ELSEVIER

Bioresource 7’echnology 65 (1998) 73-82 0 1998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain 0960-8524198 $19.00

PII:SO960-8524(98)00011-X

EFFECTS OF HEAVY METALS ON PsEUD0lW0AL4S AERUG.lNOSA AND BACILLUS THVRlNGIENSIS

A. Hassen,a * N. Saidi,” M. Cherifh & A. Boudabous”

“Institut National de Recherche Scientifique et Technique, URNE-Eau, BJ? 15 - 1082, Cite Mahrajkne, Tunis, Tunisia hlnstitut National Agronomique de Tunisie, 43 Avenue Charles Nicolle, 1082 Citk Mahra&e Tunis, Tunisia

’ Faculti des Sciences de Tunis, Laboratoire de microbiologic, Campus universitaire, 1060 Tunis, Tunisia

(Received 9 March 1997; revised version received 10 December 1997; accepted 24 December 1997)

Abstract INTRODUCTION The biosorption of the heavy metals most frequently

found in polluted environments by Pseudomonas aeruginosa and Bacillus thuringiensis was studied. The efsects of these metals on bacterial growth, quantity of dry cells, ammonium assimilation, pigment production, and protein synthesis were investigated. At lower concentrations than the minimal inhibitory concentration (MC), the metals partially limited bacterial growth and caused an inhibition proportional to the metal concentration applied. The production of bacterial biomass varied according to the nature and concentration of the metals, and to the bacterial strain studied. The biosolption of metals by P. aeruginosa and B. thuringiensis was variable. Mercury and copper appeared to be the elements most adsorbed by bacteria. Citrate noticeably increased the biosorption of chromium by P. aeruginosa (0.07-45.9%) and copper by B. thuringiensis (18.7-33.8%). Metallic cations exerted variable effects on protein synthesis. Zinc stimulated protein synthesis in P. aeruginosa, and cadmium inhibited it significantly in B. thuringiensis Mercury and cobalt, at concentrations below the MC, always inhibited the synthesis of pigments in P. aerugi- nosa The strong interactions of mercuy and copper with organic matter suggest that these undesirable elements might be removed from the environment by bacterial trapping and sequestration. A better under- standing of the different forms of metals actually existing in polluted environments (speciation) would be of great interest. 0 1998 Elsevier Science Ltd. All rights reserved

Interactions of heavy metals with microorganisms have been of increasing interest. The study of these interactions has been especially focused on bacterial transformation and conversion of metallic ions by reduction in different polluted environments (Chang et al., 1993); the selection of metal-resistant micro- organisms from polluted environments (Hiroki, 1994; Nieto et al., 1989) and the use of resistant microorganisms as indicators of potential toxicity to other forms of life (Doelman et al., 1994; Olson and Thornton, 1982); mechanisms, determinants and the genetic transfer of microbial metal-resistance (De Rore et al., 1994; Goblenz et al., 1994, Guzzo et al., 1994, Rajini Rani and Mahadevan, 1994); mobility of these metals in terms of bioavailability to plants, leachability to organic matter fractions and specia- tion of the metals (Holm et al., 1995; Lun and Chris- tensen, 1989; Mullen et al., 1989); the possibility of using these bacteria for detoxifying polluted environ- ments (Rohit and Sheela, 1994).

The complexation of metallic ions with numerous organic acids is well known. The case of citric acid is well studied and the biodegradation of the citric acid-metal complex is known to vary according to the nature of metal and microorganisms (Brynhildsen and Rosswall, 1989).

Key words: heavy metals, biosorption, cell mass, proteins, pigments, Pseudomonas aeruginosa, Bacillus thuringiensis.

