Tolerance of TBT-resistant Bacteria Isolates to Methylmercury
Four strains of selected TBT-resistant bacteria were tested for growth in the presence of methylmercury. Results indicate their potential of detoxification not only of TBT but also were resistant to MeHg. The EC50 of these four is bacterial isolates; Pseudomonas fluorescens, Enterobacter cloacae, Citrobacter braakii and Alcaligenes faecalis were 0.32, 0.39, 0.34 and 0.35 μM MeHg, respectively. Enterobacter cloacae has been shown in this study to exhibit high resistance to the toxic effects of MeHg as it is previous reported to show similar high resistance capability to TBT. These bacteria species were also examined for their biodegradability and it has found that they shows capability of degrading MeHg even in the absence of primary nutrient-glycerol, suggesting that these microorganism can utilise the carbon-source in the pollutant in order to mineralise the organic compound. This study has successively proven for the first time that these four selected TBT-resistant bacteria species were both efficient MeHg resistant and degrader. Enterobacter cloacae is the most preferable as highly effective resistant TBT and MeHg degrader and thus recommended for future use.
Received: October 28, 2011;
Accepted: December 14, 2011;
Published: February 15, 2012
Methylmercury (MeHg) has been known to be a highly toxic compound which poses
significant risks on both human and aquatic organisms. Owing to its high toxicity,
persistence and the ease of their bioaccumulation in trophic chains, methylmercury
in the environment have been of major concern for some years. Known in mythology
for its fleet-footedness, mercury-a primary source of MeHg production-swiftly
spreads all over the globe from its natural and anthropogenic sources. They
can be released in aquatic media as pesticides, or as side products of catalytic
processes in industry but can also be produced from mineral mercury through
bacterial activity in sediments, as reported by Jensen and
Jernelov (1969). The main point source of mercury contamination in marine
environment is through the loading of the atmospheric inorganic mercury. Anthropogenic
activities further added to the increasing level of total mercury in aquatic
environment. Methylation and demethylation processes are the major biotic processes
that occur in lakes and marine sediments and the chemical equilibrium of these
two is significantly influenced by environmental factors such as sulphate and
sulphide concentrations, pH and sediment organic matter level (Choi
et al., 1994; Okoronkwo and Olasehinde, 2007).
Emissions from anthropogenic activities account for the increase in the total
mercury levels in the atmosphere. MeHg enters the food chain through lower organism
like the phytoplankton and others and biomagnified along this trophic level
especially in larger aquatic organism.
However, consumption of fish and sea foods are the major route by which humans
are exposed to MeHg contamination (Choi and Ceeh, 1998).
In 1952, one of the worst anthropogenically-orchestrated disaster recorded in
human history occurred at Minamata Bay in Japan where industrial waste containing
mercury compounds frequently dumped into nearby aquatic bodies led to the poisoning
of several thousands of people (Kojima and Fujita, 1973;
Greenwood, 2006). The growing number of incidents with
huge loss of human life and destruction to aquatic ecosystem prompted regulatory
attention globally and is mainly administered by the United Nation for Environment
Programme, UNEP (Weiss, 1995; Gray,
2002; UNEP, 2003). In recent years, there have been
various global agreements and treaties on mercury emission cuts and this reflects
global cooperation and commitment towards the abatement of anthropogenic mercury
emissions from scientific and policy development standpoints. Several authors
have successfully demonstrated robust studies on the assessment of the MeHg
exposure to humans, wildlife and aquatic organism and its complex interaction
with the environment (Arnot and Gobas, 2003; Boudou
and Ribeyre, 1997; Cabana et al., 1994; Clarkson
and Magos, 2006). However, fewer works have been carried out in investigating
extensively, the use of bacteria in detoxification of MeHg especially by bacterial
strains previously without MeHg exposure (Mergler et
al., 2007; Hedayati, 2012).
For the past decade, numerous studies have propounded many methods for MeHg
detoxification. More importantly, degradation by various types of bacteria have
shown higher efficiency and this paves a major pathway for sustainable remediation
solutions (Czuba et al., 1987; Bending
and Rodriguez-Cruz, 2007; Gupta and Ali, 2004; Marvin-DiPasquale
and Oremland, 1998). However, it is so obvious that typical contaminated
sediment would contain mixed pollutants. Therefore, a need arise for a microorganism
that can effectively remediate such environment. Rather, bacteria species capable
of degrading a particular toxicant is generally believed among several reports
to be isolated from a sediments or surface waters contaminated with the toxicant
(Spangler et al., 1973; Wuertz
et al., 1991; Sakultanitimetha et al.,
2009). From previous studies, most researchers only examined the degradability
efficiency of bacteria species against a single pollutant like MeHg (Kawai
et al., 1998; Bernat and Dlugonski, 2002;
Spangler et al., 1973; Wuertz
et al., 1991; Sakultanitimetha et al.,
2009). Clearly, though some authors suggested that bacteria can exhibit
a multi-resistant mechanisms to detoxify various organic pollutant and heavy
metals (Mergler et al., 2007; De
et al., 2003, 2008; Khoramabadi
et al., 2008), there have been no reports (to our knowledge) recorded
where TBT-resistant bacteria were equally investigated for MeHg tolerance. Organometals
(like MeHg) can stall a variety of energy-linked reactions in bacteria; these
include growth, solute transport and biosynthesis of macromolecules (Wuertz
et al., 1991). Therefore, this makes remediation effort ineffective
as the additive effect of the variety of these organic pollutants adversely
affects the microbial activity and hence lowers degradation efficiency.
