isolation, screening and selection of dechlorinating...
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103
Isolation, Screening and Selection of Dechlorinating
Cultures
5.1 Introduction
It is well known that microorganisms play an important role in degradation of
contaminants thereby restoring the contaminated sites. However, the success of
any biological treatment depends on the degradation capabilities of the particular
microbial community in a given environment. There are chances that total number
of desired microorganisms is very low. In addition, the substrate concentration may
be inhibitory to potential degraders, or the degradation products may be toxic to
the cells. One possible way to overcome these limitations is to use inoculants that
are selected or adapted to degrade the target compound by a strategy referred to as
bioaugmentation. This requires isolation of efficient cultures capable of degrading
the pollutants.
There are reports on isolation of bacteria degrading different organochlorines.
Marks et. al. (1984) isolated Arthrobacter sp, a 4-Chlorobenzoic acid degrading
bacteria from sewage sludge. Apajalahti et. al. (1986) isolated a novel species of
chlorophenol mineralizing actinomycete which was named as Rhodococcus
chlorophenolicus. Fava et. al. (1995) reported degradation of 2CP (2-
Chlorophenol), 3CP and 4CP by a Pseudomonas pickettii strain isolated from a
consortium maintained on 2CP. Steinle et. al. (1998) isolated 2,6DCP mineralizing
Ralstonia sp strain RK1 from the sediment of a freshwater pond close to a
contaminated site. Gallizia et. al. (2003) isolated nine bacterial strains from a
mixed culture containing phenol and 2,4DCP as substrate and further characterized
one of the strain identified as Micrococcus sp. All these studies have been carried
out on pure compounds as substrate. There are hardly any reports on cultures
utilizing AOX barring one or two reports. Fulthorpe and Allen (1995) reported a
comparative account of AOX removal from bleached kraft PAP mill effluent
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(BKME) by dehalogenating Pseudomonas, Ancylobacter and Methylobacterium
strains.
Though microbial degradation of various organochlorines has been investigated for
many years, there is still a considerable interest in isolating new naturally
occurring microorganisms which are capable of degrading organochlorines
generally found in wastewater and soil.
In this chapter isolation of bacteria capable of degrading AOX was performed. The
isolates were then characterized with respect to their morphological, physiological
and biochemical characters. They were identified using these characters and 16S
rRNA sequencing. The isolates were then screened for their spectrum of
chlorophenol degradation and AOX degradation. Selected isolates were tested for
AOX degradation at different pH.
5.2 Materials and methods
5.2.1 Sample Collection
Samples collected from agricultural fields irrigated with treated effluent of PAP
industry, as described in Chapter II section 2.2.1, were used for setting up
enrichment cultures and isolation.
5.2.2 Enrichment
The soil samples collected from different agricultural fields were pooled together
to make a uniform sample. Three enrichments were set up using two different
media. DMM (as described in Chapter II, section 2.2.2) was used for enrichment of
sets E1 and E9 whereas Freedman Medium (FM) for set E2. FM had the following
composition (gL-1
): KH2PO4; 0.05, K2HPO4; 0.1, MgCl2.6H2O; 0.2, CaCl2.2H2O;
0.2, NH4Cl; 0.2, yeast extract; 0.5, sodium acetate; 5.0, glucose; 1.0 and trace
metal solution; 1.0 mlL-1
having following composition (gL-1
): MnCl2.4H2O; 0.1,
CoCl2.6H2O; 0.17, ZnCl2; 0.1, CaCl2; 0.2, H3BO4; 0.019, NiCl2.6H2O; 0.05,
Na2MoO4.2H2O; 0.2, pH adjusted to 7.0 with 1N NaOH. In enrichment culture E1
25 ppm each of 2CP and metachlorobenzoate were added. In enrichment culture
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E2 10 ml effluent of anaerobic digester and 2.5 ppm each of 2CP and 3 chloro 2
benzoate (3C2B) were added. In enrichment culture E9 10 ml each of chlorine
filtrate and BCWW was added along with 25 ppm each of 3CP, 4CP, 2,3DCP and
3,4DCP. The enrichment was initiated in 250 ml Erlenmeyer flask containing 90
ml medium and 10% w/v composite soil sample. The flasks were incubated at 30 ±
2oC on shaker at 120 rpm. After every seven days samples of enrichment cultures
were analyzed microscopically and on HPLC for chlorophenol degradation. The
sample for HPLC analysis was first extracted with acetonitrile (1:1 v/v) by
vortexing vigorously for 1 min and then extract was centrifuged at 10,000 rpm for
10 mins at 4oC. The supernatant was taken for analysis. The HPLC system
(Dionex, UK) had UV-IR detector and a P680 pump. A Reverse Phase C18 column
(LichroCARTTM
4.6 mm x 250 mm, Merck, Germany) was used. Mobile phase
was methanol: water: acetic acid (60: 38: 2 by volume) at a flow rate of 1 mlmin-1
run at room temperature. Chlorophenols were analyzed using UV detector set at
285 nm (Becker, 1999). Data interpretation of all the analysis was done using
Chromeleon software. Two successive transfers (10% v/v inoculum) were given to
the enrichment culture in fresh medium. Isolation was then carried out.
Enrichments were maintained by transferring 10% v/v enrichment culture after
every seven days in fresh medium, supplemented with respective organochlorines.
5.2.3 Isolation
After two successive transfers to the enrichment cultures, phase contrast
microscopy revealed thin and long rod shaped bacteria in E1 and thick, small rods;
thin, long rods and cocci shaped bacteria in E9. In enrichment E2 no growth was
observed. Pure cultures were obtained from the last enrichment culture (0.1 ml) by
streak plate method with DMM agar supplemented with 2.5 ppm of 2CP for E1
and 3CP and 4CP, separately, for E9. Morphologically different colonies were
transferred in tubes containing DMM broth supplemented with 2.5 ppm of
respective chlorophenol. The isolation and transfer was carried out aseptically in
laminar air flow cabinet. After incubation of tubes at 30 ± 2oC, purity of the
isolates was determined by observing cells under phase contrast microscope
(Nikon Eclipse 80i, Japan) at 400X magnification and Gram staining. The isolates
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were maintained on DMM agar slope tubes with 2.5 ppm of respective
chlorophenols at 4oC and transferring to fresh medium after every 1 month.
5.2.4 Morphological and physiological characterization
Characterization of the isolates was carried out in DMM with respective
chlorophenols except where mentioned otherwise. Incubation for all the
characterization studies was done at 30 ± 2oC, under shake culture conditions
unless mentioned otherwise. Absorbance for determination of increase in cell
density of the culture was measured spectrophotometrically at 600 nm.
5.2.4.1 Colony characteristics
Colony of the isolates was characterized visually and results were recorded.
5.2.4.2 Morphology
Morphology of the isolates was observed using phase contrast microscope (Nikon
Eclipse 80i, Japan).
5.2.4.3 Gram staining
Gram character of the isolates was determined using standard Gram staining kit
(HiMedia) and observing under oil immersion lens of phase contrast microscope
(Nikon Eclipse 80i, Japan) at a magnification of 1000X. Cultures of B. subtilis and
E. coli from MACS culture collection were used as the reference cultures.
5.2.4.4 Motility
Motility of the isolates was determined by observing wet mounts of the cultures in
cavity slides using phase contrast microscope (Nikon Eclipse 80i, Japan) at a
magnification of 400X.
