Ultrastructure of the cell surface of heteromorphic Nostoc muscorum CALU 304 cocultured with Rauwolfia tissue]

The ultrastructure of the heteromorphic cells (HMCs) of the cyanobacterium Nostoc muscorum CALU 304 grown in pure culture, monoculture, and a mixed culture with the Rauwolfia callus tissue was studied. The comparative analysis of the cell surface of HMCs, the frequency of the generation of cell forms with defective cell walls (DCWFs), including protoplasts and spheroplasts, and the peculiarities of the cell surface ultrastructure under different growth conditions showed that, in the early terms of mixed incubation, the callus tissue acts to preserve the existing cyanobacterial DCWFs, but begins to promote their formation in the later incubation terms. DCWFs exhibited an integrity of their protoplasm and were metabolically active. It is suggested that structural alterations in the rigid layer of the cell wall may be due to the activation of the murolytic enzymes of cyanobacteria and the profound rearrangement of their peptidoglycan metabolism caused by the Rauwolfia metabolites diffused through the medium. These metabolites may also interfere with the functioning of the universal cell division protein of bacteria, FtsZ. In general, the Rauwolfia callus tissue promoted the unbalanced growth of the cyanobacterium N. muscorum CALU 304 and favored its viability in the mixed culture. The long-term incubation of the Rauwolfia tissue with the N. muscorum CALU 304 cells led to their transformation to L-forms.     

Stimulating and inhibiting effect of Spirulina platensis on microorganisms

The blue-green microalga (cyanobacterium) S. platensis and the complex of its metabolites in the culture fluid being added into nutrient agar in doses of 0.01, 0.1 and 10 mg/ml, may produce a stimulating and inhibiting effect on microorganisms. In Spirulina biomass and in culture fluid substances which may be associated with the stimulation or inhibition of the growth of some microorganisms are supposedly contained. The manifestation of stimulating or inhibiting properties depended on the concentration of S. platensis and the complex of its metabolites, as well as on sterilization methods of nutrient media supplemented with these substances.     

Herbicide effects on the population growth of some green algae and cyanobacteria

Six herbicides were tested for their effects on the population growth of a range of green algae and cyanobacteria by an easily replicated low-volume liquid culture technique using Repli-dishes. Diuron, propanil and atrazine were most inhibitory, chlorpropham was intermediate and MCPA and glyphosate were least inhibitory. Chlorpropham was more inhibitory to green algae than to cyanobacteria. The effects of chlorpropham and 3-chloroaniline, a metabolite, on populations of the cyanobacterium Anacystis nidulans and the alga Chlamydomonas reinhardii were monitored in larger scale batch cultures. Both compounds reduced the growth rate although in some cases there was partial recovery. 3-Chloroaniline was less inhibitory than the parent herbicide chlorpropham.     

Surface Ultrastructure of the Heteromorphic Cells of Nostoc muscorumCALU 304 in a Mixed Culture with the RauwolfiaCallus Tissue

The ultrastructure of the heteromorphic cells (HMCs) of the cyanobacterium Nostoc muscorumCALU 304 grown in pure culture, monoculture, and a mixed culture with the Rauwolfiacallus tissue was studied. The comparative analysis of the cell surface of HMCs, the frequency of the generation of cell forms with defective cell walls (DCWFs), including protoplasts and spheroplasts, and the peculiarities of their ultrastructure under different growth conditions showed that, in the early terms of mixed incubation, the callus tissue acts to preserve the existing cyanobacterial DCWFs, but begins to promote their formation in the later incubation terms. DCWFs exhibited an integrity of their protoplasm and were metabolically active. It is suggested that structural alterations in the rigid layer of the cell wall may be due to the activation of the murolytic enzymes of cyanobacteria and the profound rearrangement of their peptidoglycan metabolism caused by the Rauwolfiametabolites diffused through the medium. These metabolites may also interfere with the functioning of the universal cell division protein of bacteria, FtsZ. In general, the Rauwolfiacallus tissue promoted the unbalanced growth of the cyanobacterium N. muscorumCALU 304 and favored its viability in the mixed culture. The long-term mixed cultivation substantially augmented the probability of the formation of L-forms of N. muscorumCALU 304.     

