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Abstract This study examines the in vitro growth and ex vitro establishment of Brassavola tuberculata in relation to the micropropagation system and sucrose concentration employed in the in vitro culture. A completely randomized experimental design was utilized, employing a 2 x 5 factorial arrangement. The experimental period began with seedlings cultivated in vitro for 180 days, which were subsequently transferred to Murashige and Skoog culture media containing sucrose concentrations of 0, 15, 30, 45, or 60 g L-1. The cultures were subjected to two micropropagation systems: conventional and gas exchange. After 90 days of in vitro cultivation, the plants were evaluated, transplanted into a substrate, and placed in a screened nursery for ex vitro cultivation. After 300 days of ex vitro cultivation, the survival and initial characteristics of the plants were assessed. The micropropagation system allowing gas exchange and sucrose concentrations up to 30 g L-1 enhanced the shoot and root growth of in vitro propagated plants. No noticeable anatomical differences were observed after 90 days of in vitro culture among the different sucrose concentrations and micropropagation systems used. In the ex vitro establishment, irrespective of sucrose concentration, the micropropagation system facilitating gas exchange positively influenced all evaluated characteristics.

• In vitro plantlets of Origanum majorana exhibit photomixotrophic cultivation. • The sucrose concentration affected the in vitro accumulation of arbutin. • Ventilation and wavelength affected the growth, pigments and arbutin content. • Arbutin accumulation was higher in the 30%B:70%R and where there is a red light. • Monochromatic light blue, green and yellow have lower arbutin content. The ventilation system using porous membranes allows for better plantlet growth and increased survival during the acclimatization phase. The objective was to investigate the effect of alternative membrane system and wavelength on the growth and accumulation of arbutin in vitro plantlets of Origanum majorana. Nodal segments containing a pair of leaves were cultivated in vitro using MS culture medium and an alternative membrane system consisting of four porous membranes. Sucrose was added at concentrations of 0, 7.5, 15, and 30 g. Another experiment was conducted using four porous membranes supplemented with 15 g l -1 of sucrose and under different light-emitting diodes (LEDs): white LED (W); blue (B); green (G); yellow (Y); red (R); a combination of blue and red (50%B:50%R; 30%B:70%R; 70%B:30%R). Marjoram (Origanum majorana) plantlets exhibit photomixotrophic growth, with a medium containing 15 g of sucrose, and an alternative membrane system with 4 porous membranes promoting increased growth. The sucrose concentration influenced the accumulation of arbutin. The wavelength, in conjunction with the use of a membrane system, affected the growth, concentration of photosynthetic pigments, and accumulation of arbutin in marjoram plantlets. The highest biomass accumulation and arbutin synthesis were induced by the 30%B:70%R spectrum combination, while lower biomass and accumulation were observed under the monochromatic blue spectrum. Improving factors such as ventilation, wavelength, and sucrose can lead to more efficient resource use, reducing costs and increasing the yield of arbutin in vitro. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Background Converting carbon dioxide (CO2) into value-added chemicals using engineered cyanobacteria is a promising strategy to tackle the global warming and energy shortage issues. However, most cyanobacteria are autotrophic and use CO2 as a sole carbon source, which makes it hard to compete with heterotrophic hosts in either growth or productivity. One strategy to overcome this bottleneck is to introduce sugar utilization pathways to enable photomixotrophic growth with CO2 and sugar (e.g., glucose and xylose). Advances in engineering mixotrophic cyanobacteria have been obtained, while a systematic interrogation of these engineered strains is missing. This work aimed to fill the gap at omics level. Results We first constructed two engineered Synechococcus elongatus YQ2-gal and YQ3-xyl capable of utilizing glucose and xylose, respectively. To investigate the metabolic mechanism, transcriptomic and metabolomic analysis were then performed in the engineered photomixotrophic strains YQ2-gal and YQ3-xyl. Transcriptome and metabolome of wild-type S. elongatus were set as baselines. Increased abundance of metabolites in glycolysis or pentose phosphate pathway indicated that efficient sugar utilization significantly enhanced carbon flux in S. elongatus as expected. However, carbon flux was redirected in strain YQ2-gal as more flowed into fatty acids biosynthesis but less into amino acids. In strain YQ3-xyl, more carbon flux was directed into synthesis of sucrose, glucosamine and acetaldehyde, while less into fatty acids and amino acids. Moreover, photosynthesis and bicarbonate transport could be affected by upregulated genes, while nitrogen transport and assimilation were regulated by less transcript abundance of related genes in strain YQ3-xyl with utilization of xylose. Conclusions Our work identified metabolic mechanism in engineered S. elongatus during photomixotrophic growth, where regulations of fatty acids metabolism, photosynthesis, bicarbonate transport, nitrogen assimilation and transport are dependent on different sugar utilization. Since photomixotrophic cyanobacteria is regarded as a promising cell factory for bioproduction, this comprehensive understanding of metabolic mechanism of engineered S. elongatus during photomixotrophic growth would shed light on the engineering of more efficient and controllable bioproduction systems based on this potential chassis.