5.2.4.5 Sporulation test
Spore staining was performed using Schaeffer and Fulton‘s Spore Stain Kit
(HiMedia). Smear of the culture suspension was prepared on a glass slide and
gently heat fixed. The slide was then placed over a beaker of boiling water. When
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large droplets condensed under the slide the smear was flooded with Schaeffer and
Fulton‘s Spore Stain A for 1 min. The smear was then washed with cold water and
flooded with Schaeffer and Fulton‘s Spore Stain B for 30 secs. The slide was then
washed with water, air dried and observed under oil immersion lens of microscope
(Nikon Eclipse 80i, Japan). Simultaneously the isolates at different stages of
growth were observed as wet mount under phase contrast microscope (Nikon
Eclipse 80i, Japan) at a magnification of 400X to determine presence of spores.
5.2.4.6 Optimum temperature for growth
Optimum temperature for growth of the isolates was determined by inoculating
duplicate tubes with the isolates and incubating at different temperatures from
10.0oC to 60.0
oC. Absorbance of the broth was measured after 24 h and maximum
growth was indicated by maximum absorbance.
5.2.4.7 Optimum pH for growth
For optimum pH determination DMM was prepared with initial pH ranging from
5.0 to 10.0. Duplicate tubes of the medium for each pH were inoculated with the
isolates. Absorbance of the broth was measured after 24 h and maximum growth
was indicated by maximum absorbance.
5.2.5 Biochemical characterization
Biochemical characterization of the isolates was carried out by testing
carbohydrate utilization profile on Analytical Profiling Index (API) system and
according to Bergey‘s Manual of Systematic Bacteriology, Volume 3 (1984).
Carbohydrate utilization profiling was done using API system (bioMérieux, Lyon,
France API) in which total 32 miniaturized assimilation tests were carried out. ID
32 GN and ID 32 STAPH strips consisting of 32 cupules, each containing the
dehydrated carbohydrate substrate, were used for testing. A semisolid minimal
medium was inoculated with a suspension of the test isolates. After 48 h of
incubation growth in each cupule was detected by an automatic reader and the test
results were interpreted using APILAB Plus software version 3.3.3.
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Morphological and physiological characters were used as reference to carry out
further biochemical tests according to Bergey‘s Manual.
5.2.5.1 Starch hydrolysis
Starch hydrolysis was tested by spot inoculating the cultures on 2% starch agar
plate. After 24 h of incubation the plates were flooded with Gram‘s iodine.
Presence of clear zone around the colony against purple background was
considered as positive reaction.
5.2.5.2 Gelatin hydrolysis
Gelatin hydrolysis was tested by spot inoculating the cultures on 0.4% gelatin agar
plate. After 24 h incubation the plates were flooded with Frazier‘s solution.
Presence of clear zone around the colony against opaque background was
considered as positive reaction.
5.2.5.3 Casein hydrolysis
Casein hydrolysis was tested by spot inoculating the cultures on 5% milk agar
plate. On 7th and 14
th day of incubation the plates were observed for clearing of
casein around and underneath the growth which was considered as positive
reaction.
5.2.5.4 IMViC test
Indole test – The cultures were inoculated in 2% peptone water and incubated at
37oC for 48 h. After incubation 0.5 ml Kovac‘s reagent was added to the tubes and
gently shaken. Development of red color was considered as positive reaction.
Methyl red test – The cultures were inoculated in 2% peptone water and incubated
at 37oC for 48 h. After incubation 5 drops of methyl red indicator was added to the
tubes. Development of bright red color was considered as positive reaction.
Vogus-Proskaur test – The cultures were inoculated in 0.5% peptone water
containing 0.5% glucose. After 48 h of incubation at 37oC 1 ml of 40% potassium
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hydroxide and 3 ml of 5% α-naphthol was added to the tubes. The tubes were
shaken at intervals for maximum aeration. Development of pink color in 2-5 mins
which becomes crimson after 30 mins was considered as positive reaction.
Citrate test – The culture suspension was streaked on Simmon‘s citrate agar slant.
The tubes were incubated for 48 h at 37oC. After incubation presence of growth
and color change to blue was considered as positive reaction.
5.2.5.5 NaCl tolerance test
NaCl tolerance was tested by inoculating the cultures in nutrient broth (HiMedia)
with varying concentrations of NaCl, Viz., 0%, 2%, 5%, 7% and 10%. The tubes
were incubated for 24 h at 37oC in a slanting position to improve aeration.
Presence of growth in terms of turbidity seen visually was considered as positive
reaction.
5.2.5.6 Catalase production
Catalse production was tested by flooding nutrient agar slope tubes with freshly
grown cultures with 30% solution of hydrogen peroxide. Presence of effervescence
was considered as positive reaction.
5.2.5.7 Propionate utilization
Propionate utilization was tested by inoculating the cultures in 0.2% propionate
broth. The tubes were incubated at 37oC for 14 days. Presence of growth in terms
of increase in turbidity as seen visually was considered as positive reaction.
5.2.5.8 Urease production
Urease production was tested by streaking the cultures on Christensen‘s agar slant.
The tubes were incubated at 37oC for 96 h. Purple-pink coloration of the slants was
considered as positive reaction.
5.2.5.9 Nitrate reduction
Nitrate reduction was tested by inoculating the cultures in 0.02% nitrate broth. The
tubes were incubated at 37oC for 96 h. After incubation 0.1 ml of test reagent
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(prepared freshly by mixing solution A and solution B in equal volume) was added
to the tubes. Development of red color within few mins was considered as positive
reaction.
5.2.5.10 Lecithinase production
Lecithinase production was tested by spot inoculating the cultures on egg yolk agar
plate. The plates were incubated for 48 h at 37oC. After incubation presence of
white zone of opalescence was considered as positive reaction.
5.2.5.11 Tyrosine degradation
Tyrosine degradation was tested by spot inoculating the cultures on 0.5% tyrosine
agar plate. The plates were incubated at 37oC for 14 days. After 7 and 14 days
clearing of the tyrosine crystals around and below the colony was considered as
positive reaction.
5.2.5.12 Dihydroxyacetone production
Dihydroxyacetone production was tested by spot inoculating the cultures on
glycerol agar plate. The plates were incubated at 37oC for 10 days. After
incubation the plates were flooded with test reagent (prepared freshly by mixing
solution A and solution B in equal volume) and were observed after 2 h. Presence
of red halo around the colony was considered as positive reaction.
5.2.5.13 Deamination of phenylalanine
Deamination of phenylalanine was tested by heavily inoculating the cultures on
0.2% phenylalanine slant. The tubes were incubated for 24 h at 37oC. After
incubation a few drops of 10% ferric chloride solution was run down over the
growth. Development of green color in the solution and slant was considered as
positive reaction.
