Optimization of eicosapentaenoic acid production byPhaeodactylum tricornutumin the flat panel airlift (FPA) reactor

Biomass and eicosapentaenoic acid (EPA) productivities were investigated in a flat panel airlift loop reactor ideally mixed by static mixers. Growth with ammonium, urea and nitrate as nitrogen source were performed at different aeration rates. Cultures grew on ammonium but the decay of pH strongly inhibited biomass increase. On urea biomass productivity reached 2.35 g L^–1d^–1at an aeration rate of 0.66 vvm (24 h light per day, 1000 mol photon m^–2s^–1). Aeration rates between 0.33 vvm and 0.66 vvm and maximal productivities on urea were linearly dependent. Productivity on nitrate never exceeded 1.37 g L^–1d^–1. In the range of maximum productivity photosynthesis efficiency of 10.6% was reached at low irradiance (250 mol photon m^–2s^–1). Photosynthesis efficiency decreased to 4.8% at 1000 mol photon m^–2s^–1. At these high irradiances the flat panel airlift reactor showed a 35% higher volume productivity than the bubble column. At continuous culture conditions the influence of CO₂concentration in the supply air was tested. Highest productivities were reached at 1.25% (v/v) CO₂where the continuous culture yielded 1.04 g L^–1d^–1(16 h light per day, 1000 mol photon m^–2s^–1). The average EPA content amounted to 5.0% of cell dry weight, that resulted in EPA productivities of 52 mg L^–1d^–1(continuous culture, 16 h light per day) or 118 mg L^–1d^–1(batch culture, 24 h light per day).     

Growth and betacyanin production by hairy roots of Beta vulgaris in airlift bioreactors

Hairy roots of red beet (Beta vulgaris L.) were cultivated in different types of airlift bioreactors (cone, balloon, bulb, drum and column bioreactors of 5 l capacity and containing 3 l of half strength Murashige & Skoog medium). The cone type of airlift bioreactor gave the highest biomass of hairy roots and betacyanin accumulation. Betacyanin accumulation was 27 mg g^–1 dry wt in cultures aerated at 0.3 vvm. Light irradiation of 20 mol m^–2 s^–1 promoted hairy root growth but optimum betacyanin (34 mg g^–1 dry wt) accumulation was with the cultures grown under 60 mol m^–2 s^–1.     

Ulva rigida (Ulvales,Chlorophyta) tank culture as biofilters for dissolved inorganic nitrogen from fishpond effluents

Ulva rigida was cultivated in 7501 tanks at different densities with direct and continuous inflow (at 2, 4, 8 and 12 volumes d^–1) of the effluents from a commercial marine fishpond (40 metric tonnes, Tm, of Sparus aurata, water exchange rate of 16 m³ Tm^–1) in order to assess the maximum and optimum dissolved inorganic nitrogen (DIN) uptake rate and the annual stability of the Ulva tank biofiltering system. Maximum yields (40 g DW m^–2 d^–1) were obtained at a density of 2.5 g FW 1^–1 and at a DIN inflow rate of 1.7 g DIN m^–2 d^–1. Maximum DIN uptake rates were obtained during summer (2.2 g DIN M^–2 d^–1), and minimum in winter (1.1 g DIN m^–2 d^–1) with a yearly average DIN uptake rate of 1.77 g DIN m^–2 d^–1 At yearly average DIN removal efficiency (2.0 g DIN m^–2 d^–1, if winter period is excluded), 153 m² of Ulva tank surface would be needed to recover 100% of the DIN produced by 1 Tm of fish.Abbreviations DIN= dissolved inorganic nitrogen (NH _inf4 ^sup+ + NO _inf3 ^sup– + NO _inf2 ^sup– ); - FW= fresh weight; - DW= dry weight; - PFD= photon flux density; - V= DIN uptake rate     

Repeated elicitation enhances taxane production in suspension cultures of Taxus chinensis in bioreactors

Methyl jasmonate added at 100 M on days 8, 14 and 20 to cultures of Taxus chinensis growing in flasks and 1 l airlift bioreactors improved taxuyunnanine C (TC) production by 50–60% in flasks and by about 80% in airlift bioreactors compared with single addition of 100 M methyl jasmonate on day 8. The final TC production in the airlift bioreactors reached 565±47 mg l^–1, which is the highest reported for taxoid production in a bioreactor.     

