FEMS Microbiology Ecology 85 (1991) 183-192 © 1991 Federation of European Microbiological Societies 0168-6496/91/$03.50 Published by Elsevier ADONIS 016864969100071R

183

FEMSEC 00326

Studies on the accumulation of cadmium by a strain of Proteus mirabilis

V. A n d r e o n i 1, C. F inol i 2, p. M a n f r i n 2, M. Pelosi 3 a n d A. Vecchio 2

i lstituto Microbiologia e Industrie Agrarie, Universitgt degli Studi, Turin, Italy, 2 Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universith degli Studi, Milan, Italy, and 3 Consorzio per l'Acqua Potabile ai Comuni della Provincia di Milan, Italy

Received 2 July 1990 Revision received 7 December 1990

Accepted 17 December 1990

Key words: Cadmium tolerance; Uptake; Proteus mirabilis

1. SUMMARY 2. I N T R O D U C T I O N

A bacterium isolated from a wastewater plant sludge, identified as Proteus mirabilis, was tested for cadmium tolerance and accumulation capacity. The organism was able to grow in the presence of Cd 2+ up to 3 0 0 m g l -1 .

Accumulation of cadmium is reported for grow- ing and non-growing cells of the organism. In non-growing cultures a 70% removal of cadmium was observed when the initial concentration of Cd 2+ was 1 mg 1-1 whereas at the same con- centration the removal by growing cells was only 22%. The metal was shown to be associated with the cell envelope (80%) and accumulated in the cytoplasm (20%).

Correspondence to: V. Andreoni. Present address: Universit~ degli Studi, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, via G. Celoria 2, 20133 Milan, Italy.

In recent years research has indicated that many microorganisms are capable of growing in the presence of relatively high concentrations of toxic heavy metals by means of different detoxification mechanisms. Among the mechanisms there are chemical transformations to more volatile com- pounds or to ions with different valencies, accu- mulation of dissolved and particulate metals, for- mation of by-products which make the contami- nant insoluble, utilization of efflux systems and plasmid transfer [1-21]. These different mecha- nisms of detoxification may be exploited to re- move toxic metals from industrial effluents to reduce environmental damage. The ability to accu- mulate metals is furthermore attractive for the recovery of valuable or economically important metals [22-25].

Among the anthropogenic toxicants, cadmium, which performs no biological functions and is

184

released into the environment by wastewaters from several industries, is of major concern [26,27].

The purpose of the present study was to isolate a microorganism capable of growing in the pres- ence of cadmium, to test its sensitivity to this metal and to investigate the removal and the up- take mechanism of the metal, with a view to its eventual use in the removal of the metal in its soluble state from aqueous wastes.

3. MATERIALS A N D M E T H O D S

3.1. Isolation of metal-tolerant microorgan&m The microorganism was isolated from a sludge

sample of a wastewater treatment plant, as previ- ously described [28], and identified as Proteus mirabilis.

3.2. Media Luria Broth medium (LB) [29] and LB without

yeast extract (LB-YE) were utilized in this study. Where appropriate, cadmium acetate was incorpo- rated to obtain the required final concentration of Cd 2+. The heavy metal solutions, sterilized by filtration, were added to autoclaved nutrient broth whose final pH has been previously adjusted to 7.0 with 2N HC1.

3.3. Growth of microorganism The growth in cultural broths containing

cadmium from 0-500 mg 1-1 was monitored by a Zeiss M4 QII I turbidimiter (Zeiss, F.R.G.) at 600 nm. Cultures were also tested for inducibility to cadmium resistance by the growth for six succes- sive transfers in cadmium free LB-YE broth and by comparing them with cultures growing in the presence of 10 and 100 mg 1-1 of cadmium (adapted cells). In these experiments, actively growing cultures of induced and uninduced cells were incubated in LB-YE broth with different amounts of cadmium added.

3.4. Scanning electron microscopy The membrane transfer technique (MTT) [30]

was used for SEM investigations. Membrane filters (0.22 /~m GS Millipore) were put on LB-YE agar and on LB-YE agar supplemented with 100 mg

1-1 Cd2+ and inoculated with a cell suspension of the organism. After various periods of incubation the membranes were picked up and placed on SEM stubs. Samples were fixed in 1% osmium tetraoxide vapors for 1 night, shadowed with a thin layer of gold, and examined by a Phifips 515 scanning electron microscope (Philips, Eindhoven, The Netherlands).