5.2.5.14 Resistance to lysozyme
Resistance to lysozyme was tested by inoculating the cultures in nutrient broth
containing lysozyme at a concentration of 100 enzyme units ml-1
. The tubes were
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incubated at 37oC for 14 days. Presence of growth was considered as positive
reaction.
5.2.6 Specific growth rate
Specific growth rates of the isolates were determined as described by Joklik et. al.
(1988). Growth curve of each isolate cultivated in DMM with respective
chlorophenols at 30 ± 2oC was plotted using absorbance values at 600 nm
measured after every 1 h for 24 h.
5.2.7 Identification of isolates using 16S rDNA sequencing
To identify the isolates on the basis of 16S rDNA sequencing a single well isolated
colony of the individual isolate grown on DMM agar was inoculated in 10 ml
nutrient broth. The tubes were incubated at 30oC for 24 h. After incubation 2 ml of
culture suspension was taken in microcentrifuge tube and cell pellet was obtained
by centrifuging the tubes at 12,000 rpm for 10 mins at 4oC. The pellets were
washed twice with 1 ml PBS to remove cell debris by centrifuging at 12,000 rpm
for 5 mins at 4oC. The cell pellet was further used for extraction of total genomic
DNA.
5.2.7.1 Total gemonic DNA extraction
The cell pellet was resuspended in 250 μl of solution C (0.1M NaCl, 10mM Tris-
HCl; pH 8.0 and 10mM EDTA) and 25 μl of
20% SDS. The reaction mixture was incubated at
65oC for 1 h. Then 25 μl of proteinaseK (5 mgml
-
1 stock) was added to the reaction mixture and
incubated overnight at 37oC. After incubation
equal volume of tris saturated phenol was added
to the reaction mixture. The contents of the tube
were mixed properly and centrifuged at 12,000
rpm for 10 mins at 4oC. Upper aqueous layer was
removed in fresh microcentrifuge tube and
extracted again with tris saturated phenol by
1 2 3 4 5 6 7
Fig. 5.1: Genomic DNA of
isolates. Lane 1 – E1-1, Lane
2 – E1-2, Lane 3 – E9-1, Lane
4 – E9-2, Lane 5 – E9-3, Lane
6 – E9-4, Lane 7 – E9-5
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centrifugation. The upper aqueous layer was removed in fresh microcentrifuge
tube. Ice chilled chloroform:isoamyl alcohol (24:1) was added to it (1:1) and then
centrifuged at 12,000 rpm for 10 mins The upper aqueous layer was removed
carefully in fresh microcentrifuge tube and 0.1 volume sodium acetate, pH 4.8, and
5 volume of ice chilled 95% ethanol was added to the tube. The content of the tube
was mixed gently and incubated at 0oC for 15 mins After incubation the tube was
centrifuged at 12,000 rpm for 15 mins at 4oC. Supernatant was discarded and the
DNA pellet was washed twice with ice chilled 70% ethanol by centrifuging at
12,000 rpm for 10 mins at room temperature. The DNA pellet was air dried and
resuspended in 100 μl of sterile deionized water. RNase treatment was given to the
DNA pellet by adding 1 μl of RNase solution (1 mgml-1
stock) and incubating at
37oC for 1 h. DNA was purified by PEG-NaCl as described in Chapter III, section
3.2.5.1.
5.2.7.2 PCR amplification of 16S rRNA
PCR amplification of 16S rRNA was carried out as described in Chapter III,
section 3.2.3.
5.2.7.3 Cycle sequencing PCR, clean up and sequencing
Cycle sequencing PCR, clean up and sequencing was carried out as described in
Chapter III, section 3.2.7.
5.2.7.4 Phylogenetic analysis
The 16S rRNA sequence of each isolate was compared with those in the GenBank
DNA database by using the basic local alignment search tool (BLASTn) search
program at NCBI site (www.ncbi.nlm.nih.gov/BLAST). Closest hits obtained for
each isolate, except different strains of the same species, were used for further
analysis. All the sequences were aligned using CLUSTAL W software
(www.ebi.ac.uk) (Thompson et. al., 1994). Then using DAMBE software (Xia,
2001) unaligned positions were deleted from the aligned sequences.
Unambiguously aligned base positions were then used to construct phylogenetic
dendrogram using the neighbour-joining method in MEGA version 3.0 (Kumar et.
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al., 2004). Confidence in the tree topology was established by carrying out 1000
bootstrap replications. The sequences other than those of the isolates were obtained
from GenBank database.
5.2.8 Culture conditions
DMM was used for all the experiments with organochlorines in liquid cultures
until and unless specified. The isolates were grown in 250 ml Erlenmeyer flask,
separately, containing 100 ml DMM with respective chlorophenol. After 18 h the
cells were harvested by centrifugation at 10,000 ppm for 10 mins at 4oC and the
cell pellet was washed twice with sterile saline (0.85% NaCl) by centrifugation at
5,000 rpm for 2 mins at 4oC. The pellet was then resuspended in 2 ml saline. 250
ml Erlenmeyer flask containing 100 ml saline was inoculated with culture
suspension, separately, to give final absorbance of 0.6 at 600 nm.
5.2.9 Spectrum of chlorophenol biodegradation
The isolates were checked for their ability to dechlorinate different chlorophenols
other than those used for growth substrates. 50 ml Erlenmeyer flasks containing
13.5 ml DMM were used for the experiment. A total of seven sets of 14 flasks each
were spiked with different chlorophenols at a final concentration of 100 ppm,
separately. The chlorophenols tested were 2CP; 3CP; 4CP; 2,3DCP; 2,4DCP;
3,4DCP and 2,4,6TCP. Each set was inoculated with 1.5 ml of single isolate
suspension (10% v/v inoculum). Negative control had the flasks containing DMM
with respective chlorophenol whereas positive control had flasks containing DMM
inoculated with 10% v/v of single isolate suspension. All the flasks were kept on
rotary shaker at 120 rpm for 14 days at 30 ± 2oC. The experiment was run in
duplicate for a period of 14 days. Samples were drawn on 0, 7th
and 14th
day for
growth measurement and chlorophenol degradation as estimated by HPLC
analysis.
5.2.10 Biodegradation of extracted AOX by isolates
After checking spectrum of chlorophenol degradation it was necessary to check the
ability of the isolates to degrade AOX. 250 ml Erlenmeyer flasks containing 89 ml
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DMM were spiked with 1 ml AOX extracted in methanol (as described in Chapter
IV, section 4.2.1.1). A total of seven sets, one set per isolate, having 3 flasks each
were prepared with final concentration of 10 ppm AOX, separately. Each set was
inoculated with 10 ml of single isolate suspension (10% v/v inoculum). Negative
control had flasks containing DMM spiked with AOX. Two sets of positive
controls were kept, one set having flasks containing DMM inoculated with 10%
v/v of single isolate suspension whereas second set having flasks containing DMM
with 1 ml methanol and 10% v/v of single isolate suspension. All the flasks were
kept on rotary shaker at 120 rpm at 30 ± 2oC. The experiment was run in triplicate
for a period of 40 days. Samples were drawn on day 0 and then after every 10th
day
for growth measurement and AOX degradation by AOX analyzer.
