Culture medium influence on growth,fatty acid,and pigment composition of <Emphasis Type="Italic">Choricystis minor</Emphasis> var. <Emphasis Type="Italic">minor</Emphasis>: a suitable microalga for biodiesel production

The purposes of this study were to assess the influence of culture medium on biomass production, fatty acid, and pigment composition of Choricystis minor var. minor and to evaluate the use of this microalga as a source of fatty raw material for biodiesel production. Cultures of C. minor var. minor were grown using WC (Wright’s cryptophyte) and BBM (Bold’s Basal medium) media. BBM medium produced more biomass (984.3 mg L^?1) compared to the WC medium (525.7 mg L^?1). Despite this result, WC medium produced a higher methyl ester yield for biodiesel production than the BBM medium (170.0 and 90.2 mg g^?1 of biomass, respectively). The average percentage of fatty acids obtained using the WC medium (17.0 %) was similar to soybean (18.0 %) and with similar biomass fatty acid profile. However, for pigment production, carotenoids and chlorophyll concentrations were twice as high when using the BBM medium.     

Physiological response of Nannochloropsis sp. to saline stress in laboratory batch cultures

In the present paper, we investigated the physiological response of the marine microalga Nannochloropsis sp. to salt stress (13, 27, 54, and 81 g L^?1 NaCl). Increasing the sodium chloride concentration caused up to a 70 % decrease in the chlorophyll a concentration, cell growth, and net photosynthesis rate. The chlorophyll a fluorescence measurements indicated a strong reduction in the effective quantum yield of photosystem II (?60 %) and an increase in nonphotochemical quenching when the cells were exposed to NaCl concentrations greater than 27 g L^?1 (control). In contrast, the specific lipid content increased up to 80 % when the sodium chloride concentration was increased from 27 to 54–81 g L^?1. These results are relevant for the outdoor cultivation of this microalga using open photobioreactors, in which microalgae are subjected to strong changes in salinity concentration caused by water evaporation.     

Enhancement of Neuroprotective Effects of <Emphasis Type="Italic">Spirulina maxima</Emphasis> by a Low-temperature Extraction Process with Ultrasonic Pretreatment

17.5 ± 2.41 mg/g and 15.2 ± 2.09 mg/g of total chlorophyll and chlorophyll a, respectively, were obtained from the marine microalga, Spirulina maxima under an optimal extraction condition of 70% ethanol at 65°C for 4 h associated with ultrasonic pretreatment at 40 kHz for 8 h. In comparison, 14.5 ± 1.36 mg/g and 7.1 ± 0.99 mg/g of total chlorophyll and chlorophyll a, respectively, were obtained from a conventional extraction process using 70% ethanol at 80°C for 12 h. The extract from the optimal conditions had the highest ratio of chlorophyll a to total chlorophyll, approximately 3:4, indicating that more intact chlorophyll a was obtained at low temperature. Moreover, the extract obtained using the optimal extraction condition showed substantial neuroprotective effects such as 92.78 ± 0.04% protection against glutamate-induced mouse hippocampal neuronal cells, compared to 81.64 ± 0.07% protection with the extract from the conventional extraction process. Compared to the control, the activities of two key enzymes related to glutathione synthesis, i.e., glutathione reductase and glutathione peroxidase, were also strongly increased, up to 90%, by the extracts from the optimal conditions. Interestingly, the addition of the same concentration of chlorophyll a as in the optimized extract had lower neuroprotective effects than did the extracts. This finding indicates that the extract likely exerted a synergistic effect, showing that the extract had better neuroprotective activity than the single component (chlorophyll a) alone. This work also confirmed the neuroprotective mechanism of the extract mainly due to its high antioxidant activity, allowing it to greatly decrease accumulation of ROS and Ca²⁺ within HT22 cells. The results of this work will have implications for expanding the use of a nonthermal ultrasonic process to extract heat-sensitive bioactive substances from natural resources.     

