Fixed-bed biosorption of Cu2+ by polyacrylonitrile-immobilized dead cells of Saccharomyces cerevisiae

Dead cells of Saccharomyces cerevisiae 54 were immobilized by entrappment in polyacrylonitrile. The beads obtained were used to adsorb copper in an up-flow fixed-bed column. The effect of polymer content and cell loading were studied to optimize the porosity and the efficiency in copper removal of the biosorbent beads in a batch system. The optimal concentration of the polyacrylonitrile was assumed to be 12%(w/v) and a concentration of 0.5 g cell dry weight in 1 g polymer was most effective in adsorption of Cu²⁺. The adsorption capacity of this biosorbent was 27 mg Cu²⁺/g dry biomass at 200 mg/l initial concentration of copper ions. Adsorption of Cu²⁺ in a batch system was studied using different initial concentrations of the solute. The optimal conditions in the up-flow column of the following parameters were determined: flow rate, bed height, and initial concentration of Cu²⁺ of the solutions. Results of fixed-bed biosorption showed that breakthrough and saturation time appeared to increase with the bed height, but decrease with the flow rate and the initial concentration. The linearized form of the Thomas equation was used to describe dynamic adsorption of metal ions. As a result, the adsorption capacity of the batch system and the column system was compared. Desorption of copper ions was achieved by washing the column biomass with 0.1 M HCl at an eluent flow rate of 1 ml/min. The reusability of the immobilized biomass was tested in five consecutive adsorption-desorption cycles. The regenerated beads retained over 45% of their original adsorption capacity after five A/D cycles. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cu2+ Removal and recovery by Spi SORB: batch stirred and up-flow packed bed columnar reactor systems

The biosorption of Cu²⁺ by free and poly acrylamide gel (PAG) immobilized Spirulina platensis (SpiSORB) was characterized under batch and continuous packed bed columnar reaction systems. The biosorption of Cu²⁺ was shown to be highest at pH of 6.0 for both types of biomass. The PAG immobilization process did not interfere with the Cu²⁺ binding sites present on biomass leading to cent percent (ca. 250 mg g⁻¹ of dry biomass) retention of biosorption as compared to free cells. Transmission electron microscopy on Cu²⁺ localization revealed that majority of metal is being sequestered by the cell wall only. The infrared spectrum of metal treated S. platensis biomass indicated the possible involvement of amide, amino, and carboxyl groups in metal binding. Up-flow packed bed columnar reactor containing 2.0 g of PAG immobilized S. platensis shown a maximum of 143-fold volume reduction factor at the residence time of 4.6 min for Cu²⁺ alone and found to decrease dramatically when Zn²⁺ is present in a bimetallic solution.     

Selective control of cyanobacteria by surfactin-containing culture broth of Bacillus subtilis C1

Of several types of chemical surfactants and biosurfactants, only the culture broth of Bacillus subtilis C1 containing surfactin at 10 mg l^–1 completely inhibited the growth of Microcystis aeruginosa, a bloom-forming cyanobacterium in highly eutrophic lakes. The broth with 10 mg surfactin l^–1 also removed 85% of the maximally grown M. aeruginosa (chlorophyll-a concentration, 1000 g l^–1) within 2 d, and the removal efficiency was enhanced by Ca²⁺ over 1 mM. The growth of Anabaena affinis, another bloom-forming cyanobacterium, was also inhibited about 70% with surfactin at 10 mg l^–1 broth. However, the effect of the broth was delayed over 3 d in the green algae, Chlorella vulgaris and Scenedesmus sp., and was negligible in a diatom, Navicula sp., indicating the potential for the selective control of cyanobacterial blooms.     

Quantitative studies on phosphorus transference occuring between <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and its attached bacterium (<Emphasis Type="Italic">Pseudomonas</Emphasis> sp.)

Phosphorus release from Microcystis aeruginosa and attached bacterium (Pseudomonas sp.) isolated from Lake Taihu was examined using a phosphorus isotope tracer in order to investigate the phosphorus transference between the two species. Our results reveal that the amount of phosphorus released form ³²P-saturated M. aeruginosa is determined by its growth phase and most of phosphorus is assimilated by Pseudomonas finally while the amount of phosphorus released from ³²P-saturated Pseudomonas is also determined by the growth phase of M. aeruginosa and most of them are assimilated by M. aeruginosa. The results suggest that phosphorus transference occurs between M. aeruginosa and its attached Pseudomonas . This process makes microenvironment of mucilage of M. aeruginosa attached bacteria maintain relative high amounts of phosphorus. Attached bacteria may be a temporary phosphorus bank to the growth of M. aeruginosa, and assimilation of phosphorus by M. aeruginosa becomes easy when M. aeruginosa is in lag growth phase. Thus, the phosphorus exchange between M. aeruginosa and attached Pseudomonas in microenvironment may be important to microfood web and cyanobacteria bloom.     