5.2.11 AOX biodegradation from BCWW by isolates and their
consortium
On the basis of biodegradation spectrum and AOX degradation ability three
isolates were selected for further studies. Since AOX is mixture of over 300
different compounds it was necessary to check degradation from actual wastewater
by the isolates. A consortium of these isolates was developed by mixing the
isolates in equal proportion. The consortium was divided in two parts and one part
was autoclaved to kill the cells. The live and heat killed consortium was also tested
for their AOX degradation ability. BCWW was used as the source of AOX.
BCWW was filter sterilized (0.2 μm, Whatman, UK) and diluted with DMM to
give a final AOX concentration of 10 ppm. A total of five sets, containing three
250 ml Erlenmeyer flasks each, were used for the test. The test flasks had 90 ml
test medium which were inoculated with 10 ml (10% v/v inoculum) of the culture
suspension. Negative control had flasks containing only test medium whereas the
positive control had flasks containing DMM inoculated with the culture
suspension. Another set of the control was kept with flasks containing DMM
inoculated with dead consortium. All the flasks were kept on rotary shaker at 120
rpm at 30 ± 2oC. Samples were drawn on 0 day and then after every 10
th day for
growth measurement and AOX degradation by AOX analyzer.
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5.2.12 Plasmid DNA isolation
Prior to using the isolates for bioaugmentation studies, it was necessary to check
whether the degradation ability was plasmid borne or chromosomal. The presence
of plasmid DNA was detected by the methods described by O‘Sullivan and
Klaenhammer (1993) for miniplasmid and Hot Triton X-100 lysis method
described by Tolmasky et. al. (1987), for megaplasmid. E. coli V157 was used as
positive control. For miniplasmid 18 h old culture was pelleted by centrifugation
and the cell pellet was washed twice with PBS. The cell pellet was then
resuspended in 200 μl Solution I (25% Sucrose containing 30 mgml-1
lysozyme).
RNase (Sigma, USA) was added at a concentration of 0.1 mgml-1
and the tube was
incubated at 37oC for 15 mins Then 400 μl of freshly prepared Solution II (3%
SDS, 0.2N NaOH) was added to the tube and the contents were mixed properly.
The tube was incubated at room temperature for 7 mins After incubation 300 μl of
Solution III (3M Sodium acetate, pH 4.8) was added in tube. The contents were
mixed properly and the tube was centrifuged at 14,000 rpm for 15 mins at 4oC. The
supernatant was transferred in fresh microcentrifuge tube and 650 μl of
isopropanol was added to the tube. The contents were mixed properly and the tube
was centrifuged at 14,000 rpm for 15 mins at 4oC. The supernatant was discarded
and the pellet was resuspended in 320 μl of sterile deionized water. Then 200 μl of
Solution IV (7.5M Ammonium acetate containing 0.5 mgml-1
ethidium bromide)
was added to the tube and the contents were mixed properly. The reaction mixture
was extracted with phenol and chloroform, 350 μl each, by centrifuging at 14,000
rpm for 5 mins at room temperature. The aqueous phase was transferred to fresh
microcentrifuge tube and 1 ml ice chilled ethanol was added to the tube. The
contents were mixed properly and the tube was centrifuged at 14,000 rpm for 15
mins at 4oC. Plasmid DNA pellet was washed twice with 70% ethanol by
centrifuging at 14,000 rpm for 10 mins at room temperature. The pellet was air
dried and resuspended in 25 μl of sterile deionized water. Presence of plasmid was
confirmed by agarose gel electrophoresis (0.7%) and staining with ethidium
bromide solution.
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For megaplasmid cells from 40 ml overnight grown culture was harvested by
centrifugation. The cell pellet was resuspended in 5 ml Solution I (50mM Tris-
HCl, 5mM EDTA, 50mM NaCl, pH 8.0). The cells were centrifuged at 3000 x g
for 10 mins at 4oC and the pellet was resuspended in 2 ml Solution II (25%
Sucrose, 1mM EDTA, 50 mM Tris-HCl, pH 8.0). The tube was kept on ice for 20
mins Then 400 μl of solution III (Lysozyme 10 mgml-1
, 0.25M Tris-HCl, pH 8.0)
was added to the tube and further incubated on ice for 20 mins After incubation
800 μl of 0.5M EDTA, pH 8.0, was added to the cell suspension and the cells were
lysed by adding 4.4 ml of Solution V (Triton lytic mixture containing 1 ml 10%
Triton X-100 in 10mM Tris-HCL; pH 8.0, 25 ml 0.25M EDTA; pH 8.0, 5 ml 1M
Tris-HCl; pH 8.0, 69 ml deionized water) by mixing gently. The tube was
incubated at 65oC in water bath for 20 mins Cellular debris was removed by
centrifugation at 27,200 x g for 40 mins at room temperature and the supernatant
was transferred to fresh centrifuge tube. The supernatant was adjusted to 0.5M
NaCl and 10% PEG by adding from stock solutions of 5M NaCl and 40% PEG.
The tube was kept overnight for incubation at 0oC. Plasmid DNA precipitate was
collected by centrifuging the tube at 3,000 x g for 10 mins at 4oC. The pellet was
resuspended in 2 ml ice chilled Solution VI (0.25M NaCl, 1mM EDTA, 10mM
Tris-HCl, pH 8.0). Then 4 ml of 95% ice chilled ethanol was added to the tube and
plasmid DNA was precipitated by incubating tube at -70oC for 30 mins Plasmid
DNA was pelleted by centrifugation at 12,000 x g for 10 mins at 4oC. The pellet
was air dried and resuspended in 100 μl of sterile deionized water. Presence of
plasmid was confirmed by agarose gel electrophoresis (0.7%) and staining with
ethidium bromide solution.
5.2.13 Biodegradation of 2,4DCP at different pH
Soil samples collected from different regions showed pH in the range of 6.02 –
7.72. Since the isolates were to be used for bioaugmentation trials it was necessary
to check the ability of these isolates to degrade organochlorine at different pH. The
three isolates selected earlier based on AOX degradation were tested for their
ability to degrade 2,4DCP at different pH. Twelve sets of three flasks each
containing DMM spiked with 2,4DCP to a final concentration of 100 ppm were
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taken for the experiment. For each isolate there were four sets of flasks with
varying pH, viz., 5, 6, 7 and 8. The test flasks and the positive control flasks,
having DMM with varying pH, were inoculated with 10 ml of culture suspension.
Negative control had flasks with DMM spiked with 2,4DCP and varying pH. All
the flasks were kept on rotary shaker at 120 rpm at 30 ± 2oC. Another set of flasks
was kept at stationary condition to study the effect of aeration on the rate of
degradation by these isolates. The experiment was run for a period of 30 days.
Samples were drawn on 0 day and then after every 10th
day for growth
measurement and 2,4DCP degradation by HPLC.
5.2.14 Biodegradation of AOX at different pH
After 2,4DCP biodegradation testing at different pH the isolates were further tested
for their ability to degrade AOX at different pH. The test conditions were similar to
that described above for 2,4DCP biodegradation except that AOX was spiked in
place of 2,4DCP at a final concentration of 10 ppm. All the flasks were kept on
rotary shaker at 120 rpm at 30 ± 2oC. The experiment was run for a period of 30
days. Samples were drawn on 0 day and then after every 10th
day for growth
measurement and AOX degradation by AOX analyzer.
