BIOSORPTION USING Thiobacillus ferrooxidans

Hossain, N. Anantharaman Journal of the UniversityS.ofMasud Chemical Technology and Metallurgy, 40, 3, 2005, 227-234

STUDIES ON COPPER (II) BIOSORPTION USING Thiobacillus ferrooxidans S. Masud Hossain1, N. Anantharaman

1

Department of Chemical Engineering, Mohamed Sathak Engineering College, Kilakarai 623 806, India. 2 Department of Chemical Engineering, National Institute of Technology, Tiruchirapalli 620015, India. E-mail: [email protected]

2

Received 31 May 2005 Accepted 20 July 2005

ABSTRACT Gram-negative bacteria Thiobacillus ferrooxidans accumulates copper (II) from its aqueous solution. The maximum biosorption of copper is 94.25 % w/w within 60 h of inoculation time with optimum pH 4.5 and temperature 40°C for 700 ppm initial copper (II) loading, respectively. The optimum shaking speed is 60 rpm. 7 days age-old and 30 % v/v inoculum culture is used in the studies. The aerobic conditions are maintained by supplying atmospheric air to the biosystem. The Langmuir and Freundlich isotherms fit the experimental data reasonably well and played a major role in giving a better understanding of biosorption process simulations. The Monod equation for microbial growth shows that the specific growth rate is 0.0085 h 1. Keywords: bacteria, biosorption, copper (II), isotherm, optimum.

INTRODUCTION Certain species of microorganisms have been found to adsorb surprisingly large quantities of heavy metals. Important metals in the case include metals involved in toxicity to humans and metals of commercial economic value. The removal of heavy metals from municipal and industrial wastes by biological treatment systems has continued to be of interest. Bacterial surfaces have great affinity to sorb and precipitate metals resulting in metal concentration on the surface. There have been several reports of the uptake of toxic heavy metals by bacteria so that the metals are accumulated.

Both metabolically mediated and biosorptive phenomena can and do occur in such systems. Therefore it is not unexpected that biosorptive metal uptake is subject to environmental conditions that affect the reaction chemistry of both the receptive sites and the metals [1-5]. Biosorption involves accumulation of metals on the surface of the cells or cell fractions by adsorption or ion exchange. All microbes, which expose negatively charged groups on their cell surface, have the capacity to bind metal ions. Various compounds of bacterial walls sorb different metals, which later get precipitated. Complexation with certain proteins or organic com-

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Journal of the University of Chemical Technology and Metallurgy, 40, 3, 2005

pounds presents in the cell walls. Complexolysis is a process corresponding to microbial formation of complexing or chelating agents that solubilize metal ions. As a result of that metal-organic complexes or metalorganic chelates are formed. The chelating compounds formed by microorganisms are organic acids (citric, oxalic, 2-keto-gluconic, tartaric, 2,3-dihydrobenzoic acid, etc.) or more complex compounds such as sidrophores: M+ + H+L-----H+ + LM, H+L + LM-----L2M + H+, (L: organic legends). Some microorganisms produce specific proteins called Metallothionein which are induced by metal ions and these bind metal ions. A variety of precipitates and minerals have been found associated with bacterial surfaces, but the exact mechanism of precipitation and mineral formation is currently under investigations [1-5]. The outer membrane and murein (the usual model for the gram-negative A 1γ chemotype has revealed about 80 distinct muorpeptides) of the gram-negative surface constitute the cell wall. The arrangement and exposure of the various polymers and macromolecules in gramnegative walls produce a net electronegative charge, which allows these walls to interact with cations within their immediate surroundings. Metallic ions affect the packing order of the phospholipids and lipopolysaccharides of the outer membrane and even affect the bonding forces that hold the two faces of the membrane together. In fact, it is possible that the hydrophilicity or hydrophobicity of this membrane can be molded by its metallic ion salt form. Lipopolysaccharide normally partitioned to the outer membrane has an abundance of phosphoryl groups implicated as the primary sites of metal interaction [1-5]. The biosorption of heavy metal ions by microorganisms is affected not only by the surface properties of the organism but also by various other physico-chemical parameters of metal ion solution. The present batch investigations were undertaken to develop an effective bacterial treatment (biosorption) of copper (II) in aqueous solution using Thiobacillus ferrooxidans. Aerobic studies were conducted to optimize physical parameters [610] such as biosorption time, initial copper (II) loading, pH, temperature and shaking speed (rpm) using aerobic suspension culture of the acidophilic, mesophilic, gramnegative bacteria Thiobacillus ferrooxidans.

