Bioremediation Potential of Chlorella: Spectroscopic,Kinetics, and Sem Studies

A dead dried alga, Chlorella sp., was used for the uptake of Cr⁺³, Cr₂O₇ ^?2, Cu⁺², and Ni⁺² from the aqueous solutions of these metal ions. The equilibrium data were fitted using the Langmuir and Freundlich isotherm model and the maximum uptakes for Cr⁺³, Cr₂O₇ ^?2, Ni⁺², and Cu⁺² were 98, 104, 108, and 183 mg/g, respectively. The Freundlich model, in comparison to the Langmuir model, better represented the sorption process. The kinetics of metal ions uptake by Chlorella sp. was best described by a pseudo-second order rate equation. Infrared spectroscopic data were employed to identify the site(s) of bonding in Chlorella sp. A scanning electron microscopic (SEM) study of pure dead Chlorella sp. and the species treated with different metal ions provided an idea of the extent of metal uptake by this species. The dead Chlorella sp took up maximum Cu(II). The size of the cell of the metal-treated Chlorella sp. obtained from SEM data is in agreement with the extent of metal uptake.     

Ammonium uptake by Chlorella

The preincubation of Chlorella cells with glucose caused a tenfold increase of the maximal uptake rate of ammonium without change in the K _m (2 M). A similar stimulation of ammonium uptake was found when the cells were transferred to nitrogen-free growth medium. The time-course of uptake stimulation by glucose revealed a lag period of 10–20 min. The turnover of the ammonium transport system is characterized by a half-life time of 5–10 h, but in the presence of light 30% of uptake activity stayed even after 50 h. 6-Deoxyglucose was not able to increase the ammonium uptake rate. These data together were interpreted as evidence for induction of an ammonium transport system by a metabolite of glucose. Mechanistic studies of the ammonium transport system provided evidence for the electrogenic uptake of the ammonium ion. The charge compensation for NH ₄ ⁺ entry was achieved by immediate K⁺ efflux from the cells, and this was followed after 1 min by H⁺ extrusion. Ammonium accumulated in the cells; the rate of uptake was sensitive to p-trifluoromethoxy-carbonylcyanide-phenylhydrazon and insensitive to methionine-sulfoxime. Uptake studies with methylamine revealed that methylamine transport is obviously catalyzed by the ammonium transport system and, therefore, also increased in glucose-treated Chlorella cells.Abbreviation p.c. packed cells     

Bioaccumulation of Heavy Metals by Green Algae

The biosorption of metal ions (Cr⁺³, , Cu⁺², and Ni⁺²) on two algal blooms (designated HD-103 and HD-104) collected locally was investigated as a function of the initial metal ion concentration. The main constituent of HD-103 is Cladophora sp., while Spirulina sp. is present significantly in the bloom HD-104. Algal biomass HD-103 exhibited the highest Cu⁺² uptake capacity (819 mg/g). This bloom adsorbed Ni⁺² (504 mg/g), Cr⁺³ (347 mg/g), and (168 mg/g). Maximum of Ni⁺² (1108 mg/g) is taken by HD-104. This species takes up 306, 202, and 576 mg/g Cr⁺³, , and Cu⁺², respectively. Equilibrium data fit very well to both the Langmuir and the Freundlich isotherm models. The sorption process followed the Freundlich model better. Pseudo-first-order kinetic model could describe the kinetic data. Infrared (IR) spectroscopic data were employed to identify the site(s) of bonding. It was found that phosphate and peptide moieties participate in the metal uptake by bloom HD-103. In the case of bloom HD-104, carboxylate and phosphate are responsible for the metal uptake. The role of protein in metal uptake by HD-103 was investigated using polyacrylamide gel electrophoresis.     

