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.     

Reduced inorganic sulfur oxidation supports autotrophic and mixotrophic growth of Magnetospirillum strain J10 and Magnetospirillum gryphiswaldense

Magnetotactic bacteria are present at the oxic–anoxic transition zone where opposing gradients of oxygen and reduced sulfur and iron exist. Growth of non‐magnetotactic lithoautotrophic Magnetospirillum strain J10 and its close relative magnetotactic Magnetospirillum gryphiswaldense was characterized in microaerobic continuous culture. Both strains were able to grow in mixotrophic (acetate + sulfide) and autotrophic (sulfide or thiosulfate) conditions. Autotrophically growing cells completely converted sulfide or thiosulfate to sulfate and produced 7.5 g dry weight per mol substrate at a maximum observed growth rate of 0.09 h^?1 for strain J10 and 0.07 h^?1 for M. gryphiswaldense. The respiratory activity for acetate was repressed in autotrophic and also in mixotrophic cultures, suggesting acetate was used as C‐source in the latter. We have estimated the proportions of substrate used for assimilatory processes and evaluated the biomass yields per mol dissimilated substrate. The yield for lithoheterotrophic growth using acetate as the C‐source was approximately twice the autotrophic growth yield and very similar to the heterotrophic yield, showing the importance of reduced sulfur compounds for growth. In the draft genome sequence of M. gryphiswaldense homologues of genes encoding a partial sulfur‐oxidizing (Sox) enzyme system and reverse dissimilatory sulfite reductase (Dsr) were identified, which may be involved in the oxidation of sulfide and thiosulfate. Magnetospirillum gryphiswaldense is the first freshwater magnetotactic species for which autotrophic growth is shown.     

Effect of organic carbon,C:N ratio and light on the growth and lipid productivity of microalgae/cyanobacteria coculture

Current culture methods based on monocultures under phototrophic regimes are prone to contamination, predation, and collapse. Native cultures of multiple species are adapted to the local conditions and are more robust against contamination and predation. Growth, lipid and biomass productivity of a Louisiana native coculture of microalgae (Chlorella vulgaris) and cyanobacteria (Leptolyngbya sp.) in heterotrophic and mixotrophic regimes were investigated. Dextrose and sodium acetate at C:N ratios of 15:1 and 30:1 under heterotrophic (dark) and mixotrophic (400 μmol m^?2 s^?1) regimes were compared with autotrophic controls. The carbon source and C:N ratio impacted growth and biomass productivity. Mixotrophic cultures with sodium acetate (C:N 15:1) resulted in the highest mean biomass productivity (156 g m^?3 d^?1) and neutral lipid productivity (24.07 g m^?3 d^?1). The maximum net specific growth rate (U) was higher (0.97 d^?1) in mixotrophic cultures with dextrose (C:N 15:1) but could not be sustained resulting in lower total biomass than in mixotrophic cultures with acetate (C:N 15:1), with a U of 0.67 d^?1. The ability of the Louisiana coculture to use organic carbon for biomass and lipid production makes it a viable feedstock for biofuels and bioproducts.     

Mixotrophic growth of Thiobacillus A2 on acetate and thiosulfate as growth limiting substrates in the chemostat

During heterotrophic growth on acetate, in batch culture, the autotrophic growth potential of Thiobacillus A2, i.e. the capacity to oxidize thiosulfate and to fix carbon dioxide via the Calvin cycle, was completely repressed. The presence of thiosulfate in a batch culture with acetate as the organic substrate partly released the repression of the thiosulfate oxidizing system. Cultivation of the organism in continuous culture at a dilution rate of 0.05 h^-1 with different concentration ratios of thiosulfate and acetate in the reservoir medium led to mixotrophic growth under dual substrate limitation. Growth on the different mixtures of acetate and thiosulfate yielded upto 30% more cell dry weight than predicted from the growth yields on comparable amounts of these substrates separately. The extent to which the carbon dioxide fixation capacity and the maximum thiosulfate and acetate oxidation capacity are repressed appeared to be a function of the thiosulfate to acetate concentration ratio in the reservoir medium. The results of ¹⁴C-acetate assimilation experiments and of gas-analysis demonstrated that the extent to which acetate was assimilated depended also on the substrate ratio in the inflowing medium. Under the different growth conditions surprisingly little variation was found in some tri-carboxylic acid cycle enzyme activities. Cultivation of T. A2 at different growth rates with a fixed mixture of thiosulfate (18 mM) and acetate (11 mM) in the medium, showed that dual substrate limitation occured at dilution rates ranging from 0.03–0.20 h^-1.Abbreviations PPO 2,5-diphenoloxazol - RubPCase Ribulose-1,5-bisphophate carboxylase - Tris tris (hydroxymethyl) aminomethane - EDTA ethylenediaminetetra-acetic acid     

