World Journal of Microbiology & Biotechnology 20: 881–886, 2004.

881

2004 Kluwer Academic Publishers. Printed in the Netherlands.

Isolation and characterization of nickel-resistant microflora from serpentine soils

of Andaman

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

Received 5 November 2003; accepted 7 June 2004

Keywords: Serpentine soil, metal-resistance, nickel-resistant microorganisms, antibiotic-resistance, Pseudomonas

and Bacillus

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.

Introduction reported the occurrence of nickel-resistant bacteria in

Serpentine or ultramafic soils produced by weathering
and pedogenesis of ultramafic rocks are characterized by
high levels of the metals Ni, Cr and sometimes Co, but
contain low levels of N, P, K and Ca (Brooks 1987). The
serpentine areas have been considered as ecological
islands, which represent an interesting model for evolu-
tion of metal-resistant bacterial communities (Lefevre &
Vernet 1990), completely different from those isolated
from man-made contaminated sites (Mengoni et al.
2001). Microbial ecology of the serpentine outcrops has
so far received very little attention although they are
expected to provide newer strains and determinants for
heavy metal-resistance.
Microorganisms resistant to nickel have been mostly
isolated and characterized from anthropogenically
metal-polluted sites (Schmidt & Schlegel 1994; Singh &
Kumar 1998; Grass et al. 2001). However, they are not
uncommon in naturally occurring nickel-percolated
serpentine or ultramafic soils. Schlegel et al. (1991)
soil samples collected from the canopy of nickel-hyper-
accumulating shrubs and trees. They considered that
nickel-metallophytes could drive a ‘nickel cycle’, which
resulted in the evolution of high population of nickel-
resistant strains in the plant rhizosphere. Mengoni et al.
(2001) also found higher proportion of nickel-resistant
colony forming units (c.f.u) in proximity to the nickel
hyperaccumulating plant, Alyssum bertolonii, than in
free soil in serpentine outcrops of Central Italy. Stoppel
& Schlegel (1995) however, demonstrated strong nucle-
otide homologies between the nickel-resistant determi-
nants isolated from anthropogenically nickel-polluted
areas and those from naturally nickel-enriched soil.
The occurrence of serpentine soils in Andaman Island
(India) particularly in Saddle Hills, Chidyatapu and
Rutland has been well documented (Halder 1984; Roy
et al. 1988; Vohra et al. 1989; Jafri et al. 2003; Pal et al.
2003) but microbiological studies of these unique soil
types have not been undertaken so far. The present
study reports for the first time on the occurrence and
882
characterization of nickel-resistant microorganisms
from such naturally nickel-percolated serpentine eco-
systems of Andaman, India.
Materials and methods
Isolation of microorganisms
Serpentine soil samples from three different sites of the
South Andaman Islands, India were collected in sterile
containers and brought to the laboratory for microbi-
ological analysis. For isolation and enumeration of
microorganisms, soil samples were serially diluted in
sterile distilled water and plated on three different
media, viz. nutrient agar, glycerol asparagine agar and
Czapek-Dox agar for isolation of bacteria, actinomy-
cetes and fungi respectively. Plates were incubated at 37,
30 and 28 C respectively for 4–6 days and colonies
differing in morphological peculiarities were isolated in
pure form and maintained on slopes of respective agar
media.
Analysis of soil samples
Soil pH, moisture content, electrical conductivity and
salinity were determined following standard methods for
soil testing (Jackson 1973). Air-dried soil was seived
through a 2 mm mesh and the pH was measured in 1:2.5
soil:water and soil:1(N) KCl suspensions. Electrical
conductivity was calculated in 1:5 soil:water suspension
using Systronics Conductivity Meter (Model 304). Metal
content of soil was determined following the methods
described in American Public Health Association
(1998). Soil was dried at 80 C, sieved through a
0.2 mm mesh and digested in nitric acid–perchloric acid
(3:1). The digested samples were diluted and filtered
through Whatman No. 42 filter paper. The metal
content of the digested samples was analysed by a
Varian Atomic Absorption Spectrophotometer (Model
SpectrAA-20Plus) using Atomic Absorption Standard
solutions of respective metals (Sigma–Aldrich).
Screening for nickel-resistance
Nickel-resistance potential of the isolates were deter-
mined in 100 ml Erlenmeyer flask containing 20 ml
liquid media supplemented with increasing concentra-
tion of Ni2+ (as NiCl2, 6H2O) ranging from 25 to
400 mg/l added from a sterile stock solution. Nutrient
broth, glycerol-asparagine broth and Czapek-Dox broth
were used for bacteria, actinomycetes and fungi respec-
tively. The broth was inoculated with 0.2 ml of freshly
prepared inoculum. Bacteria and actinomycetes pre-
grown in their respective media served as the inoculum.
For fungi, a homogeneous spore suspension from a
14 days-old Czapek-Dox slant culture was prepared in
sterile Tween 80 (0.1%) solution. Inoculated flasks were
incubated at 30 C on a rotary shaker (120 rev/min) for
A. Pal et al.
48–120 h. Growth of the isolates was measured by
determining the dry weight of biomass. The biomass was
harvested by centrifugation at 10,000 rev/min using
Hitachi SRC20B Centrifuge, washed thoroughly, trans-
ferred to pre-weighed aluminium cups and dried to
constant weight at 80 C. Relative growth was calculated
as percentage of those obtained in untreated control,
which was considered as 100%.
Selection of potent strains
Based on the growth performances in nickel-containing
liquid media, potent nickel-resistant strains were se-
lected. The isolates were identified following morpho-
logical and biochemical characterization according to
Bergey’s Manual of Systematic Bacteriology (Holt &
Kreig 1989; Holt et al. 1989).
Growth kinetics
Growth of nickel-resistant bacterial isolates was studied
in 250 ml flasks containing 50 ml of nutrient broth
supplemented with 200–300 mg Ni2+/l. Flasks were
inoculated with 0.2 ml of freshly prepared inoculum
and agitated on a rotary shaker (120 rev/min) at 30 C.
Optical density changes of the culture during growth
were recorded at 540 nm wavelength using an AIMIL
Photochem 5 spectrophotometer.
Resistance to other heavy metals
Resistance of selected bacterial isolates to other heavy
metals was tested in nutrient broth supplemented with
metals. Metals that were tested include Cu2+, Cd2+,
Co2+, Cr6+, Hg2+, Mg2+, Mn2+ and Fe3+. All metals
except Cr6+ and Fe3+ were supplied as the chloride salt,
while Cr6+ was supplied as potassium chromate and
Fe3+ as ferric ammonium citrate. Stock solution of
metals were prepared in double distilled water and
sterilized by autoclaving at 15 p.s.i. for 15 min. How-
ever, ferric ammonium citrate solution was filter steril-
ized. Growth of the organisms was measured by
determining optical density of cultures at 540 nm after
48–96 h of incubation. Percent relative growth was
calculated against control set without metal supplemen-
tation, which was taken as 100%.
Sensitivity to antibiotics
Antibiotic sensitivity of nickel-resistant isolates was
determined by disc diffusion method. Antibiotic impreg-
nated discs (6 mm diameter, HIMEDIA) were placed on
nutrient agar plates seeded with individual isolates and
incubated at 30 C for 24 h. The diameter of the
inhibition zones were recorded and the antibiotic
sensitivity profile of the bacterial strains was determined
following the DIFCO Manual, 10th edition (1984). The
following antibiotic discs were used in the present study:
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).

