Growth Phase Of Culture Research Articles

Construction and Characterization of Nisin-Controlled Expression Vectors for Use inLactobacillus reuteri

The Nisin-controlled gene expression (NICE) system, which was discovered in Lactococcus lactis, was adapted to Lactobacillus reuteri by ligating nisA promoter (PnisA) and nisRK DNA fragments into the Escherichia coli-Lb. reuteri shuttle vector pSTE32. This chimerical plasmid (pNICE) was capable of expressing the heterologous amylase gene (amyL) under nisin induction. Optimization of induction factors for this Lb. reuteri/pNICE system, including nisin concentration (viz. 50 ng/ml), growth phase of culture at which nisin be added (viz. at the early exponential phase), and the best time for analyzing the gene product after inoculation (viz. at the 3rd h), allowed the amylase product to be expressed in high amounts, constituting up to about 18% of the total intracellular protein. Furthermore, the signal peptide (SP) of amyL gene (SPamyL) from Bacillus licheniformis was ligated to the downstream of PnisA in pNICE, upgrading this vector to a NICE-secretion (NIES) level, which was then designated pNIES (Sec+, secretion positive). Characterization of pNIES using an amyL-SPDelta gene (amyL gene lacking its SP) as a reporter revealed the 3rd h after induction as the secretion peak of this system, at which the secretion efficiency and the amount of alpha-amylase being secreted into the culture supernatant were estimated to reach 77.6% and 27.75 mg/l. Expression and secretion of AmyL products by pNIES in Lb. reuteri was also confirmed by SDS-PAGE and immunoblotting analysis.

Adsorption to goethite of extracellular polymeric substances from Bacillus subtilis

Extracellular polymeric substances (EPS) are heterogeneous biopolymers produced by Gram-negative and Gram-positive bacterial cells. Adsorption of EPS to minerals can alter the substrata physico-chemistry and influence initial bacterial adhesion processes via conditioning film formation, but the effects of solution chemistry on uptake of EPS remain poorly understood. In this study, the adsorption to goethite (α-FeOOH) of EPS isolated from the early stationary growth-phase culture of Bacillus subtilis was investigated as a function of pH and ionic strength ( I) in NaCl background electrolyte using batch studies coupled with Fourier transform infrared spectroscopy and size-exclusion high-performance liquid chromatography. Proteins, particularly those of higher molar mass, and phosphorylated macromolecules were adsorbed preferentially. Increasing solution I (1–100 mM NaCl) or pH (3.0–9.0) resulted in a decrease in the mass of EPS adsorbed. Batch studies and diffuse reflectance infrared Fourier transform spectra are consistent with ligand exchange of EPS phosphate groups for surface hydroxyls at Fe metal centers. The data indicate that both electrostatic and chemical bonding interactions contribute to selective fractionation of the EPS solution. Proteins and phosphate groups in phosphodiester bridges of nucleic acids likely play an important role in conditioning film formation at Fe oxide surfaces.

The biological and biogeochemical consequences of phosphate scavenging onto phytoplankton cell surfaces

Phytoplankton carbon (C) and nitrogen (N) content is commonly normalized to phosphorus (P) quotas as Redfield C:N: P ratios. We examined how surface-bound P pools affect Redfield stoichiometry and P uptake kinetics in cultures and natural blooms of diatoms, raphidophytes, dinoflagellates, pelagophytes, prasinophytes, and cyanobacteria. The amount of surface-adsorbed P on exponential growth phase cultures ranged from 14% to 57% of the total cellular P. The C: total P and N: total P ratios (surface-adsorbed P + intracellular P) for all species were near Redfield values, with lower values for the diatoms and higher ratios for the dinoflagellates and cyanobacteria. However, when corrected for the P adsorbed to exterior surfaces, intracellular or “biological” C: P and N: P ratios are 1.2–2 times higher. As with the cultured isolates, the amount of surface-adsorbed P for the natural bloom samples ranged from 15% to 46%. Carbon : total P ratios ranged from 71 to 151, whereas C: intracellular P ratios were 1.3–1.7 times higher. N: P ratios for the natural samples showed similar trends. Phosphate uptake rates into the intracellular pool were as little as half those measured for uptake into total cellular P pools for both the natural blooms and cultures. Cells can, however, access some of the surface-bound P over time to support growth, and the partitioning of P between adsorbed and interior pools is affected by growth stage, cellular P demand, and external phosphate concentrations. We conclude that, as with iron (Fe) and other particle-reactive elements, bulk particulate C:N: P ratios do not necessarily reflect the true biological P quotas of eukaryotic and prokaryotic algae and that P scavenging onto cell surfaces is likely to exert a large influence on the biogeochemical cycling of this nutrient.

