose in the medium) and heterotrophic (non-enriched CO₂ conditions and sucrose-supplemented medium) methods and under various levels of photosynthetic photon flux (PPF). Bulblet growth and net photosynthetic rate (NPR) were analyzed. CO₂₋ and PPF-enriched conditions enhanced the overall growth of bulblets, scale leaves, and roots. Heterotrophic conditions enhanced bulblet growth but higher PPF levels were inhibitory to the development of scale leaves. These results indicate the CO₂₋ and PPF-enriched conditions (photoautotrophic conditions) are beneficial for the production of high-quality bulblets of Asiatic hybrid lilies in vitro     

Extracellular degradation of phenol by the cyanobacterium Synechococcus PCC 7002

Investigations of the unicellular marine cyanobacterium Synechococcus PCC 7002 revealed its ability to metabolize phenol under non-photosynthetic conditions up to 100 mg L^–1. Under continuous light, photoautotrophic growth was reduced only slightly in the presence of this phenol concentration, but no transformation was observed. However neither under photoautotrophic nor heterotrophic conditions were the cells able to use phenol for growth. During the degradation of phenol in the dark cis,cis-muconic acid was produced as the major product, which was identified by gas chromatography/mass spectrometry. This result was confirmed by an identical absorption spectrum and an identical retention time in high performance liquid chromatographic analysis with authentic muconic acid as standard. This provides the first record for an ortho-fission of a phenolic substance by cyanobacteria. Further investigations of the breakdown mechanism of phenol have shown that the transformation is an extracellular process inhibited by heat, proteases and metal ions and is probably catalyzed by a protein.     

Fed-batch culture of astaxanthin-rich Haematococcus pluvialis by exponential nutrient feeding and stepwise light supplementation

A fed-batch culture process followed by subsequent photoautotrophic induction was established for the high density culture of astaxanthin-rich Haematococcus pluvialis using a CO₂-fed flat type photobioreactor under unsynchronized illumination. Fed-batch culture was performed with an exponential feeding strategy of the growth-limiting nutrients, nitrate and phosphate, concurrently with the stepwise supplementation of light depending on the cell concentration. During the growth phase, a biomass of 1.47 g/L was obtained at a biomass productivity of 0.33 g/L/day. Photoautotrophic induction of the well-grown vegetative cells was performed consecutively by increasing the light intensity to 400 μmol photon/m²/s, while keeping the other conditions in the CO₂-fed flat type photobioreactor fixed, yielding an astaxanthin production of 190 mg/L at an astaxanthin productivity of 14 mg/L/day. The proposed sequential photoautotrophic process has high potential as simple and productive process for the production of valuable Haematococcus astaxanthin.     

Urea degradation by picophytoplankton in the euphotic zone of Lake Biwa

The ability of photoautotrophic picoplankton Synechococcus to degrade urea was examined in the euphotic zone of Lake Biwa. Samples were divided into pico (0.2–2.0 μm) and larger (>2.0 μm) size fractions by filtration. The rates of urea degradation (the sum of the rates of incorporation of carbon into phytoplankton cells and of liberation of CO₂ into water) measured by radiocarbon urea were 8 and 17 μmol urea m⁻³ day⁻¹ in June and July, respectively, for the picophytoplankton in the surface water, and 196 and 96 μmol urea m⁻³ day⁻¹, respectively for the larger phytoplankton. The rates decreased with depth, somewhat similar to the vertical profiles of the photosynthetic rate. The urea degradation rates were obviously high under light conditions. In daylight, urea was degraded into two phases, carbon incorporation and CO₂ liberation, whereas in the dark it was degraded only into the CO₂ liberation phase. The contribution of picophytoplankton to total phytoplankton in urea degradation was high in the subsurface to lower euphotic layer. Urea degradation activity was higher in the picophytoplankton fraction than in the larger phytoplankton fraction. Shorter residence times of urea were obtained in the upper euphotic zone. The contribution of picophytoplankton to urea cycling was 4% to 35%. The present results suggest that the picophytoplankton Synechococcus is able to degrade urea and effectively makes use of regenerated urea as a nitrogen source in the euphotic layer, and that picophytoplankton play an important role in the biogeochemical nitrogen cycle in Lake Biwa. Received: June 25, 1998 / Accepted: February 10, 1999     

Complementary limiting factors of astaxanthin synthesis during photoautotrophic induction of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: C/N ratio and light intensity