Results and discussion

Soil samples collected from serpentine outcrops of

Jahaji Dera, Portman Bay (Rutland Island) and Chid-
yatapu of the south Andaman Islands of India were
analysed following standard methods for soil testing
(Jackson 1973; APHA 1998) and the physico-chemical
characteristics are presented in Table 1. It was interest-
ing to note that the nickel content of the soil samples
was exceptionally high and ranged between 4316.7 and
8033.4 mg/kg dry soil. Microbiological analysis follow-
ing dilution and plating on three different media
revealed that the bacterial population of these soils
was high compared to fungi and actinomycetes
(Table 1). In contrast, ultramafic areas of New Caledo-
nia were reported to harbour a high density of actino-
mycetes strains followed by fungi (Amir & Pineau 1998).
Screening of microorganisms for nickel-resistance
Eighty-nine isolates representing 51 bacteria, 20 actino-
mycetes and 18 fungi were isolated in pure form from
isolation plates and subjected to screening for nickel-
resistance in media containing 25–400 mg/l of Ni2+. The
majority (83.1%) of the isolates was able to grow in
50 mg Ni2+/l (0.85 mM) but their number decreased as
the metal concentration in the media was increased.
Actinomycetes were least resistant to nickel and none of
the strains showed visible growth above 100 mg Ni2+/l
(1.7 mM). Fungal strains showed moderate resistance in
nickel and 2 out of 18 isolates (11.2%) were able to grow
at 200 mg/l (3.4 mM) of Ni2+. Bacteria were found to be
most resistant to 400 mg Ni2+/l (6.81 mM), the highest
Figure 1. Nickel-resistance in microorganisms isolated from serpen-
tine soil of Andaman. (Relative resistance of microorganisms was
determined in liquid medium containing 25, 50, 100, 200 and 400 mg
Ni2+/l.)
concentration tested and only 3 isolates representing
3.4% of the total population showed visible growth at
this concentration (Figure 1). These results confirm the
nickel-resistance potential of bacterial strains isolated
from serpentine soil as reported earlier (Stoppel &
Schlegel 1995; Mengoni et al. 2001).
Growth of selected nickel-resistant isolates in Ni2+-
containing media (Table 2) revealed that relative growth
of all isolates decreased with increasing concentration of
Ni2+. Bacterial isolates AND305, AND408 and
AND603 showed a high degree of resistance with
relative growth ranging between 45 and 52% of the
control. The MIC values of Ni2+for these three isolates
were >400 mg Ni2+/l (6.81 mM), while for most of the
isolates the value ranged between 300 and 400 mg/l (5.1–
6.81 mM). Nickel-resistant species, therefore, appear to
be abundant in natural serpentine soils of Andaman,
although they differ significantly in their degree of
resistance. Schlegel et al. (1991) have reported that
bacteria from serpentines of New Caledonia were