Spangler et al. (1973) examined microcosm taken
from MeHg-contaminated sediments for the capability in demethylating MeHg at
certain concentration range. They observed that 30 isolated bacteria from the
microcosm were tolerant at the range of 0.5-10 μg mL-1. However,
it can be deduced that excessive levels of MeHg can adversely impact bacterial
growth. Decrease in bacterial population observed is known by counting the colony
forming units after bacteria exposure to MeHg in the incubator for seven days.
The chronic exposure to a toxicant can increase tolerance to that particular
toxicant as long exposure increase the bacteria memory response (Blanck
and Dahl, 1996; Bending and Rodriguez-Cruz, 2007;
Mortazavi et al., 2005). Bacteria isolated from
different sediment can shows tolerance to another toxicant in another environment.
However, very low tolerance level is observed in contrast to the sediment/site
it is isolated from. De et al. (2003) suggested
that Hg-resistant bacteria were capable of growth at far higher concentration
(i.e., 50 ppm Hg) than previously reported. It can be inferred that bacteria
can be exhibit resistance not only to Hg but also other heavy metals such as
Cd, Pb, Phenols and other xenobiotics. However, De et
al. (2003) did not examine these microorganisms for MeHg resistance
even at higher concentration. Hence, it could be presumed that bacterial isolates
may exhibit multi-resistant to different deleterious organic pollutants, for
example, like TBT and MeHg which are highly toxic and commonly found in aquatic
Organomercurial-resistant bacteria were first isolated from MeHg-contaminated
marine sediment by Spangler et al. (1973). Spangler
et al. (1973) isolated bacterial species that are capable of degrading
MeHg with the evidence of methane and Hg production as end products of the degradation
process. These microorganisms degrade MeHg by volatilising Hg out from their
cells. Pseudomonas sp., Enterobacter sp. and Citrobacter sp.
have been isolated and found to be highly resistance to MeHg (De
et al., 2003; Spangler et al., 1973;
Mirzaei et al., 2008). Most of these species
mentioned earlier are gram negative and are said to have certain resistant genes
which attribute for their defensive mechanism. Bacteria isolated from such sediment
have been reported to have develop a surprising array of resistance mechanisms
based on clustering genes in a single operon referred as “mer operon”. Ubiquity
of mer operon genes with respect to geographical location, environment
and species allows resistant strains of bacteria and other microorganisms to
thrive in presence of ionic or organic mercury compounds generally toxic to
non-resistant bacteria and other forms of life (Rasmussen
et al., 2008). The mer operon that confers mercury resistance
to bacteria is widely distributed in mercury-resistant bacterial populations
(Osborn et al., 1997; Mahmod,
2001; Barkay et al., 2003; Rezaee
et al., 2008) which is fairly highly conserved.
Some bacterial strains, Alcaligenes faecalis, Enterobacter cloacae
and pseudomonas sp. have been identified to be MeHg resistant at
about 2.5 mg L-1 CH3HgCl (Shariat
et al., 1979). Bacterial resistance to MeHg is determined by plasmids,
which in many instances also encode resistance to other heavy metals and organometallic
compounds (De et al., 2003). Single bacteria
strain can be resistant to many organometals. A bacteria strain, Enterobacter
cloacae, isolated from sediment contaminated with organotin compounds has
been found to also possess the capability of degrading both TBT and MeHg though
at different concentration relatively lower than the former. However, previous
studies revealed that the four bacteria isolates examined in this study have
been known to be highly resistant to TBT and MeHg especially when they are isolated
from their corresponding contaminated sediments (Shariat
et al., 1979; Spangler et al., 1973;
Wuertz et al., 1991; Sakultanitimetha
et al., 2009).
Interestingly, this study will for the first time attempts to examine the resistance of TBT-resistant bacteria to MeHg. Anticipated positive outcome of this research study will enable us make a reasonable suggestion for the use of this kind of bacteria species for remediating sediments contaminated with two or more organometals. By and large, it is hoped that a deeper insight of scientific role in the defining environmental problems provide a lasting solution for concrete problems associated to mercury contamination.
MATERIALS AND METHODS
Chemicals: All chemicals were used without additional purification.
Methylmercury chloride (96% purity) and Methanol (98% purity) were obtained
from Aldrich (Steinheim, Germany). All solvents were HPLC grade obtained from
Merck (Darmstadt, Germany). All reagents used were of analytical grade. All
bacteriological nutrients and agars were obtained from Oxoid (Basingstoke, UK).
All other chemicals used were obtained from BDH (Poole, UK).
Sample preparation: Nutrient media and solutions: The nutrient media
was prepared by adding 1.0 g of K2HPO4, 1.0 g of KH2PO4,
1.0 g of (NH4)2SO4, 0.4 g of MgCl2,
0.125 g of yeast extract and 1.0 mL of glycerol in 1 L of nanopure water and
subsequently autoclaved for 3 h at 55°C while the media used for the screening
experiment was made of the composition as mentioned above but the amount of
the glycerol was gradually reduced till zero (Sakultanitimetha
et al., 2009). The media was adjusted to pH 6.8 (by adding required
volume of 2 N NaOH) before been autoclaved at similar operating conditions.