绿色木霉代谢产物的植物毒性研究

通过人工固体培养和液体发酵研究发现绿色木霉的代谢产物对植物的幼苗生长有抑制作用,其代谢产物大量分泌到它所生长的环境中,在不同的营养基质中其代谢产物的抑制作用有差异．     

Inhibition of Camellia sinensis (L.) O. Kuntze on Microcystis aeruginosa and isolation of the inhibition factors

Low concentration of tea (Camellia sinensis (L.) O. Kuntze) was shown to inhibit the growth of the toxic cyanobacterium Microcystis aeruginosa. The inhibition efficiency was 40 % at 0.1 g dry tea/L and 90 % at 0.2 g/L after a 12-day culture. All varieties of tea used in the test could inhibit Microcystis growth, in which the inhibitory effect of green tea was greater than that of black tea. Antialgal allelochemicals were isolated from tea by solvent extraction, gel-chromatography and high performance liquid chromatography. Two algal-inhibition compounds were identified by liquid chromatography/mass spectrometry as epigallocatechin-3-gallate, epicatechin-3-gallate respectively. These are the main polyphenols in tea that have inhibitory effects on the growth of cyanobacteria. The combined effect of these polyphenols makes tea a promising source of algicide to inhibit the growth of algal blooms.     

Culture of the marine cyanobacterium, Lyngbya majuscula (Oscillatoriaceae), for bioprocess intensified production of cyclic and linear lipopeptides.

Cyanobacteria are an ancient and diverse group of photosynthetic microorganisms, which inhabit many different and extreme environments. This indicates a high degree of biological adaptation, which has enabled these organisms to thrive and compete effectively in nature. The filamentous cyanobacterium, Lyngbya majuscula, produces several promising antifungal and cytotoxic agents, including laxaphycin A and B and curacin A. Samples of L. majuscula collected from Moorea Island, Tahiti (French Polynesia) and from the Culture Collection of Algae and Protozoa (CCAP 1446/4) were studied and adapted to large scale laboratory culture (5 l). This constitutes a 100-fold scale-up for the culture of this particular strain of L. majuscula. The effect of culture vessel configurations, growth conditions and media compositions on growth of L. majuscula was examined. Using optimised culture conditions, two strains of L. majuscula are currently being evaluated for their production of secondary metabolites. Results will be compared with those obtained from four environmental extracts. Comparisons were made by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). It was shown that varying the culture conditions under which L. majuscula was grown had the greatest effect on secondary metabolite production, thus providing potential for future bioprocess intensified production.     

The effect of the intra- and extracellular metabolites of microorganisms isolated from various ecotopes on the catalase activity of Staphylococcus aureus 6538P

The cell extracts (i.e., intracellular metabolites) and culture liquids (i.e., extracellular metabolites) of microorganisms isolated from various ecotopes were found to inhibit the catalase activity of Staphylococcus aureus ATCC 6538P, which resulted in a considerable inhibition of the growth of metabolite-treated S. aureus cells by hydrogen peroxide. The inhibitory effect of microbial metabolites on S. aureus catalase can be considered as a mechanism of intercellular interactions responsible for the formation of microbiocenoses.     

Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 10⁶ cells mL^?1 was obtained, with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.     

Balanced nutrient fortification enables high-density hybridoma cell culture in batch culture

Cells of an in vitro culture system are not the same as for an in vivo system, metabolically and physiologically; ineffective utilization of nutrients occurs by cells in vitro. Therefore, a simpler approach is needed to examine closely and overcome differences between in vivo and in vitro cells.Recognizing the ineffectiveness of nutrient utilization in vitro, we have constructed, a balanced, fortified high-density medium based on RPMI 1640 medium previously optimized for relatively low-density cell culture. The high-density medium was used to cultivate a hybridoma line in a batch spinner flask culture. In this fortified medium, a hybridoma cell line 2c3.1 was cultivated to near 1 x 10(7) cells/mL in batch suspension culture. During the culture, glucose, glutamine, and 10 essential amino acids of concentrations five times richer than normal in the medium were almost thoroughly consumed. Combined analysis of these consumption profiles reveals that the balanced, fortified nutrient supply contributes much to cellular activity to overcome the limitations of in vitro cellular growth. Intermediate metabolites, such as ammonium ion and lactic acid, were produced over concentrations reported until now to be inhibitory. This observation suggests that the major conclusive factor against cellular growth over the critical cell density is not so-called inhibitory metabolites. As a result of the high-density culture, 5-8 times higher production of a monoclonal antibody for hepatitis B surface antigen (anti-HBs) was obtained.Active cellular consumption of all the essential nutrients and the corresponding production of MAb strongly support the potential of our approach to overcome the growth limitation of cells in vitro and to obtain high-density hybridoma cell culture.     

