Biosorption of Cd(II) and Pb(II) onto brown seaweed, Lobophora variegata (Lamouroux): kinetic and equilibrium studies

The present work deals with the biosorption performance of raw and chemically modified biomass of the brown seaweed Lobophora variegata for removal of Cd(II) and Pb(II) from aqueous solution. The biosorption capacity was significantly altered by pH of the solution delineating that the higher the pH, the higher the Cd(II) and Pb(II) removal. Kinetic and isotherm experiments were carried out at the optimal pH 5.0. The metal removal rates were conspicuously rapid wherein 90% of the total sorption occurred within 90 min. Biomass treated with CaCl₂ demonstrated the highest potential for the sorption of the metal ions with the maximum uptake capacities i.e. 1.71 and 1.79 mmol g⁻¹ for Cd(II) and Pb(II), respectively. Kinetic data were satisfactorily manifested by a pseudo-second order chemical sorption process. The process mechanism consisting of both surface adsorption and pore diffusion was found to be complex. The sorption data have been analyzed and fitted to sorption isotherm of the Freundlich, Langmuir, and Redlich–Peterson models. The regression coefficient for both Langmuir and Redlich–Peterson isotherms were higher than those secured for Freundlich isotherm implying that the biosorption system is possibly monolayer coverage of the L. variegata surface by the cadmium and lead ions. FT-IR studies revealed that Cd(II) and Pb(II) binding to L. variegata occurred primarily through biomass carboxyl groups accompanied by momentous interactions of the biomass amino and amide groups. In this study, we have observed that L. variegata had maximum biosorption capacity for Cd(II) and Pb(II) reported so far for any marine algae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sorption of Cu(II) and Cd(II) by extracellular polymeric substances (EPS) from Aspergillus fumigatus

Sorption of Cu(II) and Cd(II) onto the extracellular polymeric substances (EPS) produced by Aspergillus fumigatus was investigated for the initial pH of the solution, EPS concentrations, contact time, NaCl concentration, initial metal ion concentration and the presence of other ions in the solution. The results showed that the adsorption of metal ions was significantly affected by pH, EPS concentrations, initial metal concentration, NaCl concentration and co-ions. The sorption of Cu(II) and Cd(II) increased with increasing pH and initial metal ion concentration but decreased with an increase in the NaCl concentration. The maximum sorption capacities of A. fumigatus EPS calculated from the Langmuir model were 40 mg g⁻¹ EPS and 85.5 mg g⁻¹ EPS for Cu(II) and Cd(II), respectively. The binary metal sorption experiments showed a selective metal binding affinity in the order of Cu(II) > Pb(II) > Cd(II). Both the Freundlich and Langmuir adsorption models described the sorption of Cu(II) and Cd(II) by the EPS of A. fumigatus adequately. Fourier transform infrared spectroscopy (FTIR) analysis revealed that carboxyl, amide and hydroxyl functional groups were mainly correlated with the sorption of Cu(II) and Cd(II). Energy dispersive X-ray (EDX) system analysis revealed that the ion-exchange was an important mechanism involved in the Cu(II) and Cd(II) sorption process taking place on EPS.     

Biosorption of heavy metals by the exopolysaccharide produced by <Emphasis Type="Italic">Paenibacillus jamilae</Emphasis>

The biosorption of several toxic heavy metals (Pb, Cd, Co, Ni, Zn and Cu) by the exopolysaccharide (EPS) produced by Paenibacillus jamilae, a potential biosorbent for metal remediation and recovery was studied. Firstly, the biochemical composition of this bacterial polymer was determined. Glucose was the most abundant neutral sugar, followed by galactose, rhamnose, fucose and mannose. The polymer presented a high content of uronic acids (28.29%), which may serve as binding sites for divalent cations. The presence of carboxylic groups was also detected by infrared spectroscopy. The EPS presented an interesting affinity for Pb in comparison with the other five metals. Lead biosorption (303.03 mg g⁻¹) was tenfold higher (in terms of mg of metal adsorbed per gram of EPS) than the biosorption of the rest of metals. Biosorption kinetics, the effect of pH and the effect of competitive biosorption were determined. Finally, we found that the EPS was able to precipitate Fe(III), but the EPS-metal precipitate did not form with Fe(II), Pb(II), Cd(II), Co(II), Ni(II), Cu(II) and Zn(II).     