About 17.5 g of the Plate Count Agar (PCA) was dissolved in 1 L of nano pure
water; the mixture was shaken vigorous to ensure complete dissolution and thereafter
sterilized in an autoclave. The sterilized plate count agar solution was taken
from the autoclave at 85°C and spiked on the plates. Peptone solution used
for the serial dilution was prepared by dissolving 1.5 mL peptone in 1000 mL
nano pure water followed by sterilization in an autoclave.
Also, 1 mM methylmercurychloride (CH3HgCl) solution was prepared by dissolving 12.5 mg CH3HgCl in 50 mL methanol and this was further diluted to 20 μM CH3HgCl.
Bacterial isolates: Bacterial isolates used in this study were previously
isolated from coastal marine sediment contaminated with organometallic compounds,
particularly TBT compounds. The bacterial strains employed in this study was
isolated from sediment slurry sample previously collected at 0-15 cm depth from
Bowling Basin (Forth and Clyde Canal), Glasgow, UK (NS 450 735) in April 2009.
All isolates highly resistant to TBT were used in this study to determine their
sensitivity or tolerance to MeHg. Their biochemical characteristics have been
studied for proper identification (Sakultanitimetha et
al., 2009). The TBT-highly resistant bacteria isolated are Alcaligenes
faecalis, Citrobacter braakii, Enterobacter cloacae and Pseudomonas
fluorescens (Inoue et al., 2000). Each of
these bacteria strains are inoculated in a nutrient media for bacterial growth
before exposure to toxicant.
Resistance of bacteria using EC50: The ability of bacteria highly resistant to TBT to grow in the presence of MeHg was tested by adding CH3HgCl at different concentrations in a growth media. Glycerol medium was prepared by adding 1 g of K2HPO4, 1 g of KH2PO4, 1 g of ( NH4)2SO4, 0.4 g of MgCl2,0.125 g of yeast extract and 1 mL of glycerol in 1 L of nano pure water. The media is subsequently sterilized in an autoclave machine prior to any further use. Two set of media were prepared for the experiment, one for the inoculation of the bacteria samples and the other for addition of the stock solution of MeHg in Methanol. The media prepared was sterilized in an autoclave prior to inoculation of bacteria and subsequently incubated at 28°C shaken at 150 rpm overnight.
Furthermore,10 mL of each sample containing MeHg was prepared by adding appropriate
quantity of 20 μM CH3HgCl stock solution in methanol to make
up different concentrations 0, 0.1, 0.2, 0.3, 0.4 and 0.5 μM CH3HgCl.
Five hundred micriliter of cultured bacteria samples are enriched by inoculation
into 10 mL of the media containing the stock solution with varying concentrations
as outlined above. These samples were immediately incubated at 28°C shaken
at 150 rpm for 7 days as considerable period of time is required for the bacteria
to grow and fully acclimatized with the alien environment. After incubation
for 7 days, the samples are serially diluted in a nano pure water containing
peptone. Aliquots of 100 μL of 10-1,10-2,10-3,10-4,10-5,10-6
and 10-7 dilutions are spread onto a plate count agar in duplicates
and are all incubated at 28°C overnight. Colonies of the bacteria within
this range i.e., 20-200 were counted in order to determine the EC50 (McNaught
and Wilkinson, 1997). Reed and Muench method was employed in performing
the EC50 test (Reed and Muench, 1938).
Screening test for biodegradability: The screening medium used in this
experiment was glycerol medium, made up of 1 g of K2PO4,1
g of KH2PO4 ,1 g of (NH4)2SO4,0.4
g of MgCl2,0.125 g of yeast extract and 1 mL of glycerol in 1 L of
distilled water. Then similar glycerol media were also prepared but now with
different quantity of glycerol 1000,100,10,0,0,0 μL ; thus making up to
six samples of 50 mL each. The samples are adjusted to pH 6.8 by adding the
required volume of 2 N NaOH prior sterilization. The study bacteria are inoculated
in the samples. Thereafter, 750 μL of 20 μM CH3HgCl stock
solution in methanol was added to 50 mL of the samples to make up a final concentration
of 0.3 μM MeHg. The sample containing 1 mL of glycerol was first incubated
at 28°C shaken at 150 rpm overnight. One hundred microliter was streaked
on plate count agar in duplicates while 4 mL was transferred into the next sample
containing 100 μL of glycerol. Subsequently, it is incubated at 28°C
shaken at 150 rpm overnight. Thereafter 100 μL was also streaked on the
plate agar count in duplicates. This procedure was repeated for the other four
samples. These procedures were adopted with slight modifications as described
by Sakultanitimetha et al. (2009).
