Adsorption of bacterial cells to crystal particles of heavy metals: Role of electrostatic interaction

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 77, No. 6, 636-641. 1994

Adsorption of Bacterial Cells to Crystal Particles of Heavy Metals: Role of Electrostatic Interaction

NAOTO YOSHIDA AND YOSHIKATSU MUROOKA*

Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, 1-Kagamiyama, Higashi-Hiroshima 724, Japan

Received 4 November 1993/Accepted 14 March 1994

Several species of bacterial cells were found to adsorb to crystal particles of heavy metals, such as cadmium, zinc, copper, and iron. In particular, ThiobaciUus sp. strain 13-1, isolated from a corroded concrete sewer, was rapidly adsorbed to crystal particles of cadmium. Observation under a light microscope revealed that cells of Thiobacillus sp. strain 13-1 were adsorbed aggregately on the surface of crystal particles of cadmium. The bacterial cells that adsorbed were released into the aqueous phase by the addition of carboxylic acid containing negatively charged hydroxyl groups or compounds. Since hydroxycarboxylic acid seems to prevent the tenacious adsorption of the bacterial cells on the crystal particles of heavy metals, the adsorption of bacterial cells to such particles may be attributed to the electrostatic interaction between them.

The phenomenon of microbial adsorption plays an important role in the biosphere, e.g., the infection of various tissues (1), motility (2), interaction between pathogenic bacteria and various target cells (3), the phagocytosis of bacteria (4), dental decay (5), ship fouling (6), fermentation (7), and waste water and sewage treatment (8). The adsorption of bacteria to interfaces has become a focus of interests in recent years (9-11). Pathogenic bacteria and rhizobia of plant root cells involve highly specialized mechanisms of recognition mediated by lec- tins (sugar and protein carbohydrates) and specific receptors of the cell surfaces to adsorb specific molecules (12). In addition, many microorganisms that degrade insolu- ble substrates, such as cellulose, chitin, and lignin, adsorb target substances. However, unknown mechanisms of interaction between substances and microorganisms which adsorb nonspecifically to many different types of interfaces as well as to inert surfaces, remain.

Recently, we isolated strains of Thiobacillus species from corroded concrete of sewers and found that these strains exhibited resistance to several heavy metals (13). During the course of our work on a heavy metal-binding protein from these strains (14), we found that cells of these bacteria were adsorbed to the crystal particles of heavy metals. In this report, we demonstrated that some bacteria are specifically adsorbed on crystal particles of heavy metals and discuss the possible mechanism behind this adsorption.

MATERIALS AND METHODS

Bacterial strains and culture conditions The bacterial strains used are summarized in Table 1. These bacteria were cultured in LB medium (15) containing 1% glucose or YEM medium (16) at 28°C on a reciprocal shaker. Cells were harvested by centrifugation at 3,000 x g for 10 rain and washed twice with 50 mM Tris-HC1 buffer (pH 8.0). The cells were suspended in a small volume of 50 mM Tris-HC1 buffer (pH 8.0) and adjusted to 3 x 109 cells per ml.

* C o r r e s p o n d i n g a u t h o r .

Determination of adsorption rate of various bacterial cells to crystal particles of cadmium Bacterial cell sus- pensions were added to Klett tubes containing 0.2g of crystal particles of cadmium (100 mesh) in 4ml of 50ram Tris-HCl buffer (pHS.0) and adjusted to 400 Klett units. The mixture was vigorously shaken with a vortex mixer for 1 rain and allowed to stand for 4 to 20 rain. The optical density of the aqueous phase was measured with a Klett-Summerson colorimeter.

Estimation of numbers of cells adsorbed on heavy metal crystal particles Crystal particles of heavy metal [0.2 g in 1 ml of 50 mM Tris-HCl (pH 8.0)] were inoculated with a bacterial cell suspension, vigorously shaken for 1 min, and allowed to sediment for 20 rain at room temperature. The density of the upper phase, con- raining unadsorbed bacterial cells, was measured using a UV/Vis spectrophotometer, and the cell number was esti- mated based on a standard curve correlating cell number to absorbance.

