不同氮、磷浓度对铜绿微囊藻生长、光合及产毒的影响

对一株从野外分离得到的铜绿微囊藻产毒株进行分批培养,在不同的氮磷条件下研究其生长、光合荧光及毒素含量的变化。结果表明:正磷酸盐浓度不变时,铵氮浓度的改变对铜绿微囊藻的生长有明显影响。叶绿素a(Chl.a)含量在铵氮浓度为1.83-18.3mg/L时明显较大;微囊藻毒素(包括MC-LR和MC-RR)的含量在铵氮浓度为1.83mg/L时达到最大;当铵氮浓度为0-1.83mg/L时,随着铵氮浓度升高,可变荧光FV和MC的产量均增大,同时MC异构体的种类增多;铵氮浓度过大对M.aeruginosa的生长、生理和产毒均有抑制作用。在另一组实验中,即铵氮浓度不变而正磷酸盐浓度增大时,Chl.a含量呈总体下降的趋势,并且与FV/Fm呈显著正相关关系(P<0.01,r=0.97),MC(MC-LR和MC-RR)的含量在正磷酸盐浓度小于0.56mg/L时明显升高,MC-LR与FV/Fm呈显著正相关关系(P<0.01,r=0.967)。       

Microcystis aeruginosa toxin: cell culture toxicity, hemolysis, and mutagenicity assays

Crude toxin was prepared by lyophilization and extraction of toxic Microcystis aeruginosa from four natural sources and a unicellular laboratory culture. The responses of cultures of liver (Mahlavu and PCL/PRF/5), lung (MRC-5), cervix (HeLa), ovary (CHO-K1), and kidney (BGM, MA-104, and Vero) cell lines to these preparations did not differ significantly from one another, indicating that toxicity was not specific for liver cells. The results of a trypan blue staining test showed that the toxin disrupted cell membrane permeability within a few minutes. Human, mouse, rat, sheep, and Muscovy duck erythrocytes were also lysed within a few minutes. Hemolysis was temperature dependent, and the reaction seemed to follow first-order kinetics. Escherichia coli, Streptococcus faecalis, and Tetrahymena pyriformis were not significantly affected by the toxin. The toxin yielded negative results in Ames/Salmonella mutagenicity assays. Microtiter cell culture, trypan blue, and hemolysis assays for Microcystis toxin are described. The effect of the toxin on mammalian cell cultures was characterized by extensive disintegration of cells and was distinguishable from the effects of E. coli enterotoxin, toxic chemicals, and pesticides. A possible reason for the acute lethal effect of Microcystis toxin, based on cytolytic activity, is discussed.     

Microcystis aeruginosa toxin: cell culture toxicity, hemolysis, and mutagenicity assays.

Crude toxin was prepared by lyophilization and extraction of toxic Microcystis aeruginosa from four natural sources and a unicellular laboratory culture. The responses of cultures of liver (Mahlavu and PCL/PRF/5), lung (MRC-5), cervix (HeLa), ovary (CHO-K1), and kidney (BGM, MA-104, and Vero) cell lines to these preparations did not differ significantly from one another, indicating that toxicity was not specific for liver cells. The results of a trypan blue staining test showed that the toxin disrupted cell membrane permeability within a few minutes. Human, mouse, rat, sheep, and Muscovy duck erythrocytes were also lysed within a few minutes. Hemolysis was temperature dependent, and the reaction seemed to follow first-order kinetics. Escherichia coli, Streptococcus faecalis, and Tetrahymena pyriformis were not significantly affected by the toxin. The toxin yielded negative results in Ames/Salmonella mutagenicity assays. Microtiter cell culture, trypan blue, and hemolysis assays for Microcystis toxin are described. The effect of the toxin on mammalian cell cultures was characterized by extensive disintegration of cells and was distinguishable from the effects of E. coli enterotoxin, toxic chemicals, and pesticides. A possible reason for the acute lethal effect of Microcystis toxin, based on cytolytic activity, is discussed.     

Effects of gibberellin A(3) on growth and microcystin production in Microcystis aeruginosa (cyanophyta)

