Biotechnol. hog. 1001, 7, 427-433
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Kinetics and Mechanism of the Adsorption of Sulfolobus acidocaldarius on Coal Surfaces Virote B. Vitaya' and Kiyoshi Toda Institute of Applied Microbiology, The University of Tokyo, Yayoi, 1-1-1,Bunkyo-ku, Tokyo 113, Japan
The adsorption of Sulfolobus acidocaldarius on five types of coals (with different sulfur contents) was investigated to define the kinetics of the bacterial cell adsorption and to clarify the mechanism of the adsorption involved in biological desulfurization of coals. A kinetic model, in which two stages of adsorption are considered, is proposed on the basis of the experimental results. The estimated values of the maximum cell numbers adsorbed per unit area of coal surface (including internal surface area of pores larger than 0.5 km), [S,], showed a good correlation with the pyritic sulfur content of the coals. The mechanism of the adsorption was studied by investigating the effects of pH, f potentials of coal particles and the bacteria, a specific inhibitor of membrane-bound ATPase (20 mM NaF), a surfactant (Tween 80,0.05%), and heat treatment (121 "C, 15 min) of the bacteria. From these observations, it was concluded that the rapid adsorption in the first stage was related with the physical force of hydrophobicity and the adsorption in the second stage was strongly related with the oxidation of pyrite. An electromicrographic study on the erosion of pyrite surfaces by the bacterial cells was also conducted. The micrographs of the pyrite particles after 5 days of the incubation showed that surfaces of pyrite minerals were eroded by adsorbed cells.
Introduction Coal desulfurization by microorganisms has attracted attention; it has been reported that the operating cost is lower than those of physical or chemical desulfurization methods (Detz et al., 1979). Yet, there are no commercial microbial desulfurization plants operating. This may be due to the slowness and complexity of reactions involved in the process. In pyrite oxidation by Thiobacillus ferrooxidans, Silverman (1967) proposed that there would be two mechanisms of bacterial pyrite oxidation operating concurrently: a direct contact mechanism, which requires physical contact between the bacteria and pyrite particles for biological pyrite oxidation, and the indirect mechanism in which the bacteria regenerate the ferric ions (required for chemical oxidation of pyrite) by oxidizing ferrous ions to ferric ions. Thus it is crucial to elucidate details of each mechanism,which are necessary in designing an efficient bioreactor. Adsorption of microorganisms to solid surfaces has been extensivelyinvestigated (Corpe, 1980;Daniels, 1980;Marshall, 1985; Stotzky, 1985). Most of the research on microbial leaching and microbial activities in commercial operations deal with T. ferrooxidans. Although several studies have been conducted in which T. ferrooxidans was used to leach pyrite from coal, the data reported by Detz and Barvinchak (1979) showed that the organisms were not effective in extracting organic sulfur. Furthermore, the temperature of 60-80 O C (Beck, 1967) in leach dump environments is also a severe constraint condition for T. ferrooxidans, which optimally grows at around 30 OC. Since Sulfolobus acidocaldarius is a thermoacidophilic archaebacterium thriving at low pH (less than 3.0) and temperature of 65-90 "C (Brock et al., 1972) and can also oxidize organic sulfur (Kargi and Robinson, 1986), the organism is considered as an excellent candidate for use in microbial desulfurization. The studies on the adsorption of this organism to solid surfaces are still few. Weiss (1973) concluded that Sulfolobus attached to sulfur by means of pili which separated 8756-7938/9 1/3007-0427$02.50/0
the bacteria from the sulfur crystal. Chen and Skidmore (1988),who conducted an adsorption of S. acidocaldarius on coal surfaces, suggested that cell attachment was very rapid and that the equilibrium was reached in less than 5 min. These same authors also confirmed the concept of selective cell attachment. The objectives of this work were (1)to study the kinetics of the cell adsorption on various types of coals, (2) to construct an adsorption model based on the experimental results, (3) to find the relation between the parameters used in the developed model and the physical properties of coal, (4) to clarify the mechanism of cell adsorption through the effects of pH, rpotential of coal particles and the bacterial cells, a specific inhibitor of membrane-bound ATPase (20 mM NaF), a surfactant (Tween 80,0.05%), and heat treatment (121 "C, 15 min) of the cells, and (5) to verify physiological adsorption by conducting an experiment of microbial pyrite dissolution directly observing the surface changes of the pyrite particles and the adsorption of bacterial cells using scanning electron microscope.