We investigated the effect of carbon/nitrogen (C/N) ratio on astaxanthin synthesis in Haematococcus pluvialis during photoautotrophic induction by continuous input of both CO₂–air mixture and intense light. When H. pluvialis was induced by constant irradiance induction at 200 μmol photon m⁻² s⁻¹, there was a positive correlation with astaxanthin content and C/N ratio, which was similar to the case for heterotrophic induction. Lower C/N ratios did not retard Haematococcus encystment, but did increase culture biomass, resulting in a decrease in astaxanthin production because of light limitation. However, induction using variable irradiance showed that reduction of astaxanthin production at low C/N ratios was successfully overcome by simply increasing the light intensity from 200 to 300 μmol photon m⁻² s⁻¹ to overcome the light limitation. This resulted in a greatly enhanced astaxanthin synthesis in proportion to cell density in cultures with low C/N ratios. Our results indicate that light intensity is more critical than C/N ratio in astaxanthin production by H. pluvialis during photoautotrophic induction.     

Photoautotrophic and photomixotrophic growth of strawberry plantlets in vitro and changes in nutrient composition of the medium

Explants excised from strawberry (Fragaria x ananassa Duch.) plantlets were cultured in vitro for 21 days on half-strength MS (Murashige & Skoog 1962) basal liquid medium with 20 g l^-1 sucrose and without sugar in the vessels capped with gas permeable microporous polypropylene film. The experiments were conducted under CO₂ nonenriched (350–450 mol mol^-1 in the culture room) and CO₂ enriched (2,000 mol mol^-1 during the photoperiod in the culture room) conditions with a PPF (photosynthetic photon flux) of 200 mol m^-2 s^-1. The CO₂ concentration in the vessels decreased to approximately 200 mol mol^-1 during the photoperiod on day 21 under CO₂ nonenriched conditions. The fresh and dry weight, net photosynthetic rate (NPR) per plantlet, NPR per g leaf fresh weight, NPR per g leaf dry weight, the number of unfolded leaves, and ion uptake of PO₄ ^3-, NO₃ ^-, Ca²⁺, Mg²⁺ and K⁺ on day 21 were the greatest under photoautotrophic (no sugar in the medium) and CO₂ enriched conditions. The residual percent of PO₄ ^3- was 3% on day 21 under photoautotrophic and CO₂ enriched conditions.Abbreviations MS Murashige & Skoog (1962) basal medium composition - NPR net photosynthetic rate - PPF photosynthetic photon flux     

Stomatal characteristics and leaf anatomy of potato plantlets cultured in vitro under photoautotrophic and photomixotrophic conditions

Summary Potato plantlets (Solanum tuberosum L. cv. Benimaru) were cultured under photoautotrophic (without any sucrose in the nutrient medium and with enriched CO₂ and high photosynthetic photon flux) and photomixotrophic conditions (20 g 1⁻¹ sucrose in the medium). Leaf anatomy and stomatal characteristics of the leaves were studied in relation to stomatal size and density. Leaf diffusive resistance, transpiration rate, and wax content of the leaves were also investigated. In the photoautotrophic treatment, stomata behaved normally by closing in the dark and opening in the light. The stomatal density increased twofold compared to that of the photomixotrophic treatment. Relatively thick leaves and an organized palisade layer were observed and the epicuticulal wax content was remarkably higher in this treatment, i.e., seven times greater than that of photomixotrophic treatment. In general, higher diffusive resistance of the leaves was observed than under photomixotrophic conditions; also the resistance increased in darkness and decreased in the light. All these characteristics led the plantlets to have a normal and controlled transpiration rate, which was exceptionally high in the photomixotrophic treatment throughout the light and the dark period.     

Engineering of Synechococcus sp. strain PCC 7002 for the photoautotrophic production of light-sensitive riboflavin (vitamin B2)

Due to their capability of photosynthesis and autotrophic growth, cyanobacteria are currently investigated with regard to the sustainable production of a wide variety of chemicals. So far, however, no attempt has been undertaken to engineer cyanobacteria for the biotechnological production of vitamins, which is probably due to the light-sensitivity of many of these compounds. We now describe a photoautotrophic bioprocess to synthesize riboflavin, a vitamin used as a supplement in the feed and food industry. By overexpressing the riboflavin biosynthesis genes ribDGEABHT from Bacillus subtilis in the marine cyanobacterium Synechococcus sp. PCC 7002 riboflavin levels in the supernatant of the corresponding recombinant strain increased 56-fold compared to the wild-type. Introduction of a second promoter region upstream of the heterologous ribAB gene – coding for rate-limiting enzymatic functions in the riboflavin biosynthesis pathway – led to a further increase of riboflavin levels (211-fold compared to the wild-type). Degradation of the light-sensitive product riboflavin was prevented by culturing the genetically engineered Synechococcus sp. PCC 7002 strains in the presence of dichromatic light generated by red light-emitting diodes (λ = 630 and 700 nm). Synechococcus sp. PCC 7002 naturally is resistant to the toxic riboflavin analog roseoflavin. Expression of the flavin transporter pnuX from Corynebacterium glutamicum in Synechococcus sp. PCC 7002 resulted in roseoflavin-sensitive recombinant strains which in turn could be employed to select roseoflavin-resistant, riboflavin-overproducing strains as a chassis for further improvement.     

