Professional Documents
Culture Documents
881
2004 Kluwer Academic Publishers. Printed in the Netherlands.
Arundhati Pal1, Paramita Choudhuri1, Suman Dutta2, P.K. Mukherjee2 and A.K. Paul1,*
1
Microbiology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road,
Kolkata 700 019, India
2
Taxonomy Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road,
Kolkata 700 019, India
*Author for correspondence: Tel.: +91-33-2475-3681/2344-0509, Fax: +91-33-2474-1042/2476-4419, E-mail:
akpaul@cal3.vsnl.net.in
Summary
Serpentine soils of Andaman Islands, India characteristically contain high levels of nickel, cobalt and chromium and
are colonized by indigenous nickel-hyperaccumulating plants. Attempts have been made to isolate and characterize
nickel-resistant microorganisms from these hitherto unexplored naturally nickel-percolated soils. The majority of
the nickel-resistant organisms showed a minimum inhibitory concentration (MIC) of Ni2+ ranging from 300 to
400 mg/l and about 3.4% of the total 89 isolates representing bacterial strains were able to grow at 400 mg/l Ni2+.
The potent Ni2+-resistant strains AND305 and AND603 were tentatively identified as Pseudomonas spp. and strain
AND408 as Bacillus sp. following detailed analysis of morphological and physio-biochemical characteristics.
Growth kinetics of these Ni2+-resistant bacteria showed a prolonged lag phase in Ni2+-containing media, which
extended with increasing nickel concentration. In addition to Ni2+, these isolates were also resistant to Co2+, Cd2+,
Cr6+, Fe3+, Cu2+, Mg2+, Mn2+(50–200 mg/l) and Hg2+ (0.5–2.0 mg/l) and the multiple metal-resistance of the
isolates were also associated with the resistance to antibiotics ampicillin, cycloserine and penicillin G.
Serpentine soil samples from three different sites of the Selection of potent strains
South Andaman Islands, India were collected in sterile
containers and brought to the laboratory for microbi- Based on the growth performances in nickel-containing
ological analysis. For isolation and enumeration of liquid media, potent nickel-resistant strains were se-
microorganisms, soil samples were serially diluted in lected. The isolates were identified following morpho-
sterile distilled water and plated on three different logical and biochemical characterization according to
media, viz. nutrient agar, glycerol asparagine agar and Bergey’s Manual of Systematic Bacteriology (Holt &
Czapek-Dox agar for isolation of bacteria, actinomy- Kreig 1989; Holt et al. 1989).
cetes and fungi respectively. Plates were incubated at 37,
30 and 28 C respectively for 4–6 days and colonies Growth kinetics
differing in morphological peculiarities were isolated in
pure form and maintained on slopes of respective agar Growth of nickel-resistant bacterial isolates was studied
media. in 250 ml flasks containing 50 ml of nutrient broth
supplemented with 200–300 mg Ni2+/l. Flasks were
Analysis of soil samples inoculated with 0.2 ml of freshly prepared inoculum
and agitated on a rotary shaker (120 rev/min) at 30 C.
Soil pH, moisture content, electrical conductivity and Optical density changes of the culture during growth
salinity were determined following standard methods for were recorded at 540 nm wavelength using an AIMIL
soil testing (Jackson 1973). Air-dried soil was seived Photochem 5 spectrophotometer.
through a 2 mm mesh and the pH was measured in 1:2.5
soil:water and soil:1(N) KCl suspensions. Electrical Resistance to other heavy metals
conductivity was calculated in 1:5 soil:water suspension
using Systronics Conductivity Meter (Model 304). Metal Resistance of selected bacterial isolates to other heavy
content of soil was determined following the methods metals was tested in nutrient broth supplemented with
described in American Public Health Association metals. Metals that were tested include Cu2+, Cd2+,
(1998). Soil was dried at 80 C, sieved through a Co2+, Cr6+, Hg2+, Mg2+, Mn2+ and Fe3+. All metals
0.2 mm mesh and digested in nitric acid–perchloric acid except Cr6+ and Fe3+ were supplied as the chloride salt,
(3:1). The digested samples were diluted and filtered while Cr6+ was supplied as potassium chromate and
through Whatman No. 42 filter paper. The metal Fe3+ as ferric ammonium citrate. Stock solution of
content of the digested samples was analysed by a metals were prepared in double distilled water and
Varian Atomic Absorption Spectrophotometer (Model sterilized by autoclaving at 15 p.s.i. for 15 min. How-
SpectrAA-20Plus) using Atomic Absorption Standard ever, ferric ammonium citrate solution was filter steril-
solutions of respective metals (Sigma–Aldrich). ized. Growth of the organisms was measured by
determining optical density of cultures at 540 nm after
Screening for nickel-resistance 48–96 h of incubation. Percent relative growth was
calculated against control set without metal supplemen-
Nickel-resistance potential of the isolates were deter- tation, which was taken as 100%.