Optimization of flow rate,initial metal ion concentration and biomass density for maximum removal of Cu2+ by immobilized Microcystis

The potential of alginate-immobilized Microcystis packed in a column for maximum removal of Cu²⁺ at different flow rates, biomass, and initial metal ion concentration was assessed in a continuous flow system. Although Cu²⁺ removal did occur at all the flow rates tested, it was maximum (54%) at 0.75-ml min⁻¹ flow rate, 30 μg ml⁻¹ initial metal ion concentration and 0.016 g biomass. Cu²⁺ removal was influenced by inlet metal ion concentration and biomass density. An increase in the biomass concentration from 0.016 to 0.128 g resulted in an apparent increase in percentage removal but the Cu²⁺ adsorbed per unit dry wt. declined. When the flow rate (0.75 ml min⁻¹) and biomass density (0.064 g) were kept constant and the inlet metal ion concentration was varied from 10 to 150 μg ml⁻¹, a 68% removal of Cu²⁺ was obtained at 50 μg ml⁻¹ initial concentration in a time duration of 15 min. The metal-laden columns were efficiently desorbed and regenerated following elution with double distilled water (DDW) (pH 2) (89%). This was followed by 1 mm EDTA > 1 mm NTA > 0.1 mm EDTA > 1 mm HCl > 1 mm HNO₃ > 5 mm CaCl₂ > DDW (pH 7.0) > 1 mm NaHCO₃ > 1 mm CaCl₂. Of the total (2.83 mg) adsorbed Cu²⁺, 1.89 mg (67%) was desorbed by DDW (pH 2) within the first 20 min of elution time. Thereafter the desorption rate slowed down and only 22% (0.632 mg) desorption was obtained in the last 20 min. In contrast to water pH 2, the desorption of Cu²⁺ by 1 mm EDTA was very slow, the maximum being 8% after 40 min of elution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Extracellular polysaccharides of a copper-sensitive and a copper-resistant Pseudomonas aeruginosa strain: synthesis,chemical nature and copper binding

Extracellular polysaccharides (EPS) of a copper-sensitive (Cu^s) and a copper-resistant (Cu^r) Pseudomonas aeruginosa strain were investigated in terms of their production, chemical nature and response towards copper exposure. The extent of EPS synthesis by the resistant strain (4.78 mg mg^–1 cell dry wt.) was considerably higher over its sensitive counterpart (2.78 mg mg^–1 dry wt.). FTIR-spectroscopy and gas chromatography revealed that both the polymers were acidic in nature, containing alginate as the major component along with various neutral- and amino-sugars. Acid content in the Cu^r EPS (480.54 mg g^–1) was greater than that in the Cu^s EPS (442.0 mg g^–1). Presence of Cu²⁺ in the growth medium caused a dramatic stimulation (approximately 4-fold) in EPS synthesis by the Cu^r strain, while in a similar condition, the Cu^s failed to exhibit such response. The polymer of the resistant strain showed elevated Cu²⁺ binding (320 mg g^–1 EPS) compared to that of the sensitive type (270 mg g^–1). The overall observations show the potential of the Cu^r EPS for its deployment in metal bioremediation.     

Immobilization of blue green microalgae on loofa sponge to biosorb cadmium in repeated shake flask batch and continuous flow fixed bed column reactor system

Summary An indigenous strain of blue green microalga, Synechococcus sp., isolated from wastewater, was immobilized onto loofa sponge discs and investigated as a potential biosorbent for the removal of cadmium from aqueous solutions. Immobilization has enhanced the sorption of cadmium and an increase of biosorption (21%) at equilibrium was noted as compared to free biomass. The kinetics of cadmium biosorption was extremely rapid, with (96%) of adsorption within the first 5 min and equilibrium reached at 15 min. Increasing initial pH or initial cadmium concentration resulted in an increase in cadmium uptake. The maximum biosorption capacity of free and loofa immobilized biomass of Synechococcus sp. was found to be 47.73 and 57.76 mg g⁻¹ biomass respectively. The biosorption equilibrium was well described by Langmuir adsorption isotherm model. The biosorbed cadmium was desorbed by washing the immobilized biomass with dilute HCl (0.1 M) and desorbed biomass was reused in five biosorption–desorption cycles without an apparent decrease in its metal biosorption capacity. The metal removing capacity of loofa immobilized biomass was also tested in a continuous flow fixed-bed column bioreactor and was found to be highly effective in removing cadmium from aqueous solution. The results suggested that the loofa sponge-immobilized biomass of Synechococcus sp. could be used as a biosorbent for an efficient removal of heavy metal ions from aqueous solution.     