3.5. Preparation of resting cells Bacteria grown overnight in LB broth, without

cadmium, were harvested by centrifugation at 12000 × g for 15 rain, washed twice with 0.05 M TRIS-HC1 buffer (pH 7.2) and resuspended in fresh buffer to give an O.D. of 1.85 at 540 nm.

3.6. Bacterial dry weight Bacterial dry weights were determined by

harvesting the organism by centrifugation, wash- ing the bacterial pellet twice with water and then drying overnight at 105°C.

3. 7. Cadmium uptake by growing organisms 180 ml of LB-YE medium at 0.1, 1, 10, 100,

200, and 300 mg 1 i of Cd 2÷, respectively were inoculated with 20 ml of a pre-grown bacterial culture, at the same Cd z+ concentration, and in- cubated at 30°C on a rotary shaker. At fixed times, samples were removed and centrifuged; the cells were washed with fresh broth and subse- quently with water. Supernatants and washings were combined into a 250-ml volumetric flask and made up to 250 ml with water; cadmium was determined on the cells and on supernatant aliquots.

3.8. Cadmium uptake by resting organ&ms 100 ml of resting cell suspension were added to

100 ml of 0.05 M TRIS-HC1 buffer containing 0.2, 2, 20, and 200 mg 1-1 of Cd 2÷ and incubated on a rotary shaker at 30°C. At fixed times, samples were removed and cells collected by centrifuga- tion. The cells were washed twice with fresh buffer and the washings were combined with the super- natants, as before.

In order to investigate cadmium distribution in cells, the cells were washed with a 10 mM EDTA solution, 3 × 10 ml, for 10 min each time. The phases were separated by centrifugation and the

185

EDTA solutions were combined. After recovery the cells were disrupted in a French pressure cell (American Instruments Co., Washington, DC, U.S.A.) using two passes at 20000 psi. The broken material was centrifuged twice at 60 000 x g for 20 min at 4°C to separate the supernatant from the cell walls and membranes. Cadmium was de- termined in pellets, cytoplasmatic extracts, EDTA solutions, and supernatants.

3.9. Cadmium determination Determinations were performed by a Perkin

Elmer 2380 absorption spectrometer (Perkin Elmer, Norwalk, CT, U.S.A.) after nitric acid di- gestion of samples according to APHA standard methods [31]. When necessary the spectrometer was equipped with a Perkin Elmer HGA 400 graphite furnace (Perkin Elmer, Norwalk, CT, U.S.A.).

3.10. Chemicals All chemicals were of the highest purity avail-

able.

4. RESULTS AND DISCUSSION

4.1. Effect of cadmium on bacterial growth The growth curves of Proteus mirabilis in the

presence of different Cd 2÷ concentrations are re- ported in Fig. 1. Up to 10 mg 1-1 of Cd 2÷ the growth rate was approximately equal to that in the absence of the metal. At higher concentrations the growth occurred after a lag phase which lengthened with increases in Cd 2÷ concentration. After the lag period, the cells accommodated to the pres- ence of the metal and began to proliferate. No growth occurred after 72 h at 500 mg 1-x

From microscopic observations, the cells of Proteus mirabilis which grew in the presence of cadmium seem to be shorter than those grown without the metal (Fig. 2).

The resistance of Proteus mirabilis was con- stitutive. Cells cultured in the absence of cadmium for six transfers and then reinoculated into the Cd-containing medium showed no difference in the lag phase when compared with those of cul- tures grown in the presence of 10 and 100 mg 1-1

0.6-

0 .4 -

0 .2-

0 .0

0.6-

0.4

0.2-

0. O:

A

0.6

0.4-

0.2-

0 . 0 ~, 0 i0

A A A A

vv ©

C

/

2'0 30 40 50 60 70

Time (hours)

Fig. 1. Growth of Proteus mirabilis in LB-YE medium in the presence of different concentrations of cadmium. The cells were grown for six transfers: A without Cd z+, B in the presence of 10 mg 1-1 of Cd 2+, C in the presence of 100 mg 1-1 of Cd 2+. Cadmium concentrations (mg 1): (11) 0, (*) 1, (O)

10, ( o ) 100, (zx) 200, (v) 300, (A) 400, ( + ) 500.

