Effect of irradiation intensity on cell growth and kalata B1 accumulation in <Emphasis Type="Italic">Oldenlandia affinis</Emphasis> cultures

Light irradiation had remarkable effects on callus growth of Oldenlandia affinis with an optimum intensity of 35 μmol m⁻² s⁻¹. Biosynthesis of kalata B1, the main cyclic peptide in O. affinis, was induced and triggered with rising irradiation intensities. The highest concentration of kalata B1, 0.49 mg g⁻¹ DW characterised by the maximum productivity of 3.88 μg per litre and day was analysed at 120 μmol m⁻² s⁻¹, although callus growth was repressed. The light saturation point was established to be 35 μmol m⁻² s⁻¹, where kalata B1 productivity was in a similar order (3.41 μg per day) due to the higher growth index. O. affinis suspension cultures were shown to accumulate comparable specific kalata B1 concentrations in a delayed growth associated production pattern. These were dependent on irradiation intensity (0.16 mg g⁻¹ at 2 μmol m⁻² s⁻¹; 0.28 mg g⁻¹ at 35 μmol m⁻² s⁻¹). The batch cultivation process resulted in a maximum productivity of 27.30 μg per litre and day with culture doubling times of 1.16 d⁻¹. Submers operation represented a 8-fold product enhancement compared to callus cultivation.     

Productivity, CO2/O2 exchange and hydraulics in outdoor open high density microalgal (Chlorella sp.) photobioreactors operated in a Middle and Southern European climate

Two variants of open photobioreactors were operated at surface-to-volume ratios up to 170 m⁻¹. The mean values for July and September obtained for photobioreactor PB-1 of 224 m² culture area (length 28 m, inclination 1.7%, thickness of algal culture layer 6 mm), operated in Třeboň (49^∘N), Czech Republic, were: net areal productivity, P _net = 23.5 and 11.1 g dry weight (DW) m⁻² d⁻¹; net photosynthetic efficiency (based on PAR – Photosynthetic Active Radiation), η = 6.48 and 5.98%. For photobioreactor PB-2 of 100 m² culture area (length 100 m, inclination 1.6%, thickness of algal culture layer 8 mm) operated in Southern Greece (Kalamata, 37^∘N) the mean values for July and October were: P _net = 32.2 and 18.1 g DW m⁻² d⁻¹, η = 5.42 and 6.07%. The growth rate of the alga was practically linear during the fed-batch cultivation regime up to high biomass densities of about 40 g DW L⁻¹, corresponding to an areal density of 240 g DW m⁻² in PB-1 and 320 g DW m⁻² in PB-2. Night biomass loss (% of the daylight productivity, P _L) caused by respiration of algal cells were: 9–14% in PB-1; 6.6–10.8% in PB-2. About 70% of supplied CO₂ was utilized by the algae for photosynthesis. The concentration of dissolved oxygen (DO) increased from about 12 mg L⁻¹ at the beginning to about 35 mg L⁻¹ at the end of the 100 m long path of suspension flow in PB-2 at noon on clear summer days. Dissipation of hydraulic energy and some parameters of turbulence in algal suspension on culture area were estimated quantitatively.     

Experimental tank cultivation of Porphyra in Israel

Outdoor tank cultivation of several Porphyra (nori) species was carried out from late November 2002 through early May 2003 using 40 L (with a surface of 0.25 m²), 600 L (1 m²), and 24,000 L (30 m²) fiberglass or PVC tanks provided with continuous aeration and seawater flow. Sexual and asexual spores produced from cultured conchocelis and frozen thalli in the laboratory, respectively, were subsequently grown to produce young fronds (ca. 5-10 cm) in an average time of 8 weeks. Growth in outdoor tanks and ponds was possible for a period of up to 20 weeks (i.e. growth season), with yields above 100 g FW m⁻²d⁻¹occurring during 12-14 weeks from late December through late March, when seawater temperatures were below 20 ^∘ C. These yields correlated with the species and depended on the type of tanks in which the algae were cultivated, with the highest yields observed for Porphyra sp. and Porphyra yezoensis when fertilized twice a week with NH₄ Cl and NaH₂ PO₄in 40 L tanks. Calculations of productivity for an entire growth season based on ≥ 100 g FW m⁻²d⁻¹yields exceed the average productivities using seeded nets in open sea, for all Porphyra species tested (0.96-4.06 kg DW m⁻² season⁻¹vs. 0.7-1.0 kg DW m⁻²of net season⁻¹). Therefore, tank cultivation of Porphyra can offer an additional source of nori biomass to international markets. Land-based tank cultivation also offers an environmentally friendly practice that allows for the manipulation of growth conditions to enrich seaweeds with specific, valuable chemicals such as protein and minerals.     