Materials and Methods Organisms and Medium. The organism used in this work was isolated from an acidic spa in Beppu Hot Springs, Japan, by Professor Oshima, Tokyo Institute of Technology, and designated as Sulfolobusacidocaldarius strain 7. The organism was kindly provided by Dr. T. Wakagi of Tokyo Institute of Technology. Three kinds of media were used. Medium I contained (g/L of distilled water) NaC1,0.2; KHzP04,0.3; (N&)zSOr, 1.3;MgSO4-7Hz0,0.25; CaClz-2Hz0,0.05; yeast extract (Difco Co., Detroit, MI), 1; casamino acids, 1; glucose, 1; pH was adjusted to 2.0 with 1 N HzSO4. Medium I1 contained the same composition as medium I except yeast extract, caeamino acids, and glucose. Medium I11contained (g/L of distilled water) KC1,0.2; KzHP04,0.2; (NH4)2SO4,0.4;MgSOq7Hz0,0.4; NazMoO4,3 X loa; yeast extract, 0.02% (w/v); adjusted to pH 2.0 with 1 N HzS04.
0 1991 American Chemical Society and American Institute of Chemical Engineers
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Table I. Physical Properties of Coals Used in This Work* coal A B C D E proximate analysis, w t Oio volatile matter 27.0 29.3 35.6 33.4 32.7 fixed carbon 57.0 31.5 36.5 58.6 59.9 9.4 9.3 5.2 1.4 2.6 moisture 8.0 7.5 13.4 34.0 26.5 ash ultimate analysis, wt !Z 84.4 73.1 82.4 74.0 75.2 carbon 4.7 4.9 5.5 6.9 hydrogen 5.2 1.7 1.7 0.8 1.4 nitrogen 1.7 9.2 9.0 6.8 8.3 15.6 oxygen 0.45 3.81 2.39 2.32 1.72 sulfur pyritic 0.40 3.20 1.00 1.06 0.87 organic 0.05 0.44 1.24 1.13 0.75 1.36 1.57 1.42 1.26 1.23 apparent density, g/mL specific surface area,* m2/g 0.20 0.16 0.25 0.20 0.19
The sources of coal A, B, and C, obtained from Idemitu Co., Ltd., are not known; coal D and E are samples from Old Ben Coal Co., Ltd., Mine #24 and #26, respectively. b External surface plus internal surface of pores of which size is larger than 0.5 pm. a
Preparation of Cell Suspension. The cells were harvested after about 3 days of growth at a temperature of 70 "C (Inatomi et al., 1983) in medium I, centrifuged at 4000g for 10 min, and washed 2 times with medium I1 by centrifuging at 4000g for 10 min each. Then the cells were diluted to a definite concentration with the medium 11. Turbidity was measured at 660 nm, using a spectrophotometer (Hitachi U-1000) with 1-cm light path at a room temperature. The turbidity was then converted to cell density by a calibration curve, which correlated turbidity of cell suspensions with the number of cells directly counted under amicroscope using a Petoff Hausser counting chamber. Coal, Ion-Exchange Resins, and Pretreatment. Physical properties of coals (A, B, C, D, and E) used in this work were summarized in Table I. Five types of coal, of which the pyritic sulfur content ranges from 0.40 to 3.20 wt 70 ,were used in this work. The coals were ground, washed with distilled water, air-dried, and screened into the size of 75-106 pm. Since the size of S. acidocaldarius is 0.5-1.5 pm, the cells cannot reach pores which are less than 0.5 pm in size. Therefore it is reasonable to assume that the effectivecoal surface area is the sum of the external surface area and the internal surface area of the pores of which sizes are larger than 0.5 pm. The specific surface area was measured by a porosimeter (Poresizer 9310, Micromeritics). A strongly acidic cation exchange resin, sodium form (Amberlite CG-120), and a strongly basic anion-exchange resin, chloride form (Amberlite CG-400), were used. The resins were washed with distilled water several times, airdried, and screened into the size of 75-106 pm. It was found in the preliminary experiment that when the coals or the resins were mixed with medium I1 at 70 O C , the pH of the coal or resin slurry increased gradually with time. Thus, the pretreatment of coal and resin particles was carried out by mixing 0.5 g of coal or resin particles with 5 mL of medium I1 and shaking for ca. 60 min at 70 OC. Then the pH values of the coal or resin slurry became stable. The initial pH's of the medium I1 appropriate for obtaining the desired equilibrium pH (mostly, 2) are shown for each coal in Table 11. For coal B, the designed pH was extended to 1,3, and 5, because the pH effect on cell adsorption on coal was examined later. The reason for selecting coal B was that it contained the highest pyritic sulfur (3.2wt 75, Table I) and was most suitable for the investigation. After the mixture was