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Fig. 2. Scanning electron microphotographs of Proteus mirabilis at successive incubation times. P. mirabilis grown on LB-YE in the absence of cadmium (A, after 2 h and B, after 4 h of incubation) and in presence of 100 mg 1-1 of cadmium (C, after 16 h, D, after 24

h and, E, after 30 h of incubation).

of cadmium (Fig. 1). The presence of an extended lag phase (8, 18, and 32 h) indicated that the accommodation of the organism to cadmium was not due to selection of a mutant, but rather to physiological adaptat ion. The capabil i ty of organisms to alter their physiology is a defense mechanism for biological survival against environ- mental injury. Similar findings were observed by Mitra et al. [321 with cells of E. coli which ex- hibited an abnormally long lag phase when inoc- ulated in a medium containing 3 × 10 -6 M Cd 2+ and by Aiking et al. [33] in studies carried out with Klebsiella aerogenes in the presence of 7 × 10 -5 M Cd 2+.

The toxicity of cadmium towards P. mirabilis was pH dependent; in fact, while at p H 6 and 7 the organism was able to grow in the presence of 100 mg 1-] of Cd 2+, and at pH 8 no growth was observed (Fig. 3); this result is in agreement with the observations of Babich and Stotzy [34] and Korkela and Pekkanen [35].

4.2. Uptake by growing cells Up to 10 mg 1-1 the Cd 2+ uptake by growing

cells of Proteus mirabilis increased as the initial concentration of the metal in medium increased. After 8 h, under the same growth, the Cd 2+ amount in the biomass, at 1 and 10 mg 1-1 was 10- and 20-fold respectively than that present at 0.1 mg 1-1. Concerning the concentrations be-

06 t cz 0 . 4 -

d

0 , 2 -

0 . 0 . . . . . ', ~'~ ~ ', , ; 0 10 20 30 40 50

Time (hours)

Fig. 3. Growth of Proteus mirabilis in LB-YE medium in the presence of 100 mg 1-1 of Cd 2÷ at different pH: ([2) 6, (o) 7,

(zx) 8.

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tween 100 and 300 mg l-1 it was observed that, although the cellular growth decreased as the cadmium concentration increased, the amount of metal adsorbed however was of the same magni- tude (see Table 1).

4. 3. Uptake by resting cells Table 2 shows that the level of accumulation in

resting cells also increased as the initial cadmium concentration increased but the metal uptakes were higher than previously observed with growing cells. The uptakes were readily accomplished, and after 2 h were almost complete. This phenomenon was evidenced by the experiments at 10 mg 1 -~ of cadmium. The time course of cadmium uptake is shown in Fig. 4. It is evident that cadmium ab- sorption rate was fast during the first 2 h and slowed down subsequently.

4. 4. Cellular location of accumulated cadmium The distribution and binding states of Cd 2+

taken up by resting cells of Proteus mirabilis was investigated by washing the cells with EDTA and successively disrupting them.

Table 3 shows that about 64-75% of the ad- sorbed Cd 2+ was released by EDTA washes and that about 19-22% was transferred to the cyto- plasm. The remaining amount was associated to the pellets (membranes and cell walls). These data suggest that most cadmium accumulation depends on physico-chemical adsorption at the cell surface with ligands easily substituted by EDTA. Also Horikoshi et al. [23] and Dunn and Bull [36], in experiments carried out with Gram-negative and Gram-posit ive bacteria, observed that EDTA re- moved the metal and concluded that metals were extracellularly bounded.

4.5. General remarks Aqueous effluent streams originating from pro-

duction processes can contain heavy metals which, due to their chemical and toxicological properties, can' t be discharged directly into the environment. To reduce the concentration of these metals con- ventional physico-chemical procedures, i.e. pre- cipitation, reduction and oxidation, ion exchange, reverse osmosis, and evaporation can be applied. However, because such processes may not be suit-

188

Table 1

Cadmium uptake by growing cells of Proteus mirabilis

Cadmium Time a Biomass Distribution of Cd

(mg 1 1) dry weight Biomass Supernatant (mg) (mg) (mg)

0.1 0 h 25 0.020

8 h 229 0.005 0.015 17 h 231 0.007 0.012 7 days 231 0.008 0.012

1 0 h 26 0.200 8 h 226 0.043 0.157

17 h 219 0.064 0.136 7 days 221 0.078 0.122

10 0 h 25 2.10 8 h 235 0.12 2.02

17 h 227 0.31 1.78 7 days 197 0.77 1.37

100 0 h 18 20.00 17 h 162 0.29 19.40 24 h 162 0.36 19.80

7 days 116 1.11 19.00

200 0 h 16 40.00

30 h 132 0.26 40.20 40 h 124 0.34 40.00

7 days 94 1.87 38.80

300 0 h 15 60.60 45 h 59 0.23 59.20 54 h 62 0.33 60.60

7 days 58 1.87 58.40

a Times have been established from growth curves, b n.d. = not determined.

