The Bromotyrosine Derivative Ianthelline Isolated from the Arctic Marine Sponge Stryphnus fortis Inhibits Marine Micro- and Macrobiofouling

The inhibition of marine biofouling by the bromotyrosine derivative ianthelline, isolated from the Arctic marine sponge Stryphnus fortis, is described. All major stages of the fouling process are investigated. The effect of ianthelline on adhesion and growth of marine bacteria and microalgae is tested to investigate its influence on the initial microfouling process comparing with the known marine antifoulant barettin as a reference. Macrofouling is studied via barnacle (Balanus improvisus) settlement assays and blue mussel (Mytilus edulis) phenoloxidase inhibition. Ianthelline is shown to inhibit both marine micro- and macrofoulers with a pronounced effect on marine bacteria (minimum inhibitory concentration (MIC) values 0.1–10 μg/mL) and barnacle larval settlement (IC₅₀?=?3.0 μg/mL). Moderate effects are recorded on M. edulis (IC₅₀?=?45.2 μg/mL) and microalgae, where growth is more affected than surface adhesion. The effect of ianthelline is also investigated against human pathogenic bacteria. Ianthelline displayed low micromolar MIC values against several bacterial strains, both Gram positive and Gram negative, down to 2.5 μg/mL. In summary, the effect of ianthelline on 20 different representative marine antifouling organisms and seven human pathogenic bacterial strains is presented.     

Large‐scale production of pharmaceuticals by marine sponges: Sea,cell, or synthesis?

Marine sponges are known to produce an overwhelming array of secondary metabolites with pharmaceutical potential. The technical and economical potential of using marine sponges for large-scale production of these compounds was assessed for two cases: the anticancer molecule halichondrin B from a Lissodendoryx sp., and avarol from Dysidea avara for its antipsoriasis activity. An economic and technical analysis was done for three potential production methods: mariculture, ex situ culture (in tanks), and cell culture. We concluded that avarol produced by mariculture or ex situ culture could become a viable alternative to currently used pharmaceuticals for the treatment of psoriasis. Production of halichondrin B from sponge biomass was found to not be a feasible process, mainly due to the extremely low concentration of the compound in the sponge. Technical feasibility was also analyzed for five alternatives: chemical synthesis, wild harvest, primmorph culture, genetic modification and semi-synthesis. It was concluded that the latter two approaches could prove to be valuable methods for the production of pharmaceuticals, based on chemical structures of secondary metabolites present in trace amounts in marine sponges.     

In situ pH management for microbial culture in shake flasks and its application to increase plasmid yield

Shake flasks are widely used to culture microorganisms, but they do not allow for pH control without additional infrastructure. In the presence of a carbon source like glucose, culture pH typically decreases due to overflow metabolism and can limit the growth of microorganisms in shake flasks. In this study, we demonstrate the use of magnesium hydroxide-loaded pH managing hydrogels (m-pHmH) for in situ base release to counter the decrease in culture pH in shake flasks using Escherichia coli as a model organism, in both complex and mineral salts medium. Base release from m-pHmH is shown to increase with decreasing pH (22-fold increase in release rate from pH 8 to 5), thus providing feedback from culture pH. The addition of m-pHmH resulted in better pH maintenance and higher biomass yields of E. coli K12 in media containing glucose as a carbon source. The use of m-pHmH with additional buffer resulted in pH being maintained above 6.9 while pH decreases below 5 without m-pHmH. We demonstrate one application of such in situ pH management to increase the volumetric plasmid yield from E. coli in shake flask culture. In situ glucose release through a hydrogel to mimic fed-batch culture along with the addition of m-pHmH resulted in a 395 % increase in volumetric plasmid yield to 38 μg/ml in shake flask culture.     