*Author to whom correspondance should be addressed. 73

The objectives of this study were to determine, in the presence or absence of citric acid, the metal- binding capacities of whole cells of a gram-negative bacterium, Pseudomonas aeruginosa as a well-charac- terized, ubiquitous bacterium, resistant to several anti-microbial agents, producing many intra- and extra-cellular substances and a gram-positive bacte- rium, Bacillus thuringiensis, a spore-forming and ubiquitous bacterium, aerobic, easily culturable in currently used media. The six heavy metals most frequently present in polluted aquatic and soil

74 A. Hassen et al.

environments (Cu, Cr, Hg, Cd, Zn, Co) were tested. The effects of these metals on bacterial growth, the mass of dry cells produced, ammonium assimilation, pigment production, and protein synthesis, were examined.

Effects of metals on bacterial growth

METHODS

Bacterial sorption of metals Bactetia A strain of E! aeruginosa, isolated in 1986 from a raw wastewater of the city of Tunis (Hassen et al., 1993) and a strain of B. thuringiensis (var thuringiensis, serotype 1, obtained from Dr H. De Barjac, Pasteur Institute of Paris) were used as test organisms.

p aeruginosa and B. thuringiensis were grown in 20 ml culture volume in triplicate tubes of 28 x 300 mm incubated at a temperature 37 f 1°C for 48 h. Nutrient broth (Oxoid) was used in all experi- ments. Bacterial growth was evaluated by optical density (OD) at 650 nm. For each metal concentra- tion, a non-contaminated tube served as a blank. The metal concentrations studied were chosen considering the minimum inhibitory concentration (MIC) of each culture and the concentrations that would provoke a sharp flocculation in the inoculated medium.

Pigment production

Microbial culture Bacteria were cultured in nutrient broth (Oxoi’d) at a temperature of 37+ 1°C for 36 h and in the presence or absence of the heavy metals most frequently found in polluted environments (Cu, Cr, Hg, Cd, Zn, Co). Whole-culture samples (respec- tively 80 and 180 ml for the citrate-supplemented (2%) and citrate-deficient cultures) were centrifuged (3000 x g) for 30 min. The supernatant was removed and analysed for the presence of ammonium ions by the indophenol blue reaction (Hanson and Phillips, 1981). The pellet was twice suspended in 10 ml of buffer Tris-Gly; pH 7.2 and centrifuged. Washed cells were resuspended in 10 ml sterile distilled water and divided into two homogeneous fractions, respectively l/10 and 9/10 of the washed cells. These two fractions were later dried separately at 90°C for at least 8 h until they reached constant weight (mass of dry cells).

Pigment production in l? aeruginosa was assessed qualitatively by chloroform extraction. Pyocyanin produced a blue color after addition of an equal volume of chloroform to a 4-day culture, while pyoverdin remained in the aqueous fraction (green color).

Chemicals The heavy metals used were all sulfate salts: CrS04.8H20; CuS04*5H20; ZnS04*5H20; HgSO,; CdS04.2.5H20; CoS04*7Hz0. Stock solutions were prepared in distilled water slightly acidified by the addition of 2-4 drops of 6N HCl for mercury, and were sterilized at 110°C for 15 min to prevent bacterial contamination. Autoclaving might have caused a change in the chemical properties of the metals. Salt solutions were kept at 4°C for no longer than 1 month. The glassware was leached in 2N HNOa and was rinsed several times with distilled water before use to avoid metal contamination.

Statistical analysis

Protein contents Dried samples of washed cells (first fraction of l/10) were incubated in 5 ml of 1N NaOH at 90°C for 10 min to solublize cellular protein, according to the technique of Jackson et al. (1989). Protein was measured by the method of Lowry et al. (1951) with a bovine serum albumin standard.

All experiments were replicated three times. Data were subjected to analysis of variance procedures of the SPSS statistical program, and means were separated by the least-significant-difference according to Tukey’s method. The relationship between dry cells and total protein content was determined by linear correlation studies (SPSS for Windows, SPSS Inc., Jun 17 1993).

