Mercury and organomercurial resistance in bacteria isolated from freshwater fish of wetland fisheries around Calcutta

PII:

S0269-7491

(97)00068-7

Environmental Pollution, Vol. 97, No. 1-2, pp. 71 78, 1997 © 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0269-7491/97 $17.00+0.00

ELSEVIER

M E R C U R Y A N D O R G A N O M E R C U R I A L RESISTANCE IN BACTERIA ISOLATED FROM FRESHWATER FISH OF W E T L A N D FISHERIES A R O U N D CALCUTTA Provash Chandra Sadhukhan, S. Ghosh, J. Chaudhuri, D. K. Ghosh and A. Mandal* Department of Biochemistry, University College of Science, Calcutta University, 35, Ballygunge Circular Road, Calcutta-700 019, India (Received 1 October 1996; accepted 5 May 1997)

1976; Summers and Silver, 1978; Lodenius et al., 1983; Barkay, 1992). Due to prolonged exposure to mercury compounds, aquatic and sediment bacteria may become mercuryand organomercurial-resistant. Such bacteria usually harbour plasmids having genetic factors that confer resistance to Hg +z, organomercurials, antibiotics and heavy metals (Schottel et al., 1974; Nakahara et al., 1985; Misra, 1992a). These genetic factors code for the synthesis of two inducible enzymes, namely mercuric reductase and organomercurial lyase (Silver and Misra, 1988; Misra, 1992a). Organomercurial lyase catalyses the cleavage of C-Hg bonds in organomercurial compounds to liberate Hg +2, which is then reduced by mercuric reductase in the presence of NADPH and sulfhydryl compounds to Hg ° that volatilizes out of the system due to its high vapour pressure (Schottel, 1978; Tezuka and Tonomura, 1978; Nakamura et al., 1990; Misra, 1992a; Pahan et al., 1993). There are about 4000 ha of freshwater fisheries in the eastern suburbs of Calcutta, a densely populated city in India. These fisheries produce on an average 12 000 tons of fish per day to meet local demands. These fisheries are occasionally fed with domestic effluents containing hospital wastes and some industrial wastes from the Calcutta drainage system. These fisheries are believed to function as water purifier and as a good site for fish production. It has already been reported that methylmercury may be synthesised from HgC12 in in vitro conditions by the extracts of gills and guts (Jensen and Jernelov, 1969; R u d d e t al., 1980). It is now well established that Hgresistant bacteria can detoxify Hg-compounds in the natural environment and under laboratory conditions (Misra, 1992a; Pahan et al., 1993). Fish grown in a mercury-polluted environment in wetland fisheries may contain mercury-resistant bacteria in their gills and guts. These mercury-resistant bacteria may play a significant role in the biotransformation and bioaccumulation of mercury in the aqueous environment and in fish present there. In this paper we report the total number of bacteria from gills and guts of fish from wetland fisheries, their mercury resistance, as well as the pattern of mercury detoxification by these bacterial strains.

Abstract

Mercury-resistant bacteria belonging to the genera Bacillus, Escherichia, Klebsiella, Micrococcus, Pseudomonas, Salmonella, Sarcina, Shigella, Staphylococcus and Streptococcus were isolated from gills and guts of fresh water fish collected from wetland fisheries around Calcutta, India, contaminated with mercury compounds. The total number of bacteria, as well as Hg-resistant bacteria, were always higher in guts than gills. Bottomdwelling fish contained higher number of bacteria, including Hg-resistant bacteria, than surface and middle water dwelling fish. They belonged either to narrow-spectrum or to broad-spectrum Hg-resistant groups and they also possessed other heavy metal and antibiotic resistant properties. In the presence of toxic levels of HgCI2, phenylmercuric acetate (PMA) and methylmercuric chloride (MMC), the lag in growth of the bacterial strains gradually increased with increasing concentration of Hg-compounds. Narrow-spectrum Hg-resistant bacterial strains volatilized only HgCl2from the liquid medium in the range of 64--89%, whereas the broad-spectrum group exhibited a high level of HgCI2 (80-94~), PMA (72-84%) and M M C (64-80%) volatilizing capacity with inducible mercuric reductase and organomercurial lyase enzyme activities in their cell-free extracts. Cell-free extracts prepared from narrow-spectrum Hg-resistant bacterial strains induced by HgCI2 exhibited Hg+2-dependent NADPH oxidation, indicating the presence of only mercuric reductase enzyme. © 1997 Elsevier Science Ltd