Quantitative assessment of marine sponge cells in vitro: Development of improved growth medium

Summary As sources of natural products with potential human therapeutic value, marine sponges are important subjects for cell culture studies. A critical component of any cell culture system is its growth medium. Proceeding from the hypotheses that the thawed, cryopreserved, primary cells would display detectable differential responses and that those responses could be comparatively quantified, this study has established that multiwell screening assays are useful tools for improving medium formulations in cell cultures of the marine sponge, Teichaxinella morchella. Fluorescent probe signals were correlated with known cell densities and viabilities in a 96-well format. Analysis of variance and post-test methods were applied to judge the significance of signal differences seen in a variety of medium formulations. Results from a series of experiments suggested that reducing glutamine and selenium concentrations in the standard medium would result in greater DNA, protein, and esterase activity signals. This was confirmed by the direct comparison of the standard and improved medium formulations. Significantly higher protein content and esterase activity were associated with the improved medium. DNA content was also higher, though not significantly. The result is a new medium formulation that may be more able to support cell growth and division, providing an improved cell culture system for marine sponge cell studies. The assays can be used in additional studies to further improve the in vitro conditions for marine sponge cell culture.     

Effect of sulfide on growth of marine bacteria

Severe hypoxia leads to excess production of hydrogen sulfide in marine environments. In this study, we examined the effect of sulfide on growth of four facultative anaerobic marine bacteria in minimal media under anaerobic conditions. The Gram-negative chemolithoautotrophic Marinobacter sp. tolerated sulfide concentrations up to 0.60 mM, with doubling and lag times increasing as a function of increasing sulfide concentration but with no change in maximum culture yields; growth did not occur at 1.2 mM sulfide. Similar results were obtained for the metabolically diverse Gram-negative denitrifying Pseudomonas stutzeri, except that growth occurred at 1.2 mM and culture yields at 0.60 and 1.2 mM sulfide were approximately 10-fold lower than at sulfide concentrations between 0 and 0.30 mM. Increases in doubling and lag times accompanied by an overall 10-fold decrease in maximum culture yields were found for the Gram-negative chemoheterotrophic Vibrio sp. at all sulfide concentrations tested. In contrast, growth of a Gram-positive chemoheterotrophic Bacillus sp. was resistant to all sulfide concentrations tested (0.15–1.2 mM). Our results highlight the variable responses of marine bacteria to sulfide and provide some insight into shifts that may occur in microbial community structure and diversity as a consequence of changes in sulfide levels that are the result of hypoxia.     

Mineral composition variation in the shells of freshwater mussel Anodonta woodiana at different growth stages

The levels of mineral element Na, Mg, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Mo, Al, As, Ag, Cd, and Tl were quantified in the whole shells of the freshwater bivalve Anodonta woodiana at three different growth stages (i.e. J1 juveniles of 1 month old, J2 juveniles of 3.5 months old, and adults of 36 months old). The concentrations of Na and Al were different between different growth stages (p < 0.05). The highest Na concentrations (2715 ± 86 μg/g dry weight) were found in J2 juveniles. The highest Al concentrations (303.9 ± 5.95 μg/g dry weight) were found in J1 juveniles. Manganese concentrations (517.0 ± 47.98 μg/g dry weight) were significantly higher in J2 juveniles than in J1 juveniles (432.3 ± 9.87 μg/g dry weight) (p < 0.05). Copper concentrations (27.32 ± 0.15 μg/g dry weight) were significantly higher in J1 juveniles than in J2 juveniles (26.21 ± 0.86 μg/g dry weight) and adults (24.74 ± 1.43 μg/g dry weight) (p < 0.05). Burdens of Na, Ca, Mn, Fe, Co, Cu, Mo, Ag, and Tl were positively correlated with the shell length (p < 0.05). These findings can possibly contribute to an understanding of elemental requirements for shell growth and, hence, facilitate improvement of survival and growth rates during artificial mussel culture.     