Metal content RESULTS AND DISCUSSION The total heavy metals content of the second, dried, washed cell sample (second fraction of 9/10) was determined using the HNOa-H2S04-HC104 diges- tion method, followed by atomic absorption spectro- metry with an atomic absorption spectrometer for Cd and Hg (Perkin-Elmer model 2280; K. Kolb, Scientific Technical Supplies, D-6072, Dreieich, West Germany) or with a photometric method for Cr (Lieber, 1956) and Cu (Capelle, 1960a,b) using a UV-visible spectrophotometer (Philips UV-visible 8620 Series; K. Kolb, Scientific Technical Supplies, D-6072, Dreieich, West Germany).

Growth of li aeruginosa and B. thuringiensis Growth of R aeruginosa in the presence of metals showed a lag phase much longer (on average 6-8 h) than in the absence of metals. The inhibition was variable and depended on the metal and its concen- tration in the medium. Generally, higher concentra- tions of metals, approximating the MIC (Table l), caused a higher inhibition (Fig. l(a) and (b)).

On the other hand, there was a short lag phase for B. thuringiensis (0.5-l h). Chromium, at concen- trations of 0.5 and 1 mM, caused an inhibition of

Effects of heavy metals on some bacteria

Table 1. Bacterial dry weight and metal biosorption

Strain Control Cadmium Chromium CoDDer Zinc Cobalt Mercury

P aeruginosu MIC (mM) -- 1.2 1.2 1.5 0.08 Tested -- 1 0.5 0.5 0.05

concentration (mM)

Dry weight* 98.3i17.5 (bc) 54.7k17.2 (d) 65.7k10.7 (cd) 201.6k5.5 (a) 100.3&10.8 (bc) 68.8+- 14 (cd) 108k4.8 (b) (mgi100 ml)

Metal adsorbed -- 6 40.4 1.8 ND ND 469.7 (pg mg - ’

of DW) Percentage of -- 18.3rfr5.2 0.07 f 0.02 26.8 + 3.5 ND ND 59.7k9.4

biosorptiont B. thwingicnsis MIC (mM) -- 1.2 A.5 0.5 0.5 0.05 0.06

Tested -. 0.5 0.2 0.2 0.01 0.05 concentration

(mM) Dry weight* 8X + 13 (a) 38.4k3.2 (cd) 54.6k11.2 (bc) 76.4+4 (ab) 49.3k8.6 (c) 20.5 + 1.9 (d) 50.6 + 10.6 (c)

(mg/lOO ml) Metal adsorbed -- 5.4 0.5 7.3 ND ND 925

(/Lg mg _ ’ of DW)

Percentage of -- 0.5kO.l 8.9k2.6 l&7+3.7 ND ND 42.7k1.4 biosorptiont

tBiosorption expressed as a percentage of the metal found in the pellet of washed bacterial cells compared to the quantity of metal added initially into the growth medium. *Bacteria were grown in nutrient broth (Oxoid) at 37°C for 3 days. (a, b, c, d) The one-way analysis of variance (ANOVA) and Tukey’s significant difference test were used to compare differences between means. Means in the same line with similar letters are not significantly different (P = 0.05). ND, non-detected; DW, dry weight; &, standard deviation; II = 4.

about 30% with regard to controls; whereas copper and cobalt, at 0.2 mM, inhibited the bacterial growth by approximately 30% still with regard to controls. Zinc, at 0.05 and 0.2 mM, did not have significant effects on the growth of B. thuringiensis. Mercury at 0.05 mM caused a reduction in the bacterial growth of approximately 20% (F:ig. 2(a) and (b)). The growth of B. thuringiensis appeared more sensitive to metals than that of P aeruginosa.

Effect of metals on cell production Copper, at a concentration of 0.5 mM, stimulated significantly the production of cells by Z? aeruginosa as compared to the control, around 200 mg/lOO ml (Table 1). This result seems to be in discordance with that obtained in Fig. l(a), where Cu at 0.5 mM gave approximately half the OD of the control. This apparent disagreement found between the results of growth (weight) and cell production (OD) may be related to the intrinsic properties of copper and bacterial cell wall. In the presence of El aemginosa, Cu may have formed complexes with growth medium ingredients, thus resulting in the higher values of bacterial cell production. Mercury at 0.05 mM gave a similar value to the one obtained in the control (100 mg/lOO ml of culture). Zinc, cobalt and chromium at concentrations of 0.5, 0.1 and 1 mM, respectively, had no significant effect. However, cadmium at 0.5 mM reduced the produc- tion of dry cells to about half.