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EXPERIMENTAL Collection of bacteria and growth The acidophillic, mesophilic Thiobacillus ferrooxidans, was procured from Institute of Microbial Technology (CSIR), Chandigarh, India. It was grown on a prescribed medium containing KH2PO4 - 0.4 g/l, MgSO4.7H2O - 0.4 g/l, (NH4)2SO4 - 0.4 g/l, FeSO4.7H2O - 33.3 g/l. pH of the growth medium was adjusted to 1.4 with 0.1 N H2SO4. The cultures of Thiobacillus ferrooxidans in 500 mL of the medium and 30 % v/v inoculum were inoculated at a constant temperature of 30°C in water bath with constant shaking at 60 rpm while putting non-absorbent cotton at the mouth of the flasks to prevent water loss due to evaporation. The stock and pre-inoculum cultures were maintained in the same medium under similar conditions. The stock cultures were subcultured every two weeks. General method The biosorption of copper was studied in a batch system. Experiments were carried out in 1l Erlenmeyer flasks containing 250 ml of the medium. 250 ml of 500 ppm of copper (II) salt solution and 30 % v/v inoculum were taken to the flasks. The maximum biomass concentration was measured [15] as 2.0x108 cells/ml and dry biomass was 3.6 g/l. Inoculum was taken from a seven days age-old culture. The aerobic condition of the system was maintained by putting nonabsorbent cotton to the mouth of the flasks. The dissolved oxygen (DO) level is measured to 6.4 ppm in the broth. The flasks were incubated in a constant temperature water bath maintained at 30°C with constant shaking at 20 rpm. The pH was always maintained at 2.5 by using 0.1 N H2SO4 and 1 M CaCO3 slurry [6-10]. Effect of time and initial copper loading The general method was repeated for 600, 700 and 800 ppm of initial copper (II) ion solution loading, respectively. Flasks were taken out on a regular basis i.e. after 24, 36, 48, 60, 72 and 84 h of

S. Masud Hossain, N. Anantharaman

94

500 ppm

600 ppm

700 ppm

800 ppm 92

100

90 Copper (II) biosorption, w/w %

90 Copper (II) biosorption, w/w %

80 70 60 50 40 30

88 86 84 82 80 78

20

76

10 0

74

24

36

48

60

72

84

20

96

30

40

50

Deg C

hr

Fig. 1. Effect of intial copper (II) concentration and time on biosorption.

Fig. 3. Effect of temperature on copper (II) biosorption.

88

94 84

Copper (II) biosorption, w/w%

Copper (2) biosorption, w/w%

96

500 ppm

86

82

80

78

76

92 90 88 86 84 82 80 78

74 1,5

2,5

3,5

4,5

pH

Fig. 2. Effect of pH on copper (II) biosorption.

inoculation followed by analysis for copper (II) in solution. To assess the extent of chemical reaction a set of experiments was carried out under sterile conditions (without microbe). The results are shown in Fig. 1. Effect of pH on copper biosorption The general method was repeated for various pH values such as 2.5, 3.5, 4.5, and 5.5, respectively, for 700 ppm initial copper (II) ion loading. The broths were taken out after 60 h of inoculation (optimum time) followed by analysis of copper (II) ion. The results are shown in Fig. 2.

20

40

60

80

rpm

Fig. 4. Effect of shaking speed on copper (II) biosorption.

