Elicitation with methyl-jasmonate stimulates peruvoside production in cell suspension cultures of <Emphasis Type="Italic">Thevetia peruviana</Emphasis>

Thevetia peruviana is a small tree that produces several compounds with pharmaceutical application, among which peruvoside could be highlighted. However, these compounds are produced in low concentration in the plant, making it important to develop strategies such as plant cell culture and elicitation to obtain higher quantities of the desired product. In this work, cell suspension cultures of T. peruviana were established in four different culture media: Murashige–Skoog (MS), half Murashige–Skoog (half MS), Schenk–Hildebrandt (SH), and Gamborg (B5) to study their effect on cell growth. Cell growth kinetics were studied in SH medium, and the extracellular peruvoside production during the culture time was determined. The best culture medium for the establishment of cell suspension cultures was MS with a growth index of 3.17 ± 0.2 g g⁻¹ inoculum. The cell growth kinetics showed the four characteristic growth phases of a cell culture (lag, exponential, stationary, and death), and during none of these phases was it possible to observe peruvoside production. The elicitor effect of methyl-jasmonate (MeJ) was studied in cell suspension cultures established in SH medium. The effect of MeJ concentration and the time in which it should be applied were determined. The best results were obtained at a concentration of 100 mg l⁻¹ of MeJ applied at the beginning of the culture, which induced a peruvoside production of 8.93 mg l⁻¹ medium. The current results are the first report of an in vitro peruvoside production system.     

New Phytotoxic Metabolites from Pestalotiopsis sp. HC02, a Fungus Residing in Chondracris rosee Gut

Two new phytotoxic γ‐lactones, pestalotines A and B ( 1 and 2 , resp.), along with 4‐oxo‐4H‐pyran‐3‐acetic acid ( 3 ) and 6‐hydroxyramulosin (=3,4,4a,5,6,7‐hexahydro‐6,8‐dihydroxy‐3‐methyl‐1H‐2‐benzopyran‐1‐one; 4 ), were isolateded from the culture of Pestalotiopsis sp. HC02, a fungus residing in the Chondracris rosee gut. Structures of the new metabolites were elucidated on the basis of their IR, NMR, and MS data. Pestalotines A and B ( 1 and 2 , resp.) significantly inhibited the radical growth of Echinochloa crusgalli with IC₅₀ values of 1.85×10^?4 and 2.50×10^?4 M , respectively, comparable to that of 2‐(2,4‐dichlorophenoxy)acetic acid (0.94×10^?4 M ) used as a positive control.     

Biomass and lipid enhancement in Ankistrodesmus sp. cultured with reused and minimal nutrients media

Microalgae are a promising feedstock for biofuel production. Lipid content in microalgae could be enhanced under nutrient depletion. This work investigated the effect of the nutrient on lipid accumulation in Ankistrodesmus sp. culture. Batch cultures were carried out using fresh BG11 medium, and after the harvest, the medium was reused for the next culture; this method was repeated two times. The maximum lipid productivity of 29.75 mg L^?1 day^?1 was obtained from the culture with the second reuse medium. In continuous cultures, Ankistrodesmus sp. was cultured in both fresh and modified BG11 mediums. The modified BG11 medium was adjusted to resemble the content of the first reuse medium. As a comparison between batch and continuous cultures, it was proven that the productivity in the continuous culture was better than in the batch, where the achievable maximum biomass and lipid were 188.30 and 38.32 mg L^?1 day^?1. The maximum lipid content of 34.22% was obtained from the continuous culture at a dilution rate of 0.08 day^?1, whereas the maximum saturated and unsaturated fatty acid productivities of 79.96 and 104.54 mg L^?1 day^?1 were obtained at a dilution rate of 0.16 day^?1.     