Growth of microalgae Botryococcus sp. in domestic wastewater and application of statistical analysis for the optimization of flocculation using alum and chitosan

Microalga biomass has been recognized as a sustainable bio-product to replace terrestrial biomass in biofuel production. The microalga industry has high operating costs, specifically on harvesting and biomass recovery. Therefore, the development of an efficient harvesting method is crucial to the minimization of production cost. A statistical analysis through response surface methodology was used to investigate the optimization of harvesting efficiency using alum and chitosan as a coagulant. Growth rate and biomass productivity were also determined. This research revealed that the harvesting efficiency using alum was 99.3%, with optimum dosage and pH of 177.74 mg L^?1 and 8.24, respectively. Chitosan achieved 94.2% biomass recovery at an optimal dosage of 169.95 mg L^?1 at pH of 12. Moreover, Botryococcus sp. achieved the maximum growth of 0.7551 µ_max d^?1, with an average total biomass productivity of 9.81 mg L^?1?d^?1 in domestic wastewater. Overall, this study shows that both alum and chitosan coagulants have great potential for efficient microalgal biomass recovery. It suggests that domestic wastewater as a potential growth medium for the large-scale production of microalga biomass.     

Citric acid production from extract of Jerusalem artichoke tubers by the genetically engineered yeast Yarrowia lipolytica strain 30 and purification of citric acid

In this study, citric acid production from extract of Jerusalem artichoke tubers by the genetically engineered yeast Yarrowia lipolytica strain 30 was investigated. After the compositions of the extract of Jerusalem artichoke tubers for citric acid production were optimized, the results showed that natural components of extract of Jerusalem artichoke tubers without addition of any other components were suitable for citric acid production by the yeast strain. During 10 L fermentation using the extract containing 84.3 g L^?1 total sugars, 68.3 g L^?1 citric acid was produced and the yield of citric acid was 0.91 g g^?1 within 336 h. At the end of the fermentation, 9.2 g L^?1 of residual total sugar and 2.1 g L^?1 of reducing sugar were left in the fermented medium. At the same time, citric acid in the supernatant of the culture was purified. It was found that 67.2 % of the citric acid in the supernatant of the culture was recovered and purity of citric acid in the crystal was 96 %.     

Evaluation of ethanol production by a new isolate of yeast during fermentation in synthetic medium and sugarcane bagasse hemicellulosic hydrolysate

A new xylose fermenting yeast was isolated from over-ripe banana by enrichment in xylose-containing medium. The phylogenetic analysis of ITS1-5.8S-ITS2 region sequences of ribosomal RNA of isolate BY2 revealed that it shows affiliation to genus Pichia and clades with Pichia caribbica. In batch fermentation, Pichia strain BY2 fermented xylose, producing 15 g l^?1 ethanol from 30 g l^?1 xylose under shaking conditions at 28°C, with ethanol yield of 0.5 g g^?1 and volumetric productivity of 0.31 g l^?1 h^?1. The optimum pH range for ethanol production from xylose by Pichia strain BY2 was 5–7. Pichia strain BY2 also produced 6.08 g l^?1 ethanol from 30 g l^?1 arabinose. Pichia strain BY2 can utilize sugarcane bagasse hemicellulose acid hydrolysate for alcohol production, efficiency of fermentation was improved by neutralization, and sequential use of activated charcoal adsorption method. Percent total sugar utilized and ethanol yield for the untreated hydrolysate was 17.14% w/v and 0.33 g g^?1, respectively, compared with 66.79% w/v and 0.45 g g^?1, respectively, for treated hemicellulose acid hydrolysate. This new yeast isolate showed ethanol yield of 0.45 g g^?1 and volumetric productivity of 0.33 g l^?1 h^?1 from sugarcane bagasse hemicellulose hydrolysate detoxified by neutralization and activated charcoal treatment, and has potential application in practical process of ethanol production from lignocellulosic hydrolysate.     

From crude glycerol to carotenoids by using a Rhodotorula glutinis mutant

In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280 % increase in β–carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R² = 0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P < 0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07 ± 1.45 mg l^?1) under optimized growth conditions was not statistically different from the maximal predicted (14.64 ± 1.57 mg l^?1) (P < 0.05), and it was about 100 % higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.     