INTRODUCTION Mercuric ions and organomercurials are still in use in various industries as catalysts, in hospitals as disinfectants and in agriculture as fungicides, insecticides and bacteriocides (WHO, 1976). After entering aquatic environments, all forms of mercury may be transformed biologically or abiologically to highly neurotoxic methylmercury which may accumulate in fish (Wood et al., 1968; Friberg and Vostal, 1972; WHO, *To whom correspondence should be addressed. Fax: 091 33 241 3222; e-mail: [email protected] 71

72

P . C . Sadhukhan et al.

MATERIALS AND METHODS All chemicals and reagents used in the present study were of analytical grade (E. Merck, Germany and British Drug House, UK). All mercury compounds and NADPH (tetrasodium salt) were purchased from Sigma Chemical Co., St. Louis, Missouri, USA, except methylmercury which was obtained from Wako Chemical, Japan.

Isolation of bacterial strains Fish of different kinds were collected by 1.3 cm mesh gill net from wetland fisheries. The fish were put immediately in a sterile container and brought to the laboratory within 1 h. The next part of the work was done under laminar hood. Gills and guts were removed with sterile scissors, scalpels and forceps. The wet weight of each organ was taken. The tissues were homogenised in sterilized tissue homogenizers with 0.1 M potassium phosphate buffer (pH 7.0) into a state of slurry (Austin and A1-Zahrani, 1988). The slurry was added to 50 ml sterile 0.1 u potassium phosphate buffer (pH 7.0) in a sterile BOD bottle and was shaken for 4 h. The mixture was left undisturbed overnight at 4°C, and the supernatant was used for isolating and estimating the total viable count of bacteria. Serially diluted supernatants were plated on nutrient agar media without HgC12 for estimation of the total viable count. The total number of Hg-resistant bacteria was determined by using 8.4/zg m1-1 HgC12 in nutrient agar plates (containing 1.9% agar), since this concentration of HgCI2 was reported to inhibit the growth of both gram-positive and gram-negative bacteria (Smith, 1967). Petridishes were incubated at 37°C for 24 h and the agar plates containing bacterial colonies between 30 and 300 were taken for counting. An average o f five total determinations per sample was recorded. The isolated bacterial colonies were purified and identified up to their generic level, following Bergey's Manual of Determinative Bacteriology (Buchanan et al. (eds), 1994). Determination of minimal inhibitory concentration (MIC) of HgCI2, organomercurials and other heavy metals against isolated bacteria Minimal inhibitory concentration (MIC) values of HgC12 and organomercurials, such as phenylmercuric acetate (PMA), methylmercuric chloride (MMC), merbromin (Mb), thimersol (Tm), p-hydroxy mercuric benzoate (pHMB) and fluorescein mercuric acetate (FMA), against these organisms were determined using the filter paper disc method of Ray et al. (1989). Heavy metal resistance spectra of different Hg-resistant bacteria and mercury-sensitive bacteria, e.g. Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Klebsiella aerogenes, were determined by filter paper disc method for the ions Cu +2 , Ag ÷ , Zn +2, Cd +2, Ni +2, Co +2, Bi +2, Pb + 2 CRO4-2, AsO4-2. The amounts of metal ions used were 12.5, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 and 1000 nmol per disc. In the case of Cu + 2, Zn +2, Pb +2, Ni +2, CrO4 -2 and AsO4 -2 ions, higher amounts, 1500, 2000, 2500 and 3000 nmol per disc, were also used.