5.3 Results and discussion
5.3.1 Enrichment
A total number of three enrichments were set up. At the end of second transfer
organochlorine degradation was checked using HPLC. More than 50% reduction in
the total amount of organochlorine was observed in enrichments E1 and E9.
Microscopic observation of enrichment E1 showed presence of long and short rod
shaped bacteria whereas in enrichment E9 long and short rod shaped as well as
cocci shaped bacteria were present. Enrichment E2 did not show any degradation
during any of the transfers. Therefore enrichments E1 and E9 were taken forward
for isolation of organochlorine degrading bacteria.
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5.3.2 Isolation
Many circular and irregular shaped colonies developed on DMM agar plate
containing 2CP which was streaked with sample from enrichment E1. On DMM
agar plate containing 3CP only single type of circular pinpoint colonies developed
when sample from enrichment E9 was streaked whereas when DMM agar plate
containing 4CP was streaked with sample from enrichment E9 creamish white and
yellow colored colonies developed. Two morphologically different colonies were
picked up from 2CP plate, one from 3CP plate and four from 4CP plate and were
restreaked on DMM agar plates with respective chlorophenols to confirm the
purity. Two isolates from enrichment E1 growing on 2CP were designated as E1-1
and E1-2, one isolate from enrichment E9 growing on 3CP was designated as E9-1
whereas four isolates from enrichment E9 growing on 4CP were designated was
E9-2, E9-3, E9-4 and E9-5. The isolates were then transferred in DMM broth with
respective chlorophenol for further characterization.
Initial studies carried out at our Department on the GC-MS analysis of BCWW
revealed presence of dichlorophenols as the major organochlorines in the
wastewater (data not shown). Hence, chlorophenols were selected for enrichments.
Chlorophenols have also been reported from effluent of PAP industry by
Valenzuela et. al., 1997; Tondo et. al., 1998; Andretta et. al., 2004; Yang et. al.,
2006. Chlorophenols have been reported as particularly hazardous compounds for
the Lake Baikal ecosystem. The treated effluent of the Baikalsk PAP mill is
directly discharged through aeration pond in the Lake Baikal. Total concentration
of 2CP, 4CP and 2,4DCP in Lake Baikal and aeration pond was reported to be 1.86
and 5.76 μgml-1
, respectively (Matafonova et. al., 2006). Different chlorophenols
used for establishing enrichments resulted in specific selection of bacteria. This
was evident from the isolation performed wherein we could get seven isolates
different from what was observed during culture based biodiversity studies as
described in Chapter III. DMM was found to be the most appropriate medium for
enriching and isolating dechlorinating bacteria as successful isolations were
obtained where DMM was used.
119
5.3.3 Morphological and physiological characterization
5.3.3.1 Colony characteristics
Colony characteristics of the seven isolates are given in table 5.1.
Table 5.1: Colony characteristics
Isolate Size Shape Color Margin Elevation Consistency Opacity
E1-1 2 mm Circular Cream Entire Convex Mucoid Opaque
E1-2 1 mm Irregular Cream Serrated Flat Mucoid Opaque
E9-1 Pinpoint Circular Creamish
white
Entire Convex Mucoid Translucent
E9-2 1 mm Circular Cream Entire Convex Mucoid Opaque
E9-3 Pinpoint Circular Colorless Entire Low
convex
Mucoid Opaque
E9-4 1 mm Circular Creamish
white
Entire Convex Mucoid Opaque
E9-5 0.5 mm Circular Creamish
white
Entire Convex Mucoid Translucent
5.3.3.2 Morphology
Cells of isolate E1-1 were long, thin rods occurring individually, while some in
pairs. Cells of isolate E1-2 were long, thick rods occurring individually, while
some in pairs. Cells of isolate E9-1 were long, thin rods occurring individually,
while some in pairs. Cells of isolates E9-2 and E9-5 were short, thick rods
occurring individually, while some in pairs. Cells of isolates E9-3 and E9-4 were
cocci occurring individually, while some in pairs and short chains.
120
Isolate E1-1 Isolate E1-2
Isolate E9-1 Isolate E9-2
Isolate E9-3 Isolate E9-4
Isolate E9-5
Fig. 5.2: Phase contrast photomicrograph of the isolates
121
5.3.3.3 Gram staining
All the seven isolates were Gram-positive.
5.3.3.4 Motility
Isolates E1-1, E1-2 and E9-1 were highly motile whereas isolates E9-2, E9-3, E9-4
and E9-5 were non-motile.
5.3.3.5 Sporulation test
Spore staining revealed presence of spores, as green colored oval shaped spot as
against red colored vegetative cells, in isolates E1-1, E1-2 and E9-1. No such green
colored spots were observed in smear of isolates E9-2, E9-3, E9-4 and E9-5. In
case of wet mount spores were observed after 24 h of growth in cultures of E1-1,
E1-2 and E9-1 whereas no spores were observed even after 96 h of growth in
cultures of E9-2, E9-3, E9-4 and E9-5.
5.3.3.6 Optimum temperature for growth
Three isolates namely E1-1, E1-2 and E9-1 showed growth over a temperature
range of 10oC to 60
oC with maximum growth at 30
oC. Remaining four isolates
showed growth over a temperature range of 20oC to 60
oC. Of these isolates E9-2
and E9-5 showed maximum growth at 30oC whereas isolates E9-3 and E9-4
showed maximum growth at 40oC. The observations are shown in Fig. 5.3.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
10 20 30 40 50 60
Temperature (oC)
Op
tical
Den
sit
y (
600 n
m)
E1-1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
10 20 30 40 50 60
Temperature (oC)
Op
tical
Den
sit
y (
600 n
m)
E1-2
122
0
0.2
0.4
0.6
0.8
1
1.2
1.4
10 20 30 40 50 60
Temperature (oC)
Op
tical
Den
sit
y (
600 n
m)
E9-1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
10 20 30 40 50 60
Temperature (oC)
Op
tical
Den
sit
y (
600 n
m)
E9-3
0
0.2
0.4
0.6
0.8
1
1.2
1.4
10 20 30 40 50 60
Temperature (oC)
Op
tical
Den
sit
y (
600 n
m)
E9-5
Fig. 5.3: Effect of temperature on growth of the isolates
5.3.3.7 Optimum pH for growth
Isolate E1-1 showed growth in the pH range of 5.0 – 10.0 with maximum growth at
pH 7.0. Isolate E1-2 showed growth in the pH range of 5.0 – 10.0 with maximum
growth at pH 6.0. Isolate E9-1 showed growth in the pH range of 5.0 – 10.0 with
maximum growth at pH 6.0. Isolate E9-2 showed growth in the pH range of 5.0 –
10.0 with maximum growth at pH 6.0. Isolate E9-3 showed growth in the pH range
of 5.0 – 10.0 with maximum growth at pH 8.0. Isolate E9-4 showed growth in the
123
pH range of 5.0 – 10.0 with maximum growth at pH 7.0. Isolate E9-5 showed
growth in the pH range of 5.0 – 10.0 with maximum growth at pH 8.0 and 9.0. The
observations are shown in Fig. 5.4.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
5 6 7 8 9 10
pH
Op
tical
Den
sit
y (
600 n
m)
E9-1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
5 6 7 8 9 10
pH
Op
tical
Den
sit
y (
600 n
m)
E9-3
0
0.2
0.4
0.6
0.8
1
1.2
1.4
5 6 7 8 9 10
pH
Op
tical
Den
sit
y (
600 n
m)
E9-4
124
Fig. 5.4: Effect of pH on growth of the isolates
5.3.4 Biochemical characterization
Results of carbohydrate utilization profile by API system for isolates E1-1, E1-2,
E9-1, E9-2 and E9-5 are shown in table 5.2 and for isolates E9-3 and E9-4 are
shown in table 5.3.