Edible macroalga <Emphasis Type="Italic">Ulva prolifera</Emphasis> as microelemental feed supplement for livestock: the fundamental assumptions of the production method

In the present paper, the possibility of the application of marine macroalga Ulva (Enteromorpha) prolifera, as microelemental feed supplement for livestock, was evaluated. The concept was based on two facts: the natural macroalga contains high concentrations of microelements and there is a possibility to greatly increase this content via biosorption. In order to characterize the biosorption process of metal ions by U. prolifera, preliminary experiments were conducted with Cr(III) ions. The effect of temperature, pH and the biomass concentration on the equilibrium of biosorption was investigated. For further experiments (biosorption of Mn(II), Zn(II), Cu(II), Co(II)), the following experimental conditions were chosen: pH 5, 25°C, the biomass concentration 1.0 g l⁻¹. Equilibrium of the biosorption process could be described by the Langmuir equation. The theoretical maximum biosorption capacity was also determined by potentiometric titration of the biomass. The investigation of the external structure of the macroalga and atomic concentration of elements on the surface of the biomass was analyzed using scanning electron microscopy. The content of microelements in the biomass after biosorption increased 110,555; 44,228; 21,177; 2,281 and 1,458 times for Co(II), Cr(III),Cu(II), Zn(II), Mn(II), respectively. Therefore, biomass of U. prolifera enriched with individual microelements, mixed in the proper proportion could be used as feed supplement in animal feeding to cover the nutrient requirements for microelements.     

Characterisation of acidic sites of Pseudomonas biomass capable of binding protons and cadmium and removal of cadmium via biosorption

Summary The ability of Pseudomonas aeruginosa to accumulate Cd(II) ions from wastewater industries was experimentally investigated and mathematically modelled. From the potentiometric titration and non-ideal competitive analysis (NICA) model, it was found that the biomass contains three acidic sites. The values of proton binding (pK _i =1.66±3.26×10⁻³, 1.92±1.63×10⁻⁴ and 2.16±3.79×10⁻⁴) and binding constant of cadmium metal ions (pK _M1=1.99±2.45×10⁻³ and pK _M2=1.67±4.08×10⁻³) on the whole surface of biomass showed that protonated functional groups and biosorption of Cd(II) ions could be attributed to a monodentate binding to one acidic site, mainly the carboxylic group. From the isothermal sorption experimental data and Langmuir model, it was also found that the value of Langmuir equilibrium (pK _f) constant is 2.04±2.1×10⁻⁵ suggesting that the carboxyl group is the main active binding site. In addition, results showed that the maximum cadmium capacity (q _max) and affinity of biomass towards cadmium metal ions (b) at pH 5.1 and 20 min were 96.5±0.06 mg/g and 3.40×10⁻³± 2.10×10⁻³, respectively. Finally, interfering metal ions such as Pb(II), Cu(II), Cr(III), Zn(II), Fe(II), Mn(II), Ca(II) and Mg(II) inhibited Cd(II) uptake. Comparing the biosorption of Cd(II) by various Pseudomonas isolates from contaminated environment samples (soil and sewage treatment plant) showed that maximum capacities and equilibrium times were different, indicating that there was a discrepancy in the chemical composition between biomasses of different strains.     

Biosorption of cadmium(II), lead (II) and copper(II) with the filamentous fungus Phanerochaete chrysosporium

The biosorption from artificial wastewaters of heavy metals (Cd(II), Pb(II) and Cu(II)) onto the dry fungal biomass of Phanerochaete chryosporium was studied in the concentration range of 5-500 mg l(-1). The maximum absorption of different heavy metal ions on the fungal biomass was obtained at pH 6.0 and the biosorption equilibrium was established after about 6 h. The experimental biosorption data for Cd(II), Pb(II) and Cu(II) ions were in good agreement with those calculated by the Langmuir model.     