Bioproduction of antimicrobial compounds by using marine filamentous cyanobacterium cultivation

The synthesis of bioactive compounds with antimicrobial activity, excreted by marine cyanobacteria, strongly depends on their growth conditions. Due to the wide variety of biomolecules which could show properties as growth inhibitors and their low concentrations within the culture medium, the activity of their crude extracts also seems to be related to the extraction method used. Using the marine filamentous cyanobacterium Geitlerinema strain Flo1, we demonstrate a systematic approach for identifying optimal culture conditions to obtain culture media extracts with antimicrobial activity. The changes in the culture conditions, such as the addition of NaBr to the medium, cell immobilisation in vegetable sponge pieces, or temperature, effected the production of these bioactive compounds. The crude extract, containing middle polar molecules, obtained by extraction with Amberlite XAD-1180 had a higher antifouling activity upon a number of bacteria and fungi than the extract obtained by extraction with Amberlite XAD-16. The lowest inhibitory concentration obtained upon Rhodosporidium sphaerocarpum was still ten times higher than that of bis(tributyltin)oxide, but compared to zinc pyrithione, it was two times more active.     

Identifying inhibitory threshold values of repressing metabolites in CHO cell culture using multivariate analysis methods

Elevation of lactate, ammonia, osmolality, and carbon dioxide to inhibitory levels was reported to have adverse effects on cell growth and protein productivity in mammalian cell culture. Multivariate analysis methods were used to investigate the roles of these repressing metabolites in a fed-batch CHO cell culture for antibody fusion protein B1 (B1) production. Principal Factor Analysis methodology was applied to manufacturing-scale data of 112 cell culture runs, which identified threshold values of four repressing metabolites as follows: (1) ammonium levels above 5.1 mM inhibit cell growth; (2) both lactate and osmolality levels above 58 mM and 382 mOsm/kg affect cell viability; and (3) carbon dioxide levels at or above 111 mmHg reduce protein quality. These threshold values were then verified by simulations using Monod-type equations and Canonical Correlation. These results suggest that adverse effects on cell growth, productivity, and product quality may be minimized under the ideal cell culture condition, in which the peak values of all four repressing metabolites are maintained below the threshold values. This strategy was evaluated in 45 cell culture runs in 50-L bioreactors. Eight out of 45 runs were operated under the ideal condition, while the remaining 37 runs had at least one repressing metabolite with peak value at or above the threshold. In comparison to the remaining runs, the eight cell culture runs under the ideal condition had 17%, 40%, and 11% higher values in peak viable cell density, final B1 titer, and quality attribute, respectively. The unique methodology used in this study may be generally applicable in characterizing cell culture processes.     

First report on the allelopathic effect of Tychonema bourrellyi (Cyanobacteria) against Microcystis aeruginosa (Cyanobacteria)

The allelopathic effect of the cyanobacterium Tychonema bourrellyi against the cyanobacterium Microcystis aeruginosa is reported for the first time in this paper. The filtrate of T. bourrellyi CHAB663 culture showed strong inhibitory effect on M. aeruginosa NIES-843, but the inhibitory effect was weakened by shaking culture, and such results implied that the allelopathic effect was probably mediated by the volatile substances secreted by T. bourrellyi. β-Ionone was identified as a major ingredient in the volatile substances in the cultures of T. bourrellyi, and it may act as an important allelochemical responsible for this allelopathic activity. The filtrates of T. bourrellyi culture were shown to decrease the maximum electron transport rate (ETR_max) and elevate the reactive oxygen species (ROS) levels in the cells of M. aeruginosa NIES-843.     

The Effect of the Intra- and Extracellular Metabolites of Microorganisms Isolated from Various Ecotopes on the Catalase Activity of Staphylococcus aureus ATCC 6538 P

The cell extracts (i.e., intracellular metabolites) and culture liquids (i.e., extracellular metabolites) of microorganisms isolated from various ecotopes were found to inhibit the catalase activity of Staphylococcus aureus ATCC 6538 P, which resulted in a considerable inhibition of the growth of metabolite-treated S. aureus cells by hydrogen peroxide. The inhibitory effect of microbial metabolites on S. aureus catalase can be considered as a mechanism of intercellular interactions responsible for the formation of microbiocenoses.     

EFFECT OF SODIUM AND NITRATE ON GROWTH OF ANABAENA FLOS-AQUAE (CYANOPHYTA)1

The growth response of a nitrogen fixing cyanobacterium, Anabaena flos-aquae (Lyng) Bréb., to sodium and nitrate was examined in batch culture under controlled laboratory conditions. Sodium (range 0-12 mg Na⁺· L^-1) enhanced growth of the cyanobacterium under nitrate-sufficient (5.7 mg NO³-N · L^-1) but not nitrate-limited (0.49 mg NO³- N · L^-1) conditions. The magnitude of the growth response was related to the nutritional history of the culture. No significant effect of sodium on nitrate utilization was observed. The increase in ambient sodium levels in many lakes may provide a competitive advantage to cyanobacteria.     