Determination of antibiotic resistance spectra of Hgresistant bacteria Antibiotic sensitivity test for the organisms were performed according to the method of Griffith (1970) using the following amounts of antibiotic in filter paper discs: Ampicillin (10/~g), Carbenicillin (10/~g), Chloramphenicol (30/~g), Penicillin G (10u = 6/~g), Streptomycin (10/zg) and Tetracycline (30/zg). Growth of mercury-resistant bacteria in liquid broth containing mercury compounds Growth of Bacillus sp. (strain GT2) and E. coli (strain GL3) were examined in nutrient broth supplemented with 31/zM, 62/zM and 124/ZM HgC12. Growth of Bacillus sp. (Strain GT2) in the presence of organomercury compounds, like PMA and MMC, was also studied in liquid media supplemented with 16/ZM and 31/zM PMA and MMC, respectively, according to Pahan et al. (1990). The cultures were incubated with shaking at 37°C and growth was followed turbidimetrically in a Klett-Summerson photoelectric colorimeter using a red filter. Bacterial growth was recorded in this way at hourly intervals. Volatilization of HgCI2, PMA and MMC by mercuryresistant isolates Six narrow-spectrum and four broad-spectrum bacterial strains were employed in the volatilization experiment. E. coli (strain GL3), Bacillus sp. (strain GT2), Bacillus sp. (strain GT7) and Sarcina sp. (strain GLl3) were classed as broad-spectrum and the other mercury-resistant bacteria were narrow-spectrum. In three separate control flasks, 3.36mg HgC12, 0.84mg PMA and 0.84 mg MMC were added to 200 ml of nutrient broth. In the experimental flask, an overnight culture of the bacterial cells was diluted (1:10) with nutrient broth to a final volume of 200ml and 3.36mg HgC12, 0.84mg PMA and 0.84mg MMC were added. The organisms were grown with shaking (150 revmin -1) at 37°C on a rotary shaker for 24h and the control flask was also shaken similarly. Thereafter, the cells were harvested by centrifugation at 6000g for 10min at 4°C and washed three times with deionized water. Weighed amounts of wet cells, 1 ml of the supernatant after each cell harvesting and 1 ml of control medium containing HgCI2, PMA and MMC were separately taken in 100ml volumetric flasks and digested to bring all the mercury into ionic form. The mercury contents of the solutions so prepared were then measured by cold vapour atomic absorption spectrometry (Bradenberger and Bader, 1967). Preparation of cell-free extracts Bacterial cells were induced three times with 10/ZM HgCI2 and cell-free extracts were prepared following the method of Summers and Silver (1972). Cells were grown overnight in nutrient broth at 37°C on a rotary shaker (150revmin -1) and supplemented with 10/xM HgC12 for the induction of mercury detoxifying enzymes. The bacterial cultures were diluted 10-fold with the

Hg-resistant bacteria in freshwater fish same medium and the concentration of HgC12 was maintained at 10/zM. A third addition of 10/ZM HgCI2 was made at early log phase. Cells were harvested at late log phase by centrifugation at 4°C and washed three times with cold 50mM sodium-phosphate buffer (pH 7.35). Washed cells were disrupted mechanically with sea sand in a pestle and mortar at 4°C. Disrupted cells were suspended in the same cold buffer and centrifuged at 15 000g for 30min at 4°C. Most of the Hg-reductase and organomercurial lyase activities in the cell-free extracts were precipitated with 0--50% (NH4)2 SO4 cut at 4°C and the precipitate was dissolved in a minimum volume of 50mM sodium-phosphate buffer (pH 7.35) containing 0.25 mr,t Na2 E D T A and then dialysed overnight at 4°C. The dialysate was used for the assay of Hg-reductase and organomercurial lyase. Hg-reductase and organomercurial lyase activities were determined by measuring Hg+2-dependent N A D P H oxidation spectrophotometrically at 340nm (Komura and Izaki, 1971). Protein content was determined by the method of Lowry et al. (1951).