RESULTS AND DISCUSSION
Resistance of heterotrophic bacteria: The bacterial isolates show variation
in resistance to MeHg. Colonies formations were observed 24 h after incubation
in all except for Alcaligenes faecalis which took 48 h before colonies
were counted. MeHg concentrations which inhibited colony formation by 50% (EC50)
were calculated by the method of Reed and Muench. The EC50 results
for the isolates are shown in Fig. 1. Clearly, result reveals
that Enterobacter cloacae with the highest EC50 value of 0.39
μM have the highest resistance to MeHg while Pseudomonas fluorescens
with the lowest EC50 is the least resistant to MeHg. Notably, Enterobacter
cloacae and Citrobacter braakii shows a better bacterial response
by exhibiting rapid growth within 24 h while the other two takes about 48 h
before noticeable colony formation were observed. This depicts the bacteria
behavioural properties and activity in the medium. The former were happy with
the toxicant while the latter were quite unhappy in terms of ease of growth
rate. These microorganism also exhibit different morphological shapes which
are unique to each bacteria isolates in terms of their biochemical characterizations.
Biodegradation potentiality: Not all microorganisms isolated from marine or freshwater sediment that show resistance or sensitivity to a xenobiotic can also degrade it. Most especially, in this scenario where the bacterial isolates used were not taken from a MeHg-contaminated sediment but TBT-contaminated sediment. However, results from the experiment shows that these bacteria isolates were able to degrade MeHg. This was confirmed by the bacteria growth (in colonies) on PCA plates observed for each samples at three successive days interval. Bacteria growth was observed in all sample media prepared, however, Table 1 shows their degree of relative turbidity (i.e., the cloudiness of the media solution).
The number of plus signs denotes the relative turbidity of the growth medium containing 0.3 μM for all bacteria strains.
|| Effective concentrations (EC50) of isolated bacteria
|| Growth of microorganisms in nutrient media as a function
Tolerance of bacteria isolates to MeHg: Microorganisms have been reported
to have the capability of resisting xenobiotics and other heavy metals, particularly
in natural environment where mixed pollutants exist (Agarry
et al., 2010; Sakultanitimetha et al.,
2009; Wuertz et al., 1991). From this study,
TBT-resistant bacteria have been shown to be tolerant to MeHg. The result presented
in Fig. 1 shows that the EC50 s of the selected
bacteria-Pseudomonas fluorescens, Enterobacter cloacae, Citrobacter
braakii and Alcaligenes faecalis to be 0.32, 0.39, 0.34 and 0.35
μM, respectively. It can be inferred that the variation of the EC50
values depends on the nature of the medium, biological properties of the bacteria
isolates. In natural environment, other factors such as the salinity, pH, temperature,
presence of other xenobiotics and heavy metals contribute to the sensitivity
or resistivity of these microorganisms. Wuertz et al.
(1991) reported that EC50 of TBT in freshwater is higher than
in estuarine sediments.
|| Bacterial growth of the four selected isolates in nutrient
media containing MeHg
Furthermore, EC50 values are a measure of the resistivity or tolerance
of a microorganism to a particular toxicant or mixed toxicants. The lower the
EC50, the greater the sensitivity of the bacteria and thus the less
effective it is at withstanding the toxicity of the pollutant. From Fig.
1 and 2, it was observed that that Pseudomonas fluorescens
shows a greater sensitivity to MeHg relative to others. This observation agrees
with findings reported by Shariat et al. (1979).
Therefore, tolerance level exhibited by these bacteria strain is in the decreasing
order; Enterobacter cloacae> Alcaligenes faecalis>Citrobacter braakii>Pseudomonas
fluorescens. From Fig. 2, it could be clearly seen that
both Enterobacter cloacae and Alcaligenes faecalis have high bacterial
population which in measured in terms of CFU values. The distinct feature further
complements the reason for their high EC50 values, suggesting that
these bacterial isolates possess high tolerance to MeHg relatively to others.
The MeHg contamination level in most aquatic environment is found to be estimated
at 4.59 ppb (equivalent to 0.02 μM) (Horvat et al.,
2004). In comparison with the EC50 of these two bacteria species-Enterobacter
cloacae and Alcaligenes faecalis, it indicates that the level of
MeHg contamination in environment would not cause major inhibition of bacteria
growth (or suppress its activity) and degradability even in mixed polluted sediments.
However, it is noteworthy comparing the EC50 values of these bacteria
with respect to two different pollutants i.e., TBT and MeHg respectively. For
Enterobacter cloacae and Citrobacter Braakii, their EC50
values in TBT are 200 and 280 times higher than MeHg when compared with results
reported by Sakultanitimetha et al. (2009). This
indicates that their tolerance to MeHg is relatively lower to that of TBT. We
can deduce that MeHg is highly toxic than TBT. One of the reasons for this discrepancy
might be linked to their chemical structure thus making such compound more bioavailable
(Morel et al., 1998; Agarry
et al., 2011). In organic chemistry, the reactivity of an organic
compound decreases down the homologous series as the number of alkyl group increase
(i.e., as molecular weight increases). The bulkiness of the methyl group attached
to the central metal atom coupled with increasing number of ligands causes a
phenomenon known as steric hindrance effect, thus lowers the reactivity
of the compound and making it to be more hydrophobic. From literature, the toxicity
of MeHg is linked to its liposolubility properties which TBT lacks (Morel
et al., 1998; Ekpenyong et al., 2007).
TBT is not readily liposoluble and thus it is hardly taken up by microorganism.