Regeneration of plants through somatic embryogenesis in Thymus hyemalis Lange,a potential medicinal and aromatic plant

A protocol for somatic embryogenesis was developed for Thymus hyemalis, a wild species in the Mediterranean region. First, the effects of explant type, plant growth regulators [kinetin (KIN) and 2,4-dichlorophenoxyacetic acid (2,4-D)], and genotype on callus induction were tested. For callus induction, the node was the best explant; Murashige and Skoog (MS) medium supplemented with 1.8 μM 2,4-D and 0.5 μM KIN was the best medium, and the genotype had a highly significant effect. To induce production of somatic embryos, the effects of KIN, 6-benzylaminopurine (BAP), and naphthalene acetic acid (NAA) were evaluated. After 5 wk of culture in the dark, MS medium supplemented with 4.44 μM BAP, 0.54 μM NAA, and 4.65 μM KIN gave the highest percentage (85%) of embryogenic callus and the highest number of somatic embryos (27.00) per 45 mg of callus. For germination and plant recovery, somatic embryos were transferred to MS medium without plant growth regulators and plantlet conversion from developed somatic embryos was 90%. In vitro plants with adequate growth and sufficient root systems were subsequently transplanted into a mixture of peat and vermiculite (2:1?v/v) under greenhouse conditions. The survival rate of the plantlets under ex vitro conditions was 80%.     

Sponge bioerosion accelerated by ocean acidification across species and latitudes?

In many marine biogeographic realms, bioeroding sponges dominate the internal bioerosion of calcareous substrates such as mollusc beds and coral reef framework. They biochemically dissolve part of the carbonate and liberate so-called sponge chips, a process that is expected to be facilitated and accelerated in a more acidic environment inherent to the present global change. The bioerosion capacity of the demosponge Cliona celata Grant, 1826 in subfossil oyster shells was assessed via alkalinity anomaly technique based on 4 days of experimental exposure to three different levels of carbon dioxide partial pressure (pCO₂) at ambient temperature in the cold-temperate waters of Helgoland Island, North Sea. The rate of chemical bioerosion at present-day pCO₂ was quantified with 0.08–0.1 kg m^?2 year^?1. Chemical bioerosion was positively correlated with increasing pCO₂, with rates more than doubling at carbon dioxide levels predicted for the end of the twenty-first century, clearly confirming that C. celata bioerosion can be expected to be enhanced with progressing ocean acidification (OA). Together with previously published experimental evidence, the present results suggest that OA accelerates sponge bioerosion (1) across latitudes and biogeographic areas, (2) independent of sponge growth form, and (3) for species with or without photosymbionts alike. A general increase in sponge bioerosion with advancing OA can be expected to have a significant impact on global carbonate (re)cycling and may result in widespread negative effects, e.g. on the stability of wild and farmed shellfish populations, as well as calcareous framework builders in tropical and cold-water coral reef ecosystems.     

Algicidal activity of the thiazolidinedione derivative TD49 against the harmful dinoflagellate Heterocapsa circularisquama in a mesocosm enclosure

To assess the algicidal effects of the thiazolidinedione derivative TD49 on the unarmored dinoflagellate Heterocapsa circularisquama and to evaluate the response of the planktonic community and the environment to this chemical, we undertook mesocosm (1,300 L) and small-scale experiments. The reduction ratio for H. circularisquama in each experiment was dependent on the concentration of TD49. At a TD49 concentration >0.4 μM, the abundance of H. circularisquama decreased by 99 % in the small-scale experiment and by 84 % in the mesocosm during the initial 2 days. At 0.2 μM TD49, the abundance of H. circularisquama decreased by up to 85 % in the small-scale experiment, whereas the abundance in the mesocosm increased, implying the absence of an algicidal effect. The decrease in planktonic organisms, including H. circularisquama, following TD49 treatment was correlated with abrupt declines in culture pH and dissolved oxygen concentration. Following addition of TD49, there was a significant increase in the abundance of diatoms and cryptophyta species, and after 8 days, the dominant species in the TD49 treatments shifted to small pennate diatoms including Cylindrotheca and Entomoneis species. The growth of some species among the zooplankton community was promoted at low TD49 concentrations (≤0.4 μM), whereas high concentrations (≥1.0 μM) had a negative effect. This study demonstrates that TD49 is an effective agent for the control for H. circularisquama blooms and that large-scale mesocosms play a crucial role in assessing the application of algicides such as TD49 in natural environments.     