Table 11. Initial DHof Medium I1 and Eauilibrated RH initial equilibrated
coal' A B B B B D E ion-exchange resin CG-120 CG-400 a
PH
PH
1.5 0.3 0.5 0.7 1.0 1.0 2.0 2.0
2.0 1.o 2.0
1.5
2.0 2.0
2.0
3.0 5.0 2.0 2.0 2.0
Two replicates per sample.
centrifuged at 60g for 2 min, 4 mL of the supernatant was discarded. The remaining 1mL of coal or resin slurry was subsequently used in the experiment. Adsorption Experiment. In two series of test tubes, one containing the coal particles of A, B, C, D, or E equilibrated to pH 2 and the other containing the coal particles of B equilibrated to pH 1,2,3, and 5,4 mL each of the cell suspension in medium I1 (pH was adjusted to each equilibrated pH) was added. The test tubes were capped and immediately set onto the shaker (120 strokes/ min) incubated in the water bath controlled at 70 OC. Some test tubes were taken out after shaking times of 0.5,1,5, 10, 30,60, and 120 min and centrifuged at 60g for 2 min to remove the coal particles without precipitating the cells. The supernatant including the nonadsorbing free cells was filtered through a Whatman No. 2 filter paper to remove the remaining fine coal particles. The turbidity of the filtrate was measured and recorded. However, there existed some amount of micro coal particles in the filtrate. A mixture of 0.8 M NaOH and 5% (w/v) of sodium dodecy1 sulfate in a ratio of 1:2 was employed to lyse the cells in the filtrate so that only the micro coal particles were suspended in the solution. By subtracting the turbidity of the suspension of the micro coal particles from that of the previous measurement, the actual turbidity of the nonadsorbed cells was obtained. In this adsorption experiment two replicates per coal type and per adjusted pH were conducted. When a mixture of NaOH and SDS (1 mL) was added into a cell suspension (2 mL), cell lysis occurred instantaneously in a few seconds. The cell density of the suspension converted from the turbidity of the samples before and after the lysis were as follows: cell density X initial 1.39 1.37 4.96 4.98
lo+
(cells/mL) after lysis 0.05 0.05 0.06 0.06
In the above preliminary experiments, we have confirmed that the effects of the DNA/RNA or the cell wall fragments, etc., of the lysed cells on the adsorption data were insignificant and could be neglected. The effects of the specific inhibitor NaF (Wakagi et al. 1985) of S. acidocaldarius cell membrane ATPase and the surfactant Tween 80 on cell adsorption was assessed. NaF (20 mM) or Tween 80 (0.05% final concentration) was added into the cell suspension (in medium 11, pH 2). After mixing about 5 min, they were then used in the adsorption with coal B. In the adsorption experiment with heat-inactivated S. acidocaldarius, the bacteria cultured in medium I were
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adsorbed cells desorbed into the suspension, probably owing to the shearing stress of the continuous shaker (120 strokes/min). In a preliminary adsorption experiment where the shaking rate was so low (30 strokes/min) that coal particles were all deposited on the bottom of the test tubes, the desorption of cells in the second stage was almost negligible. On the basis of the above results, a kinetic model was proposed as follows: first stage
-3
second stage
-Vd[XI a dt
-
o[
I[Sml
Time (min)
Figure 1. Time courses of adsorption of S. acidocaldarius on various types of coal particles at pH 2. Symbols of coal type: O,A;A,B;O,C;V,D,O,E.