Microphotographs Crystal particles of cadmium [0.2 g in 1 ml of 50 mM Tris-HC1 (pH 8.0)] were mixed with Thiobacillus sp. strain 13-1 (3 x 109cells/test tube) and shaken at room temperature for 1 rain. The sedi- ments were rinsed twice with 50 mM Tris-HCl buffer (pH 8.0), mounted on specimen stubs, and examined under a microscope (Nicon AFM).

Displacement of bacteria adsorbed to crystal particles of cadmium Bacterial cells (3 x 109) were displaced from 0.2 g of crystal particles of cadmium in 500/d of 50raM Tris-HCl (pH 8.0) by the addition of various chemical compounds on a descending gradient concentration. Displacement of bacterial cells was conducted in the minimum concentration of the chemical compounds added as sodium or potassium salt.

Chemicals Crystal particles of cadmium and zinc were obtained from Katayama Chemical Co. Inc., Osaka. Crystal particles of copper and iron (100 mesh) were pur- chased from Wako Pure Chemical Co. Industries Ltd., Osaka. /3-Glucuronidase was supplied by Sigma Co. Ltd., St. Louis, MO, USA. All other chemicals were standard commercial products of guaranteed grade.

636

VoL 77, 1994

TABLE 1. Bacterial strains used in this study

Strain Characteristics Source a

Agrobacterium Rod, Tuft of polar flagella, IFO radiobacter Gram-, Crown gall bacteria IFO 12665b 1

Agrobacterium Rod, Tuft of polar flagella, Isolated from tumefaciens Gram-, Crown gall bacteria soil B-2-1-1

Bacillus Rod, Tuft of polar flagella, HUT subtilis Gram ÷ HUT8052

Enterobacter Rod, Peritrichous, Gram- cloacae bv. renge AI05

Erwinia Rod, Tuft of polar flagella, carotovora Gram-, plant pathogenic IFO3380 bacteria

Escherichia Rod, (Single) polar coli JM109 flagellum, Gram

Klebsiella aerogenes W70

Pseudomonas aeruginosa IFO3448

Pseudomonas fluorescens IFO3081

Rhizobium huakuii bv. renge B3

Rod, Non-flagellum, Gram -

Rod, Single polar flagellum, Gram-

Rod, Single polar flagellum, Gram-

Rod, Subpolar flagellum, Gram-, Root nodule bacteria

Rhizobium leguminosarum KLL 1

Thiobacillus sp. strain 13-1

Rod, Subpolar flagellum, Gram-, Root nodule bacteria

Rod, Single polar flagellum, Gram-, Facultative autotroph

Thiobacillus sp. Rod, Non-flagellum, strain 3S-1 Gram-, Facultative

autotroph

Isolated from root of Astragalus sinicus(26)

IFO

Obtained from Takara Shuzo Co., Ltd. (27)

Isolated from soil (28)

IFO

IFO

Isolated from root nodule of Astragalus sinicus (29)

Isolated from root nodule of pea

Isolated from corroded concrete (13)

Isolated from corroded concrete (13)

a lFO, Institution for Fermentation, Osaka. HUT, Hiroshima University Type Culture.

RESULTS

Adsorption of bacterial cells to crystal particles of cadmium Thiobacillus sp. strain 13-1 was added to a test tube containing crystal particles of cadmium. The suspension was vigorously mixed and allowed to stand. After 20 min, the bacterial cells sedimented together with the crystal particles of cadmium, resulting in a clear upper layer (Fig. 1). Since it was thought that polysaccharides present on the cell surface might have been responsible for the adsorption to the crystal particles of cadmium, Agrobacterium radiobacter IFO12665bl, an e x t r a p o l y s a c c h a r i d e - p r o d u c i n g b a c t e r i u m , was tes ted . Howeve r , n o s e d i m e n t a t i o n o f A . rad iobac ter I F O 1 2 6 6 5 b l a n d c rys ta l par t ic les o f c a d m i u m was o b s e r v e d , even a f t e r s t a n d i n g fo r 20 m i n , s h o w i n g t h a t p o l y s a c c h a r i d e s o n the bac t e r i a l s u r f ace d o n o t p a r t i c i p a t e in a d s o r p - t i o n to m e t a l par t i c les . W h e n cells o f A . r a d i o b a c t e r I F O 1 2 6 6 5 b l were t r e a t e d w i th ~ - g l u c u r o n i d a s e , w h i c h