Effects of Ca2+ and Temperature on Potassium Uptake along Roots of Wheat, Rice and Cucumber

The differences in K⁺ uptake of different segments of excised roots of two thermophilic plants (rice, Oryza sativa L. cv. Dunghan Shali and cucumber, Cucumis sativus L. cv. Csemege Fürtös) and a non-thermophilic plant (wheat, Triticum aestivum L. cv. Aurora) were investigated in the presence and absence of Ca²⁺, at 0 and 25°C, using radiotracer K⁺(⁸⁶Rb⁺) technique. The K⁺ uptake exhibited different temperature- and Ca²⁺-dependent distributions along the root axis for the different species studied. In the case of rice and cucumber an extraordinarily large K⁺ uptake occurred in the apical root portion at 0°C if Ca²⁺ was omitted. The presence of Ca²⁺ diminished this anomaly. For wheat normal K⁺ uptake patterns were observed under similar conditions. At 25°C Ca²⁺-stimulated K⁺ uptake may appear in each root segment, depending upon species and composition of the uptake solution. The results indicate that there may be considerable differences in the compositions of the cell walls and membranes of root cells of thermophilic and non-thermophilic plants, and in their ion-exchange properties, especially in the apical region.     

Effect of nickel on certain physiological and biochemical behaviors of an acid tolerantChlorella vulgaris

This study concerns the inhibitory effects of acid pH and nickel on growth, nutrient (NO₃ ^- and NH₄ ⁺) uptake, carbon fixation, O₂ evolution, electron transport chain and enzyme (nitrate reductase and ATPase) activities of acid tolerant and wild-type strains of Chlorella vulgaris. Though a general reduction in all these variables was noticed with decreasing pH, the tolerant strain was found to be metabolically more active than the wild-type. A reduced cation (NH₄ ⁺, Na⁺, K⁺ and Ca²⁺) uptake, coupled with a facilitated influx of anions (NH₄ ⁺, PO₄ ^3- and HCO₃ ^-), suggested the development of a positive membrane potential in acid tolerant Chlorella. Nevertheless, a tremendous increase in ATPase activity at decreasing pH revealed the involvement of superactive ATPase in exporting H⁺ ions and keeping the internal pH neutral. A difference in Na⁺ and K⁺ efflux of the two strains at decreasing pH suggests there is a difference in membrane permeability. The low toxicity of Ni in the acid tolerant strain may be due to the low Ni uptake brought about by a change in membrane potential as well as in permeability. Hence, the development of superactive ATPase and a change in both membrane potential and permeability not only offers protection against acidity, but also co-tolerance to metals.     

The Occurrence of the Glucose-inducible Transport Systems for Glucose,Proline, and Arginine in Different Species of Chlorella

Incubation of the green alga Chlorella vulgaris (strain K, Tanner and Kandler, 1967) with glucose leads to the induction of a glucose transport system and of two amino acid transport systems. Because it was not clear whether the regulation of 3 different transport systems by glucose is specific to our strain of Chlorella or whether it is a general property of the genus Chlorella, 11 other free living and symbiotic Chlorella species and strains were tested for glucose-inducible glucose, arginine and proline transport. It was found that nearly all Chlorella species possess glucose and amino acid uptake systems. Often they were constitutive, although in some species they were induced or stimulated by glucose. According to the transport activities of the different Chlorella species and strains, a physiological classification of Chlorella was constructed, resulting in 3 groups: the C. fusca vacuolata, the C. vulgaris and the symbiotic Chlorella group. Our Chlorella (strain K) obviously belongs to the C. vulgaris group and forms a link to symbiotic Chlorella strains. This suggests that the possession of the glucose-regulated transport systems is of advantage for Chlorella in symbiotic situations, whereas the constitutive systems are useful for free living Chlorella.     

Screening of algal strains for metal removal capabilities

Eight algal species were tested for their ability to remove five toxic metalsduring 30-min exposures to single-metal (1 mg L^-1) solutions at pH7. Efficacy of metal bioremoval varied according to algal species and metal. Al⁺³ was best removed by the thermophilic blue-green alga(cyanobacterium) Mastigocladus laminosus, Hg⁺² and Zn⁺² by the thermophilic and acidophilic red alga Cyanidiumcaldarium, and Cd⁺² by C. caldarium and the green alga Scenedesmus quadricauda. All of these alga/metal combinations resultedin >90% metal removal. However, none of the eight algal speciesremoved more than 10% of Cr⁺⁶. Results indicate that some toxicmetals are more readily removed than others are by algae and that selectionof appropriate strains could potentially enhance bioremoval of specificmetals from wastewater at neutral pH.     