Table 1. Physico-chemical and microbiological analysis of some serpentine soil samples of Andaman.

Characteristic Location of soil samples

Jahaji Dera Portman Bay Chidyatapu

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

Each value represents the average of duplicate sets.

884 A. Pal et al.
Table 2. Relative growth of some selected nickel-resistant isolates in Ni2+ supplemented media and minimum inhibitory concentration of Ni2+.

Isolate No. % Relative growth in Ni2+, mg/l MIC of Ni2+, mg/l

200 300 400

AND305 68.9 57.8 45.0 >400

AND306 69.0 – – 200–300
AND308 67.0 11.3 – 300–400
AND404 91.7 14.3 – 300–400
AND407 76.3 27.2 – 300–400
AND408 69.2 60.5 52.6 >400
AND603 66.7 60.0 46.8 >400
AND605 67.8 13.7 – 300–400
AND607 59.0 13.4 – 300–400
AND613 46.7 12.7 – 300–400
ANDF104 42.5 – – 200–300
ANDF604 55.2 21.6 – 300–400

– 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.

resistant to 10–20 mM NiCl2 (587–1174 mg Ni2+/l), Characterization of potent nickel-resistant bacteria

whereas those from nickel-free soils could barely resist
1 mM NiCl2 (58.7 mg Ni2+/l). The majority of bacterial
isolates from serpentine outcrops of Central Italy were
also resistant to 7 mM Ni (411.05 mg Ni2+/l) and about
40.5% of the Pseudomonas strains grew on 10 mM Ni
(Mengoni et al. 2001).
Bacterial strains AND305, AND408 and AND603
showing more than 40% relative growth in 400 mg/l
Ni+2 and MIC values >400 mg/l were selected as potent
nickel-resistant strains. The isolates were characterized
morphologically and biochemically (Table 3) and iden-

Table 3. Morphological and biochemical characteristics of selected nickel-resistant bacteria.

Character Response

Bacterial isolate

AND305 AND408 AND603

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 + + +

+Positive response; ) negative response.

Nickel-resistant microbes from serpentine soil 885
tified tentatively as strains of Pseudomonas sp. AND305, Table 4. Tolerance of other heavy metals by some selected nickel-
Bacillus sp. AND408, and Pseudomonas sp. AND603 resistant bacterial isolates.
following Bergey’s Manual of Systematic Bacteriology Metal Concentration, Percent relative growth
(Holt & Kreig 1989; Holt et al. 1989). mg/l
Bacterial isolate
Growth kinetics AND305 AND408 AND603

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

Resistance to other heavy metals Mg2+ 50 95.0 91.5 97.1

100 92.0 85.7 93.5
200 90.6 82.7 88.0
The selected nickel-resistant strains were tested for their
resistance to other heavy metals such as Cu2+, Cd2+, Fe3+ 50 96.0 80.6 93.5
Co2+, Cr6+, Hg2+, Mg2+, Mn2+ and Fe3+. Relative 100 92.7 68.6 91.0
200 89.0 59.0 90.0
growth of isolates in different metal-containing media is
shown in Table 4. It was evident that isolates AND305 Hg2+ 0.5 56.0 5.5 50.3
and AND603 were resistant to all metals at low 1.0 44.8 3.5 39.7
2.0 7.5 2.6 15.5
concentration but the metals appear to be toxic at
higher concentration. Isolate AND408 was sensitive to – No growth.
Cd2+ and Hg2+ even at the lowest concentration tested Percent relative growth of bacterial isolates were measured by
though it showed a relatively high degree of resistance determining the attenuance of cultures at 540 nm in metal-amended
towards Cr6+, Fe3+, Cu2+, Mg2+ and Mn2+. Cobalt broth as compared to control set without metal that was taken as
100.
appeared to be most toxic and inhibitory to growth of Each value represents average of duplicate sets.
all isolates at 200 mg/l. A large majority of bacterial
strains from serpentine outcrops of Italy show resistance
to 3 mM Cr, 2 mM Co, 10 mM Zn and 5 mM Cu ultramafic soil were resistant to Co, Cu and Zn in
(Mengoni et al. 2001). Bacteria from New Caledonian addition to Ni (Stoppel & Schlegel 1995).

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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