RESULTS Table 1 shows the total number of bacteria and Hgresistant bacteria in the gills and guts of different kinds of fish dwelling in the surface, middle and the bottom parts of the wetland fisheries. In all cases, the total number of bacteria, as well as the total number of mercury-resistant bacteria, were always higher in the guts than in the gills. Moreover, bottom-dwelling fish like Koi, Shol and Telapia contained higher numbers of bacteria, including mercury-resistant bacteria, than surface (Catla) and middle (Rohu) water-dwelling fish. The percentage of mercury-resistant bacteria varied from 2.08x 1 0 -3 to 1.13x 10 -1 and 3.75x 10 -3 to 3.84× 10 -2 in gills and guts, respectively (Table 1). Among the 20 isolated bacterial strains, three were mercury-sensitive, 12 were in the narrow-spectrum group and five were in the broad-spectrum Hg-resistant group. These bacterial strains belonged to the following genera, Bacillus,

73

Escherichia, Klebsiella, Micrococcus, Pseudomonas, Salmonella, Shigella, Staphylococcus, Streptococcus and Sarcina (Table 2). MIC of mercury, organomercuricals and different antibiotics, against some Hg-resistant strains and the three Hg-sensitive bacterial strains are shown in Table 2. Among these strains GL3 (E. coli), GT1 (Bacillus sp.), GT2 (Bacillus sp.), GT7 (Bacillus sp.) and GL13 (Sarcina sp.) were highly resistant to all the mercury compounds used and therefore they belonged to the broad-spectrum group of Hg-resistant bacteria (Table 2). Strains GL4 (Staphylococcus sp.), GLs (Bacillus sp.), GL6 (Pseudomonas sp.), GL7 (Bacillus sp.) and GLll (E. coli) displayed resistance to HgCI2, pHMB, F M A and Tm but not to PMA, and therefore they belonged to the narrow-spectrum subclass (Table 2). The bacterial strains GLI4 (E. coli), GT5 (Streptococcus sp.) and GL15 (Klebsiella sp.) were sensitive to all the mercury compounds. Organisms were considered to be resistant to a particular mercury compound if the growth was inhibited at the following amount: (i) 12.5nmoles per disc for HgCI2, thimersoi, pHMB, merbromine and F M A and (ii) 3 nmoles per disc for PMA. Intrinsic antibiotic resistance spectra of 17 different Hg-resistant bacterial strains and three Hg-sensitive bacterial strains have also been given in Table 2. Broad-spectrum mercury-resistant bacterial strains, such as GL3, GT~, GT2, GTT, and GLI3, were multiple drug-resistant, but the patterns of resistance to different antibiotics were not similar. Growth patterns of many Hg-resistant bacterial strains were also studied using 31, 62, 124 #M HgC12 and 16/zra, 31/zt,l PMA and M M C separately in liquid media. The growth pattern of HgC12-treated cells was similar to that of control cells without HgC12, but with increased HgCI2 and organomercurial concentrations the lag in growth of the organism increased gradually (Fig. 1). Bacillus sp GT2 showed nearly no lag in growth in the absence of mercury compounds, whereas in the presence of 31, 62 and 124#ra HgC12 the organism had a lag of 0.5 h, I h and 2 h, respectively. In the presence of 16 and 31 # u PMA, the lag in growth was 1 and 3 h, respectively, and when grown in the presence of 16 and

Table 1. Hg-resistant bacteria in gills and guts of fish from wetland fisheries of Calcutta suburbs

Total no. of Average Dwelling place Name body of fish in of bacteria* per gm of organ wt (gm) water column organ

Sample no.