Interspecies variation in nitrogen uptake kinetic responses of temperate forest species to elevated CO2: potential causes and consequences

Despite the recognition that the capacity to acquire N is critical in plant response to CO₂ enrichment, there is little information on how elevated CO₂ affects root N uptake kinetics. The few available data indicate a highly variable pattern of response to elevated CO₂, but it is presently unclear if the observed inconsistencies are caused by differences in experimental protocols or by true species differences. Furthermore, if there are interspecific variations in N uptake responses to elevated CO₂, it is not clear whether these are associated with different functional groups. Accordingly, we examined intact root‐system NH₄⁺ and NO₃^– uptake kinetic responses to elevated CO₂ in seedlings of six temperate forest tree species, representing (i) fast‐ vs. slow‐growers and (ii) broad‐leaves vs. conifers, that were cultured and assayed in otherwise similar conditions. In general, the species tested had a higher uptake capacity (V_max) for NH₄⁺ than for NO₃^–. Species substantially differed in their NO₃^– and NH₄⁺ uptake capacities, but the interspecific differences were markedly greater for NO₃^– than NH₄⁺ uptake. Elevated CO₂ had a species‐dependent effect on root uptake capacity for NH₄⁺ ranging from an increase of 215% in Acer negundo L. to a decrease of about 40% in Quercus macrocarpa Michx. In contrast, NO₃^– uptake capacity responded little to CO₂ in all the species except A. negundo in which it was significantly down‐regulated at elevated CO₂. Across species, the capacity for NH₄⁺ uptake was positively correlated with the relative growth rate (RGR) of species; however, the CO₂ effect on NH₄⁺ uptake capacity could not be explained by changes in RGR. The observed variation in NH₄⁺ uptake response to elevated CO₂ was also inconsistent with life‐form differences. Other possible mechanisms that may explain why elevated CO₂ elicits a species‐specific response in root N uptake kinetics are discussed. Despite the fact that the exact mechanism(s) for such interspecific variation remains unresolved, these differences may have a significant implication for competitive interactions and community responses to elevated CO₂ environment. We suggest that differential species responses in nutrient uptake capacity could be one potential mechanism for the CO₂‐induced shifts in net primary productivity and species composition that have been observed in experimental communities exposed to elevated levels of CO₂.     

Temporal patterns of inorganic nitrogen uptake by mature sugar maple (Acer saccharum Marsh.) and red spruce (Picea rubens Sarg.) trees using two common approaches

Background: Plant uptake of nitrogen influences many ecosystem processes, yet uptake by trees in northern forests of the United States has not been quantified throughout the growing season.

Aims: To measure NH₄ ⁺ and NO₃ ^? uptake by mature sugar maple (Acer saccharum) and red spruce (Picea rubens) trees during the early, mid and late growing season.

Methods: At Hubbard Brook Experimental Forest, New Hampshire, we used two approaches to measure nitrogen uptake capacity by mature trees: an in situ depletion method using intact roots and an ex situ ¹⁵N tracer method using excised roots.

Results: NH₄ ⁺ uptake was greater than NO₃ ^? for both methods and tree species (P < 0.05). NH₄ ⁺ uptake was lowest during the early growing season, while NO₃ ^? uptake was lowest during the late growing season. Measured rates of NH₄ ⁺ uptake were 2–3 orders of magnitude greater using the in situ depletion method compared with the ex situ ¹⁵N tracer method.

Conclusions: These results demonstrate seasonal differences in nitrogen uptake by two dominant tree species in a northern forest and show that the method employed can significantly impact measured rates of uptake, which could have implications for understanding the magnitude of plant nitrogen uptake and for cross-study comparisons of this process.     