Development of media for the mixotrophic/heterotrophic culture ofBrachiomonas submarina

The marine algaBrachiomonas submarina var.pulsifera (Droop) CCAP 7/2A, is employed as a food organism in aquaculture; it can be cultured heterotrophically or mixotrophically. Growth rates and productivity under heterotrophic conditions were lower than those achieved under mixotrophic conditions. By reducing the osmotic potential of the medium, whilst simultaneously increasing the levels of nitrogen and phosphorus and using sodium acetate as a carbon source, a 20-fold increase in final yield was attained. This corresponded to a maximum culture of 9.02times 10⁶ cells ml^–1 and a dry weight of 2.51 g l^–1.Author for correspondence     

The influence of acetate on the oxidation of sulfide by Rhodopseudomonas capsulata

The capacity to oxidize sulfide and the influence of the simultaneous presence of acetate in heterotrophically (acetate) and autotrophically (sulfide/CO₂) grown Rhodopseudomonas capsulata was investigated.Sulfide oxidation of acetate-limited cultures was found inversely related to the specific growth rate. Upon acetate deprevation (metering pump stopped) increased rates of sulfide oxidation were observed. This points to the existence of a constitutive acceptor for the electrons from sulfide. It is suggested that a carrier functional in the light-induced cyclic electron flow operates as such. The rate of sulfide oxidation, however, is low when compared to autotrophically-grown cells. This is probably due to the low levels of Calvin cycle enzymes present in the acetate-grown cells.In cells growing on sulfide/CO₂, the addition of acetate resulted in less sulfide being oxidized. Upon depletion of the acetate, the rate of sulfide oxidation again increased, however, insufficiently to maintain the accelerated growth rate. This indicates that under mixotrophic conditions the enzymes of the Calvin cycle are being synthesized to a far lesser extent.Non-Standurd Abbreviations PHB poly--hydroxybutyric acid - D dilution rate - TCA Tri carboxylic acid cycle - RubPcase ribulose 1,5-bisphosphate carboxylase - RP reducing power     

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

EFFICIENCY OF GROWTH AND NUTRIENT UPTAKE FROM WASTEWATER BY HETEROTROPHIC,AUTOTROPHIC, AND MIXOTROPHIC CULTIVATION OF CHLORELLA VULGARIS IMMOBILIZED WITH AZOSPIRILLUM BRASILENSE1

Heterotrophic growth of microalgae presents significant economic advantages over the more common autotrophic cultivation. The efficiency of growth and nitrogen, phosphorus, and glucose uptake from synthetic wastewater was compared under heterotrophic, autotrophic, and mixotrophic regimes of Chlorella vulgaris Beij. immobilized in alginate beads, either alone or with the bacterium Azospirillum brasilense. Heterotrophic cultivation of C. vulgaris growing alone was superior to autotrophic cultivation. The added bacteria enhanced growth only under autotrophic and mixotrophic cultivations. Uptake of ammonium by the culture, yield of cells per ammonium unit, and total volumetric productivity of the culture were the highest under heterotrophic conditions when the microalga grew without the bacterium. Uptake of phosphate was higher under autotrophic conditions and similar under the other two regimes. Positive influence of the addition of A. brasilense was found only when light was supplied (autotrophic and mixotrophic), where affinity to phosphate and yield per phosphate unit were the highest under heterotrophic conditions. The pH of the culture was significantly reduced in all regimes where glucose was consumed, similarly in heterotrophic and mixotrophic cultures. It was concluded that the heterotrophic regime, using glucose, is superior to autotrophic and mixotrophic regimes for the uptake of ammonium and phosphate. Addition of A. brasilense positively affects the nutrient uptake only in the two regimes supplied with light.     