Respiratory terminal oxidases alleviate photo-oxidative damage in photosystem I during repetitive short-pulse illumination in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Oxygenic phototrophs are vulnerable to damage by reactive oxygen species (ROS) that are produced in photosystem I (PSI) by excess photon energy over the demand of photosynthetic CO₂ assimilation. In plant leaves, repetitive short-pulse (rSP) illumination produces ROS to inactivate PSI. The production of ROS is alleviated by oxidation of the reaction center chlorophyll in PSI, P700, during the illumination with the short-pulse light, which is supported by flavodiiron protein (FLV). In this study, we found that in the cyanobacterium Synechocystis sp. PCC 6803 P700 was oxidized and PSI was not inactivated during rSP illumination even in the absence of FLV. Conversely, the mutant deficient in respiratory terminal oxidases was impaired in P700 oxidation during the illumination with the short-pulse light to suffer from photo-oxidative damage in PSI. Interestingly, the other cyanobacterium Synechococcus sp. PCC 7002 could not oxidize P700 without FLV during rSP illumination. These data indicate that respiratory terminal oxidases are critical to protect PSI from ROS damage during rSP illumination in Synechocystis sp. PCC 6803 but not Synechococcus sp. PCC 7002.     

High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in vitro

Summary In vitro plantlets of Phalaenopsis ‘Happy Valentine’, Neofinetia falcate Hu, Cymbidium kanran Makino, and Cymbidium goeringii Reichb. f. were grown under photoautotrophic [high photosynthetic photon flux (PPF), high CO₂ concentration, and increased number of air exchanges] and heterotrophic (low PPF, low CO₂ concentration, no air exchanges) culture conditions. After 40 d of culture, a significant difference in plantlet growth was observed between the two cultures. Total fresh and dry mass were on average 1.5 times greater in photoautotrophic culture than in heterotrophic culture. Higher net photosynthetic rates were also observed for Phalaenopsis in photoautotrophic culture. In photoautotrophic culture, little difference was observed in air temperature between the inside and outside of the culture vessel, whereas in heterotrophic culture, air temperature inside the culture vessel was 1–2°C higher than that outside the culture vessel. Relative humidity inside the culture vessel was remarkably different between the two cultures: 83–85% in photoautotrophic culture and 97–99% in heterotrophic culture. These results indicated that growth and net photosynthetic rate of in vitro orchid plantlets were susceptible to the culture environments such as PPF, CO₂ concentration, relative humidity (RH), and the number of air exchanges, which would allow a more efficient micropropagation system for these orchid plants.     

Sugar metabolism, photosynthesis, and growth of in vitro plantlets of Doritaenopsis under controlled microenvironmental conditions

Suboptimal environmental conditions inside closed culture vessels can be detrimental to in vitro growth and survival of plantlets during the acclimatization process. In this study, the environmental factors that affected Doritaenopsis plantlet growth and the relationship between growth and sugar metabolism were investigated. Cultures were maintained under heterotrophic, photoautotrophic, or photomixotrophic conditions under different light intensities and CO₂ concentrations. Photoautotrophic growth of Doritaenopsis hybrid plantlets could be promoted significantly by increasing the light intensity and CO₂ concentration in the culture vessel. The concentration of different sugars in the leaves of in vitro-grown plantlets varied with different cultural treatments through a 10-wk culture period. Starch, reducing sugars, and nonreducing sugar contents were higher in plantlets grown under photoautotrophic and photomixotrophic conditions than in heterotrophically grown plantlets. Net photosynthesis rates were also higher in photoautotrophically and photomixotrophically grown plantlets. These results support the hypothesis that pyruvate, produced by the decarboxylation of malate, is required for optimal photoautotrophy under high photosynthetic photon flux density. Growth was greatest in plantlets grown under CO₂-enriched photoautotrophic and photomixotrophic conditions with high photosynthetic photon flux density. The physiological status of in vitro-grown Crassulacean acid metabolism (CAM)-type Doritaenopsis showed a transition from C3 to CAM prior to acclimatization.     