Nickel(II) biosorption by <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The present study reports the feasibility of using Rhodotorula glutinis biomass as an alternative low-cost biosorbent to remove Ni(II) ions from aqueous solutions. Acetone-pretreated R. glutinis cells showed higher Ni(II) biosorption capacity than untreated cells at pH values ranging from 3 to 7.5, with an optimum pH of 7.5. The effects of other relevant environmental parameters, such as initial Ni(II) concentration, shaking contact time and temperature, on Ni(II) biosorption onto acetone-pretreated R. glutinis were evaluated. Significant enhancement of Ni(II) biosorption capacity was observed by increasing initial metal concentration and temperature. Kinetic studies showed that the kinetic data were best described by a pseudo-second-order kinetic model. Among the two-, three-, and four-parameter isotherm models tested, the Fritz-Schluender model exhibited the best fit to experimental data. Thermodynamic parameters (activation energy, and changes in activation enthalpy, activation entropy, and free energy of activation) revealed that the biosorption of Ni(II) ions onto acetone-pretreated R. glutinis biomass is an endothermic and non-spontaneous process, involving chemical sorption with weak interactions between the biosorbent and Ni(II) ions. The high sorption capacity (44.45 mg g⁻¹ at 25°C, and 63.53 mg g⁻¹ at 70°C) exhibited by acetone-pretreated R. glutinis biomass places this biosorbent among the best adsorbents currently available for removal of Ni(II) ions from aqueous effluents.     

Biosorption of cadmium and lead ions by ethanol treated waste baker's yeast biomass

The biosorption of cadmium and lead ions from artificial aqueous solutions using waste baker's yeast biomass was investigated. The yeast cells were treated with caustic, ethanol and heat for increasing their biosorption capacity and the highest metal uptake values (15.63 and 17.49 mg g(-1) for Cd(2+) and Pb(2+), respectively) were obtained by ethanol treated yeast cells. The effect of initial metal concentration and pH on biosorption by ethanol treated yeast was studied. The Langmuir model and Freundlich equation were applied to the experimental data and the Langmuir model was found to be in better correlation with the experimental data. The maximum metal uptake values (qmax, mg g(-1)) were found as 31.75 and 60.24 for Cd(2+) and Pb(2+), respectively. Competitive biosorption experiments were performed with Cd(2+) and Pb(2+) together with Cu(2+) and the competitive biosorption capacities of the yeast biomass for all metal ions were found to be lower than in non-competitive conditions.     

Biosorption of Heavy Metals from Wastewater by Biosolids

In a study where the removal of heavy metals from wastewater is the primary aim, the biosorption of heavy metals onto biosolids prepared as Pseudomonas aeruginosa immobilized onto granular activated carbon was investigated in batch and column systems. In the batch system, adsorption equilibriums of heavy metals were reached between 20 and 50 min, and the optimal dosage of biosolids was 0.3 g/L. The biosorption efficiencies were 84, 80, 79, 59 and 42 % for Cr(VI), Ni(II), Cu(II), Zn(II) and Cd(II) ions, respectively. The rate constants of biosorption and pore diffusion of heavy metals were 0.013–0.089 min^–1 and 0.026–0.690 min^–0.5. In the column systems, the biosorption efficiencies for all heavy metals increased up to 81–100 %. The affinity of biosorption for various metal ions towards biosolids was decreased in the order: Cr = Ni > Cu > Zn > Cd.     

Comparison of the Heavy Metal Biosorption Capacity of Active,Heat‐Inactivated and NaOH‐Treated Phanerochaete chrysosporium Biosorbents

Three different kinds of Phanerochaete chrysosporium (NaOH‐treated, heat‐inactivated and active) biosorbent were used for the removal of Cd(II) and Hg(II) ions from aquatic systems. The biosorption of Cd(II) and Hg(II) ions on three different forms of Phanerochaete chrysosporium was studied in aqueous solutions in the concentration range of 50–700 mg/L. Maximum biosorption capacities of NaOH‐treated, heat‐inactivated and active Phanerochaete chrysosporium biomass were found to be 148.37 mg/g, 78.68 mg/g and 68.56 mg/g for Cd(II) as well as 224.67 mg/g, 122.37 mg/g and 88.26 mg/g for Hg(II), respectively. For Cd(II) and Hg(II) ions, the order of affinity of the biosorbents was arranged as NaOH‐treated > heat‐inactivated > active. The order of the amount of metal ions adsorbed was established as Hg(II) > Cd(II) on a weight basis, and as Cd(II) > Hg(II) on a molar basis. Biosorption equilibriums were established in about 60 min. The effect of the pH was also investigated, and maximum rates of biosorption of metal ions on the three different forms of Phanerochaete chrysosporium were observed at pH 6.0. The reusability experiments and synthetic wastewater studies were carried out with the most effective form, i.e., the NaOH‐treated Phanerochaete chrysosporium biomass. It was observed that the biosorbent could be regenerated using 10 mM HCl solution, with a recovery of up to 98%, and it could be reused in five biosorption‐desorption cycles without any considerable loss in biosorption capacity. The alkali‐treated Phanerochaete chrysosporium removed 73% of Cd(II) and 81% of Hg(II) ions from synthetic wastewater.     