Mixotrophic Growth of Hydrogenomonas eutropha

Mixotrophic growth conditions were established by the addition of lactate to cultures of Hydrogenomonas eutropha growing autotrophically in a gaseous environment of H(2), O(2), and CO(2) (6:2:1). The specific growth rate of mixotrophic cultures was double that of the autotrophic cultures, and lactate disappearance paralleled growth. Growth yields in mixotrophic cultures were significantly greater than those in heterotrophic cultures for equal quantities of lactate consumed. The magnitude of the increase in yield was directly proportional to the absolute growth rate at the time of lactate addition to the starting autotrophic culture and to the time under mixotrophic conditions. The specific activities of hydrogenase and ribulose diphosphate carboxylase decreased during mixotrophic growth; the total activities increased somewhat. The results suggested that the complete autotrophic and heterotrophic physiologies functioned simultaneously under mixotrophic contions.     

Mixotrophic and autotrophic growth of Thiobacillus acidophilus on tetrathionate

Thiobacillus acidophilus can grow in batch and chemostat culture as a heterotroph on glucose, a chemolithoautotroph on tetrathionate and CO₂, or as a mixotroph. Mixotrophically it obtains energy from the simultaneous oxidation of tetrathionate and glucose, and carbon from both glucose and CO₂. Mixotrophic cultures contain lower activities of ribulose 1,5-bisphosphate carboxylase and exhibit lower specific rates of tetrathionate oxidation than do autotrophic cultures. Mixotrophic cultures with low concentrations of glucose have growth rates that are intermediate between slow autotrophic growth and fast heterotrophic growth. Slightly more glucose-carbon is assimilated by mixotrophic cultures than by heterotrophic ones provided with the same concentrations of glucose. Mixotrophic yield in the chemostat is also slightly greater than predicted from autotrophic and heterotrophic yields. These observations indicate that there is preferential assimilation of glucose, at the expense of energy from tetrathionate oxidation, during mixotrophy, resulting in an overall energy saving that produces enhanced growth yield. These observations are relevant to understanding the regulatory behaviour of T. acidophilus in its acidic, mineral-leaching habitats.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Phosphate assimilation by Chlorella and adjustment of phosphate concentration in basal medium for its cultivation

Assimilation of phosphate by Chlorella pyrenoidosa was 0.81-8.1 mg PO(4)-P/g dry weight for heterotrophic cultures and 0.81-16.1 mg/g for mixotrophic cultures. Optimal carbon:phosphorous (C/P) ratios were 206:1-2060:1 and 103:1-2060:1 for heterotrophic and mixotrophic cultivations, respectively. These requirements for phosphate for growth of C. pyrenoidosa under either heterotrophic or mixotrophic conditions are much less (6.25-62.5 or 3.12-62.5-fold at 10 g glucose/l) than its concentration in basal medium.     

Optimization of growth and fatty acid composition of a unicellular marine picoplankton,Nannochloropsis sp., with enriched carbon sources

A unicellular marine picoplankton, Nannochloropsis sp., was grown under CO₂-enriched photoautotrophic or/and acetate-added mixotrophic conditions. Photoautotrophic conditions with enriched CO₂ of 2800 l CO₂ l^–1 and aeration gave the highest biomass yield (634 mg dry wt l^–1), the highest total lipid content (9% of dry wt), total fatty acids (64 mg g^–1 dry wt), polyunsaturated fatty acids (35% total fatty acids) and eicosapentaenoic acid (EPA, 20:53) (16 mg g^–1 dry wt or 25% of total fatty acids). Mixotrophic cultures gave a greater protein content but less carbohydrates. Adding sodium acetate (2 mM) decreased the amounts of the total fatty acids and EPA. Elevation of CO₂ in photoautotrophic culture thus enhances growth and raises the production of EPA in Nannochloropsis sp.     

Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria

1. Dry weight yields from mixed cultures ofProsthecochloris aestuarii orChlorobium limicola with the sulfur reducingDesulfuromonas acetoxidans were determined on different growth limiting amounts of acetate, ethanol or propanol. The obtained yields agreed well with values predicted from stoichiometric calculations. 2. From mixed cultures of twoChlorobium limicola strains withDesulfovibrio desulfuricans orD. gigas on ethanol as the growth limiting substrate, dry weight yields were obtained as calculated for the complete utilization of the ethanol by the mixed cultures. 3. Dry weight yield determinations for two pure cultures ofChlorobium limicola with different growth limiting amounts of sulfide in the absence and presence of excess acetate confirmed that acetate is incorporated byChlorobium in a fixed proportion to sulfide; compared to the yield in the absence of acetate the yield is increased two to threefold in the presence of acetate. 4. The lowest possible sulfide concentrations necessary for optimal growth of mixed cultures of eitherProsthecochloris orChlorobium withDesulfuromonas on acetate were 7–8 mg H₂S per liter of medium. 5. Doubling times at the growth rate limiting light intensities of 5, 10, 20, 50, 100 and 200 lux were determined under optimal growth conditions for the following phototrophic bacteria:Prosthecochloris aestuarii, Chlorobium phaeovibriodes, Chromatium vinosum andRhodopseudomonas capsulata. Reasonably good growth was still obtained withProsthecochloris at 10 and 5 lux light intensity at which no growth of the purple bacteria could be observed.     