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.     

Enhanced iturin A production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its effect on suppression of the plant pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The behavior of Streptomyces peucetius var. caesius N47 was studied in a glucose limited chemostat with a complex cultivation medium. The steady-state study yielded the characteristic constants μ _max over 0.10 h⁻¹, Y _XS 0.536 g g⁻¹, and m_S 0.54 mg g⁻¹ h⁻¹. The product of secondary metabolism, ɛ-rhodomycinone, was produced with characteristics Y _PX 12.99 mg g⁻¹ and m _P 1.20 mg g⁻¹ h⁻¹. Significant correlations were found for phosphate and glucose consumption with biomass and ɛ-rhodomycinone production. Metabolic flux analysis was conducted to estimate intracellular fluxes at different dilution rates. TCA, PPP, and shikimate pathway fluxes exhibited bigger values with production than with growth. Environmental perturbation experiments with temperature, airflow, and pH changes on a steady-state chemostat implied that an elevation of pH could be the most effective way to shift the cells from growing to producing, as the pH change induced the biggest transient increase to the calculated ɛ-rhodomycinone flux.     

Hydrophobic response of the fungus <Emphasis Type="Italic">Rhinocladiella similis</Emphasis> in the biofiltration with volatile organic compounds with different polarity

Rhinocladiella similis biodegraded volatile organic compounds (VOCs) of different polarity in gas-phase biofilters. Elimination capacities, (EC) of 74 g_hexane m⁻³ h⁻¹, 230 g_ethanol m⁻³ h⁻¹, 85 g_toluene m⁻³ h⁻¹ and 30 g_phenol m⁻³ h⁻¹ were obtained. EC values correlated with the solubility of the VOCs. R. similis grown with n-hexane or ethanol in biofilters packed with Perlite showed that the surface hydrophobicity was higher with n-hexane than ethanol. The hydrophobin-like proteins extracted from the mycelium produced with n-hexane (15 kDa) were different from those in the ethanol biofilter (8.5 kDa and 7 kDa).     

Quantitative analysis of phenolics in selected crop species and biological activity of these compounds evaluated by sensitivity of Echinochloa crus-galli

The purpose of this study was to determine the content of selected phenolic compounds in white mustard, buckwheat, spring barley, oat and rye grown under field conditions. Moreover, the allelopathic efficiency of these compounds was evaluated by sensitivity of Echinochloa crus-galli. The aromatic acids: trans-cinnamic, salicylic, ferulic, chlorogenic, p-hydroxybenzoic, protocatechuic, p-coumaric and vanillic were separated from crop plants by TLC and determined spectrophotometrically. Differences in concentrations of analysed compounds were observed for most of the examined plant species. The highest concentration was noticed for cinnamic acid and ranged from 360 μg·g⁻¹ DW in rye to 2770 μg·g⁻¹ DW in spring barley. The relatively high concentration was noticed for ferulic acid (from 73.8 μg·g⁻¹ DW in buckwheat to 1046 μg·g⁻¹ DW in spring barley) and p-coumaric acid (from 50 μg·g⁻¹ DW in oat to 1499 μg·g⁻¹ DW in buckwheat). The observed differences in the phenolics content between two successive vegetation seasons can reflect the effect of abiotic and biotic environmental factors on the phenolics level in studied plants. In the greenhouse experiment the effect of particular compounds on the growth of Echinochloa crus-galli was also studied. It has been found that the examined phenolics, and especially trans-cinnamic acid and mixture of phenolic compounds, significantly inhibit the growth of Echinochloa crus-galli. The obtained results may contribute to the explanation of the biological activity of some phenolic compounds.     