甘肃黑蛋巢抗松梢枯病菌物质的分离纯化及抑菌活性*

采用MS培养基培养甘肃黑蛋巢Cyathus gansuensis,以生测为导向,乙酸乙酯和氯仿从甘肃黑蛋巢MS培养液中萃取出抗菌活性组分。Rp-18反相色谱柱和高效液相色谱仪相结合,从抗菌代谢产物分离纯化出一种抗松梢枯病菌(Sphaeropsis sapinea)的活性物质CXL-I,高效液相色谱检测表明CXL-I为一纯物质。CXL-I对松梢枯病菌的菌丝生长和孢子萌发有较强的抑制活性,当CXL-I浓度为50礸/ml时对菌丝生长抑制率可达80%,对孢子萌发抑制率达95%以上。     

Characteristics of a plant deleterious rhizosphere pseudomonad and its inhibitory metabolite(s)

A cell-free culture filtrate of the plant growth inhibitory bacterial isolate Å313, identified as Pseudomonas fluorescens, was tested for its effect on wheat root elongation in vitro, with and without various pretreatments. The filtrate showed a strongly inhibitory effect on root elongation and could be heated to 100°C for 5 min or incubated at a pH within the range of 4–10 without losing its activity. Unlike other root growth-inhibitory bacterial metabolites the effect of the filtrate was not reversed by methionine, nor was any inhibitory activity present in the water phase after extraction with chloroform. The inhibitory agent(s) was formed when the bacterium was grown in an artificial medium as well as in root exudates from wheat. Two wheat cultivars differing in reaction to inoculation with living cells of Å313 showed the expected difference in growth reduction when exposed to culture filtrate, indicating a cultivar difference in sensitivity to the metabolite(s) formed by the bacterium. Isolate Å313 invaded intercellular spaces of the root cortex of gnotobiotically grown wheat plants, but did not produce pectolytic enzymes in vitro, nor induce a hypersensitive response in tobacco plants.     

Isolation and Biological Activity of Cyl-2, a Metabolite of Cylindrocladium scoparium

Three metabolites designated Cyl–1, –2 and –3 were isolated as plant growth regulators from culture filtrate of Cylindrocladium scoparium Morgan, a phytopathogenic fungus. Cy1–2 was obtained as pure crystals, and it revealed marked inhibitory activity on the root growth of lettuce seedlings. Cyl–1 showed inhibition on the same organ, while Cyl–3 showed promotion.     

Metabolism of phenanthrene by the marine cyanobacterium Agmenellum quadruplicatum PR-6.

Under photoautotrophic growth conditions, the marine cyanobacterium Agmenellum quadruplicatum PR-6 metabolized phenanthrene to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) and 1-methoxyphenanthrene as the major ethyl acetate-extractable metabolites. Small amounts of phenanthrols were also formed. The metabolites were purified by high-pressure liquid chromatography and identified from their UV, infrared, mass, and proton magnetic resonance spectral properties. A. quadruplicatum PR-6 formed phenanthrene trans-9,10-dihydrodiol with a 22% enantiomeric excess of the (-)-9S,10S-enantiomer. Incorporation experiments with 18O2 showed that one atom of oxygen from O2 was incorporated into the dihydrodiol. Toxicity studies, using an algal lawn bioassay, indicated that 9-phenanthrol and 9,10-phenanthrenequinone inhibit the growth of A. quadruplicatum PR-6.     

Metabolism of phenanthrene by the marine cyanobacterium Agmenellum quadruplicatum PR-6.

Under photoautotrophic growth conditions, the marine cyanobacterium Agmenellum quadruplicatum PR-6 metabolized phenanthrene to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) and 1-methoxyphenanthrene as the major ethyl acetate-extractable metabolites. Small amounts of phenanthrols were also formed. The metabolites were purified by high-pressure liquid chromatography and identified from their UV, infrared, mass, and proton magnetic resonance spectral properties. A. quadruplicatum PR-6 formed phenanthrene trans-9,10-dihydrodiol with a 22% enantiomeric excess of the (-)-9S,10S-enantiomer. Incorporation experiments with 18O2 showed that one atom of oxygen from O2 was incorporated into the dihydrodiol. Toxicity studies, using an algal lawn bioassay, indicated that 9-phenanthrol and 9,10-phenanthrenequinone inhibit the growth of A. quadruplicatum PR-6.