Accumulation of phycocyanin in heterotrophic and mixotrophic cultures of the acidophilic red alga Galdieria sulphuraria

The relationship between light intensity, nitrogen availability and pigmentation was investigated in mixotrophic and heterotrophic cultures of the unicellular red alga Galdieria sulphuraria 074G, a potential host for production of the blue pigment, phycocyanin (PC). During the exponential growth phase of batch cultures, G. sulphuraria 074G contained 2–4 mg phycocyanin per g dry weight. In carbon-limited and nitrogen-sufficient batch cultures grown in darkness, this value increased to 8–12 mg g −1 dry weight during the stationary phase, whereas the phycocyanin content in nitrogen-deficient cells decreased to values below 1 mg g −1 dry weight during stationary phase. Light intensities between 0 and 100 μmol photons m −2 s −1 had no influence on phycocyanin accumulation in mixotrophic cultures grown on glucose or fructose, while light stimulated phycocyanin synthesis in cultures grown on glycerol, in which the phycocyanin content in stationary phase was increased from 10 mg g −1 dry weight in darkness to 20 mg g −1 dry weight at a light intensity of 80 μmol photons m −2 s −1. At higher light intensities, less phycocyanin accumulated than at lower intensities, irrespective of the carbon substrate used. In carbon-limited continuous flow cultures grown on glucose or glycerol at a dilution rate of 0.63 day −1, corresponding to 50% of the maximum specific growth rate, the highest steady-state phycocyanin content of 15–28 mg g −1 dry weight was found at 65 μmol photons m −2 s −1. In contrast to the apparent glucose repression of light-induced PC synthesis observed in batch cultures, no glucose repression of the light stimulation was observed in continuous flow cultures because the glucose concentration in the culture supernatant always remained at limiting levels. Despite the fact that G. sulphuraria 074G contains less phycocyanin than some other microalgae and cyanobacteria, the ability of G. sulphuraria 074G to grow and synthesize phycocyanin in heterotrophic or mixotrophic cultures makes it an interesting alternative to the cyanobacterium, Spirulina platensis presently used for synthesis of phycocyanin.

Generation and analysis of expressed sequence tags from poplar ( Populus alba × P. tremula var. glandulosa) suspension cells

We have analyzed 8962 expressed sequence tags (ESTs) obtained from poplar cell suspension culture to gain an insight into the changes in gene expression during their growth phase in suspension culture. Single pass sequencing from 5′-end of 8962 randomly selected cDNA clones resulted in 7586 high quality ESTs with average reading length of 517 bp. A total of 3378 unigenes, consisting of 1337 contigs and 2041 singletons, were formed after assembly using CAP3 program. Among them, 2091 unigenes (61.9%) were found to significantly match (BLASTX score more than 80) the proteins with known function and 949 (28.1%) matched significantly with those having unknown function in the public databases. The remaining 338 unigenes (10.0%) failed to show significant homology to any proteins in the public databases, suggesting that they represent novel sequences. A significant portion of highly expressed genes in poplar cells encodes cell wall-related proteins, such as extensin, polygalacturonase inhibiting protein and proline-rich protein. The potentiality of the ESTs resource in studying of the regulation of cell growth was proved by growth phase-dependent expression of several selected genes.