Biosorption Performance of Multimetal Resistant Fungus Penicillium chrysogenum XJ-1 for Removal of Cu2+ and Cr6+ from Aqueous Solutions

Adsorption for heavy metals via biomaterials such as fungal biomass presents a practical remediation technique for polluted water. Among all known filamentous fungi, Penicillium chrysogenum is widespread in nature and can serve as a biosorbent for heavy metals. In the current study, the ability of P. chrysogenum XJ-1 to remove copper (Cu²⁺) and chromium (Cr⁶⁺) from water was evaluated. The maximum biosorption capacity of XJ-1 for Cu²⁺ reached 42.83 ± 0.57 mg g^?1 dry biomass at pH 5.0 after the equilibrium time of 1.5 h. The maximum biosorption capacity for Cr⁶⁺ at pH 3.0 reached 52.69 ± 1.68 mg g^?1 dry biomass after the equilibrium time of 1.5 h. The biosorption data of XJ-1 biomass were well fitted to the Freundlich isotherm model and the pseudo-second-order Lagergren kinetic model. Laundry powder-treated and HCl-treated XJ-1 biomass significantly enhanced its adsorption capacity to Cu²⁺ and Cr⁶⁺, respectively. HCl and NaOH were suitable desorbents for Cu²⁺/Cr⁶⁺ loading biomass, respectively. Fourier transform infrared spectroscopy analyses revealed that hydroxyl, amine, and sulfonyl groups on the biosorbent contributed to binding Cu²⁺ and Cr⁶⁺ and that carbonyl and carboxyl groups were also vital binding sites of Cu²⁺. Scanning electron microscopy and energy-dispersive x-ray (SEM-EDX) analyses confirmed that considerable amounts of metals were precipitated on the cell surface of XJ-1. Our results suggested that XJ-1 might be used to purify multimetal-contaminated water. This low-cost and eco-friendly biomass of XJ-1 seems to have a broad use in the restoration of metal-contaminated water.     

Antialgal activity of a hepatotoxin-producing cyanobacterium,Microcystis aeruginosa

Antimicrobial activity of toxin produced by a freshwater bloom-forming cyanobacterium Microcystis aeruginosa has been studied. When tested against certain green algae, cyanobacteria, heterotrophic bacteria and fungi, the toxin inhibited growth of only green algae and cyanobacteria. The toxin has been partially purified employing Thin layer chromatography (TLC) and High-performance liquid chromatography (HPLC) techniques and appears to be microcystin-LR (leucine–arginine). Both crude and purified toxins showed toxicity to mice, the clinical symptoms in test mice being similar to those produced by hepatotoxin. Purified toxin at a concentration of 50 g ml^–1 caused complete inhibition of growth followed by cell lysis in Nostoc muscorum and Anabaena BT1 after 6 days of toxin addition. Addition of toxin (25 g ml^–1) to the culture suspensions of the Nostoc and Anabaena strains caused instant and drastic loss of O₂ evolution. Furthermore a marked reduction (about 87%) in the ¹⁴CO₂ uptake was also observed at a concentration of 50 g ml^–1. Besides its inhibitory effects on photosynthetic processes, M. aeruginosa toxin (50 g ml^–1) also caused 90% loss of nitrogenase activity after 8 h of its addition. Experiments performed with ¹⁴C-labelled toxin indicate that the toxin uptake by cyanobacterial cells occurs both in light and dark. These results demonstrate that the toxin is strongly algicidal and point to the possibility that it may have an important role in establishment and maintenance of toxic blooms of M. aeruginosa in freshwater ecosystems. The relative significance of the hepatotoxic effect and the algicidal effect of the toxin is discussed with reference both to survival and dominance of M. aeruginosa in nature.     