Large-scale cultivation of adventitious roots of Echinacea purpurea in airlift bioreactors for the production of chichoric acid, chlorogenic acid and caftaric acid

Adventitious roots of Echinacea purpurea were cultured in airlift bioreactors (20 l, 500 l balloon-type, bubble bioreactors and 1,000 l drum-type bubble bioreactor) using Murashige and Skoog (MS) medium with 2 mg indole butyric acid l⁻¹ and 50 g sucrose l⁻¹ for the production of chichoric acid, chlorogenic acid and caftaric acid. In the 20 l bioreactor (containing 14 l MS medium) a maximum yield of 11 g dry biomass l⁻¹ was achieved after 60 days. However, the amount of total phenolics (57 mg g⁻¹ DW), flavonoids (34 mg g⁻¹ DW) and caffeic acid derivatives (38 mg g⁻¹ DW) were highest after 50 days. Based on these studies, pilot-scale cultures were established and 3.6 kg and 5.1 kg dry biomass were achieved in the 500 l and 1,000 l bioreactors, respectively. The accumulation of 5 mg chlorogenic acid g⁻¹ DW, 22 mg chichoric acid g⁻¹ DW and 4 mg caftaric acids g⁻¹ DW were achieved with adventitious roots grown in 1,000 l bioreactors.     

Malate degradation by Schizosaccharomyces yeasts included in alginate beads

Schizosaccharomyces yeasts can be used for deacidification of grape musts. To this aim, we studied malic acid degradation by yeasts included in double layer alginate beads in a bubble column reactor. Use of immobilized micro-organisms allowed a continuous process with high dilution rates giving a deacidification capacity of 0.032 g of malate/hour/dm³/g of beads. The pneumatic agitation was very convenient in this case.List of Symbols D h^–1 Dilution rate for continuous culture - h Residence time for continuous culture - dM/dt kg/(m³ · h) Rate of degradation of malic acid - dS/dt kg/(m³ · h) Rate of consumption of glucose - _max h^–1 Maximal specific rate of growth     

Quantification of the contribution of surface outgrowth to biocatalysis in sol-gels: oxytetracycline production by <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

A technique was developed for differentiating the activity of microbes solely within sol gels by using the contribution of biomass outgrowth. Streptomyces rimosus was immobilised in colloidal silica gels and biomass growth, oxytetracycline synthesis, pH and carbohydrate consumption were compared for UV surface-sterilised gels, untreated gels, and liquid cultures. Absolute and biomass specific oxytetracycline yields were higher for non-sterile gels than for liquid culture. Biomass solely within colloidal silica gels (1.7 mg ml^–1), and gels obtained from colloidal silica modified by addition of larger silica particles (1.2 mg ml^–1) yielded 27 and 21 g ml^–1 oxytetracycline compared with 97 and 104 g ml^–1 for unsterilised gels (3.6 and 5.2 mg ml^–1 biomass) displaying outgrowth. It was therefore apparent that biomass and antibiotic production within the gels was limited and that optimisation requires gel modification.     

Optimization of agitation for production of swainsonine from Metarhizium anisopliae in stirred tank and airlift reactors

The optimal agitation rate for production of swainsonine from Metarhizium anisopliae grown in batch stirred tank reactors (2 to 20 l) was 400 rpm with a mixed hyphal and pelleted morphology where the specific swainsonine production rate was 9×10^–2 mg g^–1 cell dry wt h from 87 to 142 h. Culture of the fungus in a 6-l airlift reactor produced loose pellets and the production of swainsonine started at least 24 h earlier than in the stirred tank reactor. The final yield (5.9 mg swainsonine g^–1 cell dry wt) after 168 h in the airlift reactor was 18% less than those obtained in the stirred tank reactor with an agitation rate of 400 rpm.     

Overall assessment of Monodus subterraneus cultivation and EPA production in outdoor helical and bubble column reactors

The outdoor production of Monodus subterraneus wasstudied in bubble column and helical reactors, mainly analysing the influenceofdilution rate, air flow rate and solar irradiance on growth rate andbiochemicalcomposition. Photoinhibition and photo-oxidation phenomena were also analysed.The cultures were stressed at high solar irradiance and dissolved oxygenconcentrations. A clear relationship between stress of the cultures and thefluorescence from PSII measurements was observed, the Fv/Fm ratio being lowerinthe helical reactor than in the bubble column. Growth rate and biomassproductivity were both a function of the average irradiance and the Fv/Fmratio;maximum values of 0.040 h^–1 and 0.54 gL^–1 d^–1 were measured. The influenceofphotoinhibition and average irradiance was modelled, the model also fitting theexperimental data reported by another author. The chlorophyll contenthyperbolically decreased, whereas the carotenoid content decreased linearlywiththe average irradiance. The higher the dilution rate the higher the protein andcarbohydrate content of the biomass, and the lower the lipid content. Theeicosapentaenoic acid (EPA) content ranged from 2.3 to 3.2% d.wt, the higherthe dilution rate, the lower EPA content, although the higher the EPAproportion. Maximum EPA productivity was only 9 mg L^–1d^–1, due to the stress to which the cultures wereexposed.     