while the other metals inhibited the bacterial production (Table 1). The result obtained for copper could have been due to the reduction in heavy metal toxicity caused by complexation of the metal in the medium or by its adsorption to bacterial cells and precipitation at the cell surface. On the other hand, the relatively high pH of the medium (pH 7.4) could produce a change in the chemical properties of the metal and indirectly reduce its toxic effect. Mercury, zinc and chromium gave similar quantity of dry cells, about 50 mg/lOO ml of culture. Cadmium and cobalt showed respectively the lowest quantities of dry cells (20.5 and 38.4 mg/lOO ml).

Heavy metal biosorption Bacterial sorption of metals (Cd, Cr, Cu, Hg) was expressed as the metal quantity found in the pellet of washed bacterial cells or, as a percentage of the metal added initially to the medium. Results are given in Table 1.

In P aeruginosa, the highest percentage of bacterial sorption was observed with mercury, while the lowest percentage was obtained with chromium. Cadmium and copper showed very close percentages.

In B. thuringiensis, as in P aemginosa, mercury gave the highest percentage of sorption, followed by copper. Cadmium and chromium gave the lowest percentages of sorption.

Inhibitory effects on B. thuringiensis appeared The major part of added mercury was found in different from those observed in I! aemginosa. cell pellets in both of Z? aemginosa and B. thurin- Copper at 0.2 mM did not have a significant effect, giensis. However, mercury had a strong affinity to

76 A. Hasten et al.

the organic matter of the medium and had a tendency to cause flocculation.

Our results are in good agreement with those reported by Chang et al. (1993) and Behel et al. (1983) who had underlined the metal-binding power of organic matter to mercury and copper. Mercury is reputed to be a highly toxic metal and its toxicity is partially compensated by its strong adsorption, which facilitates its removal from a polluted environ- ment by trapping and sequestration. Mercury and

copper showed a high affinity for bacterial cells, but these metals also seemed to interact with compo- nents of the growth medium and to precipitate as metal-organic matter complexes. Besides metal- to-surface adsorption, active uptake of metals into the cytoplasm and their precipitation at the cell surface may occur (Bender et al., 1994). The term ‘biosorption’ or ‘bacterial sorption’ has been used in the present study to indicate that metal was removed by one or more of these processes. Potential applica-

2 4 6 8 10 13 16 20 24 28

Time (II)

196 -

096 --

2 4 6 8 10 13 16 20 24 28

Time (h)

0 r I I I I I I I /

2 4 6 8 10 13 16 20 24 28

Time (h)

Fig. 1. (a) Growth of Pseudomonas aeruginosa in the presence of cobalt, copper and cadmium

Effects of heavy metals on some bacteria 71

tions of the microbial heavy-metal uptake have been described by some authors (Bender et al., 1994).

thuringiensis. The ratio (total protein/dry cells) was 20.8 and 12.5%, respectively, in copper-supple- mented and copper-deficient cultures of I? aerugi-

Copper and chromium biosorption in the presence of citric acid Results of the bacterial sorption are presented in Tables 2 and 3. Citrate increased the sorption of chromium by I! aeruginosu and of copper by B.

nosa. For B. thuringiensis, the ratios were similar. Correlations between dry cell weight and total

protein content were investigated. This study did not show any linear correlation between the content of protein and dry cell weights in the presence of

a- ,

2 4 6 8 10 13 16 20 24 28

Time (h) 1,6

194 --

1,2 --

1 ~-

8 098 --

076 --

0,4 ~~

0,2 --

0 I ,

4 6 8 10 13 16 20 24 28

Time (h)

Fig. 1. (b) Growth of Pseudomonas aeruginosa in the presence of mercury, chromium and zinc.