autoclaved at 121 OC for 15 min and then the cell suspension was prepared by the same procedure as mentioned above. t Potentials. After the pH treatment of coal particles (A, B, C, D, and E) and ion-exchange resins (CG-120 and CG-400) to the equilibrated pH 2, then their potentials were measured by a {potentiometer, Lazer Zee Model 501 (Pen Kem Inc.). The fpotential of the bacterial cells was determined by adjusting the cell suspension to pH 1,2,3, 5, or 7. Each sample was then measured by a { potentiometer, System 3000 (Pen Kem Inc.). ScanningElectron Microscopy (SEM). The samples of pyrite particles adsorbed with cells were gently rinsed with 0.11 N sulfuric acid three times and fixed for 2.5 h with 2.5% glutaraldehyde solution in 0.11 N sulfuric acid. After the fixation, the samples were gently rinsed three times with sulfuric acid, dehydrated, and air-dried (Grishin et al. 1989). All samples were mounted on specimen stubs with a colloidal graphite paint, sputtercoated with a layer of gold ca. 300 A thick, and examined with a JEOL JSM-840 scanning electron microscope operated at 15 kV. Pyrite Dissolution Experiment. In the pyrite dissolution experiment, 1 g of natural pyrite (Fe = 46.7 f 0.1 % , S = 52.7 f 0.1 % , particle size = 75-106 rm) was added in a cylindrical glass vessel containing 500 mL of medium 111 in which S. acidocaldarius cells were suspended with an initial concentration of 1.0 X 107 cells/ mL. Air with 5% (v/v) CO2 was fed into the reactor at a rate of 200 mL/min. The liquid was mixed with a magnetic stirrer at a rate of ca. 300 rpm. The experiment was conducted in a water bath at 70 “C. Samples of the pyrite particles were taken out after 1 and 5 days of incubation. The samples were treated and observed by SEM following the procedure described above.
r
where V is the volume of the cell suspension, [XI is the cell density of the suspension, a is the effective surface area of coals, [Sm] is the maximum number of bacterial cells that can adsorb per unit area of coal surface, ko is the rate constant for the adsorption in the first stage, kl and k2 are the rate constants for the adsorption and desorption, respectively, and X is the fraction of coverage of adsorption sites, defined as follows: (3) where [XO]is the initial cell density in suspension. At equilibrium, eq 2 can be rearranged as follows, considering that [Sei = M S m l 1 1 a =--+-1 (4) where the subscript e indicates equilibrium and K is the equilibrium constant defined as kllkz. A plot of 1/[S,] vs 1/[&I gives a straight line of intercept l/[Sm] and slope l/lC[S,] (see Figures 2 and 3). From the estimated [Sm] and the integration of eq 1,ko can be evaluated: (5) where t was taken as 0.5 min. To evaluate kl, eq 3 and k2 = kl/K are substituted into eq 2 and rearranged: -= -d[XJ dt
kl([x]2 + ([~,]a/ v - [x,]+
1/” [&I/N Integration and rearrangement of eq 6 yields
(2[&]
+ b - D)(2[XI + b + D)
I
Results and Discussion Kinetics of Adsorption and the Model. Time courses of the adsorption of S. acidocaldarius on coal A, B, C, D, and E at pH 2 are shown in Figure 1. There seemed to be a tendency for all types of coals that the cell density of the suspensions decreased drastically and rapidly in 1 min, then gradually increased, and reached equilibrium after 30 min. The results implied that in the first stage cell adsorption took place at a fast rate within a short period (ca. 1 min), and then in the second stage, some
where
6 = [Sm]a/V- [X,]
+ 1/K
A plot of the right side of eq 7 against t gives a straight line of slope kl.
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1.01
"
"
I
"
I
"
"
L
"
1
/1
5
0
10
I X, I-'X 10"
15
(mL/cell)
Figure 2. Variation of the slopes and intercepts in the plots of [XJ* against [SJl for coals. Lines (A, B, C, D, and E) are the regression of data. Symbols are as in Figure 1.
/
e -
/