Inhibitory effects of copper on bacteria related to the free ion concentration

Cu²⁺ ion determinations were carried out in complex and in inorganic salts-glycerol media, to which increasing amounts of Cu(II) had been added, with the ion-specific Cu(II)-Selectrode. Likewise, complexing capacity of bacterial suspensions was estimated by titration with CuSO₄.Copper-sensitive bacteria, e.g.,Klebsiella aerogenes, were inhibited in their growth and survival in the range of 10^–8–10^–6 M Cu²⁺ ion concentrations. In copper-buffered complex media, high copper loads could be tolerated, as growth proceeded with most of the copper bound to medium components. In low-complexing mineral salts media, in which high Cu²⁺ ion concentrations exist at low copper loads, there was competition of Cu²⁺ for binding sites of the cells. Total allowed copper was then determined by the ratio of copper to biomass.Copper-resistant bacteria could be isolated from a stock solution of CuSO₄, containing 100 ppm Cu(II). They were of thePseudomonas type and showed a much higher tolerance towards Cu²⁺, up to 10^–3 M.     

Effects of Zn2+ and Cu2+ on growth,lignin degradation and ligninolytic enzymes in Phanerochaete chrysosporium

The influence of Zn²⁺ (6.0 × 10^–3 –18.0 × 10^–3 M) and Cu²⁺ (4 × 10^–4 –1.2 × 10^–4 M) in the basal medium on mycelial growth (dry weight), activities of lignin peroxidase (Lip), manganese peroxidase (Mnp), solubilization, and mineralization (¹⁴CO₂ evolution) of lignin during a period of 3 weeks was studied in Phanerochaete chrysosporium strain MTCC-787. Highest mycelial growth was obtained at 0.6 M Zn²⁺ and 0.4 M Cu²⁺ levels. Enzyme activities were found to increase up to the highest levels of both the trace elements. However, Zn²⁺ had a relatively more stimulatory effect on Lip production and the reverse was true in case of Cu²⁺. [¹⁴C]Lignin solubilization was also promoted by higher levels of both trace elements. Mineralization of [¹⁴C]lignin was optimal at 6.0 M Zn²⁺ and 1.2 M Cu²⁺. The stimulatory effect of Zn²⁺ on Lip production was correlated with higher rates of [¹⁴C]lignin mineralization.     

Mercury removal by immobilized algae in batch culture systems

The accumulation and volatilization of mercury by non-immobilized and immobilizedChlorella emersonii have been studied in batch culture systems. Reduction in the mercury concentration in the growth medium by non-immobilized cells was highly dependent on inoculum density, whilst reduction in mercury concentration by immobilized cells was rapid at all inoculum densities. Mercury accumulation by immobilized cell biomass was significantly greater than by non-immobilized cells with 10⁶ and 10⁵ cells bead^–1 or ml^–1. Volatilization of mercury by non-immobilized cell systems was greatest at higher inoculum densities, whereas more mercury was volatilized from immobilized cell systems at lower inoculum densities, and was greatest with unstocked alginate beads. Thus, in immobilized systems, mercury removal from solution is complex and involves mercury accumulation by the cells and volatilization by the matrix and cells. Further studies of mercury accumulation and volatilization by unstocked immobilization matrices revealed that agarose volatilized much less mercury than alginate or agar. The precise mechanism of mercury volatilization by alginate remains unclear, though it is thought to be a chemical effect.     

A comparative study of antioxidative defense system in the copper and temperature acclimated strains of <Emphasis Type="Italic">Anabaena doliolum</Emphasis>

This study provides first hand comparative account of growth and antioxidative defense system of the wild type, Cu²⁺ and temperature treated wild type and acclimated strains of Anabaena doliolum Bharadwaja against Cu²⁺ and high temperature. The acclimated strains showed perceptible growth at 250 μM Cu²⁺ and 47°C temperatures, respectively. In contrast to this the wild type strain on exposure to 50 μM Cu²⁺ and 47°C temperature depicted almost complete inhibition of growth. However, the peroxide content was significantly higher in the acclimated strains than the wild type. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) showed maximum activity at high temperature followed by Cu²⁺ acclimated and minimum in the wild type strains. The ascorbate (ASC) and glutathione (GSH) contents were increased by 2.3 and 43.3, and 15.5 and 36.5-fold in Cu²⁺ and 47°C acclimated strains, respectively. However, when the wild type strain was subjected to Cu²⁺ and temperature all antioxidative enzymes except SOD showed inhibition of their activity. In case of wild type the GSH content was inhibited by 0.39-fold at 50 μM Cu²⁺ but the ASC content registered increase by 2 and 2.7-fold on subjecting to Cu²⁺ and temperature, respectively. Thus increased activity of enzymatic antioxidants as well as accumulation of ascorbate and glutathione in both the acclimated strains suggests that enzymatic and non-enzymatic antioxidants help in the acclimation of A. doliolum Bharadwaja against Cu²⁺ and high temperature. However, inhibition of antioxidative defense system of wild type under Cu²⁺ and heat stress appears to be the reason for its non survival. In view of the appreciable increase in the level of antioxidants as well as greater inhibition of specific growth rate in temperature than Cu²⁺ acclimated strains, temperature (47°C) is proposed to be is more deleterious to the organism than copper (250 μM).     