Kinetic,thermodynamic, and equilibrium biosorption of Pb(II), Cu(II), and Ni(II) using dead mushroom biomass under batch experiment

In this study, a low-cost biosorbent, dead mushroom biomass (DMB) granules, was used for investigating the optimum conditions of Pb(II), Cu(II), and Ni(II) biosorption from aqueous solutions. Various physicochemical parameters, such as initial metal ion concentration, equilibrium time, pH value, agitation speed, particles diameter, and adsorbent dosage, were studied. Five mathematical models describing the biosorption equilibrium and isotherm constants were tested to find the maximum uptake capacities: Langmuir, Freundlich, Redlich-Peterson, Sips, and Khan models. The best fit to the Pb(II) and Ni(II) biosorption results was obtained by Langmuir model with maximum uptake capacities of 44.67 and 29.17 mg/g for these two ions, respectively, whereas for Cu(II), the corresponding value was 31.65 mg/g obtained with Khan model. The kinetic study demonstrated that the optimum agitation speed was 400 rpm, at which the best removal efficiency and/or minimum surface mass transfer resistance (MSMTR) was achieved. A pseudo-second-order rate kinetic model gave the best fit to the experimental data (R² = 0.99), resulting in MSMTR values of 4.69× 10^?5, 4.45× 10^?6, and 1.12× 10^?6 m/s for Pb(II), Cu(II), and Ni(II), respectively. The thermodynamic study showed that the biosorption process was spontaneous and exothermic in nature.     

Differential pulse polarography: a method for the direct study of biosorption of metal ions by live bacteria from mixed metal solutions

The technique of differential pulse polarography is shown here to be applicable to the monitoring directly the biosorption of metal ions from solution by live bacteria from mixed metal solutions. Biosorption of Cd(II), Zn(II) and Ni(II) by P. cepacia was followed using data obtained at the potential which is characteristic of the metal ion in the absence and presence of cells. Hepes buffer (pH 7.4, 50 mM) was used as a supporting electrolyte in the polarographic chamber and metal ion peaks in the presence of cells of lower amplitude were obtained due to metal-binding by the cells. Well defined polarographic peaks were obtained in experiments involving mixtures of metal ions of Cd(II)-Zn(II), Cu(II)-Zn(II), Cu(II)-Cd(II) and Cd(II)-Ni(II). Biosorption of Cd(II), Zn(II) increased with solution pH. The method was also tested as a rapid technique for assessing removal of metal ions by live bacteria and the ability of the polarographic technique in measuring biosorption of metal ions from mixed metal solutions is demonstrated. Cu(II) was preferentially bound and removal of metals was in the order Cu(II) > Ni(II) > Zn(II), Cd(II) by intact cells of P. cepacia. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biosorption of hexavalent chromium using biofilm of <Emphasis Type="Italic">E. coli</Emphasis> supported on granulated activated carbon

The optimization of hexavalent chromium biosorption has been studied by using three different biosorbents; biofilm of E. coli ASU 7 supported on granulated activated carbon (GAC), lyophilized cells of E. coli ASU 7 and granulated activated carbon. Supporting of bacteria on activated carbon decreased both the porosity and surface area of the GAC. Significant decrement of surface area was correlated to the blocking of microspores as a result of the various additional loads. The experimental data of adsorption was fitted towards the models postulated by Langmuir and Freundlich and their corresponding equations. The maximum biosorption capacity for hexavalent chromium using biofilm, GAC and E. coli ASU 7 were 97.70, 90.70, 64.36 mg metal/g at pH 2.0, respectively. Biosorption mechanism was related mainly to the ionic interaction and complex formation. Based on the experimental conditions, the presence of bacteria could be enhanced the capacity of activated carbon to adsorb hexavalent chromium ions from aqueous solutions.     