Local name of fish

Scientific name

1

Katla

Catla catla

550

Surface

2

Mrigel

Cirrhinamrigala

450

Bottom

3

Rohu

Labeo rohita

400

Middle

75

Bottom

Shol

Oreochromis mossambicus Channamaurilus

500

Bottom

Koi

Anabas testudineus

75

Bottom

4

Telapia

5 6

gill gut gill gut gill gut gill gut gill gut gill gut

4.42x 109 5.98x 109 3.92× 109

1.92x10 l~ 7.72x 109

2.24x 10I° 2.33x 10I° 1.64x1011 1.88× 10IJ 4.72x 10ll 6.92x 10Jl 8.56x 1011

Total no. of Hg-resistant bacteria per gm of organ

Percentage of Hg-resistant bacteria

1.36× 107 2.78x 107

3.07x 10 3 4.67x 10-3 1.13× 10 i 3.84x 10 2 2.08x 10 3 5.89x 10-3 3.51 x 10-2 1.92x10 -2 6.75x 10-3 6.52×10 3 7.45× 10-3 3.75x10 -3

4.46× 108 7.38x 109 1.61 x 107 1.32x 108 8.19x 108 3.15×109 1.27x 109 3.08x 109

5.16×109 3.21x109

*Average of five separate determinations is presented. Average Hg content of water of the fishery was 0.038 • 0.0004/zg ml-l and that of the sediment was 0.622 ± 0.011/zg gm -~.

P. C. Sadhukhan et al.

74

[G -

0

o~

z ~ ~ o~ ~-z ~ Z ~ ~r~O~r..)

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48~44,~4~4.~4484~6

I

e~ 0

I Z

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0 e~

o

<

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t~

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Hg-resistant bacteria in freshwater fish 140 120 100 80-

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40-

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2o-

o

2

4

6 8 Time in hours

10

12

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o

o Control

A

* 16 gmol 1-I PMA

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~ 31 pmol I i HgCI 2 '~ 62 /zmol 1-r HgC[ 2

~ *

31/~mol 1-t PMA * 16/~mol i-I MMC

=

= 124 ~moll -I HgCI 2 --

; 31pmoll -j MMC

Fig. 1. Representation of the growth pattern of Bacillus sp. GT2 in the presence of different concentrations of HgC12, phenylmercuric acetate (PMA) and methylmercuric chloride (MMC). Q---® control in absence of any mercury compounds; x - - × in presence of 31/zM HgCI2; I-3--O in presence of 62/zM HgCI2; I1--11 in presence of 124/zM HgCI2; A - - A in presence of 16/z~l PMA; A - - A in presence of 31/zM PMA; © - - © in presence of 16/zM MMC; 0 - - 0 in presence of 31/zM MMC.

31/zM M M C , the organism showed a lag in growth of 1.5 h and 4 h, respectively. Table 3 shows heavy met~,l resistance properties of these bacteria. E. coli, K. aerogenes, B. subtilis and S. aureus are mercury-sensitive bacteria and showed a low level of resistance to other metals. However, some bacterial isolates are mercury-resistant and show significant levels of resistance to other metals (Ray et al., 1993).