Enhanced sorption of Cu2+ and Ni2+ by acid-pretreated Chlorella vulgaris from single and binary metal solutions

The influence of HCl pretreatment (0.1 mM) on sorption ofCu²⁺ and Ni²⁺ by Chlorella vulgariswas tested using single and binary metal solutions. The optimal initial pH forsorption was 3.5 for Cu²⁺ and 5.5 for Ni²⁺. Second orderrate kinetics described well sorption by untreated and acid-pretreated cells.The kinetic constant q_e (metal sorption at equilibrium) for sorptionof test metals from single and binary metal solutions was increased afterpretreatment of the biomass with HCl. The Langmuir adsorption isotherm wasdeveloped for describing the various results for metal sorption. In single metalsolution, acid pretreatment enhanced q_max for Cu²⁺ andNi²⁺ sorption by approximately 70% and 65%, respectively.Cu²⁺ and Ni²⁺ mutually interfered with sorption of theother metal in the binary system. The combined presence of Cu²⁺ andNi²⁺ led to their decreased sorption by untreated biomass by 19% and88%, respectively. However, acid-pretreated biomass decreased Cu²⁺and Ni²⁺ sorption by 15 and 22%, respectively, when both the metalswere present in the solution. The results suggest a reduced mutual interferencein sorption of Cu²⁺ and Ni²⁺ from the binary metal systemdue to the acid pretreatment. Acid-pretreated cells sorbed twice the amount ofCu²⁺ and ten times that of Ni²⁺ than the untreated biomassfrom the binary metal system. Acid pretreatment more effectively enhanced thesorption of Ni²⁺ form the binary metal solution. The total metalsorption by untreated and acid-pretreated biomass depended on theCu²⁺ : Ni²⁺ ratio in the binary metal system. Acidpretreatment of C. vulgaris could be an effective andinexpensive strategy for enhancing Cu²⁺ and Ni²⁺ sorptionfrom single and binary metal solutions.     

Distribution and toxicity of a new colonial Microcystis aeruginosa bloom in the San Francisco Bay Estuary,California

The first distribution, biomass and toxicity study of a newly established bloom of the colonial cyanobacteria Microcystis aeruginosa was conducted on October 15, 2003 in the upper San Francisco Bay Estuary. Microcystis aeruginosa was widely distributed throughout 180 km of waterways in the upper San Francisco Bay Estuary from freshwater to brackish water environments and contained hepatotoxic microcystins at all stations. Other cyanobacteria toxins were absent or only present in trace amounts. The composition of the microcystins among stations was similar and dominated by demethyl microcystin-LR followed by microcystin-LR. In situ toxicity computed for the >75 m cell diameter size fraction was well below the 1 g l^–1 advisory level set by the World Health Organization for water quality, but the toxicity of the full population is unknown. The toxicity may have been greater earlier in the year when biomass was visibly higher. Toxicity was highest at low water temperature, water transparency and salinity. Microcystins from the bloom entered the food web and were present in both total zooplankton and clam tissue. Initial laboratory feeding tests suggested the cyanobacteria was not consumed by the adult copepod Eurytemora affinis, an important fishery food source in the estuary.     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: I. Necessity of Ca2+

Summary The relationship between the external Ca²⁺ concentrations [Ca²⁺]₀ and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J. 5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca²⁺]₀. Increasing [Ca²⁺]₀ caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La³⁺ solution. [Mg²⁺]₀ depressed the membrane electrical tolerance which was supposed to be due to competition with Ca²⁺ at the Ca²⁺ binding site of the membrane. Such a depressive effect of Mg²⁺ was almost masked when the [Ca²⁺]₀/[Mg²⁺]₀ ratio was roughly beyond 2.     