Notably, there have been no reports found in the literature where this experiment
has been attempted. Most authors often use bacteria species isolated from a
contaminated sediments to study either the tolerance or degradability potential
of such isolated microorganism. Nonetheless, results of this study have demonstrated
for the first time that TBT-resistant bacteria that have not been exposed to
MeHg can tolerate it though at a very low concentration when compared to their
normal tolerance of 10 μM when isolated from MeHg-heavily contaminated
sediment (Shariat et al., 1979; Spangler
et al., 1973). Therefore, it follows that these organism have
genetically conferred resistance that makes them to resist both compounds and
possibly degrade them. In corroboration, Pain and Cooney
(1998) reported that most of the TBT-resistant bacteria are also resistant
to other organometals and six heavy metals (Hg, Cd, Zn, Sn, Cu and Pb), which
suggest that resistance (either plasmid or chromosomally-mediated) to variety
of xenobiotics may be present in the same organism. This also suggests that
these bacteria strain should be tested for other noxious organometals that are
of major threat to aquatic environment. All the more, the combined toxicity
from different pollutants could badly influence the growth rates of these bacterial
For MeHg, two main resistance genes are found in most Hg resistant bacteria.
They are classified into two namely; narrow and broad spectrum bacteria. The
narrow -spectrum bacteria possesses mer A -mercuric reductase- which
convert Hg2+ to Hg0 but cannot degrade organomercury compounds
since they lack the gene for organomercurial lyase (Summers
and Silver, 1978; Osborn et al., 1997; Ravel
et al., 2000). However, the broad-spectrum type possesses the
mer B-organomercurial lyase-which is responsible for the cleavage of the
mercury-carbon bond. They are capable of degrading all mercury chemical species
(Nakamura et al., 1990; Brown
et al., 1991).
Furthermore, all the isolates tested in this study were gram-negative which
was previously reported by Ravel et al. (2000)
to be far more resistant than gram-positive strain. It therefore suggests that
such environmental strains are of practical interest to microbial ecologists
not only to reiterate current concepts of MeHg and TBT resistance by native
microflora but also to comprehend the evolution and importance of their resistance.
The bacteria strain-Enterobacter Cloacae exhibits the greatest resistance
to Hg of any gram-negative environmental isolates (De et
In TBT, the resistance genetic mechanism is not fully understood, however it
has been reported that oxidative enzymes-cytochrome P 450-common in most aquatic
organism were responsible for the catalysis of the first step reaction which
is the hydroxylation followed by debutylation (Sakultanitimetha
et al., 2009; Wuertz et al., 1991).
MeHg-resistant bacteria isolates differed in their tolerance properties (Fig.
1).Tolerance properties are measured in terms of their EC50s.
This implies that the efficiency of mercury detoxifying systems may varies in
different Hg-resistant bacteria even as observed in this study. This finding
also corroborates observations by different authors (Pahan
et al., 1995; Ray et al., 1993; Sadhukhan
et al., 1997).
Effect of medium on MeHg-resistant bacteria: The plating medium used
might also affect the bacterial resistances as medium-organometal interaction
may affect the metal toxicities (Wuertz et al.,
1991; Ekpenyong et al., 2007). However,
it is difficult to relate the metal concentration used in agar medium to environmental
concentrations. Nonetheless, the reason for this is unknown as their interaction
mechanisms are yet to be unfolded (Duxdury, 1985). For
instance, Wuertz et al. (1991) found that EC50s
were higher on TSA than on estuarine-salts agar the EC50s on estuarine
agar for the polluted estuarine site and the unpolluted site differed significantly.
Biodegradation potentiality: Most bacteria that show some degree of
resistance to certain xenobiotics often at times not possess the capability
of degrading them. However, even though all MeHg-degrading bacteria are all
MeHg-resistant bacteria, not all MeHg-resistant bacteria are MeHg-degraders.
They can either degrade them by using carbon source from the nutrient media
or from the toxicant itself (Billen et al., 1974;
Sakultanitimetha et al., 2009). Biodegradation
can be effected by organisms either able to utilize the pollutant for their
energy and carbon requirements or only able to modify it enzymatically without
using it as a nutritional source- i.e.cometabolism (Bending
and Rodriguez-Cruz, 2007; Ekpenyong et al., 2007).
For MeHg, potential microbes can utilise methyl (CH3-) group in the compound as its growth substrate with incorporation of carbon. In an instant of limited carbon content, MeHg will become a sole source of carbon for bacterial survival. The results from this study shows that selected TBT-degrading bacteria isolates were not only resistant to MeHg but also possess the tendency to degrade MeHg in the nutrient media. However, Table 1 shows a gradual decrease in the turbidity and colony formation as the amount of carbon source (i.e., glycerol) is gradually lowered systematically. This implies that the carbon source have a significant impact on the microbial metabolism and hence the degradability.