The effect of sodium chloride on the Ca2+, K+ and Na+ concentrations of the seed coat and embryo of Acacia tortilis and A. coriacea

The uptake of Na⁺ and the loss of Ca²⁺ and K⁺ by seeds of Acacia tortilis (Forsk.) Hayne (salt tolerant) and A. coriacea DC. (salt sensitive) were determined after 24 h soaking in 250 mol m^-1,3 NaCl or in distilled water. Na⁺ uptake was higher by the seed coat than by the embryo of both species and higher by A. coriacea than by A. tortilis. The greater Na⁺ uptake by A. coriacea was associated with greater Ca and K⁺ leakage. The Na⁺ concentration of solution imbibed by the embryo of both species was lower than the Na⁺ concentration in the external solution, indicating an exclusion of Na⁺. When A. tortilis and A. coriacea seeds were treated with a series of NaCl concentration (0–400 mol m^-1,3), the exclusion mechanism was particularly clear with A. tortilis at lower concentrations (50 and 150 mol m^-1,3) of NaCl. In contrast, the seed coat of both species accumulated Na⁺. Thus the seed coat may play an important role in ion exchange. These results show that it is important to consider the seed coat and embryo separately rather than the whole seed when considering ion exchange in relation to salinity tolerance.     

Algal bioassays to determine toxicity of metal mixtures

The shortcomings of many established toxicity criteria for metals have resulted from a simplification of bioassays performed with single metals. A more comprehensive approach was needed to diagnose the effects of metal mixtures on aquatic organisms. Using Chlorella, Ankistrodesmus and Scenedesmus as test organisms, we experimented on a number of factors which affected metal toxicity bioassays. These included metal interactions, algal competitions, species sensitivities, the ratio of an excess metal to other metals and nutrient levels. An alternative technique was finally established which involved an evaluation of individual assays of Chlorella and Ankistrodesmus in separate media and a tissue-metal analysis on Chlorella. Chlorella fusca, commonly found in lakes with high metal concentrations, showed high tolerance to mixed-metal solutions, while Ankistrodesmus proved to be very sensitive. By determining the maximum yield ratio between Ankistrodesmus and Chlorella (i.e. the A/Ch ratio) it was possible to compare the toxic strength of harmful metals according to an established standard curve of A/Ch ratio versus mixed-metal concentrations. The levels of tissue-metal analysed in Chlorella also gave some indication as to which metals were responsible for the toxicity.     

Characterization of cadmium removal by Rhodotorula sp. Y11

Experiments were conducted studying the removal of Cd²⁺ from water via biosorption using Rhodotorula sp. Y11. The effects of temperature and initial pH of the solution on biosorption were studied. Caustic and heat treatments showed different influences on the biosorption capacity, and the highest metal uptake value (19.38 mg g⁻¹) was obtained by boiling treated yeast cells. The presence of competing cations, such as Ag⁺, Cu²⁺, and Mg²⁺, except Na⁺ ions, significantly interfered with the metal uptake. Results indicate that the Langmuir model gave a better fit to the experimental data than the Freundlich equation. The q ₁₀ value was 11.38 mg g⁻¹ for Cd²⁺ uptake by Y11. Chemical modifications of the biomass demonstrated that carboxyl and amide groups play an important role in Cd²⁺ biosorption.     

Immobilization of microbial cells and enzymes with hydrophobic photo-crosslinkable resin prepolymers

Summary The accumulation of cadmium from aqueous systems by various green microalgae was investigated with focus, on Chlorella regularis as it is known to concentrate large amounts of heavy metals. The amount of cadmium absorbed by Chlorella cells was rapid during the first 30 min following addition of cadmium and then continued to be absorbed more slowly. The uptake of cadmium by Chlorella was not markedly affected by temperature or metabolic inhibitors. Most of the cadmium absorbed by Chlorella cells was easily released by EDTA. The amount of cadmium absorbed differed markedly with the pH value of the solution and was inhibited by the presence of other divalent cations. Heat-killed Chlorella cells took up cadmium to a greater degree than living ones. From these results, it was considered that the uptake of cadmium into Chlorella cells was not directly mediated by metabolic processes, rather it appeared completely dependent upon physico-chemical adsorption on the cell surface.The ability to accumulate cadmium was species specific and found to be (in decreasing order); Chlamydomonas reinhardtii>Chlorella regularis> Scenedesmus bijuga>Scenedesmus obliquus>Chlamydomonas angulosa> Scenedesmus chlorelloides.Studies on the Accumulation of Heavy Metal Elements in Biological Systems Part XIV     

