a new approach for purification of recombinant human ...

BIOSORPTION OF COOPER (II) BY IMMOBILIZED DEAD BIOMASS OF SACCHAROMYCES CEREVISIAE V. Gochev1, Z. Velkova2 and M. Stoytcheva2 1 “Paisii Hilendarski” University of Plovdiv, Plovdiv, Bulgaria 2 University of Food Technologies, Plovdiv, Bulgaria Correspond to: velizar Gochev E-mail: [email protected]

ABSTRACT At present study the biosorption potential of dead biomass of Saccharomyces cerevisiae immobilized in Ca-alginate and coimmobilized in Ca-alginate and bentonite and Ca-alginate and activated carbon for removal of Cu (II) from model solutions was investigated. The highest biosorption potential demonstrated the biosorbent of S. cerevisiae co-immobilized in Ca-alginate and activated carbon. The effect of pH, biosorbent concentration, contact time and initial metal concentration on the Cu (II) removal was studied. The optimal pH value for metal removal was 4,0 and biosorption equilibrium was reached for about 30 min. The increasing of biosorbent concentration increased metal removal by the selected biosorbent. At the equilibrium maximum metal uptake qmax 47,04 mg/g was reached. The biosoption data fitted better to the Langmuir adsorption model. Key words: biosorption, Cu (II), immobilized biosorbent, Saccharomyces cerevisiae

Introduction The pollution of the environment with toxic metals has become one of the most serious environmental problems nowadays. It has been proven that large amounts of many heavy metals seriously affect human health. One of such heavy metal is Cu (II), thought it is essential to higher life, and is toxic at higher concentrations (8). The excessive intake of Cu (II) by man leads to serious health problems such as severe mucosal irritation, capillary, hepatic, renal and central nervous damages (15). Traditional physical and chemical methods for removing dissolved heavy metal ions from waste waters have significant disadvantages, because of incomplete metal removal, expensive equipment and potential risk of the generation of hazardous by-products (3,6,7,11-14,23). The biosorption of metallic ions by dried microbial biomass offers an alternative to the remediation of industrial effluents as well as the recovery of metals contained in other media (1,10,14,16). Currently one of the major research directions of biosorption is focused on screening of biosorbents (2,4,9,19,22-25). According to Volesky (23) it is necessary to

continue to search for and select the promising types of waste and inexpensive biomaterials for biosorbents. Sorption capacity of various dried microbial biomasses was studied for Cu (II) removal. One of the most intensively studied biosorbents are the yeasts S. cerevisiae (13,14,20,21). Many publications discussed the possibilities for heavy metals removal by waste biomasses, but less are dedicated to application of immobilized biosorbents. According to Arica et a. (2), Bayramoglu et al. (5), and Godjevargova et al. (10) live and dead immobilized white-rot fungus, algae and yeasts are prospective biosorbents for Cu (II), but biosorbents prepared by dead biomass were more effective in comparison with live biomass. Primary objective of the present study was to compare the Cu (II) biosorption capacity of dead biomass of S. cerevisiae immobilized in three different procedures in batch experiments and also to optimize the process parameters for Cu (II) biosorption from aqueous solutions

Materials and Methods Preparation of heat inactivated biomass Heat inactivated cells were prepared by mixing 10 g of S. cerevisiae biomass with 200 cm3 of distilled water and then the suspension was sterilized for 15 minutes at 121 °C in

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autoclave. The heat inactivated biomass was separated by centrifugation at 4000 rpm for 15 minutes and dried in a hot air oven at 80 °C for 8 hours and stored until further use. Preparation of immobilized biosorbents Immobilization in Ca-alginate beads Sodium alginate was dissolved in a hot distilled H2O at concentration of 2 % (w/v). Cell suspension of heat inactivated yeast biomass was mixed with sodium alginate solution at ratio 1:1 on a magnetic stirrer until complete homogenization. Then using the syringe-method, the suspension was extruded dropwise to a gelling solution of 0,1 M CaCl2, previously cooled to 4° C. The beads were left to polymerize for 30 minutes and then were separated from the solution by filtration using Bűchner funnel and washed twice with distilled H2O. The Ca-alginate beads without cells were also prepared as control sample. The beads were stored in distilled H2O at until biosorption. Co-immobilization in Ca-alginate beads with bentonite 6 % (w/v) bentonite clay was added to sodium alginate solution to reach final concentration of 3 % (w/v). Then the same procedure as listed above was followed. The Caalginate-bentonite beads without cells were also prepared as control sample. The beads were stored in distilled H2O at until biosorption. Co-immobilization in Ca-alginate beads with activated carbon Activated carbon was added to sodium alginate solution to reach final concentration of 3 % (w/v). Then the same procedure as listed above was followed. The Ca-alginateactivated carbon beads without cells were also prepared as control sample. The beads were stored in distilled H2O at until biosorption. Biosorption studies The stock solution of Cu (II) (1000 mg/dm3) was prepared by dissolving a weighed quantity of CuSO4.5H20 (Merck, p.a) in deionised water. Cu (II) solutions of different concentrations were prepared by adequate dilution of the initial solution. The effect of Cu (II) initial concentrations was studied at 34,06; 58,68; 107,97; 157,26 and 206,56 mg/dm3. The effect of pH was studies in the range of 2,0-6,0. The effect of biosorbent amount was studies in the range of 1-10 g/ dm3.