Moreover, it is evident that the bacteria isolates were able to survive even
in the absence of any organic source due to the fact that they derived their
energy from the toxicant .With the aid of their resistance enzymes, they were
able to metabolize MeHg into methane and mercury vapour (Hg0) (Oremland
et al., 1991). It took longer periods, usually more than 48hrs (an
indication of extended lag phase) before bacterial growth were observed. This
result shows that Enterobacter and Citrobacter exhibit faster acclimation period
and bacteria response. In the absence of glycerol, we observed that all the
bacteria strains except Enterobacter cloacae grew very happily suggesting
they utilise the carbon-energy source from MeHg. However, before the nutritional
source was totally withdrawn, bacteria isolates breakdown MeHg as cometabolite
by relying on the utilisation of the primary substrate to metabolise the secondary
substrate (which is usually the toxicant).The primary substrate which is the
glycerol in the media support bacteria growth and/or cell replication. Faster
growth rate was observed which is confirmed by the turbidity of the media solution
and the high Coliform Forming Unit (CFU) values recorded.
Consequently, co-metabolic process will be obviously depended on the growth
of the microorganism (Errecalde et al., 1995).
Thus, the addition of nutritional source enhances biodegradation process (Kawai
et al., 1998; Bernat and Dlugonski, 2006).
It often happens that the addition of a pollutant suitable as an energy source
in a medium induces an increase in the degradation capacity of the microbial
community, because of a "sociological adaptation"(Wuhrman,
1964) due to positive selection by the pollutant whose presence favours
the growth of bacterial strains able to use it.
It has been successfully demonstrated that isolated TBT-resistant bacteria
were able to show a significant resistance to MeHg even though it was assumed
that these organisms have not been exposed to MeHg at any time. Interestingly,
this is a very first time this was attempted and gives a new scientific approach
to bioremediation technologies. Enterobacter cloacae and Alcaligenes
faecalis have been shown in this study to exhibit high resistance to the
toxic effects of MeHg.
Furthermore, these bacteria species were also examined for their biodegradability and it has found that they shows capability of degrading MeHg even in the absence of primary nutrient-glycerol. It can be concluded that these microorganism can utilise the carbon-source in the pollutant in order to mineralize the organic compound. Again, it invalidates previous studies which from their observation suggest that cometabolism as the possible means of microbes degrading MeHg. Again, this study has successively proven that these four selected bacteria species isolated from TBT-contaminated sediment were able of utilising the organic pollutant as a nutritional substrate to carry out mineralisation activities.
Agarry, S.E., B.O. Solomon and T.O.K. Audu, 2010.
Optimization of process variables for the batch degradation of phenol by Pseudomonas fluorescence
using response surface methodology. Int. J. Chem. Technol., 2: 33-45.CrossRef | Direct Link |
Agarry, S.E., B.O. Solomon and T.O.K. Audu, 2011.
Bioenergetics of binary mixed culture of Pseudomonas aeruginosa
and Pseudomonas fluorescence
growth on phenol in aerobic chemostat culture. Int. J. Chem. Technol., 3: 1-13.CrossRef | Direct Link |
Arnot, J.A. and F.A.P.C. Gobas, 2003.
A generic QSAR for assessing the bioaccumulation potential of organic chemicals in aquatic food webs. QSAR Comb. Sci., 22: 337-345.CrossRef | Direct Link |
Barkay, T., S.M. Miller and A.O. Summers, 2003.
Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol. Rev., 27: 355-384.CrossRef | PubMed | Direct Link |
Bending, G.D. and M.S. Rodriguez-Cruz, 2007.
Microbial aspects of the interaction between soil depth and biodegradation of the herbicide isoproturon. Chemosphere, 66: 664-671.PubMed |
Bernat, P. and J. Dlugonski, 2002.
Degradation of tributyltin by the filamentous fungus Cunnunghamella Eelegams
, with involvement of cytochrome P-450. Biotechnol. Lett., 24: 1971-1974.Direct Link |
Bernat, P. and J. Dlugonski, 2006.
Acceleration of tributyltin chloride (TBT) degradation in liquid cultures of the filamentous fungus Cunninghamella elegans
. Chemosphere, 62: 3-8.PubMed |
Billen, G., C. Joiris and R. Wollast, 1974.
A bacterial methylmercury-mineralizing activity in river sediment. Water Res., 8: 219-225.CrossRef | Direct Link |
Blanck, H. and B. Dahl, 1996.
Pollution-Induced Tolerance (PICT) in marine perifyton in a gradient of Tri-n-Butyltin (TBT) contamination. Aquat. Toxicol., 35: 59-77.
Boudou, A. and F. Ribeyre, 1997.
Mercury in the food wed: Accumulation and transfer mechanism. Met. Ions Biol. Syst., 34: 289-319.PubMed |
Brown, N.L., J. Camakaris, B.T. Lee, T. Williams, A.P. Morby, J. Parkhill and D.A. Rouch, 1991.
Bacterial resistance to mercury and copper. J. Cells Biochem., 46: 106-114.PubMed |
Cabana, G., A. Tremblay, J. Kalff and J.B. Rasmussen, 1994.
Pelagic food chain structure in Ontario Lakes: A determinant of mercury levels in lake trout (Salvelinus namaycush
). Can. J. Fish Aquatic. Sci., 51: 381-389.
Choi, M.H. and J.J. Cech, 1998.
Unexpectedly high mercury level in pelleted commercial fish feed. Environ. Toxicol. Chem., 17: 1979-1981.CrossRef | Direct Link |
Choi, S.C., T. Chase Jr. and R. Bartha, 1994.