Environmental factors that affect the growth and microcystin production of microcystis have received worldwide attention because of the hazards microcystin poses to environmental safety and public health. Nevertheless, the effects of organic anthropogenic pollution on microcystis are rarely discussed. Gibberellin A(3) (GA(3)) is a vegetable hormone widely used in agriculture and horticulture that can contaminate water as an anthropogenic pollutant. Because of its common occurrence, we studied the effects of GA(3) on growth and microcystin production of Microcystis aeruginosa (M. aeruginosa) PCC7806 with different concentrations (0.001-25mg/L) in batch culture. The control was obtained without gibberellin under the same culture conditions. Growth, estimated by dry weight and cell number, increased after the GA(3) treatment. GA(3) increased the amounts of chlorophyll a, phycocyanin and cellular-soluble protein in the cells of M. aeruginosa PCC7806, but decreased the accumulation of water-soluble carbohydrates. In addition, GA(3) was observed to affect nitrogen absorption of the test algae, but to have no effect on the absorption of phosphorus. The amount of microcystin measured by enzyme-linked immunosorbent assay (ELISA) increased in GA(3) treatment groups, but the stimulatory effects were different in different culture phases. It is suggested that GA(3) increases M. aeruginosa growth by stimulating its absorbance of nitrogen and increasing its ability to use carbohydrates, accordingly increasing cellular pigments and thus finally inducing accumulation of protein and microcystin.     

Mathematical modeling of Microcystis aeruginosa growth and [D-Leu1] microcystin-LR production in culture media at different temperatures

The effect of temperature (26 °C, 28 °C, 30 °C and 35 °C) on the growth of native CAAT-3-2005 Microcystis aeruginosa and the production of Chlorophyll-a (Chl-a) and Microcystin-LR (MC-LR) were examined through laboratory studies. Kinetic parameters such as specific growth rate (μ), lag phase duration (LPD) and maximum population density (MPD) were determined by fitting the modified Gompertz equation to the M. aeruginosa strain cell count (cells mL⁻¹). A 4.8-fold increase in μ values and a 10.8-fold decrease in the LPD values were found for M. aeruginosa growth when the temperature changed from 15 °C to 35 °C. The activation energy of the specific growth rate (Eμ) and of the adaptation rate (E₁/LPD) were significantly correlated (R² = 0.86). The cardinal temperatures estimated by the modified Ratkowsky model were minimum temperature = 8.58 ± 2.34 °C, maximum temperature = 45.04 ± 1.35 °C and optimum temperature = 33.39 ± 0.55 °C.Maximum MC-LR production decreased 9.5-fold when the temperature was increased from 26 °C to 35 °C. The maximum production values were obtained at 26° C and the maximum depletion rate of intracellular MC-LR was observed at 30–35 °C. The MC-LR cell quota was higher at 26 and 28 °C (83 and 80 fg cell⁻¹, respectively) and the MC-LR Chl-a quota was similar at all the different temperatures (0.5–1.5 fg ng⁻¹).The Gompertz equation and dynamic model were found to be the most appropriate approaches to calculate M. aeruginosa growth and production of MC-LR, respectively. Given that toxin production decreased with increasing temperatures but growth increased, this study demonstrates that growth and toxin production processes are uncoupled in M. aeruginosa. These data and models may be useful to predict M. aeruginosa bloom formation in the environment.     

Photosynthesis and carbon metabolism in photoautotrophic cell suspensions of Chenopodium rubrum from different phases of batch growth

Rapidly dividing photoautotrophic cell suspensions from Chenopodium rubrum L. assimilated about 85 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. During the late stationary phase of culture growth, CO₂ fixation rate was reduced to about 60 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. Actively dividing cells characteristically incorporated a smaller proportion of ¹⁴C into starch than cells from non-dividing stationary phases. In rapidly dividing cells, [¹⁴C]-turnover from free sugars, sugar-phosphates, organic and amino acids was substantially higher compared to non-dividing cells from stationary growth phase. Higher proportions of photosynthetically fixed carbon were channelled into proteins, lipids and structural components in actively dividing cells than in non-dividing cells. In the latter. ¹⁴C was preferentially channeled into starch, and a striking increase in starch accumulation was observed. The transfer of non-dividing, stationary growth-phase cells into fresh culture medium resulted in an increase in the maximum extractable activities of some enzymes involved in the glycolytic and dark respiratory pathways and in the citric acid cycle. In contrast, the maximum extractable activities of the chloroplastic enzymes, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.38) and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were highest after the cells had reached the stationary growth phase.     

Comparative protein expression in different strains of the bloom-forming cyanobacterium Microcystis aeruginosa

Toxin production in algal blooms presents a significant problem for the water industry. Of particular concern is microcystin, a potent hepatotoxin produced by the unicellular freshwater species Microcystis aeruginosa. In this study, the proteomes of six toxic and nontoxic strains of M. aeruginosa were analyzed to gain further knowledge in elucidating the role of microcystin production in this microorganism. This represents the first comparative proteomic study in a cyanobacterial species. A large diversity in the protein expression profiles of each strain was observed, with a significant proportion of the identified proteins appearing to be strain-specific. In total, 475 proteins were identified reproducibly and of these, 82 comprised the core proteome of M. aeruginosa. The expression of several hypothetical and unknown proteins, including four possible operons was confirmed. Surprisingly, no proteins were found to be produced only by toxic or nontoxic strains. Quantitative proteome analysis using the label-free normalized spectrum abundance factor approach revealed nine proteins that were differentially expressed between toxic and nontoxic strains. These proteins participate in carbon-nitrogen metabolism and redox balance maintenance and point to an involvement of the global nitrogen regulator NtcA in toxicity. In addition, the switching of a previously inactive toxin-producing strain to microcystin synthesis is reported.     