An Organic Acid Based Counter Selection System for Cyanobacteria

Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the production of fuels and chemicals. This work describes a novel counter selection method for the cyanobacterium Synechococcus sp. PCC 7002 based on organic acid toxicity. The organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards Synechococcus sp. PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which is annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method. DNA fragments of interest were inserted into the acsA locus and strains harboring the insertion were isolated on selective medium containing acrylate. This methodology was also used to introduce DNA fragments into a pseudogene, glpK. Application of this method will allow for more advanced genetics and engineering studies in Synechococcus sp. PCC 7002 including the construction of markerless gene deletions and insertions. The acrylate counter-selection could be applied to other cyanobacterial species where AcsA activity confers acrylate sensitivity (e.g. Synechocystis sp. PCC 6803).     

Remarks on mixotrophic and autotrophic carbon nutrition of Vitis plantlets cultured in vitro

Two Vitis species were cultured in vitro under photoautrophic (sucrose-free culture medium) and photomixotrophic (sucrose 15 g l^-1) conditions during the period following microcutting rooting (day 34 to day 120). Several parameters were measured at the end of the culture: growth, plant dry weight, carbohydrate uptake from the medium and rates of photosynthesis and dark respiration. The two species behaved very differently. Under photoautotrophic conditions, dark respiration, net photosynthesis and daily CO₂ fixation were higher in Vitis vinifera than in Vitis rupestris. Culture under mixotrophic conditions caused increase in growth, respiration and photosynthesis in Vitis rupestris. In contrast, photosynthesis decreased in Vitis vinifera under the same conditions.     

Growth of In vitro banana (Musa SPP.) shoots under photomixotrophic and photoautotrophic conditions

Summary In vitro banana (Musa spp.) shoots were cultured under photomixotrophic (30 gl⁻¹ sucrose and 0.2 h⁻¹ number of air exchanges of culture vessels) and photoautotrophic (0 gl⁻¹ sucrose and 3.9 h⁻¹ number of air exchanges) conditions for 28 d in 370 cm³ Magenta boxes (GA7-type) containing 70 ml of half-strength Murashige and Skoog (MS) medium with 22.2 μM N⁶-benzyladenine (BA). The effects of varying CO₂ concentration (475 or 1340 μmol mol⁻¹) and light intensity (photosynthetic photon flux (PPF) of 100 or 200 μmol m⁻² s⁻¹) were investigated. Fresh and dry weights of banana shoots grown photomixotrophically were significantly greater on day 28 than those grown photoautotrophically. Photoautorophic shoots had a larger number of unfolded leaves and greater leaf area than photomixotrophic plants by days 14 and 28, regardless of CO₂ concentration. The shoot fresh and dry weights on day 14 in photoautotrophic conditions were significantly greater at PPF of 200 μmol m⁻² s⁻¹ than at 100 μmol m⁻² s⁻¹. The increase in net photosynthetic rate of photoautotrophic banana shoots was significant compared with photomixotrophic shoots. The multiplication ratio of in vitro banana shoots grown photoautotrophically in a 28-d culture period was the greatest at 100 μmol m⁻² s⁻¹ PPF and 475 μmol mol⁻¹ CO₂.     

Optimization of production of C-phycocyanin and extracellular polymeric substances by Arthrospira sp.

The key factors influencing the production of C-phycocyanin (C-PC) and extracellular polymeric substances (EPS) by photoautotrophic culture of Arthrospira sp. were optimized using Taguchi method. Six factors were varied at either three or two levels as follows: light intensity at three levels; three initial culture pHs; two species of Arthrospira; three concentrations of Zarrouk’s medium; three rates of aeration of the culture with air mixed with 2% v/v carbon dioxide; and two incubation temperatures. All cultures ran for 14 days. The optimal conditions for the production of C-PC and EPS were different. For both products, the best cyanobacterium proved to be Arthrospira maxima IFRPD1183. The production of C-PC was maximized with the following conditions: a light intensity of 68 µmol photons m⁻² s⁻¹ (a diurnal cycle of 16-h photoperiod and 8-h dark period), an initial pH of 10, the full strength (100%) Zarrouk’s culture medium, an aeration rate of 0.6 vvm (air mixed with 2% v/v CO₂) and a culture temperature of 30 °C. The concentration of Zarrouk’s medium was the most important factor influencing the final concentration of C-PC. The optimal conditions for maximal production of EPS were as follows: a light intensity of 203 µmol photons m⁻² s⁻¹ with the earlier specified light–dark cycle; an initial pH of 9.5; a 50% strength of Zarrouk’s medium; an aeration rate of 0.2 vvm (air mixed with 2% v/v CO₂); and a temperature of 35 °C. Production of C-PC and EPS in raceway ponds is discussed.