Effect of temperature on copper biosorption The general method was performed accordingly for various temperatures such as 20, 30, 40, and 50°C, respectively, for 700 ppm initial copper (II) ion loading. The pH was maintained at 4.5 (optimum). The broths were taken out after 60 h of inoculation (optimum time) followed by analysis of copper (II) ion. The results are shown in Fig. 3. Effect of shaking on copper biosorption The general method was performed accordingly for different shaking speed such as 20, 40, 60 and 80 rpm, respectively, for 700 ppm initial copper (II) ion loading. The

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pH value was maintained at 4.5 (optimum). The temperature maintained at 40°C (optimum). The broths were taken out after 60 h of inoculation (optimum time) and were analyzed for copper (II). The results are shown in Fig. 4.

0.025

0.02

Ce/qe

0.015

Determination of copper (II)

0.01

At the end of the specific biosorption time, 100 mL of the solution were taken out of the flasks and centrifuged at 1000 rpm for 10 min in Sorvall RC-5 super speed centrifuge at room temperature to remove biomass and unsolubilized materials followed by filtration [6-10]. The supernatant solution was digested with 1:1 H2SO4 and after digestion the solution was diluted with demineralised water and filtered to separate silica. The filtrate was made to mark in a 250 ml volumetric flask. The amount of cupric copper (II) was determined by direct titration method using Fast Sulphon Black F indicator with EDTA [11]. Calculation: 1 mol EDTA = 1 mol Cu (II) = 1 mol Cu (I)

0.005

0

0

0.5

1

1.5

Ce

2

2.5

Fig 5. Langmuir isotherm for copper (II) biosorption.

0.35 0.3

log (X/m)

0.25 0.2

Lackey drop method for microbial counting

0.15 0.1 0.05 0 1.37

1.38

1.39

1.4

1.41

1.42

1.43

1.44

1.45

log Ce Fig. 6. Frendlich isotherm for copper (II) biosorption.

Growth of T.ferrooxidans, no. of cells/mL. hr

0.03 0.025 0.02 0.015 0.01

RESULTS AND DISCUSSION 0.005

Effect of initial copper loading and time on biosorption

0 8.33

7.55

6.37

5.1

3.82

2.62

2.05

FeSO4 concentration, g/l

Fig. 7. Monod growth model on bacterial growth and rate.

230

1.46

Exactly 0.1ml volume of the sample was put by using a calibrated medicinal dropper onto a glass slide. A cover slip of known area was placed, avoiding any air bubble. The slide was put under a microscope and measured the width of the high power microscopic field. Suppose the area visible at one time is one micro transect. Now the slide moved from one corner to another counting planktons in each microscopic field visible. It was counted several fields by moving the slide in horizontal and vertical directions. Counting must be quick to avoid drying of the sample. Calculation of the phytoplankton as follows: Number of planktons per mL = (No. of organisms counted in all fields x Area of cover slip, mm2)/ (Area of one macroscopic field, mm2 x No. of field counted x volume of sample in the cover slip).

The effect of the initial copper (II) loading and biosorption time are shown in Fig.1. Initial copper (II)