Enzymatic catalysis of mercury methylation by Desulfovibrio
desulfuricans LS. Applied Environ. MIcrobiol., 60: 1342-1346.Direct Link |
Clarkson, T.W. and L. Magos, 2006.
The toxicology of mercury and its chemical compounds. Crit. Rev. Toxicol., 36: 609-662.PubMed |
De, J., N. Ramaiah and L. Vardanyan, 2008.
Detoxification of toxic heavy metals by marine bacteria highly resistant to mercury. Mar. Biotechnol., 10: 471-477.CrossRef | PubMed | Direct Link |
De, J., N. Ramaiah, A. Mesquita and X.N. Verleker, 2003.
Tolerance to various toxicants by marine bacteria highly resistant to mercury. Mar. Biotechnol., 5: 185-193.CrossRef |
Duxdury, T., 1985.
Ecological aspects of heavy metal responses in microorganisms. Adv. Microbiol. Ecol., 8: 185-235.
Ekpenyong, M.G., S.P. Antai, J.P. Essien and G.D. Iwatt, 2007.
pH-dependent Zinc toxicity differentials in species of Penicillium
and rhodotorula during oil biodegradation. Int. J. Biol. Chem., 1: 54-61.CrossRef | Direct Link |
Errecalde, O., M. Astruc, G. Maury and R. Pinel, 1995.
Biotransformation of butyltin compounds using pure strains of microorganisms. Applied Organomet. Chem., 9: 23-28.CrossRef | Direct Link |
Gray, J.S., 2002.
Biomagnification in marine systems: The perspective of an ecologist. Mar. Pollut. Bull., 45: 46-52.CrossRef | PubMed |
Greenwood, M.R., 2006.
Methylmercury poisoning in Iraq: An epidemiological study of the 1971-1972 outbreak. J. Appied Toxicol., 5: 148-159.PubMed |
Gupta, N. and A. Ali, 2004.
Mercury volatilization by R factor system in Escherichia
coli isolated from aquatic environments of India. Curr. Microbiol., 48: 88-96.PubMed |
Hedayati, A., 2012.
Effect of marine mercury toxicity on immunological responses of seabream. Asian J. Anim. Sci., 6: 1-12.CrossRef | Direct Link |
Horvat, M., V. Mandic, L. Liang, N.S. Bloom and S. Padberg et al
Working method paper: Certification of methylmercury compounds concentration in sediment reference material, IAEA-356. Applied Organomet. Chem., 8: 533-540.CrossRef | Direct Link |
Inoue, H., O. Takimura, H. Fuse, K. Murakami, K. Kamimura and Y. Yamaoka, 2000.
Degradation of triphenyltin by a fluorescent pseudomonad. Applied Environ. Microbiol., 66: 3492-3498.PubMed |
Jensen, S. and A. Jernelov, 1969.
Biological methylation of mercury in aquatic organisms. Nature, 233: 753-754.CrossRef | Direct Link |
Kawai, S., Y. Kurokawa, H. Harino and M. Fukushima, 1998.
Degradiation of Tributyltin by a bacterial strain isolated from polluted river water. Environ. Pollut., 102: 259-263.Direct Link |
Khoramabadi, G.S., A. Jafari and J.H. Jamshidi, 2008.
Biosorption of mercury (II) from aqueous solutions by zygnema fanicum algae. J. Applied Sci., 8: 2168-2172.CrossRef | Direct Link |
Kojima, K. and M. Fujita, 1973.
Summary of recent studies in Japan on methyl mercury poisoning. Toxicology, 1: 43-62.PubMed |
Mahmod, A.A., 2001.
Investigation of the reversible inhibition of butrylcholinesterase by mercury chloride. J. Med. Sci., 1: 251-254.CrossRef | Direct Link |
Marvin-DiPasquale, M.C. and R.S. Oremland, 1998.
Bacterial methylmercury degradation in floride everglades peat sediment. Environ. Sci. Technol., 32: 2556-2563.CrossRef | Direct Link |
McNaught, A.D. and A. Wilkinson, 1997.
IUPAC Gold Book: Compendium of Chemical Terminology. 2nd Edn., Blackwell Scientific Publications, London, UK
Mergler, D., H.A. Anderson, L.H.M. Chan, K.R. Mahaffey, M. Murray, M. Sakamoto and A.H. Stern, 2007.
Methylmercury exposure and health effects in humans: A worldwide concern. AMBIO, 36: 3-11.Direct Link |
Mirzaei, N., F. Kafilzadeh and M. Kargar, 2008.
Isolation and identification of mercury resistant bacteria from Kor River, Iran. J. Boil. Sci., 8: 935-939.CrossRef | Direct Link |
Morel, F.M.M., A.M.L. Kraepiel and M. Amyot, 1998.
The chemical cycle and bioaccumulation of mercury. Annu. Rev. Ecol. Sys., 29: 543-566.Direct Link |
Mortazavi, S., A. Rezaee, A. Khavanin, S. Varmazyar and M. Jafarzadeh, 2005.
Removal of mercuric chloride by a mercury resistant Pseudomonas putida
strain. J. Biological Sci., 5: 269-273.CrossRef | Direct Link |
Nakamura, K., M. Sakamoto, H. Uchiyama and O. Yagi, 1990.