Enhancement of carotenoid biosynthesis in the green microalga Dunaliella salina with light-emitting diodes and adaptive laboratory evolution

There is a particularly high interest to derive carotenoids such as β-carotene and lutein from higher plants and algae for the global market. It is well known that β-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased β-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.     

Evaluation of antioxidant capacities of green microalgae

Three strains of green microalgae, Chlorococcum sp.C53, Chlorella sp. E53, and Chlorella sp.ED53 were studied for their antioxidant activities. Crude extracts of these microalgae in hot water and in ethanol were examined for their total phenolic contents and for their antioxidant capacities. In order to determine their phenolic contents, the Folin–Ciocalteu method was used. As for the determination of their antioxidant capacities, four different assays were used: (1) total antioxidant capacity determination; (2) DPPH radical scavenging assay; (3) ferrous ion chelating ability assay; and (4) inhibition of lipid peroxidation (using thiobarbituric acid reactive substance). For all the strains we have studied, their ethanolic extract showed more antioxidant activities than their hot water extract. Categorically, the ethanolic extract of Chlorella sp.E53 exhibited both the highest total phenolic content of 35.5?±?0.14 mg gallic acid equivalent (GAE) g^?1 dry weight and the highest DPPH radical scavenging of 68.18?±?0.38 % at 1.4 mg mL^?1 (IC₅₀ 0.81 mg mL^?1), whereas Chlorella sp.ED53 showed both the highest ferrous ion chelation activity of 42.78?±?1.48 % at 1 mg mL^?1 (IC₅₀ 1.23 mg mL^?1) and the highest inhibition of lipid peroxidation of 87.96?±?0.59 % at 4 mg mL^?1. This high level of inhibition is comparable to 94.42?±?1.39 % of butylated hydroxytoluene, a commercial synthetic antioxidant, at the same concentration.     

Abscisic Acid-Induced Starch Accumulation in Bioenergy Crop Duckweed <Emphasis Type="Italic">Spirodela polyrrhiza</Emphasis>

Spirodela polyrrhiza, a fast-growing duckweed with high starch and low lignin content, shows promise as a feedstock for bioenergy. Abscisic acid (ABA) is a biological hormone that controls plant growth and stress response. The effects of different ABA concentrations (0, 1.0 × 10^?5, 1.0 × 10^?4, 1.0 × 10^?3, 1.0 × 10^?2, and 1.0 × 10^?1 mg/L) on duckweed biomass growth, carbon dioxide fixation, formation of photosynthetic pigments (Chlorophyll a (Chla), Chlorophyll b (Chlb), and carotenoids), the activities of soluble starch synthase (SSS) and starch branching enzyme (SBE), and the starch content of biomass were investigated in this study. ABA at concentrations lower than 1.0 × 10^?3 mg/L promoted carbon dioxide fixation, whereas it inhibited carbon dioxide fixation at concentrations over 1.0 × 10^?3 mg/L. ABA enhanced SSS and SBE activities at concentrations lower than 1.0 × 10^?2 mg/L. ABA treatment increased the content of Chla, Chlb, and carotenoids and resulted in the enhancement of starch content. Chla content gradually increased with the increasing concentration of ABA (1.0 × 10^?5 to 1.0 × 10^?2 mg/L). After culturing for 10 days, starch content in 1.0 × 10^?2 mg/L ABA medium reached 35.3% of dry weight (DW), which was the highest level in this study. This suggests that there is a great potential to develop a technology to increase starch accumulation in duckweed which can be used as an alternative to corn, sugarcane, or other food crops as a starch source.     