Batch biosorption experiments were carried out in Erlenmeyer flasks at 30 °C on a shaker for 180 minutes. Samples were taken in intervals and the residual Cu (II) concentration was determined. Determination of the concentration of Cu (II) solutions The concentration of Cu (II) in the solution before and after biosorption was measured using Spectroquant® Cooper Test (Merck) following the manufacturer’s instructions. Determination of Cu (II) uptake The Cu (II) uptake was calculated by the simple difference method (23):

q

C

i

 C f V

(1)

W

where q is the Cu(II) uptake, mg/g; V is the volume of the solution in the flask, dm3; Ci and Cf – initial and final concentration of Cu (II) in the solution, mg/ dm3; W is the mass of biosorbent, g. Removal efficiency of Cu (II) The removal efficiency was calculated as:

R

c

i

cf ci



(2)

.100,

%

where: ci and cf denote respectively initial concentration of Cr(VI) and final residual concentration of Cr(VI) at the moment t, in mg L-1. Sorption isotherms - Langmuir and Freundlich models The Langmuir model corresponds to the following equation:

bC e qe  q m 1  bC e

(3)

where Ce - metal concentration in solution at equilibrium, mg/dm3; qm – maximum amount of metal adsorbed per unit of dry biomass, mg/g; b – the binding stability constant, dm3/mg. The Freundlich models correspond to the following equation:



qe  KC 1 / n

(4)

where K and 1/n are Freundlich adsorbent constant and exponent characterizing the system

Results and Discussion Screening of prospective immobilized biosorbent To select the most appropriate immobilized biosorbent for Cu (II) removal from aqueous solutions, metal uptake capacity of dead biomass of S. cerevisiae, immobilized in Ca-alginate, co-immobilized in Ca-alginate-bentonite and Ca-alginateactivated carbon was investigated. The results are shown in Fig. 1. As seen the highest metal uptake 39,06 mg/g was reached with dead S. cerevisiae cells co-immobilized in Ca-alginateactivated carbon. Immobilized biosorbents in Ca-alginate and co-immobilized Ca-alginate-bentonite demonstrated lower and equal Cu (II) uptake 13,94 mg/g. At all of the studied cases control beads without immobilized cells demonstrated lower Cu (II) uptake in comparison with beads with immobilized cells. Probably the highest Cu (II) uptake of dead cells of S.cerevisiae co-immobilized in Ca-alginateactivated carbon dues to the accumulation of biosorption potential of microbial cells and activated carbon, which is well known as prospective sorbent. For all of the studied biosorbents equilibrium was reached for about 30 minutes. On the basis of the results obtained biosorbent of dead S. cerevisiae cells co-immobilized in Ca-alginate-activated carbon was selected as the most promising and all of the following experiments were carried out with it.

values the affinity with the proton at the binding site of yeast is much greater than that of the metal ion, compared with that at higher pH values. To the end of the biosorption process pH retained almost unchanged which means that the electrostatic attraction to negative charged functional groups may be was the major biosorption mechanism. The results obtained are in accordance with published by other authors (7,9,13,14). All of the following experiments were carried out at pH = 4,0. Influence of biosorbent concentration on the Cu (II) biosorption by dead S. cerevisiae cells co-immobilized in Ca-alginate-activated carbon To evaluate the effect of biosorbent concentration experiments ranging from 0,1 to 1 g/100 cm3 were carried out. The results are shown in Fig. 3. The results obtained indicated that the increasing of biosorbent concentration from 0,1g to 0,6 g increased Cu (II) removal. This fact may be attributed to the higher number of active groups available for Cu (II) adsorption on the biosorbent. At concentrations higher than 0,6 g the increasing effect of biosorbent was insignificant. As seen the increasing of initial Cu (II) concentration increased metal uptake by the biosorbent, but decreased removal efficiency. Maximum removal 63,5 % was reached at the lowest Cu (II) concentration 34,06 mg/cm3 and at the highest Cu (II) concentration 206,56 mg/cm3 removal decreased to 20,49 %. The results demonstrated that the selected biosorbent was effective at lower Cu (II) concentrations, which is in accordance with published data (10, 15, 18, 22).