Background: Converting carbon dioxide (CO2) into value-added chemicals using engineered cyanobacteria is a promising strategy to tackle the global warming and energy shortage issues. However, most cyanobacteria are autotrophic and use CO2 as a sole carbon source, which makes it hard to compete with heterotrophic hosts in either growth or productivity. One strategy to overcome this bottleneck is to introduce sugar utilization pathways to enable photomixotrophic growth with CO2 and sugar (e.g., glucose and xylose). Advances in engineering mixotrophic cyanobacteria have been obtained, while a systematic interrogation of these engineered strains is missing. This work aimed to fill the gap at omics level. Results: We first constructed two engineered Synechococcus elongatus YQ2-gal and YQ3-xyl capable of utilizing glucose and xylose, respectively. To investigate the metabolic mechanism, transcriptomic and metabolomic analysis were then performed in the engineered photomixotrophic strains YQ2-gal and YQ3-xyl. Transcriptome and metabolome of wild-type S. elongatus were set as baselines. Increased abundance of metabolites in glycolysis or pentose phosphate pathway indicated that efficient sugar utilization significantly enhanced carbon flux in S. elongatus as expected. However, carbon flux was redirected in strain YQ2-gal as more flowed into fatty acids biosynthesis but less into amino acids. In strain YQ3-xyl, more carbon flux was directed into synthesis of sucrose, glucosamine and acetaldehyde, while less into fatty acids and amino acids. Moreover, photosynthesis and bicarbonate transport could be affected by upregulated genes, while nitrogen transport and assimilation were regulated by less transcript abundance of related genes in strain YQ3-xyl with utilization of xylose. Conclusions: Our work identified metabolic mechanism in engineered S. elongatus during photomixotrophic growth, where regulations of fatty acids metabolism, photosynthesis, bicarbonate transport, nitrogen assimilation and transport are dependent on different sugar utilization. Since photomixotrophic cyanobacteria is regarded as a promising cell factory for bioproduction, this comprehensive understanding of metabolic mechanism of engineered S. elongatus during photomixotrophic growth would shed light on the engineering of more efficient and controllable bioproduction systems based on this potential chassis. [ABSTRACT FROM AUTHOR]