The citric acid production from raw glycerol by Yarrowia lipolytica yeast and its regulation

The optimal cultivation conditions ensuring the maximal rate of citric acid (CA) biosynthesis by glycerol-grown mutant Yarrowia lipolytica NG40/UV7 were found to be as follows: growth limitation by inorganic nutrients (nitrogen, phosphorus, or sulfur), 28 °C, pH 5.0, dissolved oxygen concentration (pO₂) of 50 % (of air saturation), and pulsed addition of glycerol from 20 to 80 g L^?1 depending on the rate of medium titration. Under optimal conditions of fed-batch cultivation, in the medium with pure glycerol, strain Y. lipolytica NG40/UV7 produced 115 g L^?1 of CA with the mass yield coefficient of 0.64 g g^?1 and isocitric acid (ICA) amounted to 4.6 g L^?1; in the medium with raw glycerol, CA production was 112 g L^?1 with the mass yield coefficient of 0.90 g g^?1 and ICA amounted to 5.3 g L^?1. Based on the activities of enzymes involved in the initial stages of raw glycerol assimilation, the tricarboxylic acid cycle and the glyoxylate cycle, the mechanism of increased CA yield from glycerol-containing substrates in Y. lipolytica yeast was explained.     

Characterization of an evolved carotenoids hyper-producer of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> through bioreactor parameter optimization and Raman spectroscopy

An evolutionary engineering approach for enhancing heterologous carotenoids production in an engineered Saccharomyces cerevisiae strain was used previously to isolate several carotenoids hyper-producers from the evolved populations. β-Carotene production was characterized in the parental and one of the evolved carotenoids hyper-producers (SM14) using bench-top bioreactors to assess the impact of pH, aeration, and media composition on β-carotene production levels. The results show that with maintaining a low pH and increasing the carbon-to-nitrogen ratio (C:N) from 8.8 to 50 in standard YNB medium, a higher β-carotene production level at 25.52 ± 2.15 mg β-carotene g^?1 (dry cell weight) in the carotenoids hyper-producer was obtained. The increase in C:N ratio also significantly increased carotenoids production in the parental strain by 298 % [from 5.68 ± 1.24 to 22.58 ± 0.11 mg β-carotene g^?1 (dcw)]. In this study, it was shown that Raman spectroscopy is capable of monitoring β-carotene production in these cultures. Raman spectroscopy is adaptable to large-scale fermentations and can give results in near real-time. Furthermore, we found that Raman spectroscopy was also able to measure the relative lipid compositions and protein content of the parental and SM14 strains at two different C:N ratios in the bioreactor. The Raman analysis showed a higher total fatty acid content in the SM14 compared with the parental strain and that an increased C:N ratio resulted in significant increase in total fatty acid content of both strains. The data suggest a positive correlation between the yield of β-carotene per biomass and total fatty acid content of the cell.     

Alkaline-sulfite pretreatment and use of surfactants during enzymatic hydrolysis to enhance ethanol production from sugarcane bagasse

Sugarcane bagasse is a by-product from the sugar and ethanol industry which contains approximately 70 % of its dry mass composed by polysaccharides. To convert these polysaccharides into fuel ethanol it is necessary a pretreatment step to increase the enzymatic digestibility of the recalcitrant raw material. In this work, sugarcane bagasse was pretreated by an alkaline-sulfite chemithermomechanical process for increasing its enzymatic digestibility. Na₂SO₃ and NaOH ratios were fixed at 2:1, and three increasing chemical loads, varying from 4 to 8 % m/m Na₂SO₃, were used to prepare the pretreated materials. The increase in the alkaline-sulfite load decreased the lignin content in the pretreated material up to 35.5 % at the highest chemical load. The pretreated samples presented enhanced glucose yields during enzymatic hydrolysis as a function of the pretreatment severity. The maximum glucose yield (64 %) was observed for the samples pretreated with the highest chemical load. The use of 2.5 g l^?1 Tween 20 in the hydrolysis step further increased the glucose yield to 75 %. Semi-simultaneous hydrolysis and fermentation of the pretreated materials indicated that the ethanol yield was also enhanced as a function of the pretreatment severity. The maximum ethanol yield was 56 ± 2 % for the sample pretreated with the highest chemical load. For the sample pretreated with the lowest chemical load (2 % m/m NaOH and 4 % m/m Na₂SO₃), adding Tween 20 during the hydrolysis process increased the ethanol yield from 25 ± 3 to 39.5 ± 1 %.     