Selenium and Mercury in Native and Introduced Fish Species of Patagonian Lakes,Argentina

A survey of mercury (Hg) and selenium (Se) contents was performed in fish collected from lakes located in two National Parks of the northern patagonian Andean range. Two native species, catfish (Diplomystes viedmensis) and creole perch (Percichthys trucha), and three introduced species, brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss), and brook trout (Salvelinus fontinalis), were caught from lakes Nahuel Huapi, Moreno, Traful, Espejo Chico, and Guillelmo belonging to Nahuel Huapi National Park and from lakes Futalaufquen and Rivadavia, Los Alerces National Park. In lake Moreno, fish diet items were analyzed and rainbow trout grown in a farm. Hg and Se were measured in muscle and liver tissues by instrumental neutron activation analysis. The average concentrations in muscle of Hg for all species, ages, and lakes are between 0.4 to 1.0 μg g⁻¹ dry weight (DW) with a few fish, mainly native, exceeding the United States Environmental Protection Agency health advisory for freshwater fish limited consumption, and from 0.8 to 1.5 μg g⁻¹ DW for Se. Average concentrations in liver of Hg in all species range from 0.4 to 0.9 μg g⁻¹ DW. Brown trout, the top predator in these lakes, showed the lowest average Hg burden in both tissues. Se concentrations in the liver of brown and rainbow trout, up to 279 μg g⁻¹ DW, are higher than those expected for nearly pristine lakes, exceeding 20 μg g⁻¹ DW, the threshold concentration associated with Se toxicity. These species show lower Hg contents in muscle, suggesting a possible detoxification of Hg by a Se-rich diet. Creole perch and velvet catfish livers have lower Se concentrations, with a narrower span of values (2.3 to 8.5 μg g⁻¹ and 3.3 to 5.5 μg g⁻¹ DW respectively).     

Cultivation of the edible green seaweed <Emphasis Type="Italic">Gayralia</Emphasis> (Chlorophyta) in southern Brazil

During the last two decades, the monostromatic green seaweed Gayralia sp. has been harvested sporadically by local fishermen on the Paraná coast of southern Brazil and sold to Japanese restaurants. However, the production is erratic and its economic impact very small. This paper provides basic information about a technique to cultivate this seaweed on suspended nets in Paranaguá Bay, southern Brazil, aiming to develop a more reliable and sustainable source of income for impoverished coastal dwellers. Gayralia sp. occurs year round in the region, usually growing on mangrove stems and roots. Polypropylene nets (10 m long × 1 m wide with 16 cm mesh) were placed close to the mangrove fringe. Recruitment occurred year round reaching a peak of 500 recruits m⁻² during early spring. Higher recruitment occurred at periods of low temperature (21–23°C) and high salinity (30–33 psu). Growth rates of Gayralia sp. ranged from 5.75 ± 0.56% to 6.50 ± 0.43% day⁻¹ during the winter and from 1.43 ± 1.65% to 4.65 ± 2.17% day⁻¹, during the summer. Production ranged from 22 ± 6 g m⁻² DW in June to 58 ± 21 g m⁻² DW in September 2004 in 45 days after zooid settlement. The simplicity of the cultivation method, reasonable growth rates and extensive favorable area for cultivation suggest that mariculture of Gayralia sp. may become a good alternative of income for the local inhabitants.     