Culture ofGelidium sesquipedale (Clem.) Born.et Thur. in a chemostat system. Biomass production and metabolic responses affected by N flow

Gelidium sesquipedale is the most important raw material used for extraction of agar in Spain. Based on chemostats, a system of culture for macroalgae with a continuous flow of culture medium has been developed. A stressed morphotype from the South of Spain was cultured, and the effects of different rates of NO ₃ ^– flow on growth and internal constituents were investigated in the laboratory. Cultivation was successful after optimizing factors affecting growth, such as irradiance level, renewal rate and water movement. Mass production was dependent on N supply. With a flow of 35 mol NO₃ ^– g^–1 DW d^–1, optimal values of growth (2.1% d^–1) and biomass yield were obtained. In these conditions, biomass yield resembled the values observed in natural populations (about 500 g DW m^–2 y^–1). When the flow of N was reduced to 15 mol NO ₃ ^– g^–1 DW d^–1, growth rate and biomass yield were reduced three-fold, and were null when N was supplied as 7 mol NO ₃ ^– g^–1 DW d^–1. C:N ratio was an index of the physiological status of the tissue, remaining low when N was sufficient and raised to critical values when N supply was limited. Phycobiliproteins, kept at a constant irradiance level, were affected by N supply, acting as an internal nitrogen reserve, unlike chlorophylla. An effective phycobiliprotein synthesis took place when the flow of N was sufficient. Agar yield, on dry weight basis, was similar as a function of N flow, whereas agar yield of the culture was higher when N was sufficient as a result of growth not being limited by N.This system of culture, commonly used in microalgal studies, may have an important use in macroalgae as a system to obtain biomass of high quality as well as a good tool for physiological studies in conditions of continuous and controlled flow of nutrients.     

Feeding rate inhibition in crowded Daphnia pulex

Feeding rates of Daphnia pulex fed a range of levels of the alga Chlamydomonas reinhardi of 15 °C are strongly density-dependent. At lower densities, Daphnia (30 1^–1) fed at higher rates than crowded (270 1^–1) Daphnia which manifest a relatively depressed saturation feeding response. At 30 individuals/liter, Daphnia consumed 8.5 – 15.7 × 10⁴ cells d^–1h^–1 (on a volume basis, 12.1 – 22.2 × 10⁶ m³), at 270 L^–1 3.7 – 3.9 × 10⁴ (5.2 – 5.5 = 10⁶ m³ cells d^–1h^–1 when feeding on algae at 80 000 cells ml^–1 (11.3 × 10⁶ m³ ml^–1). The feeding rate data best fit an Ivlev feeding function. An autoallelopath might be causing the repression. Water preconditioned with crowded Daphnia completely repressed feeding in uncrowded Daphnia after six hours.     

High density culture of the freshwater rotifer,Brachionus calyciflorus

The freshwater rotifer, Brachionus calyciflorus is one of the live food organisms used for the mass production of larval fish. In this study possibility of obtaining high density cultures of the freshwater rotifer B. calyciflorus were investigated. The two culture systems used differed in their air and dissolved oxygen supplies using three temperatures in each case: 24, 28 and 32 °C. Rotifers were batch-cultured using 5 l-vessels and fed with the freshwater Chlorella. The growth rate of rotifers significantly increased with an increase in temperature. The maximum density of the rotifers with air-supply at 24 °C, 6500 ind. ml^–1, was significantly lower than those cultured at 28 and 32 °C, i.e. 8600 and 8100 ind. ml^–1, respectively. Dissolved oxygen levels decreased with time and ranged from 0.8 to 1.4 mg l^–1 when the density of freshwater rotifer was the highest at each temperature. The highest density (19200 ind. ml^–1) of freshwater rotifer was obtained in cultures with a supply of oxygen at 28 °C. Densities of 13500 and 17200 ind. ml^–1 were found at 24 and 32 °C, respectively. Levels of NH₃-N increased with time and a dramatic increase of NH₃-N was observed at high temperatures. Levels of NH₃-N at 24, 28 and 32 °C were 13.2, 18.5 and 24.5 mg l^–1, respectively. These levels coincided with the highest rotifer density at each of the three temperatures. When rotifers were cultured with an oxygen-supply and pH was adjusted to 7, the maximum density of rotifer reached 33500 ind. ml^–1 at 32 °C . These results suggested that high density culture of freshwater rotifer, B. calyciflorus could be achieved under optimal conditions with DO value of exceeding 5 mg l^–1 and NH₃-N values of lower than 12.0 mg l^–1.     