BACTERIAL ADSORPTION TO HEAVY METALS 637

FIG, 1. Adsorption of bacterial cells to crystal particles of cadmium. Test tubes containing Thiobacillus sp. strain 13-1 (T) or A. radiobacter IFO12665bl (A) were mixed with crystal particles of cadmium in 50raM Tris-HCl (pH 8.0) and left to stand for 20min. Thiobacillus sp. strain 13-1 cells adsorbed to the crystal particles of cadmium, forming a clear upper phase. A. radiobacter IFO12665bl remained dispersed in the upper aqueous phase.

removed cell-surface polysaccharides (17), the cells were adsorbed to the crystal particles of cadmium (Fig. 2).

To determine whether this phenomenon of adsorption was specific only to a certain bacterium, we tested several bacterial strains, most of which were isolated from the biosphere, including other strains of Thiobacillus sp. isolated from corroded concrete (13). Figure 3 shows that Thiobacillus sp. strain 13-1 sedimented most rapidly in the presence of crystal particles of cadmium, followed

100

~ 8O

D . ~ 6o

- i

g 4o

.~" 2O

p -

FIG. 2. Effect

0 ! I i !

0 5 10 15 20

Time (rain)

of /3-glucuronidase on adsorption of A. radiobacter IFO12665bl to crystal particles of cadmium. Treatment with /3-glucuronidase was as follows: 10 mg of/%glucuronidase was dissolved in 50 mM citric buffer (pH 5.0) containing 3 × 109 cells. The reaction mixture was incubated at 37°C with reciprocal shaking for 30min, and centrifuged at 3,000xg for 10min. Cells were washed twice in 50 mM Tris-HC1 (pH 8.0) and used as a cell suspension for adsorption to crystal particles of cadmium with ((3) or without treatment of/3-glucuronidase ( • ) .

638 YOSHIDA AND MUROOKA J. FERMENT. BIOENG.,

100

~ ao

~ 6o 0

o- 40 m

• -- 20

t- 0 0 5 10 15 20

Time (mln)

FIG. 3. Adsorption rate (ability) of various bacterial cells to crystal particles of cadmium, measured as the turbidity of the upper phase. Cells were suspended in 50 mM Tris-HCl (pH 8.0) and 0.2 g of cadmium crystal particle was added: (©), R. huakuii bv. renge B3, R. leguminosarum KLL1, E. cloacae A105, A. radiobacter IFO12665b1, A. tumefaciens B-2-1-1, E. carotovora IFO3380, and P. fluorescens IFO3081; (e), P. aeruginosa IFO3448; (z~), E. coli JM109; (A), B. subtilis HUT8052; ( • ), K. aerogenes W70; ( [] ), Thiobacillus sp. strain 3S-l; ( . ) , Thiobacillus sp. strain 13-1.

by Klebsiella aerogenes W70 and Thiobacillus sp. strain 3S-1. Escherichia coli JM109 and Bacillus subtilis HUT8052 sedimented rapidly on 5 min standing, after which there was gradual sedimentation. Pseudomonas aeruginosa IFO3448 showed gradual sedimentation over 20min . However, no significant sedimentations with crystal particles of cadmium were observed with Pseu- domonas fluorescence IFO3080 or Erwinia carotovora IFO3380. Polysaccharide-producing strains of Entero- bacter cloacae A105, Rhizobium leguminosarum KLL1,

8 . . , "

o

c

• ~- 2

o o o.1 o:2 0:3

Crystal particles of cadmium (g)

FIG. 4. Adsorption rate of Thiobacillus sp. strain 13-1 to different concentrations of crystal particles of cadmium in terms of cell numbers. After shaking for 1 min and allowing to stand for 20 rain, the numbers of cells adsorbed were determined by counting the number of bacterial cells remaining in the upper phase.

R. huakuii bv. renge B3, or A. tumefaciens B-2-1-1, like A. radiobacter IFO12665bl, were not sedimented with crystal particles of cadmium. At pH values of 6.0 to 11.0, but not at pH < 5 or >_--12, bacterial cells were adsorbed to crystal particles of cadmium (Table 2).