Influence of pH on the Cu (II) biosorption by dead S. Adsorption isotherm analysis cerevisiae cells co-immobilized in Ca-alginate-activated The application of the biosorption technique in commercial carbon scale requires proper quantification of the sorption The effectiveness of biosorption depends mainly on the pH equilibrium for process simulation. The Langmuir and (17, 18, 23). For this reason the effect of pH on the Cu (II) Freundlich isotherms are more frequently used to give the removal by the selected biosorbent was studied. The results sorption equilibrium (23). The Langmuir and Freundlich are shown in Fig. 2. equations were linearized, equations (5) and (6), respectively, The effect of initial pH on copper (II) biosorption was and sorption models were used to calculate maximum Cu (II) examined in the pH range 2, 0 - 6, 0. The highest Cu (II) biosorption capacity of dead S. cerevisiae cells couptake was found 37,67 mg/g at pH = 4,0. At pH higher that immobilized in Ca-alginate-activated carbon. 6, 0 Cu (II) ions precipitated. It was reported that the optimal pH value for Cu (II) biosorption by dead S. cerevisiae is in (5) 1 1 1 1 the range 4,0 - 5,0 (9,13,14). The optimum pH depends on  .  qe q m b Ce q m the type of metal cation and from the biosorbent. At low pH 609 BIOTECHNOL. & BIOTECHNOL. EQ. 24/2010/SE SECOND BALKAN CONFERENCE ON BIOLOGY SPECIAL EDITION/ON-LINE

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As seen the studied biosorption process fitted well to the Langmuir equation. At the equilibrium q max was 47,04 mg/g, b 0,0704 L/mg and R was 0,9954.

(6)

1 lg qe  lg K  lg Ce n

The linearized Cu (II) biosorption isotherms are shown in Fig. 5 and Fig. 6.

45 40 35 1

q, mg/g

30

2

25

3

20

4 5

15

6 10 5 0 0

20

40

60

80

100

120

140

160

180

Time, min Fig. 1. Biosorption of Cu (II) by immobilized dead cells of S. Cerevisiae, C0 = 107,97 mg/cm3, pH = 4,0, V = 100 cm3, W = 0,1 g 1 - S.cerevisiae immobilized in Ca-alginate; 2 - Control Ca-alginate beads; 3 - S.cerevisiae co-immobilized in Ca-alginate-bentonite; 4 - Control Ca-alginate-bentonite beads; 5 - S.cerevisiae co-immobilized in Ca-alginate-activated carbon and 6 - Control Ca-alginate-activated carbon beads

40 35

q, mg/g

30 25 20 15 10 5 0 2

2,5

3

3,5

4

4,5

5

5,5

6

pH Fig.2. The influence of pH on the Cu (II) biosorption by co-immobilized dead S.cerevisiae cells, C0 = 107,97 mg/cm3, V = 100 cm3, W = 0,1 g



48 46 44

R, %

42 40 38 36 34 32 30 0,1

0,3

0,5

0,7

0,9

W, g

Fig.3. The influence biosorbent concentration on the Cu (II) biosorption by dead S.cerevisiae cells co-immobilized in Ca-alginate-activated carbon, C0 = 107,97 mg/cm3, pH = 4,0, V = 100 cm3

45

70

40

60

3,8

50

3,7

35 25

40

q, mg/g

3,6

20

30

R, %

3,5

15

20

lnqe

q, mg/g

30

10 5 0 34

54

74

94

3,4

10

3,3

0

3,2

114 134 154 174 194

3,1

c0, mg/cm 3 3,0 2,5

Fig.4. The influence of initial Cu (II) concentrations on the metal uptake and removal by dead S.cerevisiae cells co-immobilized in Ca-alginate-activated carbon, pH = 4,0, V = 100 cm3, W = 0,1 g

0,045

0,040

1/qe

3,5

4,0

4,5

5,0

5,5

lnce

Fig. 6 Linear plot of the Freundlich isotherm

Conclusions

0,050

0,035

0,030

0,025

0,020 0,00

3,0

0,01

0,02

0,03

0,04

0,05

0,06

0,07

0,08

1/ce

Fig. 5 Linear plot of the Langmuir isotherm

0,09

In this study Cu (II) biosorption potential of dead S. cerevisiae cells immobilized in different procedures was investigated. Biosorbent of yeast cells co-immobilized in Caalginate-activated carbon was selected as the most prospective among the tested biosorbents. It was determined that maximum Cu (II) uptake was reached for about 30 minutes, at pH 4,0. The biosorption process was more effective at lower initial metal concentrations and increasing concentration of the biosorbent. The Langmuir adsorption model conformed very well to the adsorption equilibrium in the studied conditions. The future experiments will be 611

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focused on the possibilities for biosorbent regeneration and column experiments.

Acknowledgment The present study was financially supported by NSFB project GAMA DO 02-70 11/12/2009 of the Bulgarian Ministry of Education.

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