A simple and low-cost airlift photobioreactor for microalgal mass culture

A simple, low-cost, and efficient airlift photobioreactor for microalgal mass culture was designed and developed. The reactor was made of Plexiglas, and composed of three major parts: outer tube, draft tube and air duct. The fluid-dynamic characteristics of the airlift reactor were studied. The system proved to be well suited to the mass cultivation of a marine microalga, Chlorella sp. In batch culture, the biomass volumetric output rate of 0.21 g l^–1 d^–1 was obtained at the superficial gas velocity of 4 mm s^–1 in the draft tube.     

Menthol tolerant clones of Mentha arvensis: approach for in vitro selection of menthol rich genotypes

In vitro raised shoots of Mentha arvensis L. were screened for menthol tolerance level by growing them in media containing 0–100 g ml^–1 menthol. A total of 2850 regenerated shoots were step wise screened for menthol tolerance at the concentrations of 50 g ml^–1 followed by 60 and 70 g ml^–1. In this screening, only 30 individual regenerated shoots were able to survive. The clones from the primary screen were inoculated into rooting medium and, after rooting, transferred to pots in the greenhouse. Ultimately, these 30 menthol tolerant clones were multiplied and grown in the field in replicated plots of 2.5×2.5 m sizes. Twigs of 30 clones from the replicated trials were rechecked for tolerant phenotypes at a concentration of 70 g ml^–1 menthol wherein, these survived even after 7 days (secondary screening). These clones were checked for oil and menthol content and were found to be better than the control plants. Out of these 30 plants, five tolerated 80 g ml^–1 menthol (tertiary level screening) and were found to contain the highest amount of menthol per g leaf biomass. Molecular analysis through RAPD showed distinct variation in the profiles of these five plants, in comparison to the control. Using this method the relationship between the primer OPT 04, menthol tolerance and high menthol content character of the genotype was established. Further, a cultivar `Saksham' was released from the selections by CIMAP for superior performance.     

Accumulation of cadmium by immobilizedZoogloea ramigera 115

Summary Zoogloea ramigera 115 was immobilized into beads of calcium-alginate and placed into batch air-bubbled column reactors. In the absence of any added nutrients the immobilized bacterium adsorbed Cd from solutions containing levels of 2 and 20 g ml^–1 per day, over a period of 21 and 20 days, respectively. Adsorption of Cd from solutions containing 20 g ml^–1 Cd was better than 90% for 16 days. Beads treated with Cd at 2 g ml^–1 never adsorbed less than 95% of the metal. Alginate adsorbed Cd as well, but inclusion of cells changed the effectiveness of adsorption. Of a 250 g ml^–1 Cd solution, alginate adsorbed 70.4% Cd in 60 min whereas alginate plus cells adsorbed 90.5% in the same time span. Temperature had no effect on adsorption by immobilized cells at levels of 2 and 10 g ml^–1 Cd. However at higher concentrations, binding was enhanced as temperature increased.Z. ramigera beads were stable during all treatments and for prolonged periods of time (21 days).     

Phenol degradation by a psychrotrophic strain of Pseudomonas putida

Summary Cell growth and phenol degradation kinetics were studied at 10°C for a psychrotrophic bacterium, Pseudomonas putida Q5. The batch studies were conducted for initial phenol concentrations, S_o, ranging from 14 to 1000 mg/1. The experimental data for 14<=S_o<=200 mg/1 were fitted by non-linear regression to the integrated Haldane substrate inhibition growth rate model. The values of the kinetic parameters were found to be: _m=0.119 h^–1, K _S=5.27 mg/1 and K _I=377 mg/1. The yield factor of dry biomass from substrate consumed was Y=0.55. Compared to mesophilic pseudomonads previously studied, the psychrotrophic strain grows on and degrades phenol at rates that are ca. 65–80% lower. However, use of the psychrotrophic microorganism may still be economically advantageous for waste-water treatment processes installed in cold climatic regions, and in cases where influent waste-water temperatures exhibit seasonal variation in the range 10–30°C.Nomenclature K _S saturation constant (mg/l) - K _I substrate inhibition constant (mg/l) - specific growth rate (h^–1) - _m maximum specific growth rate without substrate inhibition (h^–1) - _max maximum achievable specific growth rate with substrate inhibition (h^–1) - S substrate (phenol) concentration (mg/l) - S_o initial substrate concentration (mg/l) - S_max substrate concentration corresponding to _max (mg/l) - t time (h) - X cell concentration, dry basis (mg DW/l) - X_f final cell concentration, dry basis (mg DW/l) - X_o initial cell concentration, dry basis (mg DW/l) - Y yield factor (mg DW cell produced/mg substrate consumed)     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.     