The biosorption of heavy metals from aqueous solution by Spirogyra and Cladophora filamentous macroalgae

The aim of this research was to develop a low cost adsorbent for wastewater treatment. The prime objective of this study was to search for suitable freshwater filamentous algae that have a high heavy metal ion removal capability. This study evaluated the biosorption capacity from aqueous solutions of the green algae species, Spirogyra and Cladophora, for lead (Pb(II)) and copper (Cu(II)). In comparing the analysis of the Langmuir and Freundlich isotherm models, the adsorption of Pb(II) and Cu(II) by these two types of biosorbents showed a better fit with the Langmuir isotherm model. In the adsorption of heavy metal ions by these two types of biosorbents, chemical and physical adsorption of particle surfaces was perhaps more significant than diffusion and adsorption between particles. Continuous adsorption-desorption experiments discovered that both types of biomass were excellent biosorbents with potential for further development.     

Equilibrium and kinetics of biosorption of cadmium(II) and copper(II) ions by wheat straw

Biosorption equilibrium and kinetics of Cd(2+) and Cu(2+) ions on wheat straw, Triticum aestivum, in an aqueous system were investigated. Among the models tested, namely the Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich isotherms, the biosorption equilibrium for both Cd(2+) and Cu(2+) was best described by the Langmuir model. The Langmuir biosorption capacity for Cd(2+) was about 27% higher than that for Cu(2+). It was also found that biosorption of Cd(2+) and Cu(2+) by wheat straw followed second-order kinetics. The equilibrium amount of metal ions adsorbed onto the wheat straw increased with increasing of pH from 4.0 to 7.0, and the effect was more pronounced for Cd(2+) than for Cu(2+). The equilibrium adsorbed amount also increased with the initial concentration of the metal ions, as expected. On the other hand, an increase of temperature from 25 to 30 degrees C only enhanced the biosorption of Cd(2+) and Cu(2+) slightly. The apparent temperature independence and the strong pH dependence of the amount of metal ions adsorbed along with moderate mean free energies of biosorption (between 8.0 and 12.9 kJ mol(-1)) altogether indicate that biosorption of Cd(2+) and Cu(2+) by wheat straw might follow a chemisorption mechanism.     

Synthesis and characterization of a crosslinked chitosan derivative with a complexing agent and its adsorption studies toward metal(II) ions

A new chitosan derivative has been synthesized by crosslinking a metal complexing agent, [6,6′-piperazine-1,4-diyldimethylenebis (4-methyl-2-formyl) phenol] (L), with chitosan (CTS). The resulting material (CCTSL) was characterized by elemental (CHN), spectral (FTIR and solid-state NMR), thermal (TGA and DTA), and structural (powder XRD and SEM) analyses. Adsorption experiments (pH dependency, kinetics, and equilibrium) of CCTSL toward various metal ions such as Mn(II), Fe(II), Co(II), Cu(II), Ni(II), Cd(II), and Pb(II) were carried out at 25 °C. The results showed that the adsorption was dependent on the pH of the solution, with a maximum capacity between pHs 6.5 and 8.5. The kinetics was evaluated by applying the pseudo-first-order and pseudo-second-order equation models and the equilibrium data were analyzed by Langmuir isotherm model. The maximum adsorption capacity was 1.21 mmol g⁻¹ for Cu(II) and the order of adsorption capacities for the metal(II) ions studied was found to be Cu(II) > Ni(II) > Cd(II)  Co(II)  Mn(II)  Fe(II)  Pb(II).     