Purification and Characterisation of the Enantiospecific Dioxygenases from Delftia acidovorans MC1 Initiating the Degradation of Phenoxypropionate and Phenoxyacetate Herbicides

After cultivation on (R,S)‐2‐(2,4‐dichlorophenoxy)propionate, two α‐ketoglutarate‐dependent dioxygenases were isolated and purified from Delftia acidovorans MC1, catalysing the cleavage of the ether bond of various phenoxyalkanoate herbicides. One of these enzymes showed high specificity for the cleavage of the R‐enantiomer of substituted phenoxypropionate derivatives: the K_m values were 55 μM and 30 μM, the k_cat values 55 min^–1 and 34 min^–1 with (R)‐2‐(2,4‐dichlorophenoxy)propionate [(R)‐2,4‐DP] and (R)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. The other enzyme predominantly utilised the S‐enantiomers with K_m values of 49 μM and 22 μM, and k_cat values of 50 min^–1 and 46 min^–1 with (S)‐2‐(2,4‐dichlorophenoxy)propionate [(S)‐2,4‐DP] and (S)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. In addition, it cleaved phenoxyacetate herbicides (i.e. 2,4‐dichlorophenoxyacetate: K_m = 123 μM, k_cat = 36 min^–1) with significant activity. As the second substrate, only α‐ketoglutarate served as an oxygen acceptor for both enzymes. The enzymes were characterised by excess substrate inhibition kinetics with apparent K_i values of 3 mM with (R)‐2,4‐DP and 1.5 mM with (S)‐2,4‐DP. The reaction was strictly dependent on the presence of Fe²⁺ and ascorbate; other divalent cations showed inhibitory effects to different extents. Activity was completely extinguished within 2 min in the presence of 100 μM diethylpyrocarbonate (DEPC).     

Trends in accumulation of ephedrine in callus cultures of <Emphasis Type="Italic">Ephedra major</Emphasis> Host

Ephedra major Host, a medicinal plant, belongs to the family of Ephedraceae. Ephedrine is the main alkaloid in Ephedra, which has different medicinal properties. However, the amount of ephedrine in plant material is low and callus culture can be a way to increase the alkaloid content. The aim of this research was to compare Murashige and Skoog (MS) and Gamborg’s B5 culture media for callus induction and ephedrine production. For this purpose, stem explants were cultured on MS or B5 media containing 0.0, 0.5, 1.0, 2.0, or 3.0 mg L^?1 of kinetin (Kin) either alone or in combination with 0.0, 0.5, 1.0, or 2.0 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D) and/or naphthalenacetic acid (NAA), in five replicates. MS medium containing 1.0 or 2.0 NAA and 0.5 mg L^?1 Kin were the most effective for callus induction. The highest percentage of callus induction (100%) on B5 culture medium was obtained with 2.0 2,4-D and 0.5 mg L^?1 Kin treatments. The results showed that there was no significant difference between MS and B5 media for callus induction, and fresh and dry weight production. High-performance liquid chromatography was conducted for the identification and quantification of ephedrine in the obtained callus. The highest level of ephedrine (7.38 mg g^?1 DW) was found in callus grown on MS medium containing 0.5 mg L^?1 of 2,4-D. The results revealed that ephedrine can accumulate in callus cultures to levels much higher than in E. major wild plants.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Growth,lipid content,productivity, and fatty acid composition of tropical microalgae for scale‐up production

Biomass and lipid productivity, lipid content, and quantitative and qualitative lipid composition are critical parameters in selecting microalgal species for commercial scale‐up production. This study compares lipid content and composition, and lipid and biomass productivity during logarithmic, late logarithmic, and stationary phase of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. grown in L1‐, f/2‐, and K‐medium. Of the tested species, Tetraselmis sp. exhibited a lipid productivity of 3.9–4.8 g m^?2 day^?1 in any media type, with comparable lipid productivity by Nannochloropsis sp. and Isochrysis sp. when grown in L1‐medium. The dry biomass productivity of Tetraselmis sp. (33.1–45.0 g m^?2 day^?1) exceeded that of the other species by a factor 2–10. Of the organisms studied, Tetraselmis sp. had the best dry biomass and/or lipid production profile in large‐scale cultures. The present study provides a practical benchmark, which allows comparison of microalgal production systems with different footprints, as well as terrestrial systems. Biotechnol. Bioeng. 2010;107: 245–257. © 2010 Wiley Periodicals, Inc.     