Enhancement of total phenolic and flavonoids extraction from <Emphasis Type="Italic">Rosmarinus officinalis</Emphasis> L using electromagnetic induction heating (EMIH) process

Electromagnetic induction heating (EMIH) assisted extraction of phenolic compounds from Rosmarinus officinalis L, and the antioxidant and antimicrobial activities of the plant extract were examined in this study. The extraction yield acquired with this process was found to be 25.1?±?2%, with maximum amounts of phenolic compounds: 127.87?±?2.1 mg Gallic acid equivalents per g dry weight and total flavonoids contents 14.48?±?1.5 mg quercetin equivalents per g dry weight, under optimum extraction conditions (extraction time 2 h, ratio of raw material to liquid 1:2 and 0% of NaCl). The antioxidant activity was assessed by the 1,1-diphenyl-2-picrylhydrazyl (DPPH); 2, 2′-azinobis (3-ethylbenzothiazoline-6- sulfonic acid) radical cation (ABTS⁺) and ferric reducing power (FRAP) methods. The results indicate the extract derived through EMIH showed a strong antioxidant ability (89.25%; EC₅₀ of 0.0148 µg/mL). Besides, the antimicrobial bioassay demonstrated that the extract possessed a good antimicrobial activity against all tested fungi and bacteria.     

Effect of Pulsed Electric Field Treatments on Permeabilization and Extraction of Pigments from Chlorella vulgaris

The effect of pulsed electric field (PEF) treatments of different intensities on the electroporation of the cytoplasmatic membrane of Chlorella vulgaris, and on the extraction of carotenoids and chlorophylls were investigated. Staining the cells with propidium iodide before and after the PEF treatment revealed the existence of reversible and irreversible electroporation. Application of PEF treatments in the range of 20–25 kV cm^?1 caused most of the population of C. vulgaris to be irreversibly electroporated even at short treatment times (5 pulses of 3 µs). However, at lower electric field strengths (10 kV cm^?1), cells that were reversibly electroporated were observed even after 50 pulses of 3 µs. The electroporation of C. vulgaris cells by PEF higher than 15 kV cm^?1 and duration is higher than 15 µs increased significantly the extraction yield of intracellular components of C. vulgaris. The application of a 20 kV cm^?1 for 75 μs increased the extraction yield just after the PEF treatment of the carotenoids, and chlorophylls a and b 0.5, 0.7, and 0.8 times, respectively. However, further increments in electric field strength and treatment time did not cause significant increments in the extraction yield. The extraction of carotenoids from PEF-treated C. vulgaris cells after 1 h of the application of the treatment significantly increased the extraction yield in comparison to the yield obtained from the cells extracted just after the PEF treatment. After PEF treatment at 20 kV cm^?1 for 75 µs, extraction yield for carotenoids, and chlorophylls a and b increased 1.2, 1.6, and 2.1 times, respectively. A high correlation was observed between irreversible electroporation and percentage of yield increase when the extraction was conducted after 1 h of the application of PEF treatment (R: 0.93), but not when the extraction was conducted just after PEF treatment (R: 0.67).     

Effect of controlled oxygen limitation on Candida shehatae physiology for ethanol production from xylose and glucose

Carbon distribution and kinetics of Candida shehatae were studied in fed-batch fermentation with xylose or glucose (separately) as the carbon source in mineral medium. The fermentations were carried out in two phases, an aerobic phase dedicated to growth followed by an oxygen limitation phase dedicated to ethanol production. Oxygen limitation was quantified with an average specific oxygen uptake rate (OUR) varying between 0.30 and 2.48 mmolO₂ g dry cell weight (DCW)^?1 h^?1, the maximum value before the aerobic shift. The relations among respiration, growth, ethanol production and polyol production were investigated. It appeared that ethanol was produced to provide energy, and polyols (arabitol, ribitol, glycerol and xylitol) were produced to reoxidize NADH from assimilatory reactions and from the co-factor imbalance of the two-first enzymatic steps of xylose uptake. Hence, to manage carbon flux to ethanol production, oxygen limitation was a major controlled parameter; an oxygen limitation corresponding to an average specific OUR of 1.19 mmolO₂ g DCW^?1 h^?1 allowed maximization of the ethanol yield over xylose (0.327 g g^?1), the average productivity (2.2 g l^?1 h^?1) and the ethanol final titer (48.81 g l^?1). For glucose fermentation, the ethanol yield over glucose was the highest (0.411 g g^?1) when the specific OUR was low, corresponding to an average specific OUR of 0.30 mmolO₂ g DCW^?1 h^?1, whereas the average ethanol productivity and ethanol final titer reached the maximum values of 1.81 g l^?1 h^?1 and 54.19 g l^?1 when the specific OUR was the highest.     