2 4 6 8 10 13 16 20 24 28

Time (b)

78 A. Hassen et al.

which, in turn, suggested an role of protein synthesis by

of chromium, ratios of total dry cell weights fluctuated only and 5.8%, which suggested that perturb cell-protein synthesis.

This result was confirmed by a statistical analysis that showed a good correlation (r = 0.8) between dry cells and total protein content.

copper and citrate eventual disruptive copper.

In the presence protein content and slightly, between 3.3 chromium did not

In conclusion, citrate had a stimulating effect on the bacterial sorption of chromium by I? aeruginosu; but had no detectable effect on B. thuringiensis. The copper-citrate association did not enhance bacterial sorption of copper by R aeruginosu; conversely, it increased copper sorption in B. thuringiensis. The results presented here for copper and its effects on metabolic activity and growth rate are consistent with the observations of Jonas et al. (1984) but contrast with those of Sadler and Trudinger (1967)

098 -- 0,7 --

076 -- Q OS -- O 0,4 --

073 --

032 --

091 --

099

098

097

096

079

078

037

076

2 4 6 8 10 13 16 20 24

Time (h)

2 4 6 8 10 13 16 20 24

Time (h)

2 4 6 8 10 13 16 20 24

Time (la)

Fig. 2. (a) Growth of Bacillus thuringiensis in the presence of cobalt, copper and cadmium

Effects of heavy metals on some bacteria 79

and Jonas (1989) which showed, in pure culture, a reduction in cell density and inhibition of cell

and 1 mM, did not affect protein synthesis in B.

division rather than inhibition of general thuringiensis but, on the other hand, Co at 0.1 mM

metabolism. showed a significant inhibitory effect on protein biosynthesis. Cadmium at 0.1 mM gave the highest

Effects of metals on protein synthesis Results presented in Table 4 suggest that with P

percentage of inhibition (about 85%). Copper and chromium gave the lowest percentages of inhibition, respectively 22 and 38%.

aeruginosa, zinc at a concentration of 0.5 mM stimu- lated protein synthesis; the quantity of protein being

It appeared from these results that metals exerted

twice that of the control. Metals such as Hg, Cu, Cr variable effects on protein biosynthesis, but the rate

and Cd, at respective concentrations of 0.2, 0.5, 0.5 of synthesis depended on the bacterial species; thus, in the same conditions of culture, the total amount

0 J- / I

2 4 6 8 10 13 16 20 24

Time (h)

0,g l-

I I I

2 4 6 8 10 13 16 20 24

Time (h)

Fig. 2. (b)

/ +Control

i

*Zn 0,05 mM +Zn 0,2 mM

2 4 6 8 10 13 16 20 24

Time (II)

Growth of Bacillus thuringiensis in the presence of mercury, chromium and zinc.

80 A. Hussen et al.

Table 2. Biosorption of copper in the presence of citrate

Strain Sample DW (mg) Protein (pg) Pr/DW (%) Copper added Copper Biosorption (mg/lOO ml) recovered (%)

(mg/pellet)

F! aeruginosa 1 41.2 9837 23.8 -

: 47.5 68.7 12325 10612 25.9 15.4 - - average 52.5 f 13.5 (a) 10925 f 1272 (b) 20.8* 6.1 (a) -

II aeruginosa (+Cu) 1 67.5 6845 10.1 2.73

: 61.2 9075 9472 14.8 14.5 3.31 3.87 average E.5+3.1 (a) 8127k 1150 (1) 12.5 f2.6 (a) 3.3 +0.56

B. thuringiensis 1 91.2 12303 10.5 - 2 92 10787 11.8 - 3 117.5 6793 7.3 average 100.2f 14.8 (b) 9961 f2846 (a) lOk5.4 (a) 1

B. thuringiensis (+Cu) 1 76.2 9062 11.8 1.38

2 66.2 9887 14.9 3 56.2 8108 14.4 ::;6 average 66.2 f 10 (a) 9018 * 890 (a) 13.6k5.2 (a) 1.67kO.28