A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L.

Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 10⁶ g fwt^–1) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 10⁴ ml^–1 while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 10⁴ ml^–1. Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl^–1 w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l^–1 sucrose, NAA (0.2–0.5 mg l^–1), zeatin riboside (0.5–2.0 mg l^–1) and GA₃ (0.5–1.0 mg l^–1). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l^–1 agar-solidified B5 medium containing 30g l^–1 sucrose, IBA (0.01 mg l^–1) and BAP (1.0 mg l^–1). Elongated shoots developed roots after transfer to 8.0g l^–1 agar-solidified, hormone-free MS medium with 30 g l^–1 sucrose.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzyladenine or benzylaminopurine - B5 medium after Gamborg et al (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - GA³ gibberellic acid - 2,i-P 6-(--dimethylallylamino) purine - MS medium after Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid     

Effect of serum concentration on production of monoclonal antibodies and on shear sensitivity of a hybridoma

The effect of serum content of culture medium on the specific production rate of monoclonal antibodies (Mab's) and on shear sensitivity has been studied with hybridoma's, cultured in a continuous stirred tank reactor (CSTR). No decrease in specific Mab-production was found when the serum concentration was reduced from 10 to 2.5%, while steady state cell concentrations were hardly affected as well. In contrast the cell death rate in a bubble column strongly increased when the serum concentration was lowered, which could be ascribed to a reduced physical protective effect by the serum.List of Symbols k _d s^–1 death-rate constant - k _g s^–1 growth-rate constant - D m diameter of bubble column - d m diameter of air bubble - H m height of bubble column - X m³ hypothetical killing volume - X (m³/m³) specific hypothetical killing volume - F m³/s volumetric air-flow rate - C cells/m³ number of viable cells - C₀ cells/m³ number of viable cells at t=0 - t s time     

Biology of Moina mongolica (Moinidae,Cladocera) and perspective as live food for marine fish larvae: review

Moina mongolica, 1.0-1.4 mm long and 0.8 mm wide, is an Old World euryhaline species. This paper reviewed the recent advances on its autecology, reproductive biology, feeding ecology and perspective as live food for marine fish larviculture. Salinity tolerance of this species ranges from 0.4–1.4 to 65.2–75.4. Within 2–50 salinity, Moina mongolica can complete its life cycle through parthenogenesis. The optimum temperature is between 25 °C and 28 °C, while it tolerates high temperature between 34.4 °C and 36.0 °C and lower temperature between 3.2 °C and 5.4 °C. The non-toxic level of unionised ammonia (24 h LC₅₀) for M. mongolica is <2.6 mg NH₃–N l^–1. Juvenile individuals filter 2.37 ml d^–1 and feed 9.45×10⁶ algal cells d^–1, while mature individuals filter 9.45 ml d^–1 and consume 4.94×10⁶ algal cells d^–1. At 28 °C, M. mongolica reaches sex maturity in 4 d and gives birth once a day afterward; females carry 7.3 eggs brood^–1 and spawn 2.8 times during their lifetime. A variety of food can be used for M. mongolica culture including unicellular algae, yeast and manure, but the best feeding regime is the combination of Nannochloropsis oculata and horse manure. Moina mongolica reproduces parthenogenetically during most lifetime, but resting eggs can be induced at temperature (16 °C) combined with food density at 2000–5000 N. oculata ml^–1. The tolerance to low dissolved oxygen (0.14–0.93 mg l^–1) and high ammonia makes it suitable for mass production. Biochemical analyses showed that the content of eicospantanoic acid (20:53) in M. mongolica accounts for 12.7% of total fatty acids, which is higher than other live food such as Artemia nauplii and rotifers. This cladoceran has the characteristics of wide salinity adaptation, rapid reproduction and ease of mass culture. The review highlights its potential as live food for marine fish larvae.