AUTOLYSIS KINETICS OF THE MARINE DIATOM DITYLUM BRIGHTWELLII (BACILLARIOPHYCEAE) UNDER NITROGEN AND PHOSPHORUS LIMITATION AND STARVATION1

Autolysis kinetics in axenic cultures of the diatom Ditylum brightwellii (West) Grunow were studied under nutrient limitation in continuous cultures and under nutrient starvation in batch-mode cultures obtained by switching off nutrient supply in the continuous cultures. Under N limitation, the specific algal autolysis rates (δ, day^?1) were found constant at 0.014 ± 0.002 day^?1over a broad range of specific dilution rates (D, day^?1) (0.09–0.56 day^?1), implying an intrinsic death factor independent of the physiologzc state of the algal cells. Under P limitation, 8 was inversely related to D and ranged between 0.067 and 0.005 day^?1 at D = 0.17–0.44 day^?1. Under conditions of nutrient stamation, the degree of algal nutrient deficiency prior to stamation affected autolysis rates (δ_b, day^?1) and subsequently survival of the algal cultures. Nitrogen-starved D. brightwellii showed highest δ_b (maximum, 0.10 day^?1) when precultured at the higher growth rates. Phosphorus stamation led to highest δ_b (maximum, 0.21 day^?1) in the cultures preconditioned at the lower steady state growth rates. The lower death rates for D. brightwellii under limitation and starvation of N compared to P suggest that D. brightwellii was better equipped to handle N than P deficiency. The present results showed that cell lysis induced by nutrient stress was a significant cause of mortality in D. brightwellii and provided more insight into the field distribution of this neritic diatom.     

In vitro production of sedative galphimine B by cell suspension cultures of Galphimia glauca

The sedative triterpene, galphimine B (1), was detected in cell suspension-batch cultures of Galphimia glauca. The effect of inoculum size, growth regulators and different concentrations of sucrose, nitrates and phosphates was evaluated. A two-stage batch process for biomass production and accumulation of compound 1 was established. Major cellular growth (15 g l^–1 dry wt) was obtained in the first stage with naphthaleneacetic acid (2 mg l^–1) + kinetin (2 mg l^–1). Adding 4 mg 2,4-dichlorophenoxyacetic l^–1 acid in the second stage resulted in the highest accumulation of 1 (0.21 mg g^–1 dry wt) which was 36% higher with respect to calluses and comparable to that obtained from wild plants.     

Effect of culture conditions, cytokinins, methyl jasmonate and salicylic acid on the biomass accumulation and production of withanolides in multiple shoot culture of Withania somnifera (L.) Dunal using liquid culture

The influence of cytokinins and culture conditions including medium volume, harvest time and elicitation with abiotic elicitors (SA/MeJ) have been studied for the optimal production of biomass and withanolides in the multiple shoot culture of Withania somnifera. Elicitation of shoot inoculum mass (2 g l^?l FW) with SA at 100 μM in the presence of 0.6 mg l^?l BA and 20 mg l^?l spermidine for 4 h exposure time at the 4th week in 20 ml liquid medium recorded higher withanolides production (withanolides A [8.48 mg g^?l DW], withanolides B [15.47 mg g^?l DW], withaferin A [29.55 mg g^?l DW] and withanone [23.44 mg g^?l DW]), which were 1.14 to 1.18-fold higher than elicitation with MeJ at 100 μM after 5 weeks of culture. SA-elicited cultures did not exhibit much variation in biomass accumulation when compared to control. This cytokinin induces and SA-elicited multiple shoot culture protocol provides a potential alternative for the optimal production of biomass and withanolides utilizing liquid culture.     

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.)

Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 26⁰C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4dichlorophenoxy acetic acid - IAA Indole acetic acid - IBA Indole butaric acid - NAA Naphthalene acetic acid     

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

Estimating the contribution of carnivorous waterbirds to nutrient loading in freshwater habitats

1. We estimated nitrogen (N) and phosphorus (P) loading into wetlands by carnivorous waterbirds with alternative physiological models using a food‐intake and an excreta‐production approach. The models were applied for non‐breeding and breeding Dutch inland carnivorous waterbird populations to quantify their contribution to nutrient loading on a landscape scale. 2. Model predictions based on food intake exceeded those based on excretion by 59–62% for N and by 2–36% for P, depending on dietary assumptions. Uncertainty analysis indicated that the intake model was most affected by errors in energy requirement, while the excretion model was dependent on faecal nutrient composition. 3. Per capita loading rate of non‐breeders increased with body mass from 0.3–0.8 g N day^?1 and 0.15 g P day^?1 in little gulls Larus minutus to 4.5–11.5 g N day^?1 and 2.1–3.2 g P day^?1 in great cormorants Phalacrocorax carbo. For breeding birds, the estimated nutrient loading by a family unit over the entire breeding period ranged between 17.6–443.0 g N and 8.6 g P for little tern Sterna albifrons to 619.6–1755.6 g N and 316.2–498.1 g P for great cormorants. 4. We distinguished between external (i.e. importing) and internal (i.e. recycling) nutrient loading by carnivorous waterbirds. For the Netherlands, average external‐loading estimates ranged between 38.1–91.5 tonnes N and 16.7–18.2 tonnes P per year, whilst internal‐loading estimates ranged between 53.1–140.5 tonnes N and 25.2–39.2 tonnes P and per year. The average contribution of breeding birds was estimated to be 17% and 32% for external and internal loading respectively. Most important species were black‐headed gull Larus ridibundus and mew gull Larus canus for external loading, and great cormorant and grey heron Ardea cinerea for internal loading. 5. On a landscape scale, loading by carnivorous waterbirds was of minor importance for freshwater habitats in the Netherlands with 0.26–0.65 kg N ha^?1 a^?1 and 0.12–0.16 kg P ha^?1 a^?1. However, on a local scale, breeding colonies may be responsible for significant P loading.     

Biomass production and fatty acid accumulation in Chlorella sp. (strain DEC1B) isolated from a petrol refinery in Huelva (Spain)

A microalgal strain was established from Cepsa's refinery wastewater treatment plant in Huelva (southwest of Spain). Genetic analysis of the chloroplastic rbcL gene encoding for the large subunit of the ribulose bisphosphate carboxylase enzyme (Rubisco) showed the strain had high homology with other known rbcL sequences of the genus Chlorella. The strain grows well autotrophically in minimum mineral medium, with a growth rate of 0.28 ± 0.012 day^?1 and a biomass productivity of 138.9 ± 6.7 mg L^?1 day^?1. N‐starvation and/or over illumination with 650 µmol photons m^?2 s^?1 of PAR light on the cultures induced a significant increase in the intracellular content of lipids in this microalga. Total lipids were extracted from the strain biomass with 2:1 chloroform‐methanol, and they accounted for approximately 50% of the dry biomass. Polyunsaturated fatty acids (PUFAs) represented 60.4% of the total fatty acids found in the strain, thus making this biomass attractive as a high added‐value product source. The strain was able to grow efficiently in the refinery treated wastewater from which it was isolated, providing an attractive advantage for further development of more sustainable algal biomass production processes at reduced costs close to a petrol refinery area.     

Proteomic analysis of urine in rats chronically exposed to fluoride

Urine is an ideal source of materials to search for potential disease‐related biomarkers as it is produced by the affected tissues and can be easily obtained by noninvasive methods. 2‐DE‐based proteomic approach was used to better understand the molecular mechanisms of injury induced by fluoride (F^?) and define potential biomarkers of dental fluorosis. Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F^? for 60 days (n = 15/group). During the experimental period, the animals were kept individually in metabolic cages, to analyze the water and food consumption, as well as fecal and urinary F^? excretion. Urinary proteome profiles were examined using 2‐DE and Colloidal Coomassie Brilliant Blue staining. A dose‐response regarding F^? intake and excretion was detected. Quantitative intensity analysis revealed 8, 11, and 8 significantly altered proteins between control vs. 5 ppm F^?, control vs. 50 ppm F^? and 5 ppm F^? vs. 50 ppm F^? groups, respectively. Two proteins regulated by androgens (androgen‐regulated 20‐KDa protein and α‐2μ‐globulin) and one related to detoxification (aflatoxin‐B1‐aldehyde‐reductase) were identified by MALDI‐TOF‐TOF MS/MS. Thus, proteomic analysis can help to better understand the mechanisms underlying F^? toxicity, even in low doses. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:8–14 2011; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20353     