Cd (~g) Removal

Biomass (mg) of Cd (%)

n.d. b

0.022 25 0.03O 35 0.035 40

n.d. 0.190 22 0.292 32 0.353 39

n.d. 0.51 6 1.36 15 3.91 37

n.d. 1.79 1 2.22 2 9.57 6

n.d. 1.97 < 1 2.74 < 1

19.89 5

n.d. 3.90 < 1 5.30 <1

32.30 3

2.0-

e-

0,

.0

0 2 1 1 I [ 1 1 1 1 1 1 1 1 1 1 I 1

4 6 8 10 12 14 16 18

Time (hours)

Fig. 4. Time course of cadmium uptake by resting cells of Proteus mirabilis in the presence of 10 mg 1-1 of cadmium.

able when the initial concentration is in the range of tens of mg 1-1 and the final concentration has to be less than 1 mg 1-1, methods using micro- organisms for this purpose are getting more atten- tion [37].

The present study shows that P. mirabilis, which we isolated from a biological treatment plant, is able to grow in the presence of cadmium up to 300 mg 1-1 and exhibits a physiological adaptation mechanism to this metal. The presence of this mechanism is confirmed by a lag phase whose length increases by increasing the concentrations of the metal in the cultural medium. P. mirabilis is capable of removing cadmium from aqueous solutions both as growing and resting cells but with different efficiency.

Table 2

Cadmium uptake by resting cells of Proteus mirabilis

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Cadmium Time Distribution of Cd

(rag 1-- 1) (h) Biomass a Supernatant

(mg) (mg)

Cd (/.t g)

Biomass (mg)

Removal of Cd (%)

0.1 0 0.020 2 0~014 0.O06 4 0.015 0.006

1 0 0.200 2 0.131 0.070 4 0.146 0.054

10 0 1.96 2 0.44 1.50 4 0.48 1.48 6 0.50 1.46

17 0.55 1.38

100 0 18.70 2 1.30 17.40 4 1.51 17.20

n.d. b 0.044 0.047

n.d. 0.410 0.460

n.d. 1.38 1.50 1.56 1.72

n.d. 4.06 4.72

70 75

65 73

22 24 26 28

7 8

a Biomass concentration, 1.6 mg ml- 1 as dry weight, b n.d. = not determined.

In the application of bacteria in metal recovery both the rate and the levels of accumulation are important. The findings obtained in this study suggest that our microorganism was appropriate only in the presence of low metal concentrations and that the non-growing cells were more suitable.

The use of non-growing cells in metal accumu- lation is an attractive possibility as the metal can

Table 3

Cadmium uptake by resting cells of Proteus mirabilis: distribu- tion in cells

Cadmium Time Distribution of Cd

(rag 1 - 1) (h) EDTA Pellet Cytoplasm (mg) (mg) (mg)

0.1 2 0.009 (64%) 0.002 (14%) 0.003 (22%) 4 0.010 (67%) 0.002 (13%) 0.003 (20%)

1 2 0.092 (70%) 0.012 (9%) 0.027 (21%) 4 0.104 (71%) 0.012 (8%) 0.030 (21%)

10 2 0.32 (74%) 0 .03 (6%) 0.09 (20%) 4 0.35 (72%) 0 .03 (7%) 0.10 (21%)

100 2 0.97 (75%) 0 .08 (6%) 0.25 (19%) 4 1.13 (75%) 0 .08 (5%) 0.30 (20%)

be supplied at concentrations lethal to growing cells and moreover the organism-metal bond is easily disrupted and the cells could be re-used in the removal process.

Our work will be a contribution in the field of microorganism utilization. However, the biotech- nological application of this organism needs fur- ther investigation especially with respect to the development and testing of practical systems.

ACKNOWLEDGEMENTS

We are very grateful to Mr. Piero Fabbri, Centro Sperimentale SNIA Fibre Ce~ano Mader- no, for the careful execution of electron mi- croscopy photos. This work was supported by the Italian Ministry of Public Instruction (60%).

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