     

Metabolic aspects of LPP cyanophage replication in the cyanobacterium Plectonema boryanum

Cyanophage LPP1G is reproduced at the same yield in heterotrophic conditions (dark, glucose) as in photoautotrophic conditions; aerobiosis is required for dark cyanophage replication. Exogenous glucose is not required for the cyanophage replication in the dark in heterotrophically grown cells. In photoautotrophically grown cells, the maximum burst size in dark and glucose is delayed for a period corresponding to glucose uptake induction. Cyanophage LPP2SPI replication occurs in conditions where only Photosystem I operates. Of photosynthesis parameters tested, only CO₂ photoassimilation is affected during cyanophage LPP1G infection under photoautotrophic conditions.     

Carbon metabolism and energy conversion of Synechococcus sp. PCC 7942 under mixotrophic conditions: comparison with photoautotrophic condition

To investigate the carbon metabolism and energy conversion efficiency of the cyanobacterium Synechococcus sp. PCC 7942 under mixotrophic conditions, we studied its growth characteristics in mixotrophic cultures with glucose and with acetate, respectively, and further discussed the carbon metabolism and energy utilization based on metabolic flux analysis. Results showed that both glucose and acetate could enhance the growth of Synechococcus sp. PCC 7942. The metabolic flux through the glycolytic pathway, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation was affected by the two organic substrates. Additionally, the cellular composition was also modulated by glucose and acetate. Under mixotrophic conditions, glucose exerts more significant impact on the diminishment of photochemical efficiency. Although the contribution of light energy was smaller, the cell yields based on total energy in mixotrophic cultures were higher compared with that of photoautotrophic one. On the basis of chlorophyll fluorescence analysis, the actual energy conversion efficiencies based on ATP synthesis in the photoautotrophic, glucose-mixotrophic, and acetate-mixotrophic cultures were evaluated to be 4.59%, 5.86%, and 6.60%, respectively.     

The cyanobacterium Synechococcus sp. PCC 7942 possesses a close homologue to the chloroplast ClpC protein of higher plants

The Clp family consists of large, ubiquitous proteins that function as molecular chaperones and/or regulators of ATP-dependent proteolysis. A single copy gene coding for one of these proteins, ClpC, was cloned from the unicellular cyanobacterium Synechococcus sp. PCC 7942. The predicted polypeptide is most similar (ca. 88%) to the chloroplast-localized ClpC protein from higher plants. Using degenerate PCR primers specific for the two distinct ATP-binding domains characteristic of all ClpA-C proteins, partial sequences homologous to clpC from Synechococcus were also identified in five other cyanobacterial strains. The Synechococcus clpC gene is transcribed under standard growth conditions as a monocistronic message of around 2.7 kb. The level of this message, however, decreases slightly after a shift from 37 to 47.5°C for 2 h, similar to expression previously observed for clpC mRNA from heat-shocked higher plants. At the protein level, the amount of ClpC remains relatively unchanged during the high temperature shift, while that of the known heat shock protein GroEL rises considerably. In contrast, the constitutive level of ClpC in Synechococcus increases considerably under conditions of rapid growth, both with increasing light intensities or CO₂ concentrations. This, and the fact that attempts to inactivate clpC expression fail to produce a viable phenotype, suggest that ClpC activity is essential for growth in this obligate photoautotrophic cyanobacterium.     

Continuous culture growth of photoautotrophic cell suspensions from Chenopodium rubrum

The construction and operation of a continuous culture system for the propagation of cell suspensions from Chenopodium rubrum under photoautotrophic conditions has been described. A dilution rate of 0.16/day gave an equilibrium culture density of 1,100,000 cells/ml and a mean doubling time of 150 hours. During continuous culture steady state conditions with respect to nutrient uptake, cell protein and chlorophyll content, starch accumulation, in vitro activities of enzymes related to different metabolic pathways could be established. Photosynthetic CO₂ assimilation of steady state cells was about 100 mol CO₂/mg chlorophyll x hour. Dark CO₂ fixation was 3% of the light values.