Effects of heavy metals on the adsorption of Thiobaci l - lus sp. strain 13-1 The numbers of cells of Thiobacil- lus sp. strain 13-1 that can be adsorbed on various heavy metals were determined. 6 × 10 ~° cells of Thiobacil- lus sp. strain 13-1 were added into crystal particles of various heavy metals. About 5.5 × 10 9 cells per 0.2 g of crystal particles of cadmium were adsorbed (Fig. 4). This adsorption capacity was 2 or 3 orders higher than those

A B C

| |

I 50 m I

FIG. 5. Microphotographs of crystal particles of cadmium inoculated with Thiobacillus sp. strain 13-1. A, Crystal particles of cadmium; B, Thiobacillus sp. 13-1; The intact cells were readily stained with 0.4% alcoholic solution of crystal violet for 1 min. C, Thiobacillus sp. strain 13-1 adsorbed to crystal particles of cadmium after standing for 20 min.

VoL 77, 1994

TABLE 2. Effect of pH on the adsorption of Thiobacillus sp. strain 13-1 cells to crystal particles of cadmium a

pH Adsorption

< 4.0 5.0 6.0 + 8.0 +

11.0 + >_--12.0

Thiobacillus sp. strain 13-1 cells (3 × 109) were mixed with 0.2 g of crystal particles of cadmium in buffers at pH values that ranged from 3.4 to 12.0, namely, 50 mM sodium acetate (pH 3.7, 5.0, 6.0), 50 mM Tris-HCl (pH 8.0, 11, 12).

of zinc, copper or i ron (Table 3). Crystal particles of cadmium have unusual ly high affinity for bacterial cells compared to copper, zinc, or iron (data not shown).

Figure 5 shows microphotographs of crystal particles of cadmium exposed to Thiobacillus sp. strain 13-1 cells in 50mM Tris-HC1, pH8.0 . The large areas covered with the cells seem to indicate that the surfaces of crystal particles of cadmium are highly heterogeneous with respect to their affinity for the bacteria.

Disp lacement of Thiobacillus sp. strain 13-1 adsorbed on crystal particles o f c a d m i u m The effects of various organic or inorganic compounds, such as carboxylic acids, surface active agents, organic solvents, a saccharide, and phosphate acid on the displacement of adsorbed Thiobacillus sp. strain 13-1 were tested. Displac- ing ligands used and the min imum concentrat ion of the ligand required to displace the bacterial cells were determined on a descending concentrat ion gradient (Table 4). The cells were displaced by compounds containing di-, tri-, or tetracarboxylic acid, such as tartaric acid, malic acid, citric acid and ethylenediaminetetraacetic acid (EDTA), at low concentrations. Compounds containing a monovalent anion, such as glycolic acid and lactic acid, displaced cells at a rather high concentrat ion (at least 100mM). Thus, trivalent or tetravalent anions were found more efficient than monovalent or divalent ones in the displacement of bacterial cells from the crystal particles of cadmium. However, no displacement of the bacteria was found with acetic or propionic acid, even at a concentrat ion of 1 M. Various heavy metal ions (200 mM), KC1 (1M) and NaC1 (1M) showed no effect on the displacement of cells from crystal particles of cadmium. However surface active agents such as sodium lauryl sulfate (SDS) did cause the displacement of bacteria, and with the addit ion of phosphate acid almost same the phenomenon was found as with carboxylic acids. Organ- lc solvents and saccharide did not have any effect. Citric acid, maleic acid, tartaric acid, and malic acid used as

TABLE 3. Adsorption of Thiobacillus sp. strain 13-1 cells on crystal particles of heavy metals

Crystal particles a Adsorbed cells b of heavy metal (number / g metal)

Cd 3 × 1010 Zn 2 × l0 s Cu 2 × 107 Fe 5 × 107

a Crystal particles of heavy metals passed through a size 100 mesh. b Crystal particles of heavy metals (0.2 g) were inoculated with the

cell suspension 00 l° cells) and shaken.