loading was 500, 600, 700, and 800 ppm in the present studies. The permissible limit of copper (II) discharge into water stream is 200 ppm. Therefore, the above mentioned concentrations of copper (II) ions are undertaken for bacterial treatment. The copper (II) ion biosorption was measured after 24 h of inoculation. This period is avoidance of lag-phase for growth of bacteria and adaptation to the environment of solid substrate i.e. copper (II) ion solution [10]. From Fig. 1, it is shown that the biosorption of copper (II) ion is maximum after 60 h of inoculation by the bacteria T. ferrooxidans. The biosorption of copper (II) was noticed as 93.75, 88.60, 75.30 and 17.65 % w/w for 500 ppm, 600 ppm, 700 ppm and 800 ppm of initial copper (II) ion loading after 60 h, respectively (Fig. 1). It can be concluded that the tolerance limit for the bacteria is 800 ppm of initial copper (II) ion loading. At this concentration (800 ppm) of copper (II) ion loading, the growth and activity of bacteria demised maximally, hence the biosorption is less. The rate of metal ions binding with microbe is more at initial stages, which gradually decreases and remains almost constant after an optimum time. At equilibrium, the removal of copper (II) ions attains a constant value, because adsorption and desorption balance each other. Therefore, 700 ppm of initial copper (II) ion is taken as optimum loading with 60 h as optimum biosorption time for studies of the other biological process parameters, respectively. It is evident from Fig. 1 that as the concentration of copper (II) ion increases the percentage removal efficiency decreases. This is because microbial populations in the system can effect copper ion removal. A sharp increase in biomass was observed up to 60 h (optimum time), then decreases in growth rate. As can be seen at the early stage of biosorption, which coincided with lag-phase of bacterial growth the biosorption of copper was slow. The extent of lag-phase was dependent on initial loading of copper and cultures initially containing high concentration of copper showed longest lagphase. The transition of bacterial activity from the lagphase to exponential phase of growth led to a notable increase in biosorption of copper, which proceeded until

the completion of stationary phase. It should be pointed out that the application of copper ions at higher concentrations only increased the extent of lag-phase. There is a real danger that these metals may poison the system stopping the biological activity and the microbial growth. It has been inferred in several instances that the accumulation of metal results from the lack of specificity in a normal metal transport system and that, at high concentrations, metals may act as competitive substrates in a transport system [12-14]. Effect of pH on copper biosorption The effect of pH on biosorption of copper (II) metal ions with T. ferrooxidans is shown in Fig. 2. The copper (II) biosorption is measured after 60 h of inoculation. An increase in percent removal with increase in pH of the medium was observed for the copper ions to a pH value of 4.5. It is observed that the maximum 87.20 % w/w reduction of copper (II) ions being occurred at pH 4.5 for 700 ppm of initial copper (II) loading (Fig. 2). The biosorption of 700 ppm of initial copper (II) ions loading are 78.65 % w/w, 83.50 % w/w and 81.25 % w/w for pH value of 2.5, 3.5 and 5.5, respectively (Fig. 2). With increase in pH (beyond 4.5), the biosorption of copper ions sharply declines (Fig. 2). pH of 4.5 is the optimum value as maximum copper (II) ions biosorption takes place. The tolerance of bacteria in low pH media probably results from effective competition by H +ions for negatively charged sites at the cell surface. The results suggest that the biosorption of copper ions to the biomass is mainly due to ionic attraction. Therefore, as pH decreases the cell surface becomes more positively charged, reducing the attraction between the biomass and the metal ions. In contrast, higher pH results in facilitation of metal uptake, since the cell surface is more negatively charged. An optimum pH of 4.5 for the adsorption of copper (II) ions was found. At that pH value, neutralization of positive and negative ions is being occurred. However, it is needless to mention T. ferrooxidans is a gramnegative, acidophilic, mesophilic and chemautotrophic bacteria. In biosorption of copper ions from its aqueous salt solution pH sharply decreases with time which results in suppression of bacterial activity. In order to avoid this negative effect lime and/or calcite was added to the medium [12-14].