Table 5.2: Carbohydrate utilization profile of isolates
Carbohydrates Code Quantity
(mg/cup.)
Isolates
E1-1 E1-2 E9-1 E9-2 E9-5
L-Rhamnose RHA 0.68 + - + - -
N-Acetyl-Glucosamine NAG 0.68 + + + - -
D-Ribose RIB 0.70 + + + + +
Inositol INO 0.70 + + + + +
D-Saccharose SAC 0.66 + + + - -
D-Maltose MAL 0.70 + + + - -
Itaconic Acid ITA 0.23 - - - - -
Suberic Acid SUB 0.35 - - - - -
Sodium Malonate MNT 1.20 - - - - -
Sodium Acetate ACE 0.55 - - - - -
125
Lactic Acid LAT 0.32 + + + - -
L-Alanine ALA 0.68 + + + ? -
K-5-Ketogluconate 5KG 0.90 - - + - -
Glycogen GLYG 0.64 + + + - -
3-Hydroxybenzoic Acid mOBE 0.23 - - - - -
L-Serine SER 0.80 - - - - -
D-Mannitol MAN 0.68 + + + - -
D-Glucose GLU 0.78 + + + + +
Salicin SAL 0.52 ? + + - -
D-Melibiose MEL 0.66 + + + - -
L-Fucose FUC 0.64 - - - + +
D-Sorbitol SOR 0.68 + + + - -
L-Arabinose ARA 0.70 + + + - -
Propionic Acid PROP 0.29 - - - - ?
Capric Acid CAP 0.11 - - - - -
Valeric Acid VALT 0.25 - - - - -
Trisodium Citrate CIT 0.57 + + + - -
L-Histidine HIS 0.80 + + + - -
K-2-Ketogluconate 2KG 0.98 - - - - ?
3-Hydroxybutyric Acid 3OBU 0.30 - - - - -
4-Hydroxybenzoic Acid pOBE 0.23 - - - - -
L-Proline PRO 0.52 + + + - -
+, Positive reaction; -, Negative reaction; ?, Variable reaction
126
Table 5.3: Carbohydrate utilization profile of isolates
Carbohydrates Code Quantity
(mg/cup.)
Isolates
E9-3 E9-4
Urea URE 1.12 - -
L-Arginine ADH 0.76 - -
L-Ornithine ODC 0.76 - -
Esculin
Ferric Citrate
ESC 0.224
0.032
+ +
D-Glucose GLU 0.56 + +
D-Fructose FRU 0.56 + +
D-Mannose MNE 0.56 + +
D-Maltose MAL 0.56 + +
D-Lactose (Bovine origin) LAC 0.56 + +
D-Trehalose TRE 0.56 + +
D-Mannitol MAN 0.56 + +
D-Raffinose RAF 0.56 - -
D-Ribose RIB 0.56 + +
D-Cellobiose CEL 0.56 + +
Potassium Nitrate NIT 0.054 + +
Sodium Pyruvate VP 0.475 - -
2-Naphthyl-βD-galactopyranoside β-GAL 0.0364 - -
L-Arginine-β-naphthylamide ArgA 0.0172 - -
2-Naphthyl Phosphate PAL 0.0123 + +
Pyroglutamic Acid-β-naphthylamide PyrA 0.0128 - -
Novobiocin NOVO 0.0018 + +
127
D-Saccharose SAC 0.56 + +
N-Acetyl-Glucosamine NAG 0.56 + +
D-Turanose TUR 0.56 + +
L-Arabinose ARA 0.56 + +
4-Nitrophenyl-βD-glucuronide βGUR 0.0158 + +
+, Positive reaction; -, Negative reaction
Table 5.4 describes biochemical characters of the isolates according Bergey‘s
Manual.
Table 5.4: Biochemical characteristics of the isolates
Tests Isolates
E1-1 E1-2 E9-1 E9-2 E9-3 E9-4 E9-5
Starch hydrolysis + + + ? - - -
Gelatin hydrolysis + + + - + + -
Casein hydrolysis + + + - + + -
IMViC test
Indole - - - - - - -
Methyl red - - - - + + -
Vogus-Proskaur ? + - - - - -
Citrate - - + - - - -
NaCl tolerance
0% + + + + + + +
2% + + + + + + +
5% + + + + + + +
7% + + + + + + +
128
10% + + + + + + +
Catalase production + + + + + + +
Propionate utilization - - - - - - +
Urea production - - - + - - +
Nitrate reduction + + + - + + -
Lecithinase production - - - - - - -
Tyrosine degradation - - - + - - +
Dihydroxyacetone
production
+ + + - - - -
Deamination of
phenylalanine
- - - - - - -
Lysozyme tolerance + + + + + + +
+, Positive reaction; -, Negative reaction; ?, Variable reaction
5.3.5 Specific growth rate
Specific growth rates of isolates E1-1, E1-2, E9-1, E9-2, E9-3, E9-4 and E9-5 in
DMM with respective chlorophenols at 30 ± 2oC were 0.20 h
-1, 0.35 h
-1, 0.13 h
-1,
0.12 h-1
, 0.26 h-1
, 0.31 h-1
and 0.16 h-1
, respectively.
5.3.6 16S rRNA sequencing
After isolating total genomic DNA, PCR amplification of 16S rRNA region was
performed using three primer pairs. The sequences obtained for each primer pair
for each isolate were aligned to form a single sequence of the length 1488, 1488,
1513, 1457, 1464, 1510 and 1401 bases for the isolates E1-1, E1-2, E9-1, E9-2,
E9-3, E9-4 and E9-5, respectively. These sequences were deposited in the
GenBank database under accession numbers FJ573169, FJ573170, FJ573171,
FJ573172, FJ573173, FJ573174 and FJ573175, respectively (Fig. 5.5 - 5.11).
5.3.6.1 Phylogenetic analysis
The BLASTn search revealed that the sequences of isolates E1-1, E1-2 and E9-1
were closely related to Bacillus subtilis, sequences of isolates E9-2 and E9-5 were
129
closely related to Brevibacterium stationis and that of isolates E9-3 and E9-4 were
closely related to Staphylococcus sciuri. The phylogenetic dendrograms based on
16S rRNA sequence analysis showing the relationships of the isolates with the
most closely related strains and with each other are shown in Fig 5.12. From the
Fig. 5.12 it is seen that isolates E1-1 and E1-2 are closely related to each other than
to the isolate E9-1. The five isolates E1-1, E1-2, E9-1, E9-3 and E9-4 belong to
Firmicutes with low GC content of DNA whereas the two isolates E9-2 and E9-5
belong to Firmicutes with high GC content of DNA.