BACTERIAL ADSORPTION TO HEAVY METALS 639

TABLE 4. Gradient elutions of bacterial cells from crystal particles of cadmium with displacing ligand

Displacing ligand a Structure Minimumb concentration

Monocarboxylic acids Acetic acid CH3-COOH N.D. c (1 M)

Propionic acid CH3CH2-COOH N.D. (1 M)

Glycolic a c i d OH-CH2-COOH 100 mM

Lactic acid CH3CH-COOH 200 mM /

OH Dicarboxylic acids

Malonic a c i d HOOC-CH2-COOH 100 mM

Maleic acid HOOC-C = C-COOH 50 mM

Malic a c i d HOOC-CH2CH-COOH 20 mM /

OH

Tartaric acid HOOC-CHCH-COOH 10 mM

6H6H Tricarboxylic acid

OH Citric a c i d HOOC-CH2~CH2-COOHv 5 mM

1 COOH

Tetracarboxylic acid

Ethylenediami- HOOC-CH2 CH2-COOH 5 mM netetraacetic acid (EDTA) NCH,CH2N

HOOC-CH2 CH2-COOH Surface active agents

O II

Sodium lauryl CH3(CH2)H-S-O-ONa 0.2% sulfate (SDS) /

Polyethylene glycol mono-p- - ~ - - ~ O ( C H 2 C H 2 0 ) n H N.D. (10%) isooctylphenyl ether

(Triton X-100)

Inorganic compound

Phosphate acid O 10 mM Ir NaO-P-OH

ONa Organic solvents

Methanol CH3-OH N.D.

Ethanol CH3CH2-OH N.D.

Ethyl acetate CHa-COOC2H5 N.D.

Saccharide OH~ D-(+)-Glucose OHOH O N.D. (1 M)

I I I J / 1 CHCHCHCHCH- CH2OH

I OH

Displacing ligands were used as sodium or potassium salt. b The displacement position of 3 × 109 cells of Thiobacillus sp.

strain 13-1 from 0.2 g of crystal particles of cadmium is given as the minimum concentration of compounds during a descending concentration gradient.

c Not displaced.

640 YOSHIDA AND MUROOKA J. F1/RM1/NT. BIOENG.,

displacing ligands contain one or more hydroxyl groups and negative charges, and they seem to compete with bacterial cells for metal binding site, resulting in the dis- persion of the bacterial cells. However, there was no difference in the minimum concentration of displacing ligands between Thiobacillus and other bacterial strains such as E. coli and K. aerogenes.

DISCUSSION

Thiobacillus ferrooxidans apparently adsorbs to solid surfaces such as sulfur, sulfide minerals, the glass wall of culture flasks, and hydrated ferric precipitates (18). How- ever, quantitative information on the adsorption of bacteria to solid surfaces is not available. In the present study, direct observation showed that specific bacterial cells, in particular Thiobacillus sp. strain 13-1, can be adsorbed to crystal particles of heavy metals, such as cadmium, zinc, copper, and iron.

Bacterial cells adsorbed to crystal particles of cadmium were released into an aqueous phase by the addition of negatively-charged compounds at pH 8.0. The minimum retention concentrations of malonic acid and maleic acid were much higher than that of citric acid, reflecting the number of negative charges and hydroxyl groups in the molecule. Citric acid had a high affinity to crystal particles of cadmium which may be due to the presence of three negative charges and hydroxyl groups forming a coordination complex with crystal particles of cadmium. Acetic acid and propionic acid, which lack hydroxyl groups, did not have any affinity to crystal particles of cadmium. The hydroxyl group and a negative charge may be necessary for the displacement of bacteria from crystal particles of cadmium. The effect of phosphate acid also supports this mechanism. A surface active agent has a high affinity to crystal particles of cadmium due to its negative charges, whereas polyethylene glycol mono-p-isooctylphenyl ether (Triton X-100), which lacks negative charges, did not bind to the crystal particles of cadmium. SDS may be act in preventing the tenacious adsorption of bacterial ceils on the surfaces of crystal particles of cadmium either by the repression of adsorption or the displacement of previously adsorbed cells from the solid surfaces, or both. Organic solvents and saccharides, which lack negative charges, did not affect the displacement of bacterial cells. The cellular polysaccharide might act in neutralizing and masking the negative charges of the cell surface, resulting in a de- crease in the capacity of adsorption. This hypothesis was supported by the treatment of Agrobacterium radiobacter IFO12665bl with /3-glucuronidase. These results sug- gest that adsorption of Thiobacillus sp. strain 13-1 to crystal particles of cadmium can be explained by the physicochemical electrostatic interaction of the surfaces of both the bacterial ceils and metal particles.