New insights on toluene biodegradation by Pseudomonas putida F1: influence of pollutant concentration and excreted metabolites

The influence of toluene concentration on the specific growth rate, cellular yield, specific CO₂, and metabolite production by Pseudomonas putida F1 (PpF1) was investigated. Both cellular yield and specific CO₂ production remained constant at 1.0 ± 0.1 g biomass dry weight (DW) g⁻¹ toluene and 1.91 ± 0.31 g CO₂ g⁻¹ biomass, respectively, under the tested range of concentrations (2–250 mg toluene l⁻¹). The specific growth rate increased up to 70 mg toluene l⁻¹. Further increases in toluene concentration inhibited PpF1 growth, although inhibitory concentrations were far from the application range of biological treatment processes. The specific ATP content increased with toluene concentration up to toluene concentrations of 170 mg l⁻¹. 3-Methyl catechol (3-MC) was never detected in the cultivation medium despite being an intermediary in the TOD pathway. This suggested that the transformation from toluene to 3-MC was the limiting step in the biodegradation process. On the other hand, benzyl alcohol (BA) was produced from toluene in a side chain reaction. This is, to the best of our knowledge, the first reported case of methyl monoxygenation of toluene by PpF1 not harboring the pWW0 TOL plasmid. In addition, the influence of 3-MC, BA, and o-cresol on toluene degradation was investigated respirometrically, showing that toluene-associated respiration was not significantly inhibited in the presence of 10–100 mg l⁻¹ of the above-mentioned compounds.     

Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Quantifying the surface characteristics and flocculability of Ralstonia eutropha

The microbial surface and flocculability were qualitatively characterized through the combination of the surface thermodynamic and the extended DLVO approaches, with Ralstonia eutropha, a polyhydroxybutyrate-producing bacterium, as an example. The negativity of the ζ potential of R. eutropha decreased from the initial −19.5 to −11 mV in its cultivation with the consumption of glucose. The total interfacial free energy (ΔG _adh) was changed from −80 to 28.5 mJ m⁻² in its entire growth process. This suggests that the bacterial surface changed from hydrophobic into hydrophilic, resulting in an alteration of its surface characteristics and flocculability in its different growth phases. As a result, the stability ratio of suspensions increased with the increasing cultivation time, indicating that the cell particles became more repulsive with each other and led to a more stable suspension of R. eutropha in its cultivation. The obtained information in this work might be useful for better understanding the surface characteristics and the flocculability and even manipulating its flocculability in the microbial growth process.     

Effect of NaCl on kinetics ofd-glucosamine uptake in yeasts differing in halotolerance

The initial rate ofd-glucosamine uptake by the non-halotolerant yeastSaccharomyces cerevisiae was approximately halved as the apparent half saturation constant (K_m) and the apparent maximum velocity (V_max) changed from 6.6mm to 16.4mm and from 22 μmol · g⁻¹ · min⁻¹ to 16 μmol · g⁻¹ · min⁻¹, respectively, when the salinity in the medium was increased from zerom to 0.68m NaCl. Corresponding changes in a high affinity transport system in the halotolerant yeastDebaryomyces hansenii were from 1.1mm to 4.6mm and from 3.1 μmol · g⁻¹ · min⁻¹ to 4.5 μmol · g⁻¹ · min⁻¹, implying a practically unchanged transport capacity. In 2.7m NaCl, K_m and V_max in this system were 24.5mm and 1.1 μmol · g⁻¹ · min⁻¹, respectively, representing a marked decrease in transport capability. Nevertheless, the degree of affinity in this extreme salinity must still be regarded as noteworthy. In addition to the high affinity transport system inD. hansenii, a low affinity system, presumably without relevance ind-glucosamine transport, was observed.     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

Abscisic acid and gibberellin-like substances in roots and root nodules ofGlycine max

Summary The content of endogenous gibberellin (GA)-like substances of roots and root nodules of SOya, and GA production byRhizobium japonicum cultures, were investigated by a combined thin layer chromatographic (TLC)-dwarf pea epicotyl bioassay technique. GAs were more concentrated in root nodules than in the roots, totalling 1.34 and 0.16 nM GA₃ equivalents g⁻¹ dry wt. respectively. GA production byR. japonicum cultures was demonstrated (1.00 nM GA₃ equivalentsl ⁻¹) and comparison of the GA components of plant and bacterial culture medium extracts, suggested that rhizobial GA production may contribute to the nodule GA content. Cis-trans abscisic acid (ABA) was identified in root and nodule extracts by TLC-gas liquid chromatography (GLC), and amounted to 0.18 and 2.21 nM g⁻¹ dry wt. respectively, whereas 0.30 and 4.63 nM ABA equivalents g⁻¹ dry wt. were detected by a TLC-wheat embryo bioassay technique. ABA was not detected in extracts of bacterial cultures.     