Chemical composition and ethanol production potential of Thai seaweed species

This study evaluated the potential use of several Thai seaweed species for ethanol production. The high biomass of the green algae Ulva intestinalis and Rhizoclonium riparium and the red algae Gracilaria salicornia and Gracilaria tenuistipitata in an earthen pond culture led us to select these species for our study. The seaweed species were analyzed for chemical composition, resulting in ash contents of 37.62?±?0.15 % and fiber of 11.93?±?0.16 %, with the highest values in R. riparium. Low lipid values were found in all species, with the highest value (p?<?0.05) in G. salicornia (1.69?±?0.07 %) and the lowest in R. riparium (0.28?±?0.01 %) and G. tenuistipitata (0.26?±?0.01 %). The highest carbohydrate contents were found in G. tenuistipitata (54.89 %), and the lowest were in R. riparium (29.53 %). G. tenuistipitata (8.58?±?0.36 %) and U. intestinalis (8.24?±?0.28 %) had higher sulfate contents compared with G. salicornia (4.69?±?0.04 %) and R. riparium (1.97?±?0.20 %). The monosugar algal tissue components were analyzed by HPLC; rhamnose, xylose, fucose, arabinose, mannose, glucose, and galactose were used as reference sugars. Total sugar was found to be highest in G. tenuistipitata (98.21 %). Arabinose, glucose, and galactose were the main sugar components in all species. Glucose obtained from G. tenuistipitata (6.55 %) and R. riparium (6.52 %) was higher than in G. salicornia (0.27 %) and U. intestinalis (2.78 %). G. tenuistipitata fermentation gave a higher yield of ethanol (4.17?×?10^?3 g ethanol g^?1 sugars; 139.12 μg ethanol g^?1 glucose) than R. riparium (0.086?×?10^?3 g ethanol g^?1 sugars; 33.84 μg ethanol g^?1 glucose), U. intestinalis (0.074?×?10^?3 g ethanol g^?1 sugars; 9.98 μg ethanol g^?1 glucose), and G. salicornia (0.031?×?10^?3 g ethanol g^?1 sugars; 1.43 μg ethanol g^?1 glucose).     

Evaluation of the in vitro methods for micropropagation of Chondracanthus acicularis (Roth) Fredericq (Gigartinales, Rhodophyta): tissue culture and production of protoplasts

Tissue was cultured and protoplasts isolated from the carrageenophyte Chondracanthus acicularis with the aim of developing micropropagation as an alternative to harvesting raw material from natural beds. Both adventitious shoots and filamentous calluses were induced by tissue culture on medium solidified with 0.4–1 % (w/v) agar. Adventitious shoots were mainly produced from discoid bases while filamentous calluses were mainly induced from basal zones and sub-apical explants. A gradient of the regeneration ability was observed from the top to the bottom of the thallus. The discoid base was the most reactive explant and produced the highest number of adventitious shoots compared to basal zones and sub-apical explants, irrespective of the concentration of agar. Protoplasts were isolated enzymatically from the whole thallus using a combination of cellulase R-10 Onozuka, macerozyme R-10, and crude extract of the gland gut of algivorous molluscs. The highest mean yield of protoplasts (1.2?×?10⁶ protoplasts g^?1 fresh weight) was obtained after 16 h of digestion with an enzyme mixture containing 2 % (w/v) cellulase R-10, 1 % (w/v) macerozyme R-10 Onozuka, 4 % (v/v) crude extract of gut gland of Haliotis, 0.8 M mannitol, 50 mM sodium citrate, 0.3 % (w/v) bovine serum albumin. Depending on the conditions, mean protoplast yields ranged from 3.14?×?10⁵ to 1.2?×?10⁶ protoplasts g^?1 fresh weight. Different factors (storage duration, mannitol, sodium citrate, crude extract of the gland gut of algivorous molluscs) were tested to improve the yield of protoplasts but none has a significantly effect.     