聚球藻7942混养培养中碳代谢与能量利用

为了考察聚球藻7942在混养条件下的能量利用效率,分别以葡萄糖和乙酸为碳源开展了聚球藻7942的混养培养研究,并在此基础上利用代谢通量分析方法对聚球藻7942混养条件下的碳代谢和能量利用进行了探讨。结果表明:葡萄糖和乙酸均能促进藻细胞生长,且乙酸促进藻细胞生长的作用更为明显;葡萄糖利用可明显增加藻细胞糖酵解途径中碳代谢流量,而乙酸利用则导致糖酵解途径中碳代谢流量减小,两种有机碳源均增加了柠檬酸循环中碳代谢流量;有机碳源导致藻细胞光化学效率下降,而葡萄糖较之乙酸降低藻细胞光化学效率更为明显。虽然混养条件下光能的贡献率要小于光自养,但基于能量的细胞得率和能量转换率均高于光自养,光自养和以葡萄糖、乙酸为碳源的混养中基于ATP生成的能量转换效率分别为6.81%、7.43%和8.77%。     

Growth and Carbohydrate Metabolism of Sulfobacilli

The moderately thermophilic acidophilic bacteria Sulfobacillus thermosulfidooxidans, strain 1269, S. thermosulfidooxidanssubsp. asporogenes, strain 41, and the thermotolerant strain S. thermosulfidooxidanssubsp. thermotolerans K1 prefer mixotrophic growth conditions (the concomitant presence of ferrous iron, thiosulfate, and organic compounds in the medium). In heterotrophic and autotrophic growth conditions, these sulfobacilli can grow over only a few culture transfers. In cell-free extracts of these sulfobacilli, key enzymes of the Embden–Meyerhof–Parnas, pentose-phosphate, and Entner–Doudoroff pathways were found. The role of a particular pathway depended on the cultivation conditions. All of the enzymes assayed were most active under mixotrophic conditions in the presence of Fe²⁺and glucose, suggesting the operation of all of the three major pathways of carbohydrate metabolism under these conditions. However, the operation of the Entner–Doudoroff pathway in strain 41 was restricted under mixotrophic conditions. After the first culture transfer from mixotrophic to heterotrophic conditions, the utilization of glucose occurred only via the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. After the first culture transfer from mixotrophic to autotrophic conditions, the activity of carbohydrate metabolism enzymes decreased in all of the strains studied; in strain K1, only the glycolytic pathway remained operative. The high activity of fructose-bisphosphate aldolase, remaining in strain 41 cells under these conditions, suggests the involvement of this enzyme in the reactions of the Calvin cycle or of gluconeogenesis.     

Biomass production in mixotrophic culture of Euglena gracilis under acidic condition and its growth energetics

When Euglena gracilis was grown in the heterotrophic condition with glucose and (NH₄)₂SO₄ as the carbon and nitrogen source, a high cell yield (4.28–4.48 g l^–1) was obtained and the culture pH decreased to 1.6–2. The biomass production in the heterotrophic culture was compared to those in the autotrophic and mixotrophic cultures. Autotrophic growth was 4.7–6.3% of the heterotrophic one, whereas about 15–19% higher growth was obtained in the mixotrophic culture. Moreover, good production of chlorophyll (39.4 mg l^–1) and carotenoids (13.8 mg l^–1) were attained in the mixotrophic culture, giving the highest fermenter productivity with respect to biomass as well as chlorophyll and carotenoids. Through an energetic analysis in the mixotrophic culture, it was estimated about 25–28% of the total ATP requirement is formed in the photochemical reactions. This resulted in an improved biomass production in the mixotrophic culture of E. gracilis.     