- - -

0.35 0.42 0.46 0.41+ 0.06 - - -

0.63 45.6 0.45 26.4 0.58 29.6 0.55 f 0.09 33.8 & 10

- -

12.8 12.6 11.9 12.4kO.5 - - -

(a, b, c, d) The one-way analysis of variance (ANOVA) and Tukey’s significant difference test were used to compare differences between means. Means in the same column with similar letters are not significantly different (P = 0.05). f , standard deviation; n = 3. (+Cu) Copper was added in the growth medium at 0.5 and 0.25 mM of Cu(S04) 5Hz0, respectively for fl aeruginosa and B. thuringiensis. Pr: protein (@pellet of 100 ml of culture), DW dry weight (mg/pellet of 100 ml of culture).

of protein produced by B. thuringiensis (190 ,ug ml-‘) was generally higher than that of I? aer~gkzosa (79 pg ml-‘).

Effects of metals on ammonium assimilation Ammonium ion concentration varied in the bacterial supernatant according to the type of treatment (Table 4). Zinc and cadmium at 0.1 and 0.5 mM, respectively gave the lowest values. A statistical

study showed that there was no evident relationship between ammonium-ion-assimilation and total protein content in the culture of I! aeruginosa (r = 0.48); but a good and positive correlation with B. thuringiensis (r = 0.78).

Production of pigments E! aeruginosa usually liberates pyocyanin and pyoverdin into the growth medium after two days of

Table 3. Biosorption of chromium in the presence of citrate

Strain Sample DW (mg) Protein (fig) Pr/DW (%) Chromium Chromium Biosorption (%) added recovered (mg/lOO ml) (mg/pellet)

P aeruginosa 1 107.5 3822 3.5 - 2 113.7 5338 4.6 - 3 147.5 7871 5.3 average 122.8 &- 21.5 (b)

Z&!&z _t 2045 (b) f 0.9 (a) 1

l? aeruginosa (+Cr) 1 226.2 4.: 2 218.7 8146 3:7

6.52 6.97

Zverage E?:: f 38.4 (a) EZkll f 50 (a) 2.8 3.3 f 0.5 (a) 7.33 6.93 + 0.4 B. thuringiensis 1 91.2 4581 5 -

2 92.5 6185 6.6 -

zverage tiy.2 + 16.2 (a) :iii f 803 (b) 4:; + 1 (a) 1 B. thuringiemis (+Cr) 1 78.8 3943 4.9 0.963

: 98.7 63.7 4106 3718 ::; 0.76 1.05 average 80.4+ 17.4 (a) 3921 _t 193 (a) 4.8 f0.9 (a) 0.92f0.22

G5 55.9 3.22 46.2 2.62 35.7 3.16kO.51 45.9+10 - -

G37 G 0.02 5.2 0.043 4.1 0.033*0.011 4.3kO.7

(a, b, c, d) The one-way analysis of variance (ANOVA) and Tukey’s significant difference test were used to compare differences between means. Means in the same column with similar letters are not significantly different (P = 0.05). k, standard deviation; n = 3. (+Cr) Chrome III was added in the growth medium at 0.7 and 0.1 mM of Cr2(S04)8H20, respectively for R aeruginosa and B. thuringiensis. Pr: protein (@pellet of 100 ml of culture), DW dry weight (mg/pellet of 100 ml of culture).

Effects of heavy metals on some bacteria 81

Table 4. Effects of heavy metals on ammonium assimilation, protein and pigment synthesis

Strains

p aeruginosa

B. thuringiensis

Metals

without metal Hg 0.02 mM Cu 0.5 mM Zn 0.5 mM Co 0.1 mM Cr 0.5 mM CdlmM without metal Hg 0.02 mM Cu 0.1 mM Zn 0.5 mM Co 0.01 mM Cr 0.5 mM Cd 0.1 mM

NHjt (mg ml ~ ‘)