Quality and intensity of light affect <Emphasis Type="Italic">Lippia gracilis</Emphasis> Schauer plant growth and volatile compounds in vitro

The aim of this study was to evaluate the effects of different intensities and quality of light and explant type on the growth of and volatile compounds in Lippia gracilis in vitro. The treatments were as follows: light intensities of 26, 51, 69, 94, or 130 µmol m^?2 s^?1 from fluorescent lamps and light-emitting diode (LED) lamps at different wavelengths, namely, white, red, blue, and combinations of red and blue light at ratios of 2.5:1 and 1:2.5, respectively, and two explant types, namely, nodal and apical segments. On the 30th day of culture on half-strength Murashige and Skoog (Physiol Plant 15(3):473–497, 1962) medium, growth, production of photosynthetic pigments, chlorophyll a and b, total chlorophyll, carotenoids, and volatile constituents (using headspace gas chromatography-mass spectrometry) were analyzed. The light quality and intensity significantly influenced the in vitro growth of L. gracilis. The apical segments were superior in all parameters evaluated compared to nodal segments. The number of segments plantlet^?1, root length, and leaf, shoot, root, and total weight were higher with increasing light intensity, especially under the 94 µmol m^?2 s^?1 treatment, for both explant types. The red light showed the highest leaf (32.28 mg plantlet^?1) and total (58.33 mg plantlet^?1) dry weight of all the light qualities. Major constituents, namely, ρ-cymene, γ-terpinene, thymol, carvacrol, and E-caryophyllene, were identified, regardless of light conditions. The amount and composition of volatile compounds varied according to light intensity and quality. Low intensity (26 µmol m^?2 s^?1) increased γ-terpinene content (12.42%) and concomitantly decreased carvacrol (38.52%). Blue LED light showed higher production of carvacrol (48.11%).     

Optimization of cultivation conditions for fatty acid composition and EPA production in the eustigmatophycean microalga Trachydiscus minutus

We determined the effects of cultivation conditions (nitrogen source, salinity, light intensity, temperature) on the composition of polyunsaturated fatty acids (PUFAs) and the production of eicosapentaenoic acid (EPA) in the laboratory cultured eustigmatophycean microalga, Trachydiscus minutus. T. minutus was capable of utilizing all nitrogen compounds tested (potassium nitrate, urea, ammonium nitrate, ammonium carbonate) with no differences in growth and only minor differences in fatty acid (FA) compositions. Ammonium carbonate was the least appropriate for lipid content and EPA production, while urea was as suitable as nitrates. Salinity (0.2 % NaCl) slightly stimulated EPA content and inhibited growth. Increasing salinity had a marked inhibitory effect on growth and PUFA composition; salinity at or above 0.8 % NaCl was lethal. Both light intensity and temperature had a distinct effect on growth and FA composition. The microalga grew best at light intensities of 470–1,070 μmol photons m^?2 s^?1 compared to 100 μmol photons m^?2 s^?1, and at 28 °C; sub-optimal temperatures (20, 33 °C) strongly inhibited growth. Saturated fatty acids increased with light intensity and temperature, whereas the reverse trend was found for PUFAs. Although the highest level of EPA (as a proportion of total FAs) was achieved at a light intensity of 100 μmol photons m^?2 s^?1 (51.1?± 2.8 %) and a temperature of 20 °C (50.9?±?0.8 %), the highest EPA productivity of about 30 mg L^?1?day^?1 was found in microalgae grown at higher light intensities, at 28 °C. Overall, for overproduction of EPA in microalgae, we propose that outdoor cultivation be used under conditions of a temperate climatic zone in summer, using urea as a nitrogen source.