75

A m o n g the mercury-resistant bacteria, E. coli (GL3), Bacillus sp. (GT2), Bacillus sp. (GT7), Bacillus sp. (GL7), Pseudomonas sp. (GL6) were highly resistant to mercury and other heavy metals. Bacillus sp. (GT2) showed highest overall resistance to different metals. Table 4 shows the pattern of volatilization of HgCI2 by different Hg-resistant bacteria. In the control medium without organisms, 10.3% of total HgC12 was volatilized abiologically during 24 h incubation at 37°C. Bacillus sp. ( G L 0 showed the lowest volatilizing capacity of 64.3%. Bacillus sp. (GTT), Bacillus sp. (GL7), E. coli (GL3) and Sarcina sp. (GL]3) showed very high mercury volatilizing capacities of 93.5, 89.4, 81.2 and 89.1%, respectively. Bacillus sp. (GT2) showed the highest volatilizing capacity, i.e. 94.0%, but in all the species of mercury-resistant strains, mercury bound with the cell mass was low. Table 5 shows the volatilization of P M A and M M C by four broad-spectrum mercury-resistant bacteria, E. coli (GL3), Bacillus sp. (GT2), Bacillus sp. (GT7) and Sarcina sp. (GLI3). F r o m the control flask without organisms, 13.09 and 17.85% of total P M A and M M C were volatilized abiologically during 24 h incubation at 37°C. Bacillus sp. (GT2) showed highest volatilizing capacity of 84.9 and 80.9% for P M A and M M C , respectively. However, the other three bacterial isolates showed nearly the same P M A and M M C volatilizing capacity. Table 6 shows the specific activity of Hg-reductase and organomercurial lyase enzymes from different Hgresistant bacteria. E. coli (GLt 1), Bacillus sp. (GT9) and Klebsiella sp. (GL2) were weakly Hg-resistant (Table 2) and had no detectable Hg-reductase and organomercurial lyase enzyme activity under our experimental conditions. In all other organisms, the Hg-reductase activity was m a x i m u m when the bacterial cells were induced three times with 10/zra HgCI2. Triple induction with a higher concentration of HgCI2 (20/zM) showed no significant change in enzyme activity. Bacillus sp. (GT2) showed the highest mercuric reductase and organomercurial lyase activities (i.e. 0.24 units and 36.26 units, respectively). The other four bacterial strains, namely E. coli (GL3), Bacillus sp. (GTT), Bacillus sp. (GT1) and Sarcina sp. (GL13) showed both mercuric

Table 3. Heavy metal resistance spectra of different mercury-resistant bacteria isolated from gills and guts of freshwater fish

Bacterial strains

Concentration (nmol per disc) of solutions of metal compounds Pb(NO3)2

Escherichia coli Klebsiella aerogenes Bacillus subtilis Staphylococcus aureus Bacillus sp. GL7 Klebsiella sp. GL2 Bacillus sp. GLI Pseudomonas sp. GL6 Bacillus sp. GL5 Streptococcus sp. GT5 Escherichia coli GL3 Bacillus sp. GT] Bacillus sp. GT2 Bacillus sp. GT7 Sarcina sp. GL] 3

100 100 200 25 1000 500 500 500 1000 200 1000 500 1500 1500 1500

CdC12 Bi(NO3)2 ZnSO4 CuSO4 NiSO4 AgNO3 12.5 12.5 25 5 100 50 100 50 50 25 400 200 500 500 400

5 12.5 25 5 50 25 500 50 100 50 200 100 500 200 100

100 200 100 50 1000 500 500 300 500 100 1000 500 500 1500 200

100 100 50 25 500 500 1000 500 500 100 1000 200 1500 1000 1000

100 200 100 50 1000 400 500 300 500 200 800 400 1000 1000 800

12.5 12.5 25 12.5 100 50 50 50 50 25 300 100 500 500 300

K2CrO4

Na2AsO4

MnSO4

100 200 200 50 1000 1000 300 500 1000 200 1000 1000 1500 1500 1500

100 200 100 50 1000 1000 1000 1000 1500 500 1500 1000 1500 1500 1000

100 200 50 50 500 500 500 1000 1000 200 1500 1000 1500 1000 1000

76

P . C . Sadhukhan et al.

Table 4. Volatilization of HgCIz from liquid media by some Hg-resistant bacteria isolated from gills and guts of freshwater fish

Bacterial strains

Without any organism E. coli GL3 Bacillus sp. GT1 Bacillus sp. GT2 Bacillus sp. GT7 Sarcina sp. GL13 Bacillus sp. GL7 Bacillus sp. GL5 Pseudomonas sp. O L 6 Bacillus sp. GL1 Staphylococcus sp. GL4

Wet wt of cells (mg)

Total HgCI2 initially present in 200 ml nutrient broth (mg)

Total HgC12 retained in 200 ml nutrient broth after volatilization (mg)

Total HgC12 bound by cells (nag)

% of HgC12 volatilization

-455 550 725 625 575 630 455 375 315 360

3.36 3.36 3.36 3.36 3.36 3.36 3.36 3.36 3.36 3.36 3.36

3.012 0.623 0.518 0.163 0.202 0.310 0.309 0.321 0.364 0.575 0.314

-0.009 0.015 0.037 0.015 0.056 0.007 0.078 0.569 0.624 0.056

10.3 81.2 84.1 94.0 93.5 89.1 89.4 88.1 72.2 64.3 89.0

reductase and organomercurial lyase activities, but Bacillus sp. (GL7), Staphylococcus Sly. (GL4) and Pseudomonas sp. (GL6) had only mercuric reductase activity.