Organomercury volatilizing bacteria in the mercury polluted sediment of Minamata bay Japan. Applied Environ. Microbiol., 56: 304-305.Direct Link |
Okoronkwo, A.E. and E.F. Olasehinde, 2007.
Investigation of lead binding by Tithonia diversifolia
. J. Applied Sci., 7: 1589-1595.CrossRef | Direct Link |
Oremland, R.S., C.W. Culbertson and M.R. Winfrey, 1991.
Methylmercury decomposition in sediments and bacterial cultures: Involvement of methanogens and sulfate reducers in oxidative demethylation. Applied Environ. Microbiol., 57: 130-137.Direct Link |
Osborn, A.M., K.D. Bruce, P. Strike and D.A. Ritchie, 1997.
Distribution, diversity and evolution of the bacterial mercury resistance (mer) operon. FEMS. Microbiol. Rev., 19: 239-262.PubMed |
Pahan, K., J. Chaudhuri, D. Ghosh, R. Gachhui, S. Rayand and A. Mandal, 1995.
Enhanced elimination of HgCl2 from natural water by a broad-spectrum Hg-resistant Bacillus
pasteurii strain DR2 in presence of benzene. Bull. Environ. Contam. Toxicol., 55: 554-561.PubMed |
Pain, A. and J.J. Cooney, 1998.
Characterization of organotin-resistance bacteria from boston harbour sediments. Arch Environ. Contam. Toxicol., 35: 412-416.PubMed |
Rasmussen, L.D., C. Zawadsky, S.J. Binnerup, G. Oregaard, S.J. Sorensen and N. Kroer, 2008.
Cultivation of hard to culture subsurface mercury-resistant bacteria and discovery of new mer
A gene sequences. Applied Environ. Microbiol., 74: 3795-3803.CrossRef | Direct Link |
Ravel, J., J. Diruggiero, F.T. Robb and R.T. Hill, 2000.
Cloning and sequence analysis of the mercury resistance operon of Streptomyces
sp. strain CHR28 reveals a novel putative second regulatory gene. J. Bacteriol., 182: 2345-2349.CrossRef | PubMed | Direct Link |
Ray, S., K. Pahan, R. Gachhui, J. Chaudhuri and A. Mandal, 1993.
Studies on the mercury volatilizing enzymes in nitrogen fixing Beijerinckia mobilis. World J. Microbiol. Biotechnol., 9: 184-186.CrossRef | Direct Link |
Reed, L.J. and H. Muench, 1938.
A simple method of estimating fifty per cent endpoints. Am. J. Epidemiol., 27: 493-497.CrossRef | Direct Link |
Rezaee, A., J. Derayat, H. Godini and G. Pourtaghi, 2008.
Adsorption of mercury from synthetic solutions by an Acetobacter xylinum
biofilm. Res. J. Environ. Sci., 2: 401-407.CrossRef | Direct Link |
Sadhukhan, P.C., S. Ghosh, J. Chaudhuri, D.K. Ghosh and A. Mandal, 1997.
Mercury and organomercurial resistance in bacteria isolated from freshwater fish of wetland fisheries around Calcutta. Environ. Pollut., 97: 71-78.PubMed |
Sakultanitimetha, A., H.E. Keenan, M. Dyer, T.K. Beattie, S. Bangkedphol and A. Songsassen, 2009.
Isolation of tributyltin-degrading bacteria Citrobacter braakii
and Enterobacter cloacae
from butyltin-polluted sediment. J. ASTM Int., 6: 1-6.Direct Link |
Czuba, M., R.W. Seagull, H. Tran and L. Cloutier, 1987.
Effect of methyl mercury on arrays of the microtubules and macromolecular synthesis in Daucus carota
cultures. Ecotoxicol. Eviron. Saf., 14: 64-72.PubMed |
Shariat, M., A.C. Anderson and J.W. Mason, 1979.
Screening of common Bacteria Capable of demethylation of methylmercuric chloride. Bull. Environ. Contain. Toxicol., 21: 255-261.PubMed |
Spangler, W.J., J.L. Spigarelli, J.M. Rose, R.S. Flippin and H.H. Miller, 1973.
Degradation of methylmercury by bacteria isolated from environmental samples. Applied Environ. Microbiol., 25: 488-493.Direct Link |
Summers, A.O. and S. Silver, 1978.
Microbial transformations of metals. Ann. Rev. Microbiol., 32: 637-672.CrossRef | Direct Link |
Global mercury assessment. United Nations Environment Programme, Geneva. http://www.chem.unep.ch/mercury
Weiss, B., 1995.
Perspectives on methyl mercury as a global health hazard. Neurotoxicology, 16: 577-578.PubMed |
Wuertz, S., C.E. Miller, R.M. Pfister and J.J. Cooney, 1991.
Tributyltin-resistant bacteria from estuarine and freshwater sediments. Applied Environ. Microbiol., 57: 2783-2789.Direct Link |
Wuhrman, K., 1964.
International vereinigung fur theoretiashe und angewandte limnologie stuttgart. Proc. Int. Assoc. Theor. Applied Limnolo., 1968: 579-604.