Production of juvenile salmonids in small Norwegian streams is affected by agricultural land use

1. We estimated the biomass and production of juvenile anadromous brown trout (Salmo trutta) and Atlantic salmon (Salmo salar) (parr) in 12 streams in the Skagerrak area of Norway to identify controlling environmental factors, such as land‐use and water chemistry. 2. Production estimates correlated positively with fish density in early summer, but not with the size of the catchment. The summer biomass of age‐0 brown trout and Atlantic salmon was smaller than that of age‐1 and constituted 27.4 and 25.7%, respectively, of the total biomass of the two groups. 3. Mean production of brown trout from July to September varied between streams, but in most cases it was below 2 g 100 m^?2 day^?1. Yearly cohort production from age‐0 in July to age‐1 in July was 10 g m^?2 or less, with mean annual production of 1.32 g 100 m^?2 day^?1, equivalent to 4.8 g m^?2 year^?1. The corresponding annual cohort production of Atlantic salmon was 0.38 g 100 m^?2 day^?1 or 1.4 g m^?2 year^?1. Annual production to biomass ratio (P/B) for brown trout of the same cohort in the various streams was between 1.47 and 4.37; the overall mean (±SD) for all streams was 2.25 ± 0.94. Mean turnover rate of Atlantic salmon was 2.73 ± 0.24. 4. Production of 0+ brown trout during the summer correlated significantly with the percentage of agricultural land and forest/bogs in the catchment, with maxima at 20 and 75%, respectively. Age‐0 brown trout production also correlated with concentration of nitrogen and calcium in the water, with maxima at 2.4 and 14 mg L^?1, respectively. 5. The results support the hypothesis that brown trout parr production reflects the quality of their habitat, as indicated by the dome‐shaped relationship between percentage of agricultural land and the concentration of nitrogen and calcium in the water.     

Using CO2 to enhance carbon capture and biomass applications of freshwater macroalgae

A major limiting factor in the development of algae as a feedstock for the bioenergy industry is the consistent production and supply of biomass. This study is the first to access the suitability of the freshwater macroalgal genus Oedogonium to supply biomass for bioenergy applications. Specifically, we quantified the effect of CO₂ supplementation on the rate of biomass production, carbon capture, and feedstock quality of Oedogonium when cultured in large‐scale outdoor tanks. Oedogonium cultures maintained at a pH of 7.5 through the addition of CO₂ resulted in biomass productivities of 8.33 (±0.51) g DW m^?2 day^?1, which was 2.5 times higher than controls which had an average productivity of 3.37 (±0.75) g DW m^?2 day^?1. Under these productivities, Oedogonium had a carbon content of 41–45% and a higher heating value of 18.5 MJ kg^?1, making it an ideal biomass energy feedstock. The rate of carbon fixation was 1380 g C m^?2 yr^?1 and 1073.1 g C m^?2 yr^?1 for cultures maintained at a pH of 7.5 and 8.5, and 481 g C m^?2 yr^?1 for cultures not supplemented with CO₂. This study highlights the potential of integrating the large‐scale culture of freshwater macroalgae with existing carbon waste streams, for example coal‐fired power stations, both as a tool for carbon sequestration and as an enhanced and sustainable source of bioenergy.     

