Abstract: Ennore creek sediments are often severely polluted with the heavy metal chromium. Several chromium-resistant bacteria were isolated and identified from the sediments and all the isolates exhibited multi-metal resistance. Chromate reduction assay showed that the isolated Bacillus cereus ECD strains were able to reduce chromate up to 76.1% at a concentration of 100 μg mL-1. Growth of the Bacillus species in the presence of increasing concentration of chromate was also determined. Since the Bacillus cereus ECD strain has high resistance to chromate and other metals and also reduces chromate under aerobic conditions, this bacterium might be potentially applicable for the treatment of metal- based industrial effluents.
INTRODUCTION
Chromium is the seventh most abundant element on the earth’s crust and exists in several oxidation states like most stable trivalent Cr (III) and hexavalent Cr (VI) species, with different chemical characteristics and biological effects (Mcgrath and Smith, 1990; Cervantes et al., 2001). Chromate is most toxic than trivalent chromium because of its high solubility, rapid permeability, ready interaction with proteins and nucleic acids, mutagenicity and carcinogenic properties. Due its many unique properties, the use and dispersion of chromate have increased vastly, resulting in extensive pollution in the soil, sediment and ground waters (Srinath et al., 2002).
Conventional methods for reduction/removal of chromate from industrial wastes include chemical reduction followed by precipitation, ion exchange and adsorption on activated coal, alum, kaolin and ash. However, most of these methods require high energy or large quantities of chemical reagents and produce secondary waste streams that require remediation (Komori et al., 1990; Viamajala et al., 2002).
Bioremediation is one of the promising technologies that are expected to play an important role in removal of chromate from industrial wastes/contaminated sites. The bioremediation strategy is to detoxify chromate in the wastes to reduce it to trivalent chromium. Microorganisms can play an important role in the detoxification and removal of chromate from the polluted sites. It has been reported that chromate is reduced to trivalent chromium by a number of bacterial species like Enterobacter cloacae, Bacillus circulans strain EB1 Pseudomonas strains (Wang et al. 1989; Silver and Walderhaug, 1992; DeLeo and Ehrlich, 1994; Shen and Wang, 1995; Rosen, 1996; Silver and Phung, 1996; Ganguli and Tripathi, 2002; Jeyasingh and Philip, 2005). The physiological mechanisms involved in chromate reduction vary widely among species. In some cases, the enzyme-catalyzed reaction is membrane associated, whereas in others the enzyme is in the soluble form. However, the availability of effective chromate reducing bacterial strain is an essential prerequisite for the bioreduction-based remediation of chromium contaminated water or soil. Hence, the present study was aimed to isolate potential aerobic chromate reducing bacterial flora from the sediments of Ennore Creek.
STUDY AREA-ENNORE CREEK
The Ennore Creek is a brackish water body located in the northeastern part of Chennai; a part of this water body also extends southwest. The total water spread of the creek is about 800 m wide and 1 to 4 m deep, being shallow near the mouth. The direct release of effluents from industries, urban wastes, depletion of the fresh water flow and regular siltation of Ennore Creek mouth enhance the accumulation of heavy metals in the sediments of the creek and pose a threat to the ecosystem. Earlier studies have reported the presence of more than the threshold level of chromium with an average of 409 ppm and maximum of 700 ppm in the sediments of Ennore Creek (Jayaprakash et al., 2005). Since the study area is being considered as a major source for variety of fishes, crustaceans, mollusks and oysters, the presence of chromium compounds in the sediments would be a primary source for the biomagnification of chromium in aquatic flora and fauna and cause ill effects in those who consume the contaminated fish.
MATERIALS AND METHODS
Sediment Sampling
Surface sediment samples at four fixed stations were collected during March
2004 using Petersen grab and transported on ice to the laboratory and processed
within 18-24 h.
Isolation and Identification of Chromate Resistant Bacteria
Aerobic chromate resistant bacteria were isolated by serially diluting 1
g of the sediment in sterile distilled water and 0.1 mL of the appropriate dilution
were plated by spread plate technique on ZoBell Marine Agar plates (Hi-media,
India) amended with 50 mg L-1 of chromate as K2CrO4.
Later, the plates were incubated at 26±1°C for 24 h and observed
for bacterial growth. Morphologically distinct colonies were picked, purified
and made glycerol stocks.
Later the isolates were identified using fatty acid fingerprinting method. Briefly, isolates were grown in Soya bean-Casein digest agar; fatty acids were extracted, methylated and analyzed by gas chromatography (Descheemaker and Swings 1995). Finally, the fatty acid profiles of unknown isolates were compared with the library entries of the Microbial Identification System (6890N GC).
Determination of Minimal Inhibitory Concentration
Minimal Inhibitory Concentration (MIC) of chromate and other heavy metals
like zinc, nickel, lead, copper, cobalt and cadmium were determined by agar
dilution method (Luli et al. 1983). The mid-log phase cultures of the
isolates grown in LB broth were inoculated into the ZoBell Marine Agar plates
(Hi-Media, India) supplemented with increasing concentrations of the aforesaid
heavy metals individually (5 μg mL-1 to 1.62 mg mL-1
with the interval of 5 μg mL-1 (Tanaka et al., 1977).
Study of Antibiotic Resistance
Chromate resistant strains were picked and transferred to series of LB agar
amended with increasing concentration of Ampicillin (Am), Chloramphenicol (Cm),
Tetracycline (Tc), Streptomycin (Sm) and Kanamycin (Kc) individually (Am, Tc
and Sm was 3.1, 6.25, 12.5, 25, 50 and 100 μg mL-1, Kc 2.15,
4.35, 8.75, 17.5, 35 and 70 μg mL-1 and Cm 1.85, 3.75. 7.5,
15, 30 and 60 μg mL-1) (Cruickshank, 1968). Later, the plates
were incubated at 26±2°C for 24 h and observed for the bacterial
growth.
Isolation and Curing of Plasmid DNA
All the Bacillus species were screened for the presence of plasmids.
Mini preps were made according to standard procedure Maniatis et al.
(1989) with minor modification. Curing of the plasmids was performed by incubating
the isolates overnight at 30°C in a LB broth containing 25 μg mL-1
of acridine orange. Samples of 0.1 mL from each culture after appropriate dilution
were separately plated on LB agar plates by spread plate technique and the plates
were incubated at 30°C for 24 h. To select the strains that lost antibiotic
resistance, the colonies were transferred to LB agar plates amended with the
respective antibiotics in appropriate concentration. The strains that had no
antibiotic resistance were then analyzed for the metal tolerance.
Chromate Reduction
All the Gram-positive isolates were employed for chromate reduction by measuring
the optical density at 380 nm using a spectrophotometer (Bopp and Ehrlich, 1988;
Viti et al., 2003; Badar et al., 2004). Mid-log phase cultures
of the isolates were aseptically transferred into the flasks containing 50 mL
of LB broth supplemented with a final concentration of 100 μg mL-1
of chromate as K2CrO4. The flasks were incubated at 28°C
and the aliquots were taken at different time intervals and cell-free filtrates
(0.45 micron pore size) were prepared and later the optical density of the cell-free
filtrates was measured at 380 nm using a spectrophotometer. Optical densities
of the chromate in the cell-free solutions were used as a control.
Growth Kinetics of Gram-Positive Isolates
Growth kinetic studies were carried out by inoculating overnight cultures
of Bacillus species in 50 mL of LB broth supplemented with final concentration
of 100 and 200 μg mL-1 of chromate (Sangeeta and Tripathi, 2001).
The flasks were incubated at 26±1°C and the growth were measured
at different time intervals in terms of increase in optical density at 480 nm
using a spectrophotometer (Model: Systronics 1304). The cultures grown in the
absence of metal served as a control in this experiment.
RESULTS
Isolation and Identification of Chromate-resistant Bacteria
Eleven morphologically distinct chromate resistant, Gram-positive and Gram-negative
bacilli were isolated from the sediment samples. Based on the fatty acid profile
the isolates were identified as Bacillus pumilus three strains designated
as ECA, ECB and ECC, two Bacillus cereus strains designated ECD, ECE,
Bacillus sphaericus, Eschericha coli, Vibrio mimicus, Vibrio
alginolyticus, Vibrio anguillarum and Vibrio parahemolyticus.
MIC to Metals
MIC of chromate and other heavy metals for the isolates was determined and
the results are presented in Table 1. B. cereus ECD
showed higher tolerance to chromate, zinc, nickel, lead and copper. The strains
B. pumilus ECC and B. cereus ECE exhibit higher tolerance to cobalt
and cadmium. Nickel and lead showed less toxic to almost all the Gram positive
isolates when compared to other heavy metals. In general Gram negative isolates
were sensitive to all the heavy metals tested. Gram positive isolates were selected
for further studies.
Growth Kinetics
Growth response of the Bacillus species in the presence of chromate
was presented in the Fig. 1a-f. All the
strains were able to grow and exhibit difference in growth phase according to
the increasing concentration, indicating that the growth of the isolates were
slightly changed in the presence of chromate. Marked difference were observed
in the lag phase of Bacillus pumilus ECC, where as, in Bacillus sphaericus
differences were observed in all the phases. Bacillus pumilus ECA strain
exhibit longer lag phase at increasing concentration. Whereas, in Bacillus
pumilus ECB and Bacillus cereus ECE difference were observed only
after the lag phase according to the increasing concentration.
Antibiotic Resistance
In antibiotic resistance assay Bacillus cereus ECD showed resistance
to Ampicllin (100 μg mL-1) and Kanamycin (50 μg mL-1)
only. Bacillus pumilus strains exhibit resistance to Chloramphenicol
(3.75 μg mL-1) and Bacillus sphaericus was resistant
to Ampicillin (12.5 μg mL-1) and Chloramphenicol (3.75 μg
mL-1).
Chromate Reduction
Kinetics of the chromate reduction was carried out under aerobic condition
with an initial concentration of 100 μg mL-1 for all the isolates.
Among the isolates, B. cereus ECD strain reduce 76.1% of chromate followed
by B. pumilus ECA (65%) and B. sphaericus (57.14%), after 72 h
of incubation (Fig. 2) with reference to the original concentration
in the medium under aerobic condition. These strains were used for further studies.
No change in chromate concentrations was observed in the control indicating
that the medium did not contain any substance to undergo abiotic chromate reduction.
| Table 1: | Minimal inhibitory concentration of the isolates to various heavy metals in μg mL-1 |
Plasmid DNA Profile of Isolate
Plasmid profile of chromate resistant Gram positive isolates showed no visible
bands, indicating the absence of lower molecular weight plasmid. After curing,
there was no loss in Ampicillin and Chloramphenicol resistance in B. sphaericus,
whereas 50.4% of B. cereus ECD strains become sensitive to Kanamycin
and cobalt (50 μg mL-1) to which it was previously resistant,
indicating plasmid borne resistance.
| Fig. 1: | a) Growth kinetics of Bacillus pumilus ECA strain in the presence of chromate. b) Growth kinetics of Bacillus pumilus ECB strain in the presence of chromate. c) Growth kinetics of Bacillus pumilus ECC strain in the presence of chromate. d) Growth kinetics of Bacillus cereus ECD strain in the presence of chromate e) Growth kinetics of Bacillus cereus ECE strain in the presence of chromate and f) Growth kinetics of Bacillus sphaericus strain in the presence of chromate |
| Fig. 2: | Chromate reduction of Bacillus strains in LB broth supplemented with 100 μg mL-1 |
DISCUSSION
Chromate resistant Gram positive and Gram negative bacilli have been isolated and identified from the Ennore Creek sediments. Bacillus species are the most abundant and also exhibit high resistance to almost all the heavy metals tested, which is similar to the results of earlier reports (Ross et al., 1981; Konopka and Zakarova, 1999) discussing the presence of Bacillus species in the heavy metal contaminated soils. All the isolated Bacillus strains were exhibited high resistance (~600 μg mL-1) to chromate. However, differences in MIC of metal will also depend upon the type of the organic constituents and negatively charged ions present in the medium (Viti et al., 2003). Growth of the isolates in the liquid medium also depends upon the bioavailability and toxicity of the chromate. Due to its high diffusion rate, bioavailability and the inhibitory effect of chromate at high concentration, the bacterial cells show fewer differences in growth rate according to increasing concentrations of chromate in liquid media (Viti et al., 2003; Yilmaz, 2003; Jeyasingh and Philip, 2005).
In chromate reduction assay, B. cereus ECD strain showed a maximum efficacy when compared with other isolates. The decrease in chromate concentration during the log phase of the cultures may indicate the metabolic reduction of chromate. It has been noted that all the chromium resistant organisms do not have the ability to reduce chromate. Chromate reduction and resistance have been considered to be unrelated (Bopp and Ehrlich, 1988; Viti et al., 2003). There are many organisms in which chromate reduction is independent from chromium resistance and mediated by plasmids, but in this study it is in controversy, both chromium resistance and chromate reduction were chromosomally mediated.
Several authors have reported that the association between the heavy metal resistance and the antibiotic resistance was generally conferred by the transmissible plasmids (Hassen et al., 1990; Ramteke, 1997; Verma et al., 2001). Bhattacherjee et al. (1988) also reported that under metal stress, the bacteria adapt faster by the spread of R-factor than by natural selection and mutation. In our case, even though isolates shows high resistance to various heavy metals, all the isolates were mostly sensitive to the antibiotics tested, lack of plasmids in all these isolates may be the reason for high sensitivity. Tanaka et al. (1977) have reported the presence of large plasmids in Bacillus species, which can be observed only by pulse field agarose gel electrophoresis. In order to confirm whether the heavy metal resistance is mediated by larger plasmids, plasmid curing was performed for the Gram positive isolates B. cereus ECD, B. sphaericus and B. pumilus. Even after the curing, the isolates B. sphaericus and B. pumilus strains showed resistance to all the heavy metals tested, suggesting chromosomal encoded the resistance whereas B. cereus ECD showed susceptibility to cobalt, indicating plasmid borne resistance.
CONCLUSIONS
To conclude, the isolated Bacillus cereus ECD showed a good chromate reduction capacity. More than 76% chromate reduction was observed for 100 μg L-1 of chromate concentration within 72 h. MIC of the isolate clearly exhibited that the strain Bacillus cereus ECD have the ability to resist various heavy metals tested. This is an added advantage for isolates while performing the desired chromate reduction in the metal based industrial effluents. Other advantages of selecting bacterial based biological treatment of industrial wastes lie in the fact that it is inexpensive, does not require high energy and does not release any additional chemical to the environment.
ACKNOWLEDGMENTS
Authors are thankful to the Director, CAS in Marine Biology, Annamalai University for providing the laboratory facility to carry out part of work. Author (S.K) is grateful to the University of Madras for the research fellowship under the UGC-UWPFE programme.