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Research Article
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Biodegradation of Low Concentration of Monochloroacetic Acid-Degrading Bacillus sp. TW1 Isolated from Terengganu Water Treatment and Distribution Plant |
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A.H. Zulkifly,
D.D. Roslan,
A.A.A. Hamid,
S. Hamdan
and
F. Huyop
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ABSTRACT
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Haloacetic acids (HAAs) are group of chemicals formed due to disinfection by products that can be detected during chlorination and chloramination of processed drinking water. In this study, Bacillus sp. strain TW1 identified by morphological/biochemical and PCR-amplified 16S rRNA gene was isolated from Kuala Terengganu water treatment and distribution plant. TW1 was isolated due to its ability to grow in low concentration of monochloroacetic acid (MCA) of 0.5 mM, 10 times lower than normal MCA as sole carbon and energy source. Bacterial cell culture was grown in liquid minimal medium, pH 6.5 at 30°C on rotary shaker (150 rpm). Degradation of monochloroacetic acid was detected by measuring the amount of chloride ion released in the liquid minimal medium. Strain TW1 degraded monochloroacetic acid at best with maximum chloride ion released of 0.32 μmol Cl mL-1 using 0.5 mM MCA concentration. Current results demonstrated that this is the first reported study on low concentration of MCA degradation by Bacillus sp. TW1.
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Received: June 30, 2010;
Accepted: August 19, 2010;
Published: October 11, 2010
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INTRODUCTION
Most halogenated compounds are major environmental pollutants. Haloacetic acids
(HAAs) are toxic organic chemicals that are frequently detected in surface waters
and in drinking water distribution systems (McRae et
al., 2004). They cause serious environmental pollution and human health
problems as a result of their toxicity, persistence and transformation into
hazardous metabolites. Most haloacetic acids (HAAs) are found throughout the
biosphere due to high consumption of modern industrial and agricultural processes.
Haloacetic acids are also used as herbicides and insecticides (Wilson
and Mabury, 2000; Hashimoto et al., 2009).
Haloacetic acids include many different compounds, such as monochloroacetic
acid (MCA), dichloroacetic acid (DCA), trichloroacetic acid (TCA) and trifluoroacetic
acids (TFA) (McRae et al., 2004; Ellis
et al., 2001). Degradation of monochloroacetic acid (MCA), monobromoacetic
acid (MBA) and fluoroacetic acid (FAA) have been reported by Olaniran
et al. (2004), Sui-Yi et al. (2007),
Donelly and Murphy (2009). Numerous studies have demonstrated
that monochloroacetic acid (MCA) is toxic to aquatic life such as fishes and
in particular to algae. These compounds, if present at high concentrations,
are poisonous to plants and could lead to carcinogens (Hanson
et al., 2002).
The destruction of organic chemicals by microorganisms may be influenced by
environmental factors or the structure of the chemical itself. One of the reasons
suggested for the lack of degradation of organic compounds by microbes is their
low concentration (Boethling and Alexander, 1979). Microorganisms
that metabolize and grow upon very low concentrations of substrates have been
designated as oligotrophs (Poindexter, 1981). Such organisms
appear to be adapted to low substrate concentrations by having high substrate
affinity (low km value) systems. Biodegradation of very low concentrations of
xenobiotic compounds has been neglected. However, it is useful to know about
growth of microorganisms in low concentrations of pollutants because of the
legal requirements. Normally this is set by a government to get the concentration
of a pollutant down to a level that is not considered harmful. Therefore, if
the microorganisms could only remove high concentration of pollutants, they
could not be used to meet requirements of the law, since there still will be
low concentrations of pollutants in the environment.
Current research focuses on the identification and partial characterization
of potential bacteria from water treatment and distribution plants that can
degrade low concentrations of MCA. Should growth be possible at low substrate
concentrations, dehalogenase(s) km values reported earlier by Huyop
et al. (2004) suggested that some dehalogenase(s) might function
satisfactorily at low substrate concentrations.
MATERIALS AND METHODS Chemicals: Various halogenated compounds of analytical grade were purchased from Sigma-Aldrich or Fluka. Bacterial agar from Difco and the rest of chemicals were of the highest purity commercially available.
Isolation and identification of organism: The bacterial isolate used
in this study was originally isolated from Kuala Terengganu water treatment
and distribution plant. About 5 mL of collected water sample were added into
250 mL shake flask liquid culture containing minimal salts medium (pH 6.5) with
5 mM MCA as the sole carbon source before sub-culturing onto a solid medium.