One of the main problems of in vitro plant tissue culture is the poor ventilation in the culture vessels. This leads to morphological and physiological anomalies in regenerated in vitro plants, a crucial issue in woody species, such as Caesalpinia spinosa (Mol.) O. Kuntz, which is a legominous tree or thorny shrub of the Andes, adequate photoautotrophic and photomixotrophic in vitro rooting systems could eliminate these problems. Therefore, the purpose of the present study was to evaluate photoautotrophic and photomixotrophic treatments to optimize the in vitro rooting of C. spinosa microshoots. Micropropagated shoots were cultured in vessels sealed with a polypropylene lid with 0, 1, or 3 ventilation holes covered with a 0.45-μm adhesive polypropylene microfilter. The vessels contained MS salts with or without 30 g L−1 sucrose or, alternatively, a porous substrate (PRO-MIX®), which facilitated rooting and produced greater fresh and dry weights, a higher concentration of photosynthetic pigments and phenolic compounds, and more developed leaves. The optimal performance and higher survival was obtained by using PRO-MIX® with three filters for increased ventilation. For treatments in agar, only the one with sucrose and highly ventilated (three filters) reached a performance approaching that with PRO-MIX®. To date, the presented results have not been reported in scientific literature for C. spinosa. [ABSTRACT FROM AUTHOR]

Xylose is a major component of lignocellulose and the second most abundant sugar present in nature after glucose; it, therefore, has been considered to be a promising renewable resource for the production of biofuels and chemicals. However, no natural cyanobacterial strain is known capable of utilizing xylose. Here, we take the fast-growing cyanobacteria Synechococcus elongatus UTEX 2973 as an example to develop the synthetic biology-based methodology of constructing a new xylose-utilizing cyanobacterial chassis with increased acetyl-CoA for bioproduction., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Optimization of mass microalgae cultures is required for the efficient production of biodiesel feedstock in terms of total fatty acid (TFA) content and a conducive fatty acid profile. A mutant strain of Nannochloropsis sp. (MT-I5), as modified via random mutagenesis and flow cytometric cell sorting, was investigated in both a single- and two-stage cultivation using 250 L laboratory raceway ponds. Culture was based on photoautotrophic biomass production (stage 1) followed by a switch to photomixotrophic growth induced by adding sodium acetate (2 mM) (stage 2). The biomass yield of the mutant in two-stage cultivation was maintained at a level similar to that of the one-stage photoautotrophic culture, but TFA content was increased by 2.3-fold. The fatty acid profile of MT-I5 also had an increased level of desirable saturated fatty acids (SFA) for use as a biodiesel feedstock, i.e. from 43 to 48 % of TFA, as well as a decreased level of less desirable polyunsaturated fatty acids (PUFA), i.e. from 22 to 7 % of TFA. The two-stage cultivation process is of interest for the mass culture of microalgae for biofuel feedstocks, as biomass productivity can be maximized during the first stage of culture until N-starvation is achieved, followed by the enhanced synthesis of SFA in the second stage of culture by adding sodium acetate as a fixed-carbon source. [ABSTRACT FROM AUTHOR]

Anaerobic digestion (AD) is an environmentally friendly approach to waste treatment, which can generate N and P-rich effluents that can be used as nutrient sources for microalgal cultivations. Modifications of AD processes to inhibit methanogenesis leads to the accumulation of acetic acid, a carbon source that can promote microalgal biosynthesis. This study tested different AD effluents from municipal wastes on their effect on d -lactate production by an engineered Synechocystis sp . PCC 6803 (carrying a novel lactate dehydrogenase). The results indicate that: (1) AD effluents can be supplemented into the modified BG-11 culture medium (up to 1:4 volume ratio) to reduce N and P cost; (2) acetate-rich AD effluents enhance d -lactate synthesis by ∼40% (1.2 g/L of d -lactate in 20 days); and (3) neutral or acidic medium had a deleterious effect on lactate secretion and biomass growth by the engineered strain. This study demonstrates the advantages and guidelines in employing wastewater for photomixotrophic biosynthesis using engineered microalgae. [ABSTRACT FROM AUTHOR]

Highlights: [•] We cultivated Doritaenopsis plantlets (CAM-type photosynthesis) in vitro under three different microenvironmental conditions and transferred in ex vitro conditions. [•] We examine the changes in growth, physiological and biochemical factors during acclimatization of those plantlets. [•] Photoautotrophically and photomixotrophically grown plants in in vitro exhibited the best growth in the ex vitro environment. [•] May be a consequence of the presence of higher sugar reserves in these plants, as well the early recovery of PEP-carboxylase activity. [ABSTRACT FROM AUTHOR]