Heterotrophic production of Chlorella sp. TISTR 8990—biomass growth and composition under various production conditions

The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon‐to‐nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L^?1 glucose, 2.5 g L^?1 KNO₃) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500‐mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L^?1 was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g^?1 and 305 mg L^?1 day^?1, respectively. A repeated fed‐batch culture with four cycles of feeding with the fivefold concentrated medium in a 3‐L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L^?1 was obtained with a TFA productivity of 223 mg L^?1 day^?1. The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1589–1600, 2017     

High‐pressure systems for gas‐phase free continuous incubation of enriched marine microbial communities performing anaerobic oxidation of methane

Novel high‐pressure biotechnical systems that were developed and applied for the study of anaerobic oxidation of methane (AOM) are described. The systems, referred to as high‐pressure continuous incubation system (HP‐CI system) and high‐pressure manifold‐incubation system (HP‐MI system), allow for batch, fed‐batch, and continuous gas‐phase free incubation at high concentrations of dissolved methane and were designed to meet specific demands for studying environmental regulation and kinetics as well as for enriching microbial biomass in long‐term incubation. Anoxic medium is saturated with methane in the first technical stage, and the saturated medium is supplied for biomass incubation in the second stage. Methane can be provided in continuous operation up to 20 MPa and the incubation systems can be operated during constant supply of gas‐enriched medium at a hydrostatic pressure up to 45 MPa. To validate the suitability of the high‐pressure systems, we present data from continuous and fed‐batch incubation of highly active samples prepared from microbial mats from the Black Sea collected at a water depth of 213 m. In continuous operation in the HP‐CI system initial methane‐dependent sulfide production was enhanced 10‐ to 15‐fold after increasing the methane partial pressure from near ambient pressure of 0.2 to 10.0 MPa at a hydrostatic pressure of 16.0 MPa in the incubation stage. With a hydraulic retention time of 14 h a stable effluent sulfide concentration was reached within less than 3 days and a continuing increase of the volumetric AOM rate from 1.2 to 1.7 mmol L^?1 day^?1 was observed over 14 days. In fed‐batch incubation the AOM rate increased from 1.5 to 2.7 and 3.6 mmol L^?1 day^?1 when the concentration of aqueous methane was stepwise increased from 5 to 15 mmol L^?1 and 45 mmol L^?1. A methane partial pressure of 6 MPa and a hydrostatic pressure of 12 MPa in manifold fed‐batch incubation in the HP‐MI system yielded a sixfold increase in the volumetric AOM rate. Over subsequent incubation periods AOM rates increased from 0.6 to 1.2 mmol L^?1 day^?1 within 26 days of incubation. No inhibition of biomass activity was observed in all continuous and fed‐batch incubation experiments. The organisms were able to tolerate high sulfide concentrations and extended starvation periods. Biotechnol. Bioeng. 2010; 105: 524–533. © 2009 Wiley Periodicals, Inc.     

Synergistic effect of methyl jasmonate and cyclodextrins on anthraquinone accumulation in cell suspension cultures of Morinda citrifolia and Rubia tinctorum

Plant in vitro culture is a platform for producing secondary metabolites that combines safety, quality and low environmental impact. Besides, it is possible to increase the accumulation of these compounds by different strategies, such as elicitation. In this work, we analyzed the effects of the combination of methyl jasmonate (MeJ) and two cyclodextrins (CDs) on the production of anthraquinones (AQs) in cell cultures of Rubiaceae (Morinda citrifolia and Rubia tinctorum). These secondary metabolites have been traditionally used as dyes and have interesting therapeutic applications. The experiments were designed according to a full factorial design of two factors (MeJ and a CD) in two levels (0 and 0.1 mM for MeJ, and 0 and 20 mM of the CD). MeJ and (2-hydroxypropyl)-β-cyclodextrin (HPCD) synergistically increased intracellular AQ content in suspension cultures of R. tinctorum, and, to a lesser extent, in suspension cultures of M. citrifolia. Combination of MeJ with another CD, 2-methyl-β-cyclodextrin, led to a more intense and later increase in AQ content in cell cultures of R. tinctorum when compared to MeJ–HPCD treatment. However, the combination of CD and MeJ failed to induce a drastic AQ release to the culture media. Nevertheless, our results show that combination of strategies (using a CD and MeJ) was successful to increase secondary metabolite accumulation in suspension cultures. To our knowledge, this is the first report of synergistic effect of MeJ and CD on AQ accumulation in plant in vitro cultures.