Modeling of typical microbial cell growth in batch culture

A mathematical model was developed, based on the time dependent changes of the specific growth rate, for prediction of the typical microbial cell growth in batch cultures. This model could predict both the lag growth phase and the stationary growth phase of batch cultures, and it was tested with the batch growth ofTrichoderma reesei andLactobacillus delbrucckii.     

Phosphorus uptake and growth of blue-green alga, Microcystis aeruginosa

The specific uptake rate Q(p) of orthophosphate (expressed throughout as phosphorus) and the specific growth rate mu of Microcystis aeruginosa were measured using batch-precultured cells, whose growth phase, and intracellular and extracellular phosphorus concentrations f(p) and P, respectively, had been changed. When the cells from phosphorus-rich precultures were used, smaller values of Q(p) (0.1-0.3 mug P mg dry wt. (-1) h (-1)) were observed. However, if phosphorusstarved cells were used, the initial value of Q(p) was enhanced to more than ten times those smaller values referred to above, but declined rapidly with time after the transfer. Q(p) leveled off at around t = 4 h, when f(p) approached the maximum value, even if phosphorus was still available in the medium. A new correlation was presented here with respect to Q(p) as a function of P and f(p) as follows: \documentclass{article}\pagestyle{empty}\begin{document}$$ Q_p = Q_{p,\max } \frac{P}{{K_p + P}}\frac{{(f_{p,\max } - f_p )}}{{(f_{p,\max } - f_p )}} $$\end{document} Although numerical values of parameters involved in the equation depend on physiological state (or preculture history) of the cells, the above equation could account not only for phosphorus uptake, during which changes in phosphorus content in the cells were observed, but also for initial rates of uptake presented previously by other workers. mu Values were confirmed to be a hyperbolic function of f(p) as has been suggested by previous workers.     

Triploid banana cell growth phases and the correlation of medium pH changes with somatic embryogenesis in embryogenic cell suspension culture

Embryogenic cell suspensions of Musa AAA and AAB genomic groups were cultured in a maintenance culture medium for 17 generations (lasting for 238 days). The cell growth phases and medium pH changes were also observed correspondingly. Three major growth phases of AAA genomic group have been focused, namely cell releasing, proliferation and globularization phases. During almost all the subculture generations the cell stocks of AAB ‘Raja’ were continuously characterized by proliferating cell aggregates while the globularization phase occurred only for short duration. The medium acidity levels of the cell stocks of AAA ‘Pei-Chiao’ and ‘Robusta’ were commonly scattered in a wider range of pH 3.3–5.3, while the AAB ‘Raja’ were deviated in a narrow range of pH 4.0–4.6. After subculture, culture medium showed biphasic pH changes, which were drastic pH falls followed by an auto-regulated steady-state level. The steady-state pH values in each of the three growth phases (i.e. cell releasing, proliferation and globularization phases) were of 3.3–4.0, 4.0–4.8 and 4.8–5.3 respectively. Morphological bipolarity and the efficiency in the formation of somatic embryos have been thoroughly discussed. Reported research indicates that the condition of pH below 4.6 may prevent the development of embryogenic cells towards polar growth.     

Effects of arsenate on the growth and microcystin production of Microcystis aeruginosa isolated from Taiwan as influenced by extracellular phosphate

Arsenic pollution and eutrophication are both prominent issues in the aquaculture ponds of Taiwan. It is important to study the effects of arsenic on algal growth and toxin production in order to assess the ecological risk of arsenic pollution, or at least to understand naturally occurring ponds. The sensitivity of algae to arsenate has often been linked to the structural similarities between arsenate and phosphate. Thus, in this study we examined the effects of arsenate (10⁻⁸ to 10⁻⁴ M) on Microcystis aeruginosa TY-1 isolated from Taiwan, under two phosphate regimes. The present study showed that M. aeruginosa TY-1 was arsenate tolerant up to 10⁻⁴ M, and that this tolerance was not affected by extracellular phosphate. However, it seems that extracellular phosphate contributed to microcystin production and leakage by M. aeruginosa in response to arsenate. Under normal phosphate conditions, total toxin yields after arsenate treatment followed a typical inverted U-shape hormesis, with a peak value of 2.25 ± 0.06 mg L⁻¹ in the presence of 10⁻⁷ M arsenate, whereas 10⁻⁸ to 10⁻⁶ M arsenate increased leakage of ∼75% microcystin. Under phosphate starvation, total toxin yields were not affected by arsenate, while 10⁻⁶ and 10⁻⁵ M arsenate stimulated microcystin leakage. It is suggested that arsenate may play a role in the process of microcystin biosynthesis and excretion. Given the arsenic concentrations in aquaculture ponds in Taiwan, arsenate favors survival of toxic M. aeruginosa in such ponds, and arsenate-stimulated microcystin production and leakage may have an impact on the food chain.     

Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture

The electrical impedance of the culture medium shows complex changes during the growth and fermentation process of yeast, and this prevents its possible application for the monitoring of certain yeast activities. Clarification of the mechanism of such changes is thus essential for practical use. As a first step toward this aim, the impedance, yeast concentration, and pH of a batch culture medium were measured using special cells with two compartments and also the usual type of cell with one compartment. In the special cells, the yeast was cultured in one compartment only. Conducting ions and nonconducting substances diffused through an intermediate porous membrane sandwiched between the two compartments. The impedances of the two compartments were measured simultaneously by the four-electrode method. The main mechanism responsible for increasing the impedance was the conducting ions produced by the yeast extract added as a nutrient to the culture broth by certain nonconducting substances during the process of growth. The increase in the yeast concentration was also a minor factor increasing the impedance. These increases surpassed the impedance decrease caused by the increase of H(+) ions produced by some accumulated acidic substances, and the impedance thus increased.     

Morphological and physiological changes in Microcystis aeruginosa as a result of interactions with heterotrophic bacteria

1. To reveal the role of aquatic heterotrophic bacteria in the process of development of Microcystis blooms in natural waters, we cocultured unicellular Microcystis aeruginosa with a natural Microcystis‐associated heterotrophic bacterial community. 2. Unicellular M. aeruginosa at different initial cell densities aggregated into colonies in the presence of heterotrophic bacteria, while axenic Microcystis continued to grow as single cells. The specific growth rate, the chl a content, the maximum electron transport rate (ETR_max) and the synthesis and secretion of extracellular polysaccharide (EPS) were higher in non‐axenic M. aeruginosa than in axenic M. aeruginosa after cell aggregation, whereas axenic and non‐axenic M. aeruginosa displayed the same physiological characteristic before aggregation. 3. Heterotrophic bacterial community composition was analysed by PCR–denaturing gradient gel electrophoresis (PCR–DGGE) fingerprinting. The biomass of heterotrophic bacteria strongly increased in the coinoculated cultures, but the DGGE banding patterns in coinoculated cultures were distinctly dissimilar to those in control cultures with only heterotrophic bacteria. Sequencing of DGGE bands suggested that Porphyrobacter, Flavobacteriaceae and one uncultured bacterium could be specialist bacteria responsible for the aggregation of M. aeruginosa. 4. The production of EPS in non‐axenic M. aeruginosa created microenvironments that probably served to link both cyanobacterial cells and their associated bacterial cells into mutually beneficial colonies. Microcystis colony formation facilitates the maintenance of high biomass for a long time, and the growth of heterotrophic bacteria was enhanced by EPS secretion from M. aeruginosa. 5. The results from our study suggest that natural heterotrophic bacterial communities have a role in the development of Microcystis blooms in natural waters. The mechanisms behind the changes of the bacterial community and interaction between cyanobacteria and heterotrophic bacteria need further investigations.     

A novel approach for estimating growth phases and parameters of bacterial population in batch culture

Using mathematical analysis, a new method has been developed for studying the growth kinetics of bacterial populations in batch culture. First, sampling data were smoothed with the spline interpolation method. Second, the instantaneous rates were derived by numerical differential techniques and finally, the derived data were fitted with the Gaussian function to obtain growth parameters. We named this the Spline-Numerical-Gaussian or SNG method. This method yielded more accurate estimates of the growth rates of bacterial populations and new parameters. It was possible to divide the growth curve into four different but continuous phases based on changes in the instantaneous rates. The four phases are the accelerating growth phase, the constant growth phase, the decelerating growth phase and the declining phase. Total DNA content was measured by flow cytometry and varied depending on the growth phase. The SNG system provides a very powerful tool for describing the kinetics of bacterial population growth. The SNG method avoids the unrealistic assumptions generally used in the traditional growth equations.     

A novel approach for estimating growth phases and parameters of bacterial population in batch culture

Traditionally, microbiologists divided bacterial growth in batch cultures into lag, exponential, station-ary and death phases[1], following the Logistic equa-tion that has been applied to the growth of human populations. The growth curves can always be ch…