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Effect of temperature on copper biosorption The effect of temperature on the biosorption of copper (II) ions with T. ferrooxidans is shown in Fig. 3. However, the effect of temperature on activity of bacteria depends on a number of factors, e.g., pH, substrate loading (copper concentration), ion concentrations of medium, etc. The optimum temperature is 40°C at which the maximum copper (II) ions biosorption is being noticed. The biosorption is 91.60 % w/w at optimum temperature 40°C for 700 ppm of initial copper (II) ions loading (Fig. 3). The biosorption of 700 ppm initial copper (II) ions loading are 80.70 % w/w, 86.75 % w/w and 83.65 % w/w for temperatures of 20, 30 and 50°C, respectively (Fig. 3). With increase in temperature (beyond 40°C), the biosorption is decreased (Fig. 3). The rates of biological oxidation increase with temperature where dissolved oxygen (DO) is least soluble. In aerobic biological process, the limited solubility of oxygen is of great importance because it governs the rate at which oxygen will be absorbed by the medium. Mesophiles grow best within temperature range of 20 to 50°C. Every type of microbes has an optimum, minimum and maximum growth temperature. Temperatures below the optimum for growth depress the rate of metabolism of cells. Above the optimal temperature, the growth rate decreases and thermal death may occur. At high temperature (beyond 40°C), death rate exceeds the growth rate, which causes a net decrease in the concentration of viable cells. When temperature is increased above the optimal (40°C), the maintenance required of cell increases. However, it is needless to maintain that the temperature optimum for growth and biosorption may be different [12-14]. Effect of shaking on copper biosorption The effect of shaking speed on biosorption of copper ion is shown in Fig. 4. The maximum copper ion biosorption is noticed as 94.25 % w/w at agitation speed of 60 rpm (Fig. 4). The biosorption of

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copper (II) ions are 85.25, 90.75, and 84.15 % w/w at agitation speed of 20, 40, and 80 rpm (Fig. 4), respectively. The biosorption of copper by the bacteria increases with increase in agitation up to 60 rpm, then it declines. This is because the binding of copper metal ions to the bacterial surface is highest as well as cell population is maximum at this optimum shaking speed. Increase in mechanical forces can disturb the elaborate shape of an enzyme molecule to such a degree that denaturation of the protein occurs and deactivates the bacterial growth [12-14]. Interpretation of experimental data with adsorption isotherms The application of biosorption technique in the commercial scale requires proper quantification of the sorption equilibrium for process simulation. The Langmuir and Freundlich isotherms are more frequently used to give the sorption equilibrium [16, 17]. The Langmuir isotherm is applied for biosorption equilibrium and can be represented as: Ce/qe = 1/Qob + Ce/Qo where: Ce is equilibrium concentration of copper ion in solution, mg/L; qe is amount of copper ion adsorbed at equilibrium, mg/L; Qo and b are Langmuir constants. The linear plot (Fig. 5) of Ce Vs qe shows that biosorption of copper (II) ion follows the Langmuir isotherm. The values of Qo and b are calculated from the linear plot (Fig. 5). Therefore, Qo=97 and b=2.95, are obtained, respectively. The Freundlich Isotherm is widely used for model of adsorption of heavy metal from an aqueous medium and represented as:


log qc = log (x/m) = log kf + 1/n log Ce where: x is amount of copper ion adsorbed at equilibrium, mg/L; m is the mass of adsorbent (dry biomass), mg/L; Ce is equilibrium concentration of copper ion in solution, mg/L; Kf and n are constants. The values of Kf and n are obtained as 0.32 and 11, respectively, from the linear plot (Fig. 6). Interpretation of bacterial growth data with Monod model The trends of microbial growth in the presence of copper ions at initial concentration of 700 ppm at optimum conditions are shown in Fig. 7. Growth rate is plotted against limiting substrate FeSO4 concentration using Monod growth mode [14] as follows: µ=µ

max

x S/ (Ks + S)

Here, Ks is limiting substrate (growth depends on FeSO4) FeSO4 concentration at which the specific growth rate (µ) is half of maximum value i.e. µ= µ max/2 and S is limiting substrate FeSO4 concentration. From Fig. 7, the value of Ks = 0.017 h-1 is obtained (up to straight line). Putting Ks 0.017 h-1, the specific growth rate (m ) of the bacteria is found to be 0.0085 h -1. CONCLUSIONS Bios orption of copper (II) ions with Thiobacillus ferrooxidans is shown to be an effective bacterial bioaccumulation process. The maximum biosorption of copper (II) ions is obtained up to 94.25 % w/w for 700 ppm of initial copper (II) ions loading by 60 hours. The optimum pH is 4.5 and optimum temperature is 40 o C for maximum biosorption of copper (II) ions. The optimum shaking speed is 60 rpm at which maximum biodegradation occurred. Results of the present study show that

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