254 + 20 (ab) 288 _t 25 (a) 279 _t 20 (a) 184+ 15 (c) 297 & 30 (a) 220+ 18 (b) 150 + 20 (d) 268 + 20 (b) 234&9 (c) 1415 18 (e) 232 f 20 (c) 373 + 37 (a) 213 f 10 (d)

94* 10 (f)

Protein (pg ml- ‘)

79& 10 (bd) 58+6 (d) 91 ~fr 12 (b)

153+ 15 (a) 60+ 10 (cd)

100+20 (b) 70f 15 (bd)

190*20 (a) 144klO (b) 148 + 22 (b)

150.7 f 7.4 (b) 181.3k9.3 (a)

1182 18 (b) 27k3 (c)

Pigments

Pv+pc - PC Pv - PC Pv - - - - -

-

(a, b, c, d) The one-way analysis of variance (ANOVA) and Tukey’s significant difference test were used to compare differences between means. Means in the same column with similar letters are not significantly different (P = 0.05). t, ammonium mg ml ’ of culture medium; &, standard deviation; n = 4. -, no pigment; Pv, pyoverdin; PC, pyocyanin.

incubation. Melanin pigments appear in older cultures (around 10 days). In the presence of cadmium, just pyocyanin appeared in the cultures. Zinc promoted only an intense pyoverdin biosynthesis. This last result has been used in field and laboratory studies in order to enhance pyoverdin production and thus to facilitate the isola- tion and the identification of Z? aeruginosa (Hassen et al., 1997). Copper and chromium inhibited pyoverdin biosynthesis and had no significant effect on the pyocyanin, while mercury (0.02 mM) and cobalt (0.1 mM) completely blocked pyocyanin and pyoverdin biosyntheses.

Capelle, R. (1960b). Dosage colorimktrique du cuivre au moyen de la bis-cyclohexanone-oxalyldihydrazone et de l’oxalyldihydrazine. Chimie Anal., 42, 127-135.

Chang, J. S., Hong, J., Oa, 0. & Bah, 0. (1993). Inter- action of mercuric ions with the bacterial growth medium and its effects on enzymatic reduction of mercury. Biotechnol., 9, 526-532.

De Rore, H., Top, E., Houwen, F.. Mergeay, M. & Verstraete, W. (1994). Evolution of heavy metal resistant transconjugants in a soil environment with a concomitant selective pressure. FEMS Microbial. Ecol., 14,263-273.

Doelman, P., Jansen, E., Michels, M. & Van Til, M. (1994). Effects of heavy metals in soil on microbial diversity and activity as shown by the sensitivity-resist- ance index, an ecologically relevant parameter. Biol. Fertil. Soil., 17, 177- 184.

ACKNOWLEDGEMENTS

This investigation was supported partly by a grant from the International Foundation for Science (Grant n” HI23294 IFS, Sweden). We thank Professor Hans-W. Ackermann, Department of Microbiology, Faculty of Medicine, Lava1 University, Quebec, Canada, for help.

Guzzo, A., Du Bow, M. & Bauda, P. (1994). Identification and characterization of genetically programmed responses to toxic metal exposure in Escherichia coli. Metals and microorganisms: relationships and applica- tions. FEMS Microbial. Revi., 14, 369-374.

Goblenz, A., Wolf, K. & Bauda, P. (1994). The role of glutathione biosynthesis in heavy metal resistance in the fission yeast Schizosaccharmyces pombe. Metals and microorganisms: relationships and applications. FEMS Microbial. Revi., 14, 303-308.

REFERENCES

Behel, D., Darrel, J. R., Nelson, W. & Sommers, L. E. (1983). Assessment of heavy metal equilibria in sewage sludge-treated soil. J. Environ. Qual., 12, 181-186.

Bender, J., Graves, B., Philips, P. & Abotsi, G. (1994). Deposit of zinc and manganese an aqueous environment mediated by microbial mats. Water Air Soil Poll., 75, 195-204.