Toxicity of heavy metals to Asellus aquaticus (L.) (Crustacea,Isopoda)

Effects of heavy metals on the isopod Asellus aquaticus (L.) are studied by static toxicity tests. Results demonstrate that the species is sensitive to Cd⁺², Cr⁺⁶, Cu⁺², Fe⁺³, Hg⁺², Ni⁺² Pb⁺² and Zn⁺², but the toxicity of each metal is different. Differences are also found between adults and between adults and juveniles. The comparative analysis of all data on the toxicity has been performed on the concentrations of metal ions and not on metal compound concentrations.Criteria for establishing water quality in order to guarantee protection of the environment are discussed.     

Rhizobium strains expressing uptake hydrogenase in different host species of cowpea miscellany

Uptake hydrogenase activity in nodules of green gram (Vigna radiata (L.) (Wilczek)), black gram (Vigna mungo (L.) (Hepper)), cowpea (Vigna unguiculata (L.) and cluster bean (Cyamopsis tetragonoloba (L.) (Taub.)), formed with two Hup⁺ (S24 and CT2014) and one Hup⁻ (M11)Rhizobium strains, was determined at different levels of external H2 in air atmosphere. Nodules of all the 4 host species formed by inoculation with strains S24 and CT2014, showed H2 uptake but not those formed with strain M11. H₂ uptake rates were higher in 1 and 2% H₂ in air atmosphere (v/v) than at 5 or 10% levels in all the host species. Variations in the relative rates of H₂ uptake were observed both, due to host species as well as due toRhizobium strains. However, no host dependent complete repression of the expression of H₂ uptake activity was observed in nodules of any of the host species formed with Hup⁺ strains.     

Short term 22Na+ and 42K+ uptake in intact, mid-vegetative plants

Spergularia marina (L.) Griseb. is. a rapidly growing, annual, coastal halophyte. Because of its small size, it is suitable for isotope studies of ion transport well beyond the seedling stage. The purpose of this report is to establish the similarities and differences between ²²Na⁺ and ⁴²K⁺ uptake in S. marina and in more commonly used mesophytic crop species. Vegetative plants were used 18 days after transfer to solution culture. Plants were grown either on Na⁺-free medium or on 0.2 × sea water. ²²Na⁺ uptake was linear with time for several hours. The rate was relatively insensitive to external concentration between 1 and 180 mol Na⁺ m?³, particularly in Na⁺-free plants. Transport to the shoot accounted for 40 to 70% of the total uptake, dependent on salinity but largely independent of time. ⁴²K⁺ uptake decreased with increasing salinity in Na⁺-free plants and increased in 0.2 × sea water plants. Both uptake and transport to the shoot were non-linear with time, upward concavity suggesting recovery from a manipulative and/or osmotic injury. Steady state root contents were compared with predicted contents based on cortical cell electrical potentials using the Nernst equation. Reasonable agreement was found in all cases except Na⁺ content of 0.2 × sea water plants, in which active efflux was indicated. Uptake studies conducted in the presence of chemical modifiers (dicyclohexylcarbodiimide, dinitrophenol and fusicoccin) showed responses of ⁴²K⁺ uptake as expected from studies on agronomic species, and implied the presence of a similar active uptake here despite the appearance of equilibrium. Active Na⁺ uptake was suggested at low Na⁺ levels. We conclude that S. marina is a promising experimental system combining the rapid nutrient acquisition strategy of agionomically important annuals with a high degree of salt tolerance.     