After several sub-culture in 5 mM MCA solid minimal media (pH 6.5), one pure
isolate was obtained by repeated streaking. The bacterial strain was characterized
by morphological/biochemical tests as described in Bergeys Manual of Systematic
Bacteriology (Holt et al., 1994) and 16S rDNA gene
analysis as described earlier (Hamid et al., 2010a,
b).
Genomic DNA extraction: Bacterial isolates were either grown in liquid minimal medium supplemented with 5 mM of MCA or grown in liquid LB medium (Luria Bertani medium). Genomic DNA was then prepared using Wizard® Genomic DNA Purification Kit (Promega).
PCR amplification of 16S rRNA gene for bacteria identification: The
primers used to amplify 16S rRNA were forward primer, FD1 (5-aga gtt tga
tcc tgg ctc ag-3) and reverse primer, rP1 (5-acg gtc ata cct tgt
tac gac tt-3) (Fulton and Cooper, 2005). The program
used for amplification of 16S rRNA gene were: initial denaturation 94°C
(5 min), denaturation 94°C (1 min), annealing 55°C (1 min) and extension
74°C (4 min).
Growth conditions: The culture was grown at 30°C on a rotary shaker
in 250 mL flasks containing 100 mL medium as described earlier by Ismail
et al. (2008). The liquid PJC minimal media was prepared as 10x concentrated
basal salts containing K2HPO4. 3H2O (42.5 g
L-1), NaH2PO4. 2H2O (10.0 g L-1)
and (NH4)2SO4 (25.0 g L-1). The
trace metal salts solution was a 10x concentrate that contained nitriloacetic
acid (NTA) (1.0 g L-1), MgSO4 (2.0 g L-1),
FeSO4. 7H2O (120.0 mg L-1), MnSO4.
4H2O (30.0 mg L-1), ZnSO4. H2O (30
mg L-1) and CoCl2 (10.0 mg L-1) in distilled
water (Hareland et al., 1975). Minimal media for
growing bacteria contained 10 mL of 10x basal salts and 10 mL of 10x trace metal
salts per 100 mL of distilled water and were autoclaved (121°C, for 15 min).
The carbon source MCA was neutralized with NaOH and sterilized by filtration and added to the autoclaved salts medium to an appropriate final concentration. The extent of growth determined by measuring the absorbance at A680 nm and the release of chloride ions.
Measuring chloride ion released in the growth medium: Chloride ion released
was detected in the growth medium by measuring the chloride ion at appropriate
time intervals. Activity of the enzyme was measured by determining the release
of chloride indicated by a colorimetric method employing mercuric thiocyanate
as previously reported by Bergman and Sanik (1957). Samples
(1.0 mL) were removed and assayed for halide ions. Each assay was carried out
in triplicates. The absorbance of the mixture was measured at A460 nm and
was proportional to the chloride ion concentration.
16S rRNA gene analysis: Following PCR, amplicons were purified using
QIAGEN PCR purification kit. The amplicons were sent for DNA sequencing to 1st
Base Laboratory, Malaysia using initial primers as described by Fulton
and Cooper (2005). The partial 16S rRNA gene sequence was analyzed using
BLASTn option (http://www.ncbi.nlm.nih.gov/BLAST/).
The partial 16S rRNA gene sequence was submitted to NCBI under accession number
HM598361.
RESULTS
Isolation of MCA degrading bacteria: A newly isolate bacterium strain
TWI which degraded MCA as sole source of carbon was isolated from Kuala Terengganu
water treatment and distribution plant. A single colony was observed on solid
minimal media supplemented with 5 mM MCA (Fig. 1). The morphological,
cultural and some physiological characteristics of the strain TWI suggested
it belongs to Bacillus sp. (Table 1). According to
Bergeys Manual of Systematic Bacteriology, the strain TWI belongs to the
genus Bacillus sp. The 16S rRNA gene fragment was also sequenced using
FD1 and rP1 primers.
| Fig. 1: |
A pure colonies (strain TW1) on solid minimal medium supplemented
with 5 mM MCA incubated at 30°C after 16 h |
Table 1: |
Some morphological and general physiological properties of
strain TWI |
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The partial nucleotide sequence of 16S rRNA gene from the isolated bacterium
also suggested that the species belongs to the genus Bacillus.