Hanson, R. S. & Phillips, J. A. (1981) Chemical composi- tion, in Gerhardt, M. R. G. E., Costilow, R. N., Nester, W. P., Wood, W. A., Krieg, N. R. & Phillips, G. B. (eds), Manual of Methods ,for General Bacteriology. American Society for Microbiology, Washington, DC, 328-364.

Brynhildsen, L. & Rosswall, T. (1989). Effects of cadmium, copper, magnesium, and zinc on the decom- position of citrate by Klebsiella sp.. Appl. Environ. Micro- biol., 55, 1375-1379.

Hassen, A., Jedidi, N., Saidi, N., Boudabous, A. & Gabteni, N. (1993). Etude experimentale des inter- actions entre ClCments mktalliques et la minCralisation de la mat&e organique d’un compost d’ordures menagkres ajoutC B un sol. Rev. I.N.A. Tunis, 8, 145-160.

Hassen, A., Kallali, H., Allani, M., Saidi, N., Jemli, M., Annabi, M., Nabli, M. & Boudabous, A. (1997). DCsin- fection des eaux u&es par irradiation ultraviolette dans une station pilote semi-industrielle - effets des faibles doses UV sur Pseudomonas aeruginosa. Vecteur Envir- onnement, 30, 75-81.

Capelle, R. (1960a). Dosage colorimCtrique du cuivre au Hiroki, M. (1994). Populations of Cd-tolerant micro- moyen de la bis-cyclohexanone-oxalyldihydrazone et de organisms in soil polluted with heavy metals. Soil Sci. l’oxalyldihydrazine. Chimie Anal., 42, 69-77. Plant Nuts: ~ 40, 515-524.

82 A. Hussen et al.

Holm, P. E., Christensen, T. H., Tjell, J. C. & McGrath, S. P. (1995). Heavy metals in the environment. Specia- tion of cadmium and zinc with application to soil solutions. J. Environ. Qual., 24, 183-190.

Jackson, M. A., Slininger, P. J. & Bothast, R. J. (1989). Effect of zinc, iron, cobalt and manganese on Fusarium monilifotrne NRRL 13616 growth and fusarin C biosynthesis in submerged cultures. Appl. Environ. Microbial., 55, 649-655.

Jonas, R. B. (1989). Acute copper and cupric ion toxicity in an estuarine microbial community Appl. Environ. Microbial., 55, 43-49.

Jonas, R. B., Gilmour, C. C., Stoner, D. L., Weir, M. M. & Tuttle, J. H. (1984). Comparison of methods to measure acute metal and organometal toxicity to natural aquatic microbial communities Appl. Environ. Micro- biol., 47, 1005-1011.

Lieber, M. (1956). Permanganate Azide test for total chromium in water. J.A. WWA., 40, 295-299.

Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193,265-275.

Lun, X. Z. & Christensen, T. H. (1989). Cadmium

complexation by solid waste leachates. Water Rex, 23, 81-84.

Mullen, M. D., Wolf, D. C., Ferris, F. G., Beveridge, T. J., Flemming, C. A. & Bailey, G. W. (1989). Bacterial sorption of heavy metals Appl. Environ. Microbial., 55, 3143-3149.

Nieto, J. J., Castillo, R. F., Marquez, M. C., Ventosa, A., Quesada, E. & Ruiz Berraquero, F. (1989). Survey of metal tolerance in moderately halophilic eubacteria Water Rex, 55, 2385-2390.

Olson, B. H. & Thornton, I. (1982). The resistance patterns to metals of bacterial populations in contami- nated land J. Soil Sci., 33, 271-277.

Rajini Rani, D. B. & Mahadevan, A. (1994). Cloning and expression of the mercury resistance genes of marine Pseudomonas sp. strain MRl plasmid pMR1 in Escheri- chia coli. Res. Microbial., 145, 121-127.

Rohit, M. & Sheela, S. (1994). Uptake of zinc in Pseudo- monas sp. strain UDG26. Appl. Environ. Microbial., 60, 2367-2370.

Sadler, W. R. 8z Trudinger, P. A. (1967). The inhibition of microorganisms by heavy metals. Mineral. Dep., 2, 351-361.