Production of the Long-Chain Alcohols Octanol, Decanol, and Dodecanol by Escherichia coli

As a follow-up to earlier studies on the emission of long-chain alcohols from broth cultures of Gram-negative enteric bacteria, E. coli was examined for the production of 1-octanol, 1-decanol, and 1-dodecanol. Ten strains of E. coli cultured in tryptic soy broth were assayed for volatile metabolites using solid-phase microextraction. Long-chain alcohols were produced by all strains with 1-decanol predominating with production ranging from 23.6 ng mL(-1) to 148 ng mL(-1). The production of long-chain alcohols followed the onset of the exponential growth phase of the broth culture. Doubling the concentration of glucose (5 g L(-1)) in the broth had no effect on the concentration of long-chain alcohols produced. Addition of octanoic, decanoic, or dodecanoic acids (as K(+) salts) to the broth (100 mg L(-1)) markedly increased the production of the corresponding alcohols by E. coli, ranging from a 13-fold increase for decanol to a 51-fold increase for dodecanol. However, decanol remained the predominant alcohol detected in all assays. These neutral volatile alcohols may have application as vapor-phase indicators for certain classes of bacteria, particularly, Gram-negative enteric bacteria.

Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues

We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors. We have assayed the k(L)a of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients consumption, and metabolites production. The medium surface oxygen transfer was shown to be higher in shaker bottles than in spinner ones, which was in direct correlation to the higher cell density obtained. Best quantitative performances of PIBs production were obtained with a SF900II medium volume/shaker-bottle volume ratio of 15% and MOI of 0.5 to 1 performed at a cell concentration at infection (CCI) of 1 to 2.5x10(6) cells/ml in a medium containing enough glucose and glutamine. Upon infection, a decrease in the cell multiplication was observed to be dependent on the MOI used, and the muX at the exponential growth phase in infected and non-infected cultures were, respectively, of 0.2832 and 0.3914 (day(-1)). The glucose consumption and lactate production were higher in the infected cultures (muGlucose and muLactate of, respectively, 0.0248 and 0.0089x10(-8) g/cellxday in infected cultures and 0.0151 and 0.0046x10(-8) g/cellxday in non infected ones). The glutamine consumption did not differ in both cultures (muGlutamine of 0.0034 and 0.0037x10(-8) g/cellxday in, respectively, infected and non infected cultures). When a virus MOI of 0.1 to 1 was used for infection, a higher concentration of PIBs/ml was obtained. This was in direct correlation to a higher cell concentration present in these cultures, where a decrease in cell multiplication due to virus infection is minimized. When a MOI of 1 was used, a more effective decrease in cell multiplication was observed and a lower concentration of PIBs/ml was obtained, but with the best performance of PIBs/cell. Correlations between MOI and CCI indicate that a MOI 0.1 to 1.4 and a CCI of 10(6) to 2x10(6) cells/ml led to the best PIBs production performances. The virulence of PIBs produced in cultures infected at low or high MOI showed comparable DL(50). Culture and infection in scaling-up conditions, performed in a bioreactor, were shown to provide the cells with a better environment and be capable of potentially improving the shaker-Schott findings. For an accurate qualitative control of PIB virulence, hemolymph from AgMNPV infected Anticarsia gemmatalis was used as starting material for passages in Sf9 cells. These led to a loss of virulence among the PIBs with an increase in the DL(50). The loss of virulence was accompanied by a loss in budded virus titer, a decreased number of PIBs produced and an altered DNA restriction pattern, suggesting the generation of defective interference particles (DIPs). Transmission electron microscopy (TEM) studies revealed that after cell passages, PIBs lacking virions were progressively synthesized. The study described here point out the biological constraints and bioprocess issues for the preparation of AgMNPV PIBs for biological control.