DISCUSSION Mercury-resistant bacteria were present in significant numbers in the gills and guts of fish from different wetland fisheries. As the wetland fisheries are occasionally fed with waste water of domestic, hospital and industrial origin, the water of the fisheries had high BOD and COD values. BOD values ranged between 15.08 and 82.31mglitre -1, whereas COD values varied from 118.44 to 254.62 mg litre- ~. The mercury content of the water in these fisheries ranged between 28 and 68 ppb (billion = 109) and that of the sediment varied between 348 and 804ppb. Unpolluted soil and water usually contains 10-20 ppb and 0.1-1.2 ppb of mercury, respectively (Kothny, 1973), so the mercury content of both water and sediment of the wetland fisheries is far above the normal level. Total numbers of bacteria, as well as mercury-resistant bacteria, were higher in bottom

dwelling fish than in surface or middle water dwelling fish, which may be due to the higher levels of organic matter and mercury in the sediment. Several workers have reported that the number of mercury-resistant bacteria in soil and aquatic environments varied according to the mercury content of the environment (Summers, 1986; Misra, 1992b; Pahan et al., 1995). In these strains heavy metal resistance properties were associated with multiple drug resistance, which supports earlier reports (Schottel et al., 1974; Bhattacharyya et al., 1988; Misra, 1992a). F r o m the growth pattern of the Hg-resistant bacterial strain Bacillus sp (GT2) which showed the highest level of mercury resistance, it appears that there was an extended lag phase in the presence of HgCI2 and different organomercurials (Fig. 1). This also corroborates observations by different workers (Nakamura, 1986; Pahan et al., 1993; Ray et al., 1993). Hg-resistant bacterial isolates differed in their Hg-tolerance properties (Table 2). This indicates that the efficiency of mercury detoxifying systems may be different in different mercury-resistant bacteria. The Hg-resistant bacterial cells are reported to volatilize mercury from mercury-containing liquid media and

Table 5. Volatilization of mercury and organomercurials by mercury-resistant bacteria isolated from gills and guts of freshwater fish

Bacterial strains

Mercury compounds used

Amount of Wet wt Mercury left after 24 h mercury compound of ceils added (mg) (mg) Cell bound (mg) Residual (mg)

% of volatilization of mercury compound

Without any organism

HgCl 2 PMA MMC

3.36 0.84 0.84

----

----

3.06 0.73 0.69

8.92 13.09 17.85

Escherichia coli GL3

HgC12 PMA MMC

3.36 0.84 0.84

425 200 150

0.010 0.045 0.061

0.631 0.190 0.211

80.91 72.00 67.66

Bacillus sp, GT2

HgC12 PMA MMC

3.36 0.84 0.84

675 270 175

0.007 0.035 0,028

0.198 0.092 0.132

93.88 84.90 80.94

Sarcina sp. GLI3

HgC12 PMA MMC

3.36 0.84 0.84

550 225 150

0.015 0.022 0.048

0.347 0.185 0.252

89.20 75.29 64.19

Bacillus sp. GT7

HgCI2 PMA MMC

3.36 0.84 0.84

650 225 175

0.010 0.098 0.015

0.234 0.119 0.278

92.72 74.17 65.16

Hg-resistant bacteria in freshwater fish Table 6. Hg + 2-reductase and organomercurial lyase activities in cell-free extracts of different mercury-resistant bacteria isolated from gills and guts of fish at different HgCi2 concentration

Bacterial strains

Concentration of HgCI2 as inducer (/zM)