Phosphorus uptake kinetics,size of root system and growth of maize and groundnut in solution culture

Phosphorus acquisition efficiency of maize (Zea mays L.) and groundnut (Arachis hypogaea L.) was investigated in a flowing nutrient solution culture at constant P concentrations of 0.2, 1 and 100 μM. To calculate the P influx and study changes in plant growth and P uptake in relation to plant age, four harvests were taken. Phosphorus uptake kinetics of the roots, i.e. maximum influx, I_\max, the Michaelis constant, K_m, and the minimum concentration, C_Lmin (the concentration at which no net uptake occurs) were estimated in a series of short-term experiments, based on the rate of depletion of P from solution over a range of concentrations. At 1 μM P, maize was more P efficient producing up to 90% of its maximum yield as compared to groundnut with only 20% of maximum yield. A 3 times faster P uptake rate was the reason for the maize P efficiency. In contrast for groundnut at 1 μM P, a net efflux was observed at some development stages of this crop indicating a much higher P requirement at the root surface for maximum growth. Maize had a 6 times higher I_\max value and a 2 times higher K_m value as compared to groundnut. The higher influx of maize was mainly because of the higher I_\max. Maize previously grown at low P concentrations had a C_Lmin of 0.1 μM, while groundnut had values of 0.2 and 0.6 μM. Furthermore groundnut previously grown at 100 μM, was not able to absorb P even at 40 μM. Acclimation to low P concentrations in solution by increasing I_\max or decreasing K_m was not evident in this study. Differences in P acquisition efficiency between maize and groundnut in solution culture were mainly because of differences in P-uptake kinetics, and to a lesser extent to the size of the root system.     

Isolation and characterization of bacteria from mercury contaminated sites in Rio Grande do Sul, Brazil, and assessment of methylmercury removal capability of a Pseudomonas putida V1 strain

Methylmercury (MeHg) is one of the most dangerous heavy metal for living organisms that may be found in environment. Given the crescent industrialization of Brazil and considering that mercury is a residue of several industrial processes, there is an increasing need to encounter and develop remediation approaches of mercury contaminated sites. The aim of this study was to isolate and characterize methylmercury resistant bacteria from soils and sludge sewage from Rio Grande do Sul, Brazil. Sixteen bacteria were isolated from these contaminated sites and some isolates were highly resistant to methylmercury (>8.7 μM). All the isolates were identified by 16S rDNA. Pseudomonas putida V1 was able to volatilize approximately 90 % of methylmercury added to growth media and to resist to copper, lead, nickel, chromate, zinc, cobalt, manganese and barium. In the presence of high concentrations of methylmercury (12 μM), cell growth was limited, but P. putida V1 was still able to remove up to 29 % of this compound from culture medium. This bacterium removed an average of 77 % of methylmercury from culture medium with pH in the range 4.0–6.0. In addition, methylmercury was efficiently removed (>80 %) in temperature of 21–25 °C. Polymerase chain reactions indicated the presence of merA but not merB in P. putida V1. The growth and ability of P. putida V1 to remove methylmercury in a wide range of pH (4.0 and 8.0) and temperature (10–35 °C), its tolerance to other heavy metals and ability to grow in the presence of up to 11.5 μM of methylmercury, suggest this strain as a new potential resource for degrading methylmercury contaminated sites.     

Effect of All-Trans Retinoic Acid on the Differentiation of U87 Glioma Stem/Progenitor Cells

GSPCs (glioma stem/progenitor cells) were isolated from U87 glioma cell lines by serum-free neural stem cell medium. Four concentrations (1, 2, 4, and 8 μmol/L) of ATRA (all-trans retinoic acid) were used to induce the differentiation of GSPCs in the medium with or without growth factors. The effect of ATRA on the differentiation of GSPCs was analyzed by flow cytometry, real-time-PCR, and immunofluorescence. The differentiation of GSPCs could be induced by 1 or 2 μmol/L ATRA when GSPCs were cultured in growth factor-free medium. The detection of real-time-PCR showed that the level of GFAP (glial fibrillary acidic protein) mRNA of differentiated GSPCs in the growth factor-free medium containing 1 μmol/L ATRA group was significantly higher than that in the control group, and there was no significant difference in the level of TUBB-3 mRNA between the two groups. The GSPCs suffered apoptosis in the growth factor-free medium containing 4 or 8 μmol/L ATRA. The differentiation of GSPCs could not be induced by ATRA when GSPCs were cultured in the medium containing growth factors. The percentage of cells in G0/G1 phase was 84.26 ± 2.24 %, and the percentage of apoptosis was 18.95 ± 2.53 % in experimental groups which was similar to those in the control group. In conclusion, ATRA has certain capacity to induce differentiation of GSPCs, while its effective concentration should be controlled strictly. The differentiation of GSPCs induced by ATRA cannot antagonize the formidable differential inhibition of epidermal growth factor and basic fibroblast growth factor.     