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, which are synthesized only by photosynthetic organisms. Due to their enormous potential to protect cells from oxidative damage, tocopherols are used, e.g., as nutraceuticals and additives in pharmaceuticals. The most biologically active form of vitamin E is α-tocopherol. Most tocopherols are currently produced via chemical synthesis. Nevertheless, this always results in a racemic mixture of different and less effective stereoisomers because the natural isomer has the highest biological activity. Therefore, tocopherols synthesized in natural sources are preferred for medical purposes. The annual sunflower ( Helianthus annuus L.) is a well-known source for α-tocopherol. Within the presented work, sunflower callus and suspension cultures were established growing under photomixotrophic conditions to enhance α-tocopherol yield. The most efficient callus induction was achieved with sunflower stems cultivated on solid Murashige and Skoog medium supplemented with 30 g l sucrose, 0.5 mg l of the auxin 1-naphthalene acetic acid, and 0.5 mg l of the cytokinin 6-benzylaminopurine. Photomixotrophic sunflower suspension cultures were induced by transferring previously established callus into liquid medium. The effects of light intensity, sugar concentration, and culture age on growth rate and α-tocopherol synthesis rate were characterized. A considerable increase (max. 230 %) of α-tocopherol production in the cells was obtained within the photomixotrophic cell culture compared to a heterotrophic cell culture. These results will be useful for improving α-tocopherol yields of plant in vitro cultures. [ABSTRACT FROM AUTHOR]

Abstract: A novel protocol for raspberry (Rubus spp.) micropropagation in photomixotrophic conditions was optimized for the commercial genotypes Heritage, Meeker and Amity. Plant cultures were established in Temporary Immersion Bioreactors (TIBs) under a controlled environment: 550ppmv CO2; light intensity 80μMm−2 s−1; sucrose concentrations 15gr/L and 30gr/L. Results showed that both CO2 flux and chlorophyll fluorescence were increased in plants cultured in TIBs with sucrose-reduced medium (15gr/L) in comparison with those plants micropropagated in TIBs+30gr/L sucrose, confirming a consistent photomixotrophic stage since the 5th day of culture. Raspberry plants multiplied in TIBs+15gr/L sucrose demonstrated the highest values of plant size, total number of internodes, and percent of acclimatization to greenhouse conditions. Studied variables could be related to an increase of the in vitro photosynthetic activity which might prime plants for the ex vitro environment. [Copyright &y& Elsevier]

In vitro propagated plants under conditions of low gas exchange generally show morphological and physiological anomalies that lead to high mortality rates during ex vitro acclimatization. The use of gas-permeable membranes increases natural ventilation in culture vessels, photosynthesis and growth rates. However, commercial membranes are expensive, which limits their application. In this study, low-cost, simple to manufacture, alternative membranes were developed to promote gas exchange in jars used for in vitro plant tissue culture. The membranes were developed using polytetrafluoroethylene film and two or three layers of microporous tape (Missner & Missner), and were designed to increase the growth of nodal cultures of Pfaffia glomerata (Brazilian ginseng). Conditions that provided higher gas exchange led to an increase in plant growth and content of photosynthetic pigments compared to a closed system without a gas-permeable membrane. The alternative membranes showed similar results for water vapor loss rate and photosynthetic pigments when compared to a commercial membrane. The alternative membranes were also an efficient barrier against contamination and remained intact after being autoclaved multiple times. Among the membranes tested, the traits of the P. glomerata in vitro-derived plants were similar when propagated using the alternative membrane with three layers of microporous tape or the commercial membrane. However, the alternative membrane has a unit cost that is ten times lower than the commercial membrane. [ABSTRACT FROM AUTHOR]