mined in 100 ml Erlenmeyer flask containing 20 ml
liquid media supplemented with increasing concentra- Sensitivity to antibiotics
tion of Ni2+ (as NiCl2, 6H2O) ranging from 25 to
400 mg/l added from a sterile stock solution. Nutrient Antibiotic sensitivity of nickel-resistant isolates was
broth, glycerol-asparagine broth and Czapek-Dox broth determined by disc diffusion method. Antibiotic impreg-
were used for bacteria, actinomycetes and fungi respec- nated discs (6 mm diameter, HIMEDIA) were placed on
tively. The broth was inoculated with 0.2 ml of freshly nutrient agar plates seeded with individual isolates and
prepared inoculum. Bacteria and actinomycetes pre- incubated at 30 C for 24 h. The diameter of the
grown in their respective media served as the inoculum. inhibition zones were recorded and the antibiotic
For fungi, a homogeneous spore suspension from a sensitivity profile of the bacterial strains was determined
14 days-old Czapek-Dox slant culture was prepared in following the DIFCO Manual, 10th edition (1984). The
sterile Tween 80 (0.1%) solution. Inoculated flasks were following antibiotic discs were used in the present study:
incubated at 30 C on a rotary shaker (120 rev/min) for ampicillin (10 lg), bacitracin (10 U), cycloserine
Nickel-resistant microbes from serpentine soil 883
(200 lg), erythromycin (15 lg), chloramphenicol
(30 lg), kanamycin (30 lg), novobiocin (30 lg), peni-
cillin G (10 U), polymyxin B (300 lg) and tetracycline
(30 lg).
Table 1. Physico-chemical and microbiological analysis of some serpentine soil samples of Andaman.
Physico-chemical
Soil pH (water) 6.14 6.23 5.13
Soil pH (1 N KCl) 5.77 5.44 4.59
Electrical conductivity 2.2 2.3 3.1
Salinity, % 0.7 0.74 0.99
Moisture content, % 25.02 44.3 18.24
Total nickel, mg/kg dry soil 5366.7 4316.7 8033.4
Total cobalt, mg/kg dry soil 533.0 400.0 534.0
Total chromium, mg/kg dry soil 4436.0 2760.0 4436.0
Microbiological analysis
Microbial content (c.f.u./g soil, ·105)
In nutrient agar 31.4 19.0 23.5
In glycerol-asparagine agar 27.6 12.0 12.34
In Czapek-Dox agar 24.7 15.5 14.0
– No growth.
Percent relative growth of isolates were measured by determining dry weight of the biomass in metal-amended broth as compared to control set
without metal that was taken as 100.
Each value represents average of duplicate sets.
Character Response
Bacterial isolate
Morphological
Gram nature Gram ()) Gram (+) Gram ())
Cell morphology Short rods in long chains Small rods mainly isolated Small rods in short chains of 2–3 cells
Endospore ) + )
Motility + + +
Biochemical
Catalase production + + +
Oxidase production + + +
Casein hydrolysis + + +
Gelatin liquefaction + + +
Nitrate reduction + + +
Indole production + ) )
Fat hydrolysis + ) )
Starch hydrolysis + + +
Anaerobic growth ) ) )
NaCl tolerance 5% 7% 5%
Fermentation of
Glucose + + +
Fructose + + +
Sucrose + + +
Lactose ) ) )
Maltose + + )
Galactose + + +
Arabinose ) ) )
Raffinose + ) )
Mannitol + + +
Growth kinetics of these selected metal-resistant isolates Cd2+ 50 92.2 6.3 59.1
in nickel-supplemented broth was monitored by atten- 100 86.0 4.2 54.0
200 77.1 3.5 19.4
uance changes over a period of 48 h (Figure 2). The
isolates showed a more or less similar pattern of growth Co2+ 50 85.1 72.5 73.5
in Ni2+-containing media and a decrease in growth yield 100 65.0 4.0 2.7
200 3.2 – –
was noted in all three isolates. Irrespective of the isolates
the lag phase of growth was prolonged with increasing Cr6+ 50 96.1 75.0 85.3
concentration of nickel and it was maximum with isolate 100 94.4 61.2 83.5
200 87.9 56.5 81.0
AND603. Such an extended lag period might be due to
acclimatization of bacterial cells with high concentration Cu2+ 50 90.5 90.0 79.0
of Ni2+ in the medium. Growth of E. coli V38 in Ni2+- 100 88.2 56.4 76.5
200 82.1 2.6 66.0
containing liquid medium also showed a prolonged lag
phase, the duration of which was dependent on nickel Mn2+ 50 84.0 100.0 97.0
concentration (Rubikas et al. 1997). 100 80.1 97.1 75.4
200 79.0 91.5 71.0
Figure 2. Growth kinetics of selected nickel-resistant bacterial isolates in nickel-containing media. [The isolates were grown in nutrient broth (m)
supplemented with 200 mg Ni2+/l (j) and 300 mg Ni2+/l (d) under shake condition at 30 C.]
886 A. Pal et al.
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a
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Acknowledgements resistance encoded by the ncc locus of Alcaligenes xyloxidans 31A.
Journal of Bacteriology 176, 7045–7054.
The authors are thankful to the Ministry of Environment Singh, K.L. & Kumar, A. 1998 Incidence of multiple heavy metal
and Forest, Government of India, New Delhi for spon- resistance in a Bacillus Species. Journal of Microbiology and
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