130
LOCUS FJ573169 1488 bp DNA linear BCT 24-MAR
-2009
DEFINITION Bacillus subtilis strain E1-1 16S ribosomal RNA gene, partial sequence.
ACCESSION FJ573169
VERSION FJ573169.1 GI:225353978
KEYWORDS SOURCE Bacillus subtilis
ORGANISM Bacillus subtilis
Bacteria; Firmicutes; Bacillales; Bacillaceae; Bacillus.
REFERENCE 1 (bases 1 to 1488)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Isolation, identification, and characterization of bacterial
isolates degrading chlorophenols and adsorbable organic halides (AOX)
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1488)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Direct Submission
JOURNAL Submitted (17-DEC-2008) Microbial Sciences Division, Agharkar Research Institute, G G Agarkar Road, Pune, Maharashtra, 411004, India
FEATURES Location/Qualifiers
source 1..1488
/organism="Bacillus subtilis"
/mol_type="genomic DNA"
/strain="E1-1"
/isolation_source="soil irrigated with effluent of pulp and paper industry"
/db_xref="taxon:1423"
rRNA <1..>1488
/product="16S ribosomal RNA"
ORIGIN 1 tcccggattc ccttttnggc agagtttgat ctggctcagg acgaacgctg gcggcgtgcc
61 taatacatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag cggcggacgg
121 gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggggcta
181 ataccggatg gttgtttgaa ccgcatggtt caaacataaa aggtggcttc ggctaccact
241 tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcaacga
301 tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc
361 tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg
421 cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtaccgt
481 tcgaataggg cggtaccttg acggtaccta accagaaagc cacggctaac tacgtgccag
541 cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt aaagggctcg
601 caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa
661 actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt 721 agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg
781 agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg
841 agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat taagcactcc
901 ccctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggg cccctcaagc
961 ggtggagcat gtggtttaat tggaagcaac gcgaagaacc ttaccaggtg ttgacatcct
1021 ttctcaatgc tagagatacg acgtgccctt ggggggctga ctgacaggtg gtgcatggtt
1081 gttgtcacct cgtgtggtga gacgttgggc taactgccgc aacgagggct acccatgatt
1141 ttagttgcca gctttcagtt gggctctata aggtgtctgc cggtgtcaaa cccgaggaag
1201 gtggggatga cgtcaaatca tcatgcccct tatgacctgg ggtacacacg tgctacaatg
1261 gacagaacaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttttcagt
1321 tcggatcgca gtttgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag 1381 catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt
1441 tgtaacaccc gaagtcggtg aggtaacctt tttggagcca gccgccga
Data Sheet 5.1: 16S rRNA nucleotide sequence of isolate E1-1
131
LOCUS FJ573170 1488 bp DNA linear BCT 24-MAR-2009
DEFINITION Bacillus subtilis strain E1-2 16S ribosomal RNA gene, partial
sequence.
ACCESSION FJ573170
VERSION FJ573170.1 GI:225353979
KEYWORDS SOURCE Bacillus subtilis
ORGANISM Bacillus subtilis
Bacteria; Firmicutes; Bacillales; Bacillaceae; Bacillus.
REFERENCE 1 (bases 1 to 1488)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Isolation, identification, and characterization of bacterial
isolates degrading chlorophenols and adsorbable organic halides
(AOX)
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1488)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Direct Submission JOURNAL Submitted (17-DEC-2008) Microbial Sciences Division, Agharkar Research
Institute, G G Agarkar Road, Pune, Maharashtra 411004, India
FEATURES Location/Qualifiers
source 1..1488
/organism="Bacillus subtilis"
/mol_type="genomic DNA"
/strain="E1-2"
/isolation_source="soil irrigated with effluent of pulp and paper industry"
/db_xref="taxon:1423"
rRNA <1..>1488
/product="16S ribosomal RNA" ORIGIN
1 tcccggattc ccttttnggc agagtttgat ctggctcagg acgaacgctg gcggcgtgcc
61 taatacatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag cggcggacgg
121 gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggggcta
181 ataccggatg gttgtttgaa ccgcatggtt caaacataaa aggtggcttc ggctaccact
241 tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcaacga
301 tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc
361 tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg
421 cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtaccgt
481 tcgaataggg cggtaccttg acggtaccta accagaaagc cacggctaac tacgtgccag
541 cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt aaagggctcg
601 caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa 661 actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt
721 agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg
781 agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg
841 agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat taagcactcc
901 ccctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggg cccctcaagc
961 ggtggagcat gtggtttaat tggaagcaac gcgaagaacc ttaccaggtg ttgacatcct
1021 ttctcaatgc tagagatacg acgtgccctt ggggggctga ctgacaggtg gtgcatggtt
1081 gttgtcacct cgtgtggtga gacgttgggc taactgccgc aacgagggct acccatgatt
1141 ttagttgcca gctttcagtt gggctctata aggtgtctgc cggtgtcaaa cccgaggaag
1201 gtggggatga cgtcaaatca tcatgcccct tatgacctgg ggtacacacg tgctacaatg
1261 gacagaacaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttttcagt 1321 tcggatcgca gtttgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag
1381 catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt
1441 tgtaacaccc gaagtcggtg aggtaacctt tttggagcca gccgccga
Data Sheet 5.2: 16S rRNA nucleotide sequence of isolate E1-2
132
LOCUS FJ573171 1513 bp DNA linear BCT 24-MAR-2009
DEFINITION Bacillus subtilis strain E9-1 16S ribosomal RNA gene, partial
sequence.
ACCESSION FJ573171
VERSION FJ573171.1 GI:225353980
KEYWORDS SOURCE Bacillus subtilis
ORGANISM Bacillus subtilis
Bacteria; Firmicutes; Bacillales; Bacillaceae; Bacillus.