Growth of Bacillus sp. TW1 on 0.5 mM monochloroacetate (MCA):
Bacillus sp. TW1 was confirmed to grow on MCA as sole source of carbon
and energy. However, growth did not occur on MCA at concentrations in excess
of 30 and 40 mM, suggesting toxicity of this compound to the organism (Zulkifly,
2008). The normal MCA concentration used to grow Bacillus sp. TW1
was 5 mM. To investigate the ability of strain TW1 to grow on low concentration
of substrate, 0.5 mM MCA, 10x lower than the normal growth concentration was
used.
An experiment was carried out to establish whether Bacillus sp. TW1
was able to grow on 0.5 mM MCA. The cell inoculum was prepared by growing TW1
in 10 mM glycolate minimal medium. Then 15 mL of an overnight culture was centrifuged
and the cells washed twice with minimal medium before inoculation into 100 mL
minimal medium supplied with 0.5 mM MCA. Growth was monitored by measuring the
amount of chloride ions released at appropriate time intervals.
| Fig. 2: |
Growth of Bacillus sp. TW1 on 0.5 mM MCA |
An uninoculated flask treated in the same way was used as a control. This
is important to make sure the chloride measured in the growth medium was due
to the cells using the MCA rather than the auto-degradation of the substrate
in the growth medium. A typical growth curve is shown in Fig.
2. with a doubling time of approximately 13 h. The maximum amount of chloride
ion released detected in liquid culture was 0.32 μmol Cl¯ mL-1
from growth at 0.5 mM MCA. The chloride ion released indicated that TW1 strain
consumed MCA. However, no chloride ion released was detected at 0.4 mM MCA concentration
suggesting the affinity of the dehalogenase is not suitable under this condition.
DISCUSSION
The identification of Bacillus sp. TW1 was based on both biochemical
tests/morphological observations and analysis of the 16S rRNA. The determination
of 16S rRNA gene sequences is a routine procedure in prokaryotic taxonomy, resulting
in large and growing databases, which improve phylogeny reconstructions, identification
results and primer specificity evaluations (Nubel et
al., 1997). The 16S rRNA gene sequence analysis confirmed that the organisms
showing the highest identity are various Bacillus species. However, the
analysis was not extensive enough to identify the species name of the organism.
According to Lane et al. (1985), in assessing
the relationship of one organism to another by the comparison of their 16S rRNA
sequence it is not important that the complete sequences be determined. The
search in the database showed the sequence from the 5 and 3 matched
the same organisms. The sequences from these organisms matched to the Bacillus
suggesting the organism used in this study is from the genus Bacillus.
There is only one recent case associated with dehalogenation which involves
the genus Bacillus. The dehalogenase from this organism was able to attack
dichloromethane (Wu et al., 2009). Generally,
it is quite rare for organisms from Bacillus sp. to be involved in hydrolytic
dehalogenation. However, this kind of organism is always associated with bacteria
of medical importance because they cause disease like food poisoning (From
et al., 2007). Current investigation is the first reported study
involving Bacillus genus able to degrade haloalkanoic acid especially
at low substrate concentration of MCA.
This study strongly suggested that Bacillus sp. TW1 was able
to grow on 0.5 mM MCA with a doubling time of approximately 13 h, similar to
the growth rate obtained at 5 mM MCA concentration indicating that the organism
was a facultative oligotroph (Zulkifly, 2008). However,
growth above 30 mM MCA was inhibited possibly due to toxicity of MCA.
The identification of so many dehalogenases and the presence of multiple dehalogenases in many genera and species so far raise the question of their environmental significance. These enzymes are specifically induced by halo-acetic and propionic acids involving complex regulatory controls which respond to the growth environment. Some dehalogenases are specific and react to carbon-halogen bonds adjacent to the carboxyl group, whether at α or β-position of halogen group. Are these dehalogenases in natural environment will be expressed as dehalogenases or less specific hydrolases? The study of a complex regulatory control which responds to the growth environment may shed light on this question. CONCLUSION In conclusion, our research demonstrates that degradation of low MCA concentration was possible using strain TW1 in natural waters and in treated drinking waters. This is the first reported study on suppressing low concentration of MCA in drinking water so far which would help protect public health. Thus, in situ studies of MCA biodegradation in natural waters and drinking water distribution systems are important especially degradation at low concentration was possible. ACKNOWLEDGMENT This project was funded by Ministry of Higher Education (Malaysia) under Vot No. 78370.
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