Pfaffia glomerata (Spreng.) Pedersen is a medicinal species of great interest because it produces the phytoecdysteroid 20-hydroxyecdysone (20E). Generally, because of atypical growing conditions, in vitro propagated plants function less efficiently as autotrophs and have poorly developed morphological structures. This study analyzed the autotrophic potential of P. glomerata propagated in vitro and evaluated the influence that this has on 20E biosynthesis. Physiological and structural parameters of plants subjected to heterotrophic, photomixotrophic and photoautotrophic growth conditions were evaluated. Levels of 20E were measured by HPLC. Plants were acclimatized in a mixture of soil, sand and substrate, in a greenhouse. Conditions that provided higher carbon input led to an increase in plant growth, and the presence of sucrose was critical, in closure systems without a gas permeable membrane, for normal anatomical development of the micropropagated plants. The absence of sucrose increased photosynthesis and conditions that enhanced photoautotrophy induced greater levels of 20E. The increase of 20E levels by the photoautotrophic system offers new prospects for increasing the commercial production of this species, and for studies that could elucidate the biosynthetic pathway of phytoecdysteroids in plants. [ABSTRACT FROM AUTHOR]

Abstract: The photomixotrophic hydrogen production was investigated in sulfur deprived Chlamydomonas reinhardtii cultures. The cultures were exposed to continuous illumination of various light intensities in 27-day batches. Light intensity of 70 × 2 μE m−2 s−1 was selected for hydrogen production. Subsequent experiments involving 27-day long light:dark cycles were conducted at the selected light intensity. The cycles consisted of hour divisions (h:h; 18:6, 14:10, 12:12) or minute divisions (min:min; 45:15, 35:25, 30:30). The results showed an adverse effect of the light:dark cycles on hydrogen production. All experiments, irrespective of the type of illumination indicated that cultures needed a lag phase for production and the highest hydrogen production was obtained during first 7–10 days of production reaching a peak in the first 5 days. [Copyright &y& Elsevier]

A forced ventilation system has been developed for large-scale photoautotrophic micropropagation of chlorophyllous plants. The major goal of the system is to provide a uniform supply of CO2 inside a large culture vessel (volume 3480 ml) to achieve uniform growth of the plantlets. The system has been designed such that sterile nutrient solution can be supplied throughout the culture period, which is essential for long-term culture. Sweet potato ( Ipomoea batatas L. Lam., cv. ‘Beniazuma’) was used as a model plant for photoautotrophic culture with stagnant and nonstagnant nutrient solution in large vessels. Growth and net photosynthetic rates of the plantlets were compared with those of the plantlets grown in a small vessel under photoautotrophic conditions (with natural ventilation) and conventional photomixotrophic conditions. The results indicated that the large vessel with the forced ventilation system was effective for improving growth and uniformity of the plantlets and the rate of net photosynthesis. The stagnant nutrient solution condition under photoautotrophic forced ventilation treatment significantly increased the fresh mass of the plantlets; however, percent dry mass was highest in the treatment with nonstagnant nutrient solution condition. The results demonstrated that the conventional photomixotrophic culture system can cause seriously inhibited growth and development. [ABSTRACT FROM AUTHOR]