Specific Specific activity activity of of Hg +2 organomercurial reductase lyase

E. coli GLII

0 10 20

0 0 0

0 0 0

Klebsiella sp. GL2

0 10 20

0 0 0

0 0 0

Bacillus sp. GT9

0 10 20

0 0 0

0 0 0

Staphylococcus sp. GL4

0 10 20

0 0.162 0.169

0 0 0

Bacillus sp. GL7

0 10 20

0 0.205 0.211

0 0 0

Pseudomonas sp. GL6

0 10 20

0 0.102 0.118

0 0 0

E. coli GL3

0 10 20

0 0.192 0.201

0 8.12 10.56

Bacillus sp. GTj

0 10 20

0 0.208 0.216

0 19.82 22.78

Bacillus sp. GTz

0 10 20

0 0.245 0.259

0 36.26 39.48

Bacillus sp. GT7

0 10 20

0 0,221 0.229

0 22.18 26.74

Sarcina sp. GLj3

0 10 20

0 0.212 0.224

0 20.66 23.62

77

(GL 0 and Bacillus sp. (GL7) have pronounced Hgreductase activity and can rapidly volatilize mercury from liquid media (Tables 4 and 5). The strain GT2, having the highest level of Hg-reductase activity (Table 6), showed highest mercury volatilizing capacity and also had a very high MIC value against mercury compounds. This finding is in agreement with earlier observations (Nakamura et al., 1986; Ray et al., 1993; Misra, 1992a). Many workers have claimed that Hg-reductase activity in Hg-resistant bacterial cells is always inducible and never constitutive (Summers, 1986). However, Nakamura (1986) showed the presence of a constitutive Hgreductase in Streptomyces sp. Bacterial strains isolated from gills and guts of fish also showed Hg-reductase and organomercurial lyase activities, only when the cells were induced with 10/zra mercuric chloride, indicating that Hg-reductase and organomercurial lyase are also inducible in all these strains. A higher concentration of the inducer could not enhance the activity of the mercury detoxifying enzymes. This observation corroborates the earlier observation of Ray et al. (1989). We have already mentioned that the mercury content of both water and sediment of these fisheries are above the normal background level, but the accumulation of mercury in both inorganic and organically-bound forms in different vital organs of edible fish grown in these wetland fisheries was much lower than the permissible limit (500 ppb wet wt basis), except in some sedimentdwelling fish. Total mercury content in surface and middle water-dwelling fish varied from 298 to 475 ppb, whereas in sediment-dwelling fish these values varied between 722 and 912ppb. The mercury-resistant bacteria present in gills and guts of fish may play a vital role in detoxifying mercury compounds and thus modulating mercury accumulation in these fish. Detailed ecological and biochemical studies are needed to assess the exact role of these bacterial strains in the process of mercury accumulation in fish. ACKNOWLEDGEMENTS

also to bind mercury with the cell constituents (Nakamura et al., 1986; Pahan et al., 1995). The E. coli (GL3), Bacillus sp. (GT2), Bacillus s p (GT7) and Sarcina sp. (GLI3) showed high mercury and organomercury volatilizing capacity in 24 h. Narrow-spectrum Hg-resistant Bacillus sp. (GL7) showed a high level of mercury volatilizing capacity (Table 4) but it could not volatilize organomercury compounds. Cell-bound mercury was also low in all Hg-resistant bacterial strains except Bacillus sp. ( G L 0 and Pseudomonas sp. (GL6). It was found that bacterial strains which had a higher level of cell-bound mercury volatilized mercury compounds less efficiently and vice versa (Table 4). The pattern of mercury volatilizing capacity of these organisms conforms with their MIC values against mercury compounds (Table 2). It has been observed that Bacillus sp. (GT2), E. coli (GL3), Bacillus sp. ( G T 0 , Bacillus sp. (GT7), Sarcina sp.

Financial assistance from the University Grants Commission, India, is highly appreciated. REFERENCES

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