Production of high-density Chlorella culture grown in fermenters

The freshwater microalga Chlorella vulgaris was grown heterotrophically in fed-batch 50–600-L fermenters at 36°C, on aerated and mixed nutrient solution with urea as a nitrogen and glucose as a carbon and energy source. Cell density increased from the initial value 6.25 to 117.18 g DW L⁻¹ in 32 h in the fermenter 50 L at a mean growth rate 3.52 g DW L⁻¹ h⁻¹. The DW increase in the fermenter 200 L was from 7.25 to 94.82 g DW L⁻¹ in 26.5 h at a mean growth rate 3.37 g DW L⁻¹ h⁻¹. Mean specific growth rate μ was about 0.1 h⁻¹ in the both fermenters, if nutrients and oxygen were adequately supplied. The DW increase in the fermenter 600 L was from 0.8 to 81.6 g DW L⁻¹ in 66.5 h at a mean growth rate 1.22 g DW L⁻¹ h⁻¹ and μ = 0.07 h⁻¹. A limitation of the cell growth rate in 600 L fermenter caused by a low dissolved oxygen concentration above cell densities higher than 10 g DW L⁻¹) occurred. Specific growth rate decreased approximately linearly with increasing glucose concentration (25–80 g glucose L⁻¹) at the beginning of cultivation and decreased with the time of cultivation. The cell yield was 0.55–0.69 g DW (g glucose)⁻¹. The content of proteins, β-carotene, and chlorophylls in the cells steadily increased and starch content decreased, by keeping aerated and mixed culture another 12 h in fermenter after the cell growth was stopped due to glucose deficiency.     

Diurnal and seasonal trends in photosynthetic performance of <Emphasis Type="Italic">Dalbergia sissoo</Emphasis> Roxb. and <Emphasis Type="Italic">Hardwickia binata</Emphasis> Roxb. from a semi-arid ecosystem

Diurnal and seasonal trends in net photosynthetic rate (P _N), stomatal conductance (g), transpiration rate (E), vapour pressure deficit, temperature, photosynthetic photon flux density, and water use efficiency (WUE) were compared in a two-year-old Dalbergia sissoo and Hardwickia binata plantation. Mean daily maximum P _N in D. sissoo ranged from 21.40±2.60 μmol m⁻² s⁻¹ in rainy season I to 13.21±2.64 μmol m⁻² s⁻¹ in summer whereas in H. binata it was 20.04±1.20 μmol m⁻² s⁻¹ in summer and 13.64±0.16 μmol m⁻² s⁻¹ in winter. There was a linear relationship between daily maximum P _N and g _s in D. sissoo but there was no strong linear relationship between P _N and g _s in H. binata. In D. sissoo, the reduction in g _s led to a reduction in both P _N and E enabling the maintenance of WUE during dry season thereby managing unfavourable environmental conditions efficiently whereas in H. binata, an increase in g _s causes an increase of P _N and E with a significant moderate WUE.     

The shikonin derivatives and pyrrolizidine alkaloids in hairy root cultures of Lithospermum canescens (Michx.) Lehm.

Hairy root cultures of Lithospermum canescens were established using three strains of Agrobacterium rhizogenes: ATCC 15834, LBA 9402 and NCIB 8196. Eight lines resulting from infection with A. rhizogenes ATCC 15834 demonstrated sufficient biomass increase and were submitted to further investigations. The contents of acetylshikonin (ACS) and isobutyrylshikonin (IBS) in transformed hairy roots made up ca. 10% of those observed in natural roots of L. canescens (24.35 and 14.48 mg g⁻¹ DW, respectively). One line, Lc1-D, produced the largest amounts of ACS (2.72 mg g⁻¹ DW) and IBS (0.307 mg g⁻¹ DW). Traces of pyrrolizidine alkaloids (PA), canescine and canescenine, were found in all lines of transformed hairy roots.     