The Göttingen minipig for assessment of retinoid efficacy in the skin: comparison of results from topically treated animals with results from organ-cultured skin

Göttingen minipigs were treated topically for 6 d with a novel retinoid (MDI 301) at concentrations ranging from 0.3% to 30% in cream vehicle. Treatment of the minipigs did not adversely affect their health (hematological and necropsy parameters) or produce changes in the skin suggestive of retinoid-induced skin irritation. After killing the animals, skin samples from each treatment site were excised and maintained in organ culture for 6 d. In addition, untreated skin was also maintained in organ culture and treated with MDI 301 (0.1–5 μg/ml). After 3 d, the culture supernatants were collected and analyzed for levels of collagen type I and for matrix metalloproteinases (MMPs). Both skin samples treated in vivo and skin samples exposed to MDI 301 in culture demonstrated increased collagen production. Only slight changes in levels of MMP-2 (gelatinase A) or MMP-9 (gelatinase B) were seen. After 6 d, the organ-cultured skin was fixed in formalin and prepared for histology. The organ-cultured skin was compared to skin that was fixed at killing after in vivo treatment. Epidermal hyperplasia was quantified at various MDI 301 concentrations. In vivo and in vitro treatments showed similar results—although the thickness was not substantially changed on average, there were focal areas of hyperplasia at higher retinoid concentrations. Taken together, these data suggest that MDI 301 enhances collagen production in minipig skin, without irritation. Furthermore, these studies suggest that minipig skin exposed to the retinoid in organ culture is equally predictive as topically treated skin. The in vitro organ culture approach may provide a cost-effective alternative model to that of the intact animal for skin retinoid testing.     

The positive effect of arabinogalactan on induction of somatic embryogenesis in Quercus bicolor followed by embryo maturation and plant regeneration

This is the first report on the successful induction of somatic embryogenesis in swamp white oak from leaf and shoot apex explants excised from in vitro shoot cultures derived from 6- to 7-year-old trees. We demonstrated that arabinogalactan from larch wood (2–4 mg/L) promoted embryogenesis in the three genotypes evaluated by increasing the frequency of somatic embryogenesis, the embryogenic sites per explant, and by speeding the onset of embryo initiation. The explants were cultured sequentially on three culture media consisting of Murashige and Skoog (MS) salts and vitamins supplemented with 500 mg/L casein hydrolysate and different concentrations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA). Somatic embryogenesis induction frequencies of up to 12.4, 4.5, and 0.7 % were obtained for the three genotypes. Clonal embryogenic lines were maintained by repetitive embryogenesis following culture on MS medium containing 0.44 μM BA with or without 0.27 μM NAA. Before germination, cotyledonary-stage embryos were cultured for 4 weeks in maturation medium (MS medium with half-strength macronutrients) containing 6 % sorbitol. Germination response was significantly improved by applying a 2-month cold storage as a post-maturation treatment. The mineral formulation and plant growth regulator content of the germination medium influenced the frequency of plantlet conversion with the best results achieved on Gresshoff and Doy medium with BA (0.25–0.44 μM). This procedure resulted in over 50–60 % of germinating embryos exhibiting continuous root growth and either epicotyl elongation or shoot development.     