Waste water treatment and metal (Pb<Superscript>2+</Superscript>, Zn<Superscript>2+</Superscript>) removal by microalgal based stabilization pond system

A case study was undertaken for the treatment of domestic wastewater generated at village of Sanghol, Distt. Fatehgarh Sahib, Punjab (India), using a schematic designed algal and duckweed based stabilization pond system, which is discussed here for winter months only (November to March) as there was no growth of duckweeds and only algae dominated the whole system. A proficient increase in pH and dissolved oxygen was observed after the treatment with reduction in chemical oxygen demand and biochemical oxygen demand by 93% and 79% respectively. Chlorella sp. was the dominating algal species in the stabilization pond water during entire period and was studied for its Zn²⁺ and Pb²⁺ metal removal efficiency. 60–70% removal of Zn²⁺ was observed from culture medium containing 5–20 mg L^?1 Zn²⁺, which declined to 42% at 50 mg L^?1. A constant decline in cell number from 538 × 10⁵ to 8 × 10⁵ cells ml^?1 was observed indicating zinc toxicity to Chlorella. Lead was maximally removed by 66.3% from culture medium containing 1 mg L^?1. The lead removal efficiency was 45 50 % at higher 5 to 20 mg L^?1 of external lead concentrations. The increase in cell number indicated no signs of Pb²⁺ toxicity up to 20 mg L^?1. The maximum uptake (q _max) by live Chlorella biomass for both Zn²⁺ and Pb²⁺ was 34.4 and 41.8 mg/g respectively.     

Examination of the substrate stoichiometry of the intestinal Na+/phosphate cotransporter

Summary The substrate stoichiometry of the intestinal Na⁺/phosphate cotransporter was examined using two measures of Na⁺-dependent phosphate uptake: initial rates of uptake with [³²P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC₃(5). Isotopic phosphate measures electrogenic and electroneutral Na⁺-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na⁺/phosphate cotransport was found to have similar Na⁺ activations (apparentK _0.5's of 28 and 25mm), apparentK _m 's for phosphate (100 and 410 m), andK _0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na⁺-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na⁺ with apparentK _0.5 for phosphate of 200 m; similar to the apparentK _m for phosphate uptake, andK _0.5 for phosphate protection against FITC-PG inhibition of Na⁺-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na⁺/phosphate cotransporter is electrogenic at pH 7.4, that H₂PO ₄ ^– is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na⁺/phosphate cotransporter.     

Sodium/potassium selectivity and pleiotropy in stl2, a highly salt-tolerant mutation of Ceratopteris richardii

The roles of Na⁺ and K⁺ (Rb⁺) uptake were further studied in a NaCl-tolerant strain of Ceratopteris richardii containing the stl2 mutation by direct comparison with the wild-type strain. In addition to Na⁺ tolerance, stl2 also confers tolerance to Mg²⁺ and sensitivity to K⁺. In addition to higher K⁺ (Rb⁺) uptake at concentrations commonly associated with low-affinity K⁺ transport, stl2 maintained higher uptake down to 0·1 mol m^–3 Rb⁺. Up to a 25-fold excess of Na⁺ had little effect in either genotype on K⁺ (Rb⁺) uptake at low concentrations, i.e. 0·2 and 0·5 mol m^–3 RbCl. Pretreatment with K⁺ (20 mol m^–3) inhibited uptake of K⁺ (Rb⁺) in the wild type, whereas concurrent inclusion of K⁺ inhibited uptake of Rb⁺ more in stl2. In the absence of K⁺, Na⁺ uptake (0·01–60 mol m^–3) was nearly identical in the wild type and stl2. K⁺ inhibited Na⁺ uptake more effectively in stl2 than the wild type, especially at 60 mol m^–3 Na⁺. Greater inhibition of K⁺ uptake in stl2 occurred with MgCl₂ or TEA (tetraethylammonium chloride) preincubation or with simultaneous inclusion of Al³⁺ (Al₂SO₄). The higher effective velocity of K⁺ uptake at a wide range of concentrations and the enhanced selectivity for K⁺ and against Na⁺ contribute to the preservation of higher cytosolic K⁺ and lower Na⁺ under salinity stress.