Identification of stress‐induced mitochondrial proteins in cultured tobacco cells

Under stress conditions, small and large heat‐shock proteins cooperatively fulfil molecular chaperone activities within many cellular compartments, including mitochondria. Here, we report the identification and characterization of 10 newly synthesized low‐molecular‐weight heat stress‐induced proteins (SIPs) found solely in the mitochondrial fraction of suspension‐cultured cells of tobacco (Nicotiana tabacum L., strain Virginia Bright Italia‐0). These SIPs were shown to be encoded by the nuclear genome and none was detected in non‐stressed cells. Mass spectroscopy analysis revealed that eight SIPs belonged to the group of small heat‐shock proteins, whilst one protein shared significant homology with transcription factors. De novo synthesis of all SIPs was detected within 10 min of the commencement of heat stress and continued for the whole 12 h stress interval. Moreover, nine of the SIPs were stable for 24 h following stress termination. SIP1, which showed the most intense synthesis profile, was found to be phosphorylated. The most intense stress response was observed mainly during the exponential growth phase of the cell culture. Native electrophoresis of mitochondrial protein complexes revealed an association of all the SIPs in high‐molecular‐weight complexes with no free SIPs left in mitochondria. These complexes were localized to the mitochondrial membrane fraction.

The effects of copper and zinc on Spirulina platensis growth and heavy metal accumulation in its cells

The effects of copper and zinc on Spirulina platensis (Nordst.) Geitl. growth and the capability of this cyanobacterium for accumulation of these heavy metals (HMs) were studied. S. platensis tolerance to HMs was shown to depend on the culture growth phase. When copper was added during the lag phase, its lethal concentration was 5 mg/l, whereas 4 mg/l were lethal during the linear growth phase. Zinc concentration of 8.8 mg/l was lethal during the linear but not lag phase of growth. HM-treated S. platensis cells were capable for accumulation of tenfold more copper and zinc than control cells. Independently of Cu2+ content in the medium and of the growth phase, cell cultures accumulated the highest amount of this metal as soon as after 1 h, which may be partially determined by its primary sorption by cell-wall polysaccharides. A subsequent substantial decrease in the intracellular copper content occurred due to it secretion, which was evident from the increased metal concentration in the culturing medium. When zinc was added during the linear growth phase, similar pattern of its accumulation was observed: the highest content after 1 h and its subsequent decrease to the initial level. When the initial density of the culture was low and the cells had much time to adapt to HM, zinc accumulated during the entire linear growth phase, and thereafter the metal was secreted to the medium. The mechanisms of S. platensis tolerance to HM related to both their sorption by the cell walls and secretion of metal excess into the culturing medium and its conversion into the form inaccessible for the cells are discussed.

Growth, nutrient uptake and proximate composition of Rhodomonas sp. cultured using f/2 medium and agricultural fertilizers

We determined the growth, nutrient (PO43–, NO3–, NH4+) uptake and proximate composition of Rhodomonas sp. cultured during seven days in batch culture using f/2 medium and agricultural fertilizers. The growth rate was similar with both media (µ = 0.37 and 0.38 day–1). Protein content in Rhodomonas sp. cultured with f/2 medium was higher than with agricultural fertilizers (10.7 and 8.96 pg cell–1, respectively). Carbohydrate and lipid contents decreased during the exponential growth phase and there were no significant differences (P = 0.027 and 0.08, respectively) between both experiments. Carbohydrate content was higher during the slow growth phase in cultures using f/2 medium, whereas lipid content showed little variation during the slow growth phase, but was higher in cells cultured using agricultural fertilizers. During the first five days, total phosphate uptake by Rhodomonas sp. was 39.9 µM (94.3%) in the f/2 medium and 40 µM (99%) in the agricultural fertilizer medium. A total of 667.3 µM of nitrate (72.2%) was consumed during the first four days in the f/2 medium, whereas in the agricultural fertilizer medium formulated with ammonium nitrate, ammonium uptake (418.1 µM, 78.7%) in the first three days was higher than nitrate uptake (37.7 µM, 7.3%); however, when ammonium concentration decreased to 1.83 µM, the nitrate uptake increased. In conclusion, the cellular quantity and quality of Rhodomonas sp. was similar when both media were used and agricultural fertilizers can be used in marine aquaculture programs.