Recovery of cowpea seedling roots from exposure to toxic concentrations of trace metals

Rhizotoxic effects of many trace metals are known, but there is little information on recovery after exposure. Roots of 3-d-old cowpea (Vigna unguiculata (L.) Walp. cv. Caloona) seedlings were grown for 4 or 12 h in solutions of 960 μM Ca and 5 μM B at two concentrations (which reduce growth by 50 or 85%) of nine trace metals that rupture the outer layers of roots. Measured concentrations were 34 or 160 μM Al, 0.6 or 1.6 μM Cu, 2.2 or 8.5 μM ?Ga, 2.3 or 12 μM Gd, 0.8 or 1.9 μM Hg, 1.0 or 26 μM In, 2.4 or 7.3 μM La, 1.8 or 3.8 μM Ru, and 1.3 or 8.6 μM Sc. Roots were rinsed, transferred to solutions free of trace metals, and regrowth monitored for up to 48 h. Recovery from exposure to Hg occurred within 4 h, but regrowth was delayed for ≥?12 h with Al, Ga, or Ru. There was poor regrowth after 4 or 12 h exposure to Cu, Gd, In, La, or Sc. Roots recovered after being grown for 12 to 48 h in 170 μM Al, 5.1 μM? Ga, 2.0 μM Hg, or 1.4 μM Ru, but the extent of recovery was reduced with longer exposure time. Microscopy showed marked differences in symptoms on roots recovering from exposure to the various trace metals. Differences in (i) concentrations that are toxic, (ii) ability of roots to recover, (iii) time for recovery to occur, and (iv) symptoms that develop, suggest that each trace metal has a unique combination of rhizotoxic effects.     

Culture medium optimization for camptothecin production in cell suspension cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley

The effect of culture medium nutrients on growth and alkaloid production by plant cell cultures of Nothapodytes nimmoniana (J. Grah.) Mabberley (Icacinaceae) was studied with a view to increasing the production of the alkaloid camptothecin, a key therapeutic drug used for its anticancer properties. Amongst the various sugars tested with Murashige and Skoog (MS) medium, such as glucose, fructose, maltose, and sucrose, maximum accumulation of camptothecin was observed with sucrose. High nitrate in the media supports the biomass, while high ammonium enhances the camptothecin content. Selective feeding of 60 mM total nitrogen with a NH₄ ⁺/NO₃ ^? balance of 5/1 on day 15 of the culture cycle results in a 2.4-fold enhancement in the camptothecin content over the control culture (28.5 μg/g DW). Furthermore, the sucrose feeding strategy greatly stimulated cell biomass and camptothecin production. A modified MS medium was developed in the present study, which contained 0.5 mM phosphate, a nitrogen source feeding ratio of 50/10 mM NH₄ ⁺/NO₃ ^? and 3 % sucrose with additional 2 % sucrose feeding (added on day 12 of the cell culture cycle) with 10.74 μM naphthaleneacetic acid and 0.93 μM kinetin. Finally, the selective medium has 1.7- and 2.3-fold higher intracellular and extracellular camptothecin content over the control culture (29.2 and 8.2 μg/g DW), respectively.     

Enhanced plasmid production in miniaturized high-cell-density cultures of Escherichia coli supported with perfluorinated oxygen carrier

A simple method for plasmid minipreps in closed 1.5 mL microcentrifuge tubes using a cultivation medium with internal substrate delivery (EnBase^®) in combination with a two-phase perfluorodecalin (PFD) system supplying additional oxygen to the E. coli culture is described. The procedure can simply be performed on a thermoshaker using only 50 μL cultivation volume. Twenty and twenty-five percent higher cell densities and plasmid concentration, respectively, were obtained with the additional oxygen delivery system when compared to cultures without PFD. Compared to standard 2 mL LB cultures ninefold higher cell densities and eightfold higher plasmid concentrations were achieved for the smaller culture volume. The μL-scale cultures can be directly utilized in further plasmid purification without any centrifugation step or the subsequent removal of the supernatant. This simplifies the routine procedure considerably. Furthermore, the new method is very robust considering the time of cultivation. Highest plasmid concentrations were already obtained after only 6 h of cultivation, but the plasmid concentration remained high (87 % of the maximum) even until 8 h of cultivation. Aside from the advantage of this method for the daily routine, we believe that it could also be applied to automated high-throughput processes.     

Development of a Polygonum minus cell suspension culture system and analysis of secondary metabolites enhanced by elicitation

Polygonum minus has been reported to contain valuable metabolites and to date, there is no report on using cell culture technique for metabolite production in P. minus. Naphthalene acetic acid (NAA) concentrations in the range of 2–6 mg L^?1 were used in a matrix of combinations with dichlorophenoxyacetic acid (2,4-D) concentrations in the range of 2–10 mg L^?1 as plant growth regulators (PGRs) to induce callus cultures. Media that were supplemented with 2 mg L^?1 2,4-D + 4 mg L^?1 NAA, 2 mg L^?1 2,4-D + 6 mg L^?1 NAA and 6 mg L^?1 2,4-D + 8 mg L^?1 NAA were effective for callus induction (93.3 % of the explants produced callus). To establish cell culture, the best growth was obtained from medium that was supplemented with 1 mg L^?1 2,4-D + 2 mg L^?1 NAA. From a 1-g inoculum size, the fresh weight increases exponentially after 5–10 days of culture, and a 26.71 g maximum fresh weight was obtained after 25 days of culture. The cell culture medium was then analyzed using gas chromatography–mass spectrometry (GC–MS). Jasmonic acid (100, 50, 25 and 5 μM), salicylic acid (100, 50, 25 and 5 μM), yeast extract (500, 250 and 100 mg L^?1) and glass beads were used in this research as elicitors. The cell cultures were then incubated with the different elicitors for 1, 2, 3 and 4 days. Several compounds with high peak area percentages were detected, including 2-furancarboxaldehyde, 5-hydroxymethyl, furfural, and 2-cyclopenten-1-one, 2-hydroxy. These results show the diversity of metabolites released by P. minus cell into the culture medium under control conditions.     

Optimization, purification and characterization of novel thermostable, haloalkaline, solvent stable protease from Bacillus halodurans CAS6 using marine shellfish wastes: a potential additive for detergent and antioxidant synthesis

A protease producing marine bacterium, Bacillus halodurans CAS6 isolated from marine sediments, was found to produce higher enzyme by utilizing shrimp shell powder. Optimum culture conditions for protease production were 50 °C, pH 9.0, 30 % NaCl and 1 % shrimp shell powder (SSP) and the protease purified with a specific activity of 509.84 U/mg. The enzyme retained 100 % of its original activity even at 70 °C, pH 10.0 and 30 % NaCl for 1 h. The purified protease exhibited higher stability when treated with ionic, non-ionic (72–94 %) and commercial detergents (76–88 %), and organic solvents (88–126 %). Significant blood stain removal activity was found with the enzyme in washing experiments. The culture supernatant supplemented with 1 % SSP showed 93.67 ± 2.52 % scavenging activity and FT-IR analysis of the reaction mixture confirmed the presence of antioxidants such as cyclohexane and cyclic depsipeptide with aliphatic amino groups. These remarkable qualities found with this enzyme produced by Bacillus halodurans CAS6 could make this as an ideal candidate to develop the industrial process for bioconversion of marine wastes and antioxidant synthesis.     

<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> cell elicitor enhances betalain content in the cell suspension culture of <Emphasis Type="Italic">Celosia cristata</Emphasis>

We started a cell suspension culture from magenta coloured calli of cockscomb to study the effect of biotic and abiotic elicitors on the biosynthesis of betalain pigments. The cultures were grown in a flask containing 30 ml MS media fortified with 13.5 μM 2,4-D and 0.44 μM BAP. These cultures were elicited during its log-phase of growth using fungal elicitors (prepared from mycelia of Fusarium oxysporum), yeast extract, copper sulphate and cobalt chloride. The elicitation reduced the cell count, cell viability and percent pigmented cell in the suspension culture. Similarly, it also resulted in reduced betalain content by all the elicitors except 0.125 × 10^?3% fungal elicitor. Rather, fungal elicitor at this concentration significantly enhanced the amaranthin, betanin, betalamic acid and betaxanthin content in the culture. Besides this, copper sulphate doubled the pigment contribution (ratio of particular pigment content to total pigment content) of betaxanthin at all the concentrations. Therefore, we conclude that fungal elicitor can further be investigated to enhance the content of betalain pigments in suspension culture at a larger scale.