Using biosorption to enrich the biomass of <Emphasis Type="Italic">Chlorella vulgaris </Emphasis>with microelements to be used as mineral feed supplement

The paper discusses biosorption of Cr(III), Cu(II), Mn(II), Zn(II) and Co(II) to the biomass of Chlorella vulgaris, to produce a biologically bound, concentrated form of microelements. The kinetics of biosorption was described with a pseudo-second order equation and equilibrium with the Langmuir isotherm. The mechanism of biosorption was identified as cation-exchange with alkaline metals. Cation-exchange capacity was evaluated as 4.07 meq g⁻¹. The effect of operation conditions, pH and temperature, on biosorption performance was investigated and the best operation conditions for biosorption were selected (pH 5, temperature 25 °C). The maximum sorption capacity of microelements was determined in single-metal system at pH 5 and 25 °C: Zn(II) 3.30 meq g⁻¹, Cu(II) 1.77 meq g⁻¹, Co(II) 1.75 meq g⁻¹, Cr(III) 1.74 meq g⁻¹, Mn(II) 0.764 meq g⁻¹. Biosorption experiments were also carried out in multi-metal system. The biomass of C. vulgaris enriched with microelements via the process of biosorption in both single- and multi-metal system was discussed in terms of preparation of feed supplement for laying hens and piglets. The experiments showed that 1 kg of conventional feed for laying hens can be supplemented with 0.20 g of the biomass enriched with microelements and for piglets with 0.15 g of the preparation.     

Biosorption of Copper by Paenibacillus polymyxa Cells and their Exopolysaccharide

Summary Biosorption of heavy metals by gram-positive, non-pathogenic and non-toxicogenic Paenibacillus polymyxa P13 was evaluated. Copper was chosen as a model element because it is a pollutant originated from several industries. An EPS (exopolysaccharide)-producing phenotype exhibited significant Cu(II) biosorption capacity. Under optimal assay conditions (pH 6 and 25 °C), the adsorption isotherm for Cu(II) in aqueous solutions obeyed the Langmuir model. A high q value (biosorption capacity) was observed with whole cells (q_max=112 mgCu g⁻¹). EPS production was associated with hyperosmotic stress by high salt (1 M NaCl), which led to a significant increase in the biosorption capacity of whole cells (q_max=150 mgCu g⁻¹). Biosorption capacity for Cu(II) of the purified EPS was investigated. The maximum biosorption value (q) of 1602 mg g⁻¹ observed with purified EPS at 0.1 mg ml⁻¹ was particularly promising for use in field applications.     

Potential capacity of Beauveria bassiana and Metarhizium anisopliae in the biosorption of Cd2+ and Pb2+

In this study Beauveria bassiana and Metarhizium anisopliae were used as inexpensive and efficient biosorbents for Pb(II) and Cd(II) from aqueous metal solutions. The effects of various physicochemical factors on Pb(II) and Cd(II) biosorption by B. bassiana and M. anisopliae were studied. The optimum pH for Cd(II) and Pb(II) biosorption by two fungal species was achieved at pH 6.0 for Pb(II) and 5.0 Cd(II) at a constant time of 30 min. The nature of fungal biomass and metal ion interactions was evaluated by Fourier transform infrared. The maximum adsorption capacities (q(max)) calculated from Langmuir isotherms for Pb(II), and Cd(II) uptake by B. bassiana were 83.33±0.85, and 46.27±0.12 mg/g, respectively. However, the q(max) obtained for Pb(II) uptake by M. anisopliae was 66.66±0.28 mg/g, and 44.22±0.13 mg/g for Cd(II). B. bassiana showed higher adsorption capacity compared to M. anisopliae. The data obtained imply the potential role of B. bassiana and M. anisopliae for heavy metal removal from aqueous solutions.     

Effect of the presence of lead on the biosorption of copper,cadmium and nickel by anaerobic biomass

Heavy metals are toxic pollutants released into the environment as a result of industrial, mining and agricultural activities. The biosorption of Pb, Cu, Cd, and Ni from single and binary metal systems were studied in equilibrium systems and in a flow-through column packed with a calcium-saturated anaerobic sludge biosorbent, respectively. The single-metal sorption uptake capacity of the biomass for Pb was slightly inhibited by the presence of Cu and Cd cations (by 6%) and by the presence of nickel (by 11%). The affinity order of anaerobic biomass for the four metals was established as: Pb > Cu > Ni > Cd. Factors such as hydration effects, hydrolysis effects and covalent binding of the metal ions may contribute to this result. By studying the breakthrough curves obtained from a fixed bed column fed with an equimolar mixture of Pb, Cd, Cu, and Ni, it was determined that lead was the last metal to break through the column at the 150 bed volume mark compared to 4, 15, 30 bed volume marks for Ni, Cd, and Cu, respectively.