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of loose smut of barley (Ustilago nuda)

Polyclonal antibodies were raised against mycelium from the logarithmic growth phase of a shake culture of Ustilago nuda, and a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) with biotinylated detection antibodies was developed. The detection limit of the assay was 15 ng total protein ml−1 for the homologous antigen and 50 ng ml−1 for a spore extract, respectively. Other species of Ustilago reacted with the antibodies. Cross-reactivity was highest with U. tritici. No signal was obtained with the tested isolates of Tilletia, Rhizoctonia, Pythium and Fusarium. With naturally infected barley seeds, the results of the ELISAs were always in good agreement with those obtained with the routinely used seed embryo test. However, when seeds grown from artificially inoculated florets were used, the ELISA indicated significantly higher infestation levels than the embryo test. Results of assays with halved seeds from the same lot showed that high amounts of mycelium were present in the non-embryo half. This and especially the relatively long duration of the assay suggested that the ELISA (as conducted here) may not be suitable as a routine method for analysing seed infection with U. nuda. With samples from barley seedlings grown from infected seeds the results of the immunoassay again corresponded very well with the infection level determined by staining of the seed embryo, irrespective of the mode of floret inoculation (natural or artificial). Potential fields of application of the ELISA include the early prediction of the efficacy of protection agents, e.g. in screenings for seed treatments, the elucidation of the biology of the fungus and characterisation of resistance mechanisms.

Auxin production by bacteria associated with orchid roots

Bacteria associated with the roots of greenhouse tropical orchids were shown to produce indole-3-acetic acid (IAA) and to excrete it into the culture liquid. The presence and activity of IAA were demonstrated colorimetrically, by thin-layer chromatography, and by biotests. The associated bacteria varied in their ability to excrete indole compounds (1-28 microg/ml nutrient broth). Addition of tryptophan to the growth medium enhanced phytohormone production. Upon addition of 200 microg/ml tryptophan, the bacteria isolated from Dendrobium moschatum roots (Sphingomonas sp. 18, Microbacterium sp. 23, Mycobacterium sp. 1, Bacillus sp. 3, and Rhizobium sp. 5) produced 50.2, 53.1, 92.9, 37.6, and 60.4 microg IAA/ml respectively, while the bacteria isolated from Acampe papillosa roots (Sphingomonas sp. 42, Rhodococcus sp. 37, Cellulomonas sp. 23, Pseudomonas sp. 24, and Micrococcus luteus) produced 69.4, 49.6, 53.9, 31.0, and 39.2 microg IAA/ml. Auxin production depended on cultivation conditions and on the growth phase of the bacterial cultures. Treatment of kidney bean cuttings with bacterial culture liquid promoted formation of a "root brush" with location height 7.4- to 13.4-fold greater than the one in the control samples. The ability of IAA-producing associated bacteria to act as stimulants of the host plant root development is discussed.

Transient Effects of Overexpressing Anthranilate Synthase α and β Subunits in Catharanthusroseus Hairy Roots

Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback-insensitive anthranilate synthase alpha subunit under the control of the glucocorticoid-inducible promoter system and the anthranilate synthase beta subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, hörhammericine, and tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h.

Growth conditions required for bacteriocin production by strains of Staphylococcus aureus

The influence of growth parameters on the production of bacteriocins (aureocins) by five strains of Staphylococcus aureus was investigated. These aureocins have a broad spectrum of activity and can inhibit important human and animal pathogens. All strains produced large inhibition zones upon the indicator strain when they were grown in rich media such as brain-heart infusion (BHI), N2GT and 2 × YT. Bacteriocin production was not influenced by the initial pH of the medium (6.0–8.0). At lower temperature (28 °C), there was a marked reduction in bacteriocin production. Incubation of the producers under anaerobiosis affected profoundly the production of two related bacteriocins, aureocins MB92 and 146L, and slightly increased the production of aureocins A53 and 215FN. Production of aureocins MB92 and 215FN was apparently abolished in media containing ≥ 2.5 g and ≥ 3.5 g Nacl/100 ml, respectively. Although production of the remaining aureocins was observed in all NaCl concentrations tested (0.5–7.5 g/100 ml), the larger inhibition zones were detected in media containing up to either 1.5 g (for aureocins A70 and 146L) or 2.5 g NaCl/100 ml (for aureocin A53). Aureocin 215FN could not be detected in the culture supernatant. For the remaining aureocins tested, the highest levels of bacteriocin production occurred in either the late-log or early stationary growth phase of cultures grown in BHI medium at 37 °C. Changes in environmental conditions can, therefore, have detrimental effects on the production of active aureocins. Such factors are relevant when considering the potential biotechnological applications of these substances and when testing new S. aureus isolates for bacteriocin production.

The vimE gene downstream of vimA is independently expressed and is involved in modulating proteolytic activity in Porphyromonas gingivalis W83.

Regulation/activation of the Porphyromonas gingivalis gingipains is poorly understood. A unique 1.3-kb open reading frame downstream of the bcp-recA-vimA transcriptional unit was cloned, insertionally inactivated with the ermF-ermAM antibiotic resistance cassette, and used to create a defective mutant by allelic exchange. In contrast to the wild-type W83 strain, the growth rate of the mutant strain (designated FLL93) was reduced, and when plated on Brucella blood agar it was nonpigmented and nonhemolytic. Arginine- and lysine-specific gingipain activities were reduced by approximately 90 and 85%, respectively, relative to activities of the parent strain. These activities were unaffected by the culture's growth phase, in contrast to the vimA-defective mutant P. gingivalis FLL92, which has increased proteolytic activity in stationary phase. Expression of the rgpA, rgpB, and kgp gingipain genes was unaltered in P. gingivalis FLL93 compared to that of the wild-type strain. Further, in extracellular protein fractions a 64-kDa band was identified that was immunoreactive with the RgpB-specific proenzyme antibodies. Active-site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone or immunoblot analysis showed no detectable protein band representing the gingipain catalytic domain. In vitro protease activity could be slightly induced by a urea denaturation-renaturation cycle in an extracellular protein fraction, in contrast to the vimA-defective mutant P. gingivalis FLL92. Expression of flanking genes, including recA, vimA, and Pg0792, was unaltered by the mutation. Taken together, these results suggest that the vimA downstream gene, designated vimE (for virulence-modulating gene E), is involved in the regulation of protease activity in P. gingivalis.

Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels.

Different plant species produce a variety of terpenoid indole alkaloids, which are of interest as plant defensive secondary metabolites and as valuable pharmaceuticals. Although significant progress has been made, the mechanisms regulating the levels of this important class of compounds require continued elucidation. Previous precursor feeding studies have indicated that alkaloid accumulation can be improved during the exponential growth phase of hairy root cultures through enhanced tryptophan availability. To test this relationship, transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of an Arabidopsis feedback-resistant anthranilate synthase alpha subunit. Enzyme assays demonstrated that the Arabidopsis alpha subunit is compatible with the native beta subunit and that anthranilate synthase activity is more resistant to tryptophan inhibition in induced than in uninduced extracts. The metabolic effects of expressing the feedback-resistant anthranilate synthase alpha subunit were also dramatic. Over a 6-day induction period during the late exponential growth phase, tryptophan and tryptamine specific yields increased from almost undetectable levels to 2.5 mg/g dry weight and from 25 microg/g to 267 microg/g dry weight, respectively. The greater than 300-fold increase in tryptophan levels observed in these studies under certain induction conditions compares favorably with the fold increases obtained in previous constitutive expression studies. Despite the large increases in tryptophan and tryptamine, the levels of most terpenoid indole alkaloids were not significantly altered, with the exception of lochnericine, which increased 81% after a 3-day induction period. These results suggest that terpenoid indole alkaloid levels are tightly controlled.

Anthraquinones production, hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from intermediary and production medium strategies.

The effects of medium strategies [maintenance (M), intermediary (G), and production (P) medium] on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H2O2) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated. These were compared with third-stage leaf and 1-month-old callus culture. With P medium strategy, cell growth at 49 g l(-1), intracellular AQ content at 42 mg g(-1) DW, and H2O2 level at 9 micromol g(-1) FW medium were the highest as compared to the others. However, the extent of lipid peroxidation at 40.4 nmol g(-1) FW and total carotenoids at 13.3 mg g(-1) FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H2O2 levels. Vitamin C content at 30-120 microg g(-1) FW in all culture systems was almost half the leaf content. On the other hand, vitamin E content was around 400-500 microg g(-1) FW in 7-day-old cultures from all medium strategies and reduced to 50-150 microg g(-1) FW on day 14 and 21; as compared to 60 microg g(-1) FW in callus and 200 microg g(-1) FW in the leaf. This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures. When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants.

Effect of ionic strength on iron oxidation with batch cultures of chemolithotrophic bacteria

Predicting the growth and activity of chemolithotrophic bacteria in process operations as a function of process water quality is beyond the capability of current models. The models in usage at present, based on substrate limitation of enzyme kinetics, are typically expressions of the Monod form. We argue that the ionic nature of bio-oxidation process solutions is a pre-determined variable that renders these models invalid to describe the solution matrix impact on cell growth. To test this proposal, a series of experiments were conducted to determine whether ionic strength of the growth media is a significant variable. Subsequently, a simple model was developed with parameters for the cell specific growth rate K d (h −1) and the specific maximum rate of iron oxidation r max (mol s −1 cell −1). This model was applied successfully to the exponential phase of culture growth. The derived values for these parameters in media of minimal ionic strength were 0.16 h −1 and 5×10 −17–2×10 −18 mol s −1 cell −1, respectively. By increasing the ionic strength whilst maintaining a constant total iron concentration both K d and r max were observed to be progressively reduced, particularly the K d. Reducing the iron concentration while holding the calculated value for the ionic strength constant produced insignificant changes in the values of K d and r max. After several growth cycles at elevated ionic strength, the values of K d and r max were 0.14 h −1 and 5×10 −17–2×10 −18 mol s −1 cell −1, respectively, demonstrating an adaptation process. We conclude that ionic strength is a significant variable in the growth of chemolithotrophic cultures.

Fermentative production of curdlan.

Curdlan was produced by pure culture fermentation using Agrobacterium radiobacter NCIM 2443. Three different carbon sources (glucose, sucrose, maltose) were selected for study. Sucrose was found to be the most efficient. Utilization of sugar during the course of fermentation was studied, and the data were correlated to the production of curdlan. Curdlan mimics a secondary metabolite, in that its synthesis is associated with the poststationary growth phase of nitrogen-depleted batch culture. This was inferred from the results obtained from utilization of nitrogen. Regulation of pH at 6.1 +/- 0.3 resulted in an increased yield of curdlan from 2.48 to 4.8 g/L, and the corresponding increase in succinoglucan production was from 1.78 to 2.8 g/L. An attempt was made to increase curdlan production by the addition of the uridine nucleotides UMP and UDP-glucose to the fermentation broth. It was found that UDP-glucose at 0.8 microg/mL and UMP at 0.6 microg/mL served as precursors for curdlan and succinoglucan production when added after 18 h of nitrogen depletion in the fermentation broth.

Catechol siderophore, produced by thermoresistent strain of Bacillus licheniformis VK21

Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for producers of siderophores. It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe3+, in response to addition of manganese(II) salts. The growth of the producer on a minimum medium containing MnSO4 under the conditions of iron deficiency is accompanied by the accumulation of a catechol product, the content of which reaches a maximum at the beginning of the stationary growth phase of culture. In the presence of FeCl3, the amount of the catechol product in the medium considerably decreases. The siderophore, called SVK21, was isolated from the cultural medium and purified by reversed phase HPLC, and its siderophore function was confirmed by the test for the restoration of growth of producer cells in a medium containing EDTA. The UV spectrum of the siderophore has absorption maxima at 248 and 315 nm. According to amino acid analysis and NMR spectrometry, the metabolite SVK21 is 2,3-dihydroxybenzoyl-glycyl-threonine. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.