Toxic blooms of cyanobacteria in the Patos Lagoon Estuary,southern Brazil

The Patos Lagoon is the largest lagoonal system in South America. Its waters are formed by a huge drainage basin (201,600 km²) situated in the most industrialized areas of the Southern state of Rio Grande do Sul. On its margins more than 3 million inhabitants live in several cities and towns. The lagoon waters are used for leisure, drinking, industry, fisheries, agriculture and navigation. A monitoring and sampling program was developed from February 1994 to January 1996 with emphasis on the estuarine area, aiming to evaluate the occurrence of algal blooms. In the last 15 years, several cyanobacterial (blue-green algal) blooms of theMicrocystis aeruginosa have been registered in the lagoon estuary. HighM. aeruginosa biomass (50 to 9,000 g chla l^–1) was observed in the whole region in late summer and autumn 1994, and early summer 1995. The LD50 of toxic bloom samples tested in mice varied from 22 to 250 mg dry weight kg body weight^–1 while levels of toxicity (LC50) in the brine shrimp varied from 0.47 to 2.44 mg ml^–1. Toxicity varied in different blooms, in the distances along the scum and with time, within the same bloom. The hepatotoxin microcystin-LR was identified in almost all samples.     

Effect of salinity and pH on cobalt biosorption by the estuarine microalgaChlorella saliva

Summary Accumulation of cobalt (⁶⁰Co) by the estuarine microalgaChlorella salina has been characterized. At cobalt concentrations ranging over 3.125–100 M, a significant amount of cobalt was bound within 1 min. This was metabolism-independent and unaffected by incubation in light or dark conditions. This initial rapid phase of biosorption was followed by a slower phase of uptake which was apparently active and inhibited by incubation in the dark, or by the uncoupler dinitrophenol and the respiratory and photosynthetic inhibitor potassium cyanide in the light. For cells suspended in 10 mM Taps pH 8, cobalt biosorption followed a Freundlich adsorption isotherm. However, in the presence of 0.5 M NaCl, biosorption deviated from the Freundlich model because of competition by Na⁺. Cobalt biosorption was decreased by increasing concentrations of Na⁺, decreasing pH and the presence of Cs⁺, Li⁺, Rb⁺, Zn²⁺. Mn²⁺ and Sr²⁺ (added as chlorides). This was a result of competition between Co²⁺ and the other cations, including H⁺, for available binding sites on the cell wall and was confirmed by increased desorption of cobalt by solutions of low pH or high salinity. Increasing cell density resulted in increased removal of cobalt from solution but decreased the specific amount of cobalt taken up by the cells.     

Wall appositions induced by ionophore A 23187, CaCl2, LaCl3, and nifedipine in characean cells

Summary The formation of wall appositions (plugs) by ionophore A 23187, CaCl₂, LaCl₃, and nifedipine was studied in mature internodal cells of characeaen algae. CaCl₂ at concentrations above 10^–2M induces thick fibrillar plugs without callose inNitella flexilis. InChara corallina andNitella flexilis ionophore A 23187 (1.25×10^–5 to 5×10^–5M) and LaCl₃ (7.5×10^–5 to 2.5×10^–4M) cause flat appositions which contain callose and have a more granular structure. Plug formation by ionophore A 23187, CaCl₂, and LaCl₃ is pH-dependent and occurs beneath the alkaline regions of the cell. Nifedipine (10^–4 to 10^–5M) induces plugs inNitella flexilis after previous injury. These callose-containing wall appositions consist of a heterogeneous granular core which is covered by a fibrillar layer. The results of this work are compared with previous studies on wound wall formation and chlortetracycline (CTC)-induced plug formation which reveal that abundant coated vesicles occur only when a thick fibrillar wall layer is formed. Neither LaCl₃ nor nifedipine inhibit the formation of CaCl₂- or CTC-plugs. The unusual effects of these substances, which normally act as Ca²⁺ antagonists and therefore should prevent and not induce plug formation, are discussed. It is suggested that La³⁺ mimicks the effects of calcium and that nifedipine binding to the Ca²⁺ channels is altered in the alkaline regions of characean internodes and allows an influx of Ca²⁺.Abbreviations AFW artificial fresh water - CTC chlortetracycline - DCMU dichlorphenyldimethylurea - DMSO dimethylsulfoxide - EGTA ethyleneglycoltetraacetic acid - MES 2-(N-morpholino) ethanesulfonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - TAPS N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid