Isolation and Characterization of Arsenic Resistant Bacteria from Different Environment in South-West Region of Bangladesh
The experiment was carried out to determine the presence and numbers of arsenic resistant bacteria population. In this study, soil samples were collected from different contaminated sites of Khulna shipyard, Rupsha, Baghmara and Ramnagor in Khulna district of Bangladesh. Twenty arsenic resistant bacterial strains were isolated from the soil samples. They were isolated by growing them on Nutrient Broth (NB) medium impregnated with high concentration of arsenic. From them, six strains Bacillus lichnefomis (1/10), Listeria murrayi (2/9), Bacillus polymyxa (3/6), Moraxella urethralis (4/9) and Planococcus citreus (5/8) and Pseudomonas fluorescens (6/8) were finally selected and studied their morphological and biochemical characters in details. All six strains were able to tolerate very high concentration (>100 ppm). The optimum pH and temperature for the growth of all six strains were 8.5 and 37°C, respectively. It was concluded that bacteria living in arsenic free environment must posses a mechanism necessary to resist other toxic levels of arsenic.
Arsenic is the 20th most abundant element in the Earth`s crust 14th in the
seawater and 12th in the human body (Woolson, 1975) and
is widely distributed throughout nature as a result of weathering, dissolution,
fire, volcanic activity and anthropogenic input (Cullen and
Reimer, 1989). The last includes the use of arsenic in pesticides, herbicides,
wood preservatives and dye stuffs as well as production of arsenic-containing
wastes during smelting and mining operations. In arsenic-enriched environments,
a major concern is the potential for mobilization and transport of this toxic
element to ground water and drinking water supplies. In Bangladesh, an estimated
57 million people have been exposed to arsenic through contaminated wells. This
incident serves as an unfortunate reminder of the toxic consequences of arsenic
mobilization and underscores the need to understand the factors controlling
the mobility and solubility of arsenic in aquatic systems (Newman
et al., 1997). The primary anthropogenic input derives from combustion
of municipal solid waste, fossil fuels in coal and oil-fired power plants, release
from metal smelters and direct use of arsenic-containing herbicides by industry
and agriculture. There are a number of ways by which the human population can
become exposed to arsenic. The most important one is probably through ingestion
of arsenic in drinking water or food. Toxicity and detoxification of heavy metal
and transition metal oxyanions in living organisms are tightly bound to membrane
transport systems of ions and oxyanions. Due to its un-ionized form at neutral
pH, arsenite can passively move across the membrane bilayer or be transported
by a carrier protein similar to those that transport un-ionized organic compounds.
Arsenate poisoning generally results from the transport of this ion by the phosphate
transport system thereby competitively inhibiting the oxidative phosphorylation
pathway. One phosphate transport system (Pit) takes up both, phosphate and arsenate,
at similar rates, whereas the other (Pst, phosphate specific transport) is highly
specific for phosphate. Bacteria defective in the Pit pathway (Pit) are usually
arsenate resistant (Cervantes et al., 1994) and
for Pit-bacteria the oxidation of As (III) to As (V) represents a potential
detoxification process allowing them to tolerate high levels of arsenate. A
number of microorganism had been isolated that could use Arsenic in their metabolism,
either using arsenate as a terminal electron acceptor in an aerobic respiration
(Ahmann et al., 1994; Stolz
and Orenland, 1999) or as a means of generating energy through chemoautotrophic
arsenite oxidation (Santini et al., 2000). Bacteria
might show resistance to an arsenite through the activity of arsenite oxidase
(Muller et al., 2003). More recently, a chemolithoautotrophic
arseniteoxidizing bacterium belonging to the proteobacteria has been isolated
from a gold mine (Santini et al., 2000).
The plasmid determined arsenic resistance system has always had the same
biochemical mechanisms, reduced uptake due to an ATPase efflux system
in both gram-negative and gram-positive bacteria. Most studies of arsenic
resistant bacteria have been conducted in environments that contain high
concentrations of arsenic. However, research shows that common microorganisms
such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus
aureus, also exhibit arsenic resistance. This goal of this research
study was to isolate bacterial from arsenic contaminated environments
that contain mechanisms of resisting arsenic and begin characterization
and identification of these isolates. We hypothesized that isolated bacteria
are highly resistant to arsenic, so they could represent good candidates
for bioremediation processes of native polluted sediments. Considering
the aspects mentioned in the present research program has been undertaken
the following objectives:
||To isolate bacterial species, which are resistant to
arsenic from arsenic polluted environment
||To characterize the putative arsenic resistant bacteria
MATERIALS AND METHODS
The experiment was conducted through August 2007 to April 2008 at Microbiology
Laboratory of Biotechnology and Genetic Engineering Discipline, Khulna University,
Bangladesh to study soil samples were collected from different arsenic contaminated
sites of Khulna shipyard (Dry soil near the ship), Bagmara (Dry soil side of
the river, Wet soil near the tube well, Wet soil near the tube pond), Ramnagar
(Wet soil near the tube well, Wet soil near the pond), Rupsha, Khulna in sterile
polyethylene bags. Soil samples were best collected from top to 20 cm. Every
time of collection hands were sterile with alcohol (95%) and then the bags were
partially tilled with materials and labeled. Special care was always taken to
avoid contamination of arsenic after collection the samples were brought to
the laboratory carefully and put it in the freeze for immediate use. For long
time preservation the sample will be storage at 4°C. Soil dilution technique
is used for the isolation of arsenic resistance bacteria. At first 34 different
bacterial colonies were isolated from the soil samples by culturing them in
Nutrient Agar (NA) medium containing 5 ppm Arsenic. From this 6 bacterial strains
were finally selected for detail study. The strains were inoculated in NA slant
using a sterile loop and incubated at 37 °C for 18-27 h. After growth the
slants were stored at 4 °C for short term storage and for uses. For long
term preservation, bacterial growth was harvested from culture slant by sterile
inoculating loop. Dense cell suspensions were prepared into 10% (v/v) aqueous
glycerol in one vial and stored at 15 °C. The selected bacterial strains
were morphological characterized through non-microscopic such as agar colony,
agar slant, broth culture and microscopic fixed stained smear such as gram stain
and spore stain modified method and biochemical characterized with the help
of Bergey`s Manual (Buchanan and Gibson, 1984).
Growth of the Strains in Different Concentrations of Arsenic (As3+)
Tubes of nutrient broth containing different concentration of arsenic
(0, 1, 5, 10, 20, 50 and 100 ppm) were equally inoculated with fresh culture
and incubated at 37 °C. After 24 h, growth on nutrient broth measured
by spectrophotometric reader (600 nm).
Growth at Different Temperature
Tubes of nutrient broth were equally inoculated with fresh culture
and incubated at 4, 20, 37 and 60 °C. After 24 h, growth on nutrient
broth measured by spectrophotometric reader (600 nm) in four times.
Growth at Different pH
Tubes of nutrient broth at different pH (4.5, 6.5 and 8.5) were prepared
in duplicates and after inoculation incubated at 37 °C. After 24 h,
growth on nutrient broth measured by spectrophotometric reader (600 nm)
in four times.
Selected strains were microscopically studied. Vegetative cells, spores
and gram reaction were observed under microscope and the results were
shown in Table 1. From the observation there are 5 strains
were gram positive and 1 strain is gram negative. In flagella reaction
4 strains were positive and 2 strains were negative.
From these morphological studies indicate that the strain number 1/10,
2/9, 3/6, 4/9, 5/8 and 6/8 may be Bacillus lichefomis,
Listeria murrayi, Bacillus polymyxa, Moraxella urethralis,
Planococcus citreus and Pseudomonas fluorescens.
Selected strains were studied for their physiological and biochemical
tests which were needed for characterization of the strains.
From these biochemical studies shows that the strain number 1/10,
2/9, 3/6, 4/9, 5/8 and 6/8 determine Bacillus lichefomis,
Listeria murrayi, Bacillus polymyxa, Moraxella urethralis,
Planococcus citreus and Pseudomonas fluorescens (Table
Growth Responses of the Selected Strains at Different Temperature
For the determination of optimum temperature, strains were grown in
nutrient broth medium. Tubes of NB were equally inoculated with inocula
and incubated at different temperature 4, 25, 37 and 60 °C. In both
cases, growth on nutrient broth was measured by spectrophometric reading
at 600 nm after 24 h. Table 3 indicated that 37 °C
are suitable for bacterial growth and then optical density is 8.7.
|| Morphological studies and staining properties of the
selected bacterial strains
|+: Indicates positive result, -: Indicates negative
|| Some biochemical characterization of selected strains
|+: Indicates positive result, -: Indicates negative
result, FA: Facultative anaerobe, OB: Obligate aerobe
|| Growth responses of the selected strains at different
|| Growth responses of the selected strains at different
|Growth responses of the selected strains at different
concentrations of Arsenic (As3+)
|| Growth responses of all strains at different concentrations
Growth Response of Different pH
Results were taken in spectrophotometer at 600 nm. Growth response
of bacterial strains depends on different pH. Table 4
indicated that 8.5 were suitable for bacterial growth.
Organisms were studied in different concentrations of Arsenic (As3+)
containing culture media e.g., nutrient broth with Arsenic (As3+)
0, 1, 5, 10, 20, 50 and 100 ppm (mg L-1) in spectrophotometer
at 600 nm. Cultural characteristics of the selected isolates were shown
in Fig. 1.
Anderson and Cook (2004) reported 17 morphologically
distinct arsenic resistant heterotrophic bacteria to be members of the genera
Exigeobacterium, Aeromonas, Bacillus, Pseudomonas,
Escherichia and Acinetobacter. Macur et al. (2001)
also found that members of the Caulobacter, Sphingomonas and
Rhizobium may be responsible for the reduction and mobilization of arsenic.
Hoeft et al. (2002) found that sulfurospirillium
and Desulfovibrio use arsenate as an electron acceptor for their growth.
This study reveals that the three genera such as Listeria, Moraxella
and Planococcus are also resistant to arsenic. The diversity of arsenic
resistance gene is probably much greater and more complex than is apparent from
studies on known arsenic resistant isolates (Jackson and
Dugas, 2003). Heavy metal toxicities and binding are pH dependent (Wood,
1983) and it appear that the strains require environmentally relevant pH
for growth. Bouchard et al. (1996), Christiansen
and Ahring (1996) and Niggemeyer et al. (2001) reported optimum pH
7.5 for arsenic resistant strains Desulfitobacterium frappieri, D.
hafniense and Desulfitobateriun strain GBFH, respectively.
Temperature is another important environmental factor, which affects bacterial
growth (Herbert and Bhakoo, 1979). The optimum temperature
(37°C) for the growth of all the strains in the present study support the
results observed by Bouchard et al. (1996), Christiansen
et al. (1996) and Niggemeyer et al. (2001).
Jackson et al. (2005) isolated numbers of culturable
arsenate (V) resistant bacteria from which some were capable to tolerate very
high (100 mM) level of arsenate, although arsenic resistance was generally much
lower. In addition, Zelibor et al. (1987) isolated
As (V)r bacteria in well water samples. These isolates tolerated
up to 2,000 pg of As (V) per mL. However, they did not test for As (III) resistance.
Honschopp et al. (1996) isolated an arsenic resistant
and arsenic methylating bacterium belonging to the Flavobacterium-Cytophaga
group, which was able to tolerate 200 ppm concentration of As in the culture
media. Plasmids also have been detected in some bacteria exhibiting high level
of resistance to arsenate, arsenite and antimonate (Cervantes
et al., 1994; Dabbs and Sole, 1988; Mobley
et al., 1983).
The bioremediation of arsenic from contaminated sites involves reduction
and oxidation of arsenic with the use of arsenic resistant microorganisms.
The successful exploitation of these bacterial strains with proper biotechnology
for bioremediation of arsenic will be beneficial. Therefore, more advance
research is required for a deeper understanding about these bacterial
strains to improve arsenic bioremediation process.
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