Under normal conditions, most metal particles are covered with hydroxyl groups (19). Since these surface hydroxyl groups seem to have a great influence on the physical and chemical properties of the surfaces of heavy metal particles, water on the surface of heavy metal may be important in the adsorbed state. The adsorbed state of water on anatase (titanium dioxide) has been investi- gated by Munuera (20), and Primet et al. (21) suggested from an infrared spectroscopic study that the surface of anatase contains hydroxyl groups. The hydrogen bond of the water molecule with the oxygen atom on a metal

Bacterial cell

~ | t | l

FIG. 6. Diagrammatic representation of the mechanism of adsorption to cadmium crystal particles in vitro by Thiobacillus sp. strain 13-1 and the effect of citric acid on adsorption.

surface is considered to be stronger than that between water molecules since the oxygen atom of the metal surface has a higher proton affinity than the oxygen atom of the water molecule (22). A water molecule coordinat- ed to crystal particles of cadmium seems to form a hydrogen bond with an adjacent surface oxygen atom, and dissociate into two hydroxyl groups by proton shift through the hydrogen bond as follows:

%. % , , i / ° , , i / ° , , i / .20 , , i /0 , j /0 , , i / , , / o , , / o , , i / Cd Cfl Cd > Cd Cd Cd > Cd Cd Cd

Crystal particles of cadmium would be covered with hydroxyl groups in water. An oxide or a hydroxide suspended in an aqueous environment can acquire its surface charge by adsorbing H ÷ or O H - ions. The pH at which a solid has no net surface charge is defined as the point of zero charge (the isoelectric point). The surface charge characteristics of Cd(OH2) at different pH levels have been examined and the isoelectric point of Cd(OH2) has been determined to be p H > 10.5 (23).

The surface charges of bacteria at different pH levels have been determined microelectrophoretically by Marshall (24). Electrophoretically determined isoelectric points of bacterial species showed no fundamental differences in the range of values for Gram-positive and Gram-negative bacteria. An increase in the net negative charge was found at pH 10.7, while a positive charge was apparent at pH 2.0. These results are consistent with an ionic surface consisting mainly of acidic (carboxyl) groups and few basic (amino) groups (25). Here, we ex-

VoL. 77, 1994 BACTERIAL ADSORPTION TO HEAVY METALS 641

amined the capacity of bacteria to adsorb to the surface of crystal particles of cadmium in terms of their cell surface charge properties.

The bacterial cells and crystal particles of cadmium were net negatively charged at pH values above their isoelectric points (pI); cells of Thiobaci l lus sp. strain 13- 1 may be net negatively charged at pH values above 6. However, crystal particles of cadmium may be net positively charged at pH values below 11.0; at higher pH values, the charges become negative in both crystal particles of cadmium and bacterial cells. We consider that the adsorption of bacterial cells onto the surfaces of crystal particles of heavy metal varies according to the surface charge of the bacterial cells. The differences in the adsorption rates may result from differences in the structure and composit ion of the surfaces of the bacterial cells; here, Thiobaci l lus sp. strain 13-1 has the highest number of negative charges. Stronger adsorption can be attributed to the number of negative charges of the surface of the bacterial cells. From the present results, we propose a hypothetical mechanism of adsorption by bacteria to crystal particles of heavy metals as shown in Fig. 6. The adsorption of bacterial cells to crystal particles of cadmium may be similar to the behavior of anions on anion exchanged resins. However, 1 M NaC1 or KC1 did not displace bacterial cells from crystal particles of cadmium, whereas 5 and 10mM citric and tartaric acids, respectively, did. Thus, the mechanism of adsorp- t ion of bacterial cells to cadmium crystal particles seems to be due to mainly the electrostatic force of attraction between the positively charged surface of the crystal particles of cadmium and the negatively charged bacterial cells. The degree of the negative charge of the bacterial cells may reflect the specificity of bacterial strains in adsorbing crystal particles of heavy metals.

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Adsorption of bacterial cells to crystal particles of heavy metals: Role of electrostatic interaction