Effects of macroelements and nitrogen source on biomass accumulation and withanolide-A production from cell suspension cultures of <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal

Withania somnifera is an important medicinal plant that contains withanolides and withaferins, both bioactive compounds. We have tested the effects of macroelements and nitrogen source in W. somnifera cell suspension cultures with the aim of optimizing the production of biomass and withanolide A. The effects of the macroelements NH₄NO_3, KNO_3, CaCl_2, MgSO₄ and KH₂PO₄ at concentrations of 0.0, 0.5, 1.0, 1.5 and 2.0× strength and of the nitrogen source [NH₄ ⁺/NO₃ ⁻ (mM/mM) ratio of: 0.00/18.80, 7.19/18.80, 14.38/18.80, 21.57/18.80, 28.75/18.80, 14.38/0.00, 14.38/9.40, 14.38/18.80, 14.38/28.20, and 14.38/37.60 (mM)] in Murashige and Skoog medium were tested for biomass and withanolide A production. The highest accumulation of biomass [147.81 g l⁻¹ fresh weight (FW) and 14.02 g l⁻¹ (dry weight (DW)] was recorded in the medium containing a 0.5× concentration of NH₄NO₃, and the highest production of withanolide A content was recorded in the medium with 2.0× KNO₃ (4.36 mg g⁻¹ DW). The NH₄ ⁺/NO₃ ⁻ ratio also influenced cell growth and withanolide A production, with both parameters being larger when the NO₃ ⁻ concentration was higher than that of NH₄ ⁺. Maximum biomass growth (110.45 g l⁻¹ FW and 9.29 g l⁻¹ DW) was achieved at an NH₄ ⁺/NO₃ ⁻ ratio of 7.19/18.80, while withanolide A production was greatest (3.96 mg g⁻¹ DW) when the NH₄ ⁺/NO₃ ⁻ ratio was 14.38/37.60 mM.     

Biofiltering efficiency, uptake and assimilation rates of Ulva clathrata (Roth) J. Agardh (Clorophyceae) cultivated in shrimp aquaculture waste water

The growth, biofiltering efficiency and uptake rates of Ulva clathrata were studied in a series of outdoor tanks, receiving waste water directly from a shrimp (Litopenaeus vannamei) aquaculture pond, under constant aeration and two different water regimes: (1) continuous flow, with 1 volume exchange a day (VE day^-1) and (2) static regime, with 1 VE after 4 days. Water temperature, salinity, pH, dissolved inorganic nitrogen (DIN), phosphate (PO₄), chlorophyll-a (chl-a), total suspended solids (TSS), macroalgal biomass (fresh weight) and tissue nutrient assimilation were monitored over 12 days. Ulva clathrata was highly efficient in removing the main inorganic nutrients from effluent water, stripping 70–82% of the total ammonium nitrogen (TAN) and 50% PO₄ within 15 h. Reductions in control tanks were much lower (Tukey HSD, P < 0.05). After 3 days, the mean uptake rates by the seaweed biomass under continuous flow were 3.09 mg DIN g DW day⁻¹ (383 mg DIN m⁻² day⁻¹) and 0.13 mg PO₄ g DW day⁻¹ (99 mg PO₄ m⁻² day⁻¹), being significantly higher than in the static regime (Tukey HSD, P < 0.05). The chl-a decreased in seaweed tanks, suggesting that U. clathrata inhibited phytoplankton growth. Correlations between the cumulative values of DIN removed from the water and total nitrogen assimilated into the seaweed biomass (r = 0.7 and 0.8, P < 0.05), suggest that nutrient removal by U. clathrata dominated over other processes such as phytoplankton and bacterial assimilation, ammonia volatilization and nutrient precipitation.