Cells of potato (Solanum tuberosum L.) were obtained which were capable of photoautotrophic growth in liquid suspension culture under a photon flux density of 90-110 μmol m-2 s-1 PAR and in an atmosphere enriched with 2% CO2. These photoautotrophic cells contained between 100 to 200 μg Chl (g fresh weight)-1 and fixed CO2 at a maximum rate of 16 μmol CO2 (g fresh weight)-1 h-1. In order to obtain cells capable of photoautotrophic growth it was necessary to adapt highly chlorophyllous heterotrophic cells (>50 μg Chl (g fresh weight)-1) for growth in medium with 2.5 g sucrose 1-1 (photomixotrophic cells). The photomixotropic cells had a Chl content of ca 10 μg Chl (g fresh weight)-1 and were capable of photosynthetic activity which allowed them to survive after sugars had been depleted from the medium. It was from the photomixotrophic cells that cells capable of photoautotrophic growth were obtained. Heterotrophic cells initially established in liquid medium with 25 g sucrose 1-1 from chlorophyllous callus contained about 50 to 150 μg Chl (g fresh weight)-1. However, after 5 to 10 passages the Chl content decreased to a maximum of 15 μg Chl (g fresh weight)-1. These cells could not be adapted to photomixotrophic or photoautotrophic growth. These cells also were not able to regain Chl or initiate high rates of CO2 fixation during the stationary phase of growth as did photomixotrophic cells or chlorophyllous heterotrophic cells. The loss of Chl exhibited by the cells during adaption to heterotrophic growth could be attributed at least in part to unbalanced growth (when cell division and growth exceeds Chl accumulation):. Sucrose appeared to have an inhibitory effect directly on photosynthesis independent of Chl accumulation. [ABSTRACT FROM AUTHOR]

Photosynthetic suspension cultures of higher plants offer an alternative approach to biomass production, potentially yielding cellulosic material and protein on a continuous year-round basis. A bench-top hybrid photobioreactor was developed to study photomixotrophic and photoautotrophic growth of Glycine max as a model system. Maximum biomass doubling times for photomixotrophic and photoautotrophic growth were 1.87 and 3.92 d, respectively. The presence of exogenous sugars resulted in photomixotrophic growth, reduced chlorophyll levels, and a reduction in photosynthetically-evolved oxygen. Depletion of carbohydrates from the medium coincided with the beginning of stationary phase and an increase in oxygen evolution by the cells. A second growth phase, prolonging cell viability, could be initiated by increasing the carbon dioxide from 2 to 5%, just before the onset of stationary phase. Biomass from bioreactor cultured cells proved resistant to enzymatic attack without pretreatment. Composition of the biomass was 7.8% lignin, 20.7% α-cellulose, 23% hemicellulose, 5.5% starch, 14.5% protein and 6.5% nucleic acids. [ABSTRACT FROM AUTHOR]

In cyanobacteria, photomixotrophic growth is considered as a promising strategy to achieve both high cell density and product accumulation. However, the conversion of glucose to acetyl coenzyme A (acetyl-CoA) in the native glycolytic pathway is insufficient, which decreases the carbon utilization and productivity of engineered cyanobacteria under photomixotrophic conditions. To increase the carbon flux from glucose to key intracellular precursor acetyl-CoA in Synechocystis sp. PCC 6803 (hereafter, Synechocystis 6803) under photomixotrophic conditions, a synthetic nonoxidative cyclic glycolysis (NOG) pathway was introduced into the wild type strain, which successfully increased the intracellular pool of acetyl-CoA by approximately 1-fold. To minimize the competition for glucose, the native Embden-Meyerhof-Parnas (EMP) and Entner-Doudoroff (ED) pathways were knocked out, respectively. Notably, eliminating the native ED pathway in the engineered strain carrying the NOG pathway further increased the intracellular pool of acetyl-CoA up to 2.8-fold. Another carbon consuming pathway in Synechocystis 6803, the glycogen biosynthesis pathway, was additionally knocked out in the above-mentioned engineered strain, which enabled an increase of the intracellular acetyl-CoA pool by up to 3.5-fold when compared with the wild type strain. Finally, the content of intracellular lipids was analyzed as an index of the productive capacity of the engineered Synechocystis 6803 cell factory under photomixotrophic conditions. The results showed the total lipids yield increased about 26% compared to the wild type (from 15.71% to 34.12%, g/g glucose), demonstrating that this integrated approach could represent a general strategy not only for the improvement of the intracellular concentration of acetyl-CoA, but also for the production of value-added chemicals that require acetyl-CoA as a key precursor in cyanobacteria.