AUTO-, HETERO-, AND MIXOTROPHIC GROWTH OF CHLAMYDOMONAS HUMICOLA (CMLOROIMIYCKAK) ON ACETATE1

Relative growth rate, isocitrate lyase activity, chlorophyll, protein, lipid, and soluble carbohydrate contents were investigated in Chlamydomonas humicola Lucksch during auto-, mixo-, and heterotrnphic growth. Mixotrophic cells have a relative growth rate of 1.66 d ^–1as compared to 0.78 d ^–1 and 0.21 d ^–1 for hetero- and autotrophic cells, respectively. Addition of acetate to autotrophic cells resulted in an increase in cell dry weight during the first day, followed by a rapid decrease and stabilization at 40 pg·cell ^–1. Cellular yield of mixotrophu cells, on a dry weight basis, was 6.6 times that of heterotrophic cells and 21.9 limes that of autotrophic ones. After 4 d, mixotrophic cells were characterized by higher chlorophyll (3.6% dry weight [d.w.]) and protein (58.6% d.w.) contents and lower lipid (4.8% d.w.) and soluble carbohydrate (1.3% d.w.) contents than those of autotrophic (2.6% d.w. chlorophyll, 31.0% d.w. protein, 10.2% d.w. lipid, and 6.5% d.w. soluble carbohydrate) and heterotrophic (1.5% d.w. chlorophyll, 36.9% d.w. protein, 5.6% d.w. lipid, and 6.0% d.w. soluble carbohydrate) cells. The ratio of chlorophyll a/b was highest in heterotrophic cells due to lower chlorophyll b content. Isocitrate lyase activity, a key enzyme in ecetate assimitation, could not be detected in autotrophic cells. Addition of 10 mM acetate to the culture medium of hetero- and mixotrophic cells resulted in increased isocitrate lyase activity with a maximum after 24 h, followed by a decline in activity over a 7-d period. After 7 d of growth, only 0.01 mM acetate was found in the culture medium of mixotrophic cells as compared to 3.2 mM in the medium of heterotrophic ones, from an initial concentration of 10 mM.     

Hydrogenase and ribulose diphosphate carboxylase during autotrophic, heterotrophic, and mixotrophic growth of scotochromogenic mycobacteria.

Two key autotrophic enzyme systems, hydrogenase and ribulose diphosphate carboxylase, were examined in Mycobacterium gordonae and two other chemolithotrophic, scotochromogenic mycobacteria under different cultural conditions. In all three organisms both enzymes were inducible and were produced in significant levels only in the presence of the specific substrate, hydrogen or carbon dioxide. M. gordonae exhibited increased growth rates and yields, indicating mixotrophic growth, in the presence of a number of single organic substrates, including acetate, pyruvate, glucose, fructose, and glycerol. In contrast to other aerobic hydrogen autotrophs, the presence of either acetate or pyruvate did not repress ribulose diphosphate carboxylase, and mixotrophic growth was rapid with these substrates. In the absence of carbon dioxide, growth in glycerol medium under an atmosphere of hydrogen and oxygen was severely inhibited, even with cells preadapted to heterotrophic growth on glycerol. Cyclic adenosine monophosphate was not effective in inducing hydrogenase or carboxylase in heterotrophic, mixotrophic, or hydrogen-inhibited cultures.     

Responses of strawberry plantlets cultured in vitro under superbright red and blue light-emitting diodes (LEDs)

Unrooted strawberry cv. `Akihime' shoots with three leaves obtained from standard mixotrophic cultures were cultured in the ``Culture Pack'-rockwool system with sugar-free MS medium under CO<sub>2</sub>-enriched condition. To examine the effect of superbright red and blue light-emitting diodes (LEDs) on in vitro growth of plantlets, these cultures were placed in an incubator, ``LED PACK', with either red LEDs, red LEDs1blue LEDs or blue LEDs light source. To clarify the optimum blue and red LED ratio, cultures were placed in ``LED PACK 3' under LED light source with either 100, 90, 80, or 70% red + 0, 10, 20, 30% blue, respectively, and also under standard heterotrophic conditions. To determine the effects of irradiation level, cultures were grown under 90% red LEDs + 10% blue LEDs at 45, 60 or 75 mol m^–2 s^–1 . Plantlet growth was best at 70% red + 30% blue LEDs. The optimal light intensity was 60 mol m^–2 s^–1. Growth after transfer to soil was also best after in vitro culture with plantlets produced were 70% red LEDs + 30% blue LEDs.