REFERENCE 1 (bases 1 to 1513)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Isolation, identification, and characterization of bacterial
isolates degrading chlorophenols and adsorbable organic halides
(AOX)
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1513)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Direct Submission JOURNAL Submitted (17-DEC-2008) Microbial Sciences Division, Agharkar Research
Institute, G G Agarkar Road, Pune, Maharashtra 411004, India
FEATURES Location/Qualifiers
source 1..1513
/organism="Bacillus subtilis"
/mol_type="genomic DNA"
/strain="E9-1"
/isolation_source="soil irrigated with effluent of pulp and paper industry"
/db_xref="taxon:1423"
rRNA <1..>1513
/product="16S ribosomal RNA" ORIGIN
1 ttcccggatt cccttttngg caagtttgat ctggctcagg acgaacgctg gcggcgtgcc
61 taatacatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag cggcggacgg
121 gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggggcta
181 ataccggatg gttgtttgaa ccgcatggtt caaacataaa aggtggcttc ggctaccact
241 tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcaacga
301 tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc
361 tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg
421 cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtaccgt
481 tcgaataggg cggtaccttg acggtaccta accagaaagc cacggctaac tacgtgccag
541 cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt aaagggctcg
601 caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa 661 actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt
721 agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg
781 agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg
841 agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat taagcactcc
901 gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc ccgcacaagc
961 ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct
1021 ctgacaatcc tagagatagg acgtccccct tcgggggcag agtgacaggt ggtgcatggt
1081 tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat
1141 cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa accggaggaa
1201 ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat
1261 ggacagaaca aagggcagcg aaaccgcgag gttaagccaa tcccacaaat ctgttctcag 1321 ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca
1381 gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt
1441 ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc agcngggaaa aagntgggac
1501 agntgattgg ggg
Data Sheet 5.3: 16S rRNA nucleotide sequence of isolate E9-1
133
LOCUS FJ573172 1457 bp DNA linear BCT 24-MAR-2009
DEFINITION Brevibacterium stationis strain E9-2 16S ribosomal RNA gene,
partial sequence.
ACCESSION FJ573172
VERSION FJ573172.1 GI:225353981
KEYWORDS SOURCE Brevibacterium stationis
ORGANISM Brevibacterium stationis
Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales;
Micrococcineae; Brevibacteriaceae; Brevibacterium.
REFERENCE 1 (bases 1 to 1457)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Isolation, identification, and characterization of bacterial
isolates degrading chlorophenols and adsorbable organic halides
(AOX)
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1457)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R. TITLE Direct Submission
JOURNAL Submitted (17-DEC-2008) Microbial Sciences Division, Agharkar Research
Institute, G G Agarkar Road, Pune, Maharashtra 411004, India
FEATURES Location/Qualifiers
source 1..1457
/organism="Brevibacterium stationis"
/mol_type="genomic DNA"
/strain="E9-2"
/isolation_source="soil irrigated with effluent of pulp and paper industry"
/db_xref="taxon:1705"
rRNA <1..>1457 /product="16S ribosomal RNA"
ORIGIN
1 gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac ggaaaggcct tgtgcttgca
61 caaggtactc gagtggcgaa cgggtgagta acacgtgggt gatctgccct gcactgtggg
121 ataagcctgg gaaactgggt ctaataccat ataggaccgc atcttggatg gtgtggtgga
181 aagcttttgc ggtgtgggat gagcctgcgg cctatcagct tgttggtggg gtaatggcct
241 accaaggcgg cgacgggtat ccggcctgag agggtgtacg gacacattgg gactgagata
301 cggcccagac tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga
361 tgcagcgacg ccgcgtgggg gatgaaggcc ttcgggttgt aaactccttt cgctatcgac
421 gaagccactt ggtgacggta ggtagataag aagcaccggc taactacgtg ccagcagccg
481 cggtaatacg tagggtgcaa gcgttgtccg gaattactgg gcgtaaagag ctcgtaggtg
541 gtttgtcgcg tcgtctgtga aatcccgggg cttaacttcg ggcgtgcagg cgatacgggc 601 ataacttgag tgctgtaggg gagactggaa ttcctggtgt agcggtgaaa tgcgcagata
661 tcaggaggaa caccgatggc gaaggcaggt ctctgggcag ttactgacgc tgaggagcga
721 aagcatgggt agcgaacagg attagatacc ctggtagtcc atgccgtaaa cggtgggcgc
781 taggtgtagg ggggcttcca cgtcttctgt gccgtagcta acgcattaag cgccccgcct
841 ggggagtacg gccgcaaggc taaaactcaa aggaattgac gggggcccgc acaagcggcg
901 gagcatgtgg attaattcga tgcaacgcga agaaccttac ctgggcttga catatacagg
961 atcgggctag agatagtctt tcccttgtgg tctgtataca ggtggtgcat ggttgtcgtc
1021 agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gtcttatgtt
1081 gccagcacgt tatggtggga actcatgaga gactgccggg gttaactcgg aggaaggtgg
1141 ggatgacgtc aaatcatcat gccccttatg tccagggctt cacacatgct acaatggtcg
1201 atacagtggg cagcgacatc gtaaggtgga gcgaatccct gaaagtcggc cttagttcgg 1261 attggggtct gcaactcgac cccatgaagt cggagtcgct agtaatcgca gatcagcaac
1321 gctgcggtga atacgttccc gggccttgta cacaccgccc gtcacgtcat gaaagttggt
1381 aacacccgaa gccagtggcc taaacttgtt agggagctgt cgaaggtggg atcggcgatt
1441 gggacgaagt cgtaaca
Data Sheet 5.4: 16S rRNA nucleotide sequence of isolate E9-2
134
LOCUS FJ573173 1464 bp DNA linear BCT 24-MAR-2009
DEFINITION Staphylococcus sciuri strain E9-3 16S ribosomal RNA gene, partial
sequence.
ACCESSION FJ573173
VERSION FJ573173.1 GI:225353982
KEYWORDS SOURCE Staphylococcus sciuri
ORGANISM Staphylococcus sciuri
Bacteria; Firmicutes; Bacillales; Staphylococcus.
REFERENCE 1 (bases 1 to 1464)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Isolation, identification, and characterization of bacterial
isolates degrading chlorophenols and adsorbable organic halides
(AOX)
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1464)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Direct Submission JOURNAL Submitted (17-DEC-2008) Microbial Sciences Division, Agharkar Research
Institute, G G Agarkar Road, Pune, Maharashtra 411004, India
FEATURES Location/Qualifiers
source 1..1464
/organism="Staphylococcus sciuri"
/mol_type="genomic DNA"
/strain="E9-3"
/isolation_source="soil irrigated with effluent of pulp and paper industry"
/db_xref="taxon:1296"
rRNA <1..>1464
/product="16S ribosomal RNA" ORIGIN
1 tccggatccg tcgacagagt ttgatctggc tcaggatgaa cgctggcggc gtgcctaata
61 catgcaagtc gagcgaacag atgagaagct tgcttctctg atgttagcgg cggacgggtg
121 agtaacacgt gggtaaccta cctataagac tgggataact ccgggaaacc ggggctaata
181 ccggataata ttttgaaccg catggttcaa tagtgaaaga cggtttcggc tgtcacttat
241 agatggaccc gcgccgtatt agctagttgg taaggtaacg gcttaccaag gcgacgatac
301 gtagccgacc tgagagggtg atcggccaca ctggaactga gacacggtcc agactcctac
361 gggaggcagc agtagggaat cttccgcaat gggcgaaagc ctgacggagc aacgccgcgt
421 gagtgatgaa ggtcttcgga tcgtaaaact ctgttgttag ggaagaacaa atttgttagt
481 aactgaacaa gtcttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc
541 cgcggtaata cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgtagg
601 cggtttctta agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg 661 ggaaacttga gtgcagaaga ggagagtgga attccatgtg tagcggtgaa atgcgcagag
721 atatggagga acaccagtgg cgaaggcggc tctctggtct gtaactgacg ctgatgtgcg
781 aaagcgtggg gatcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg
841 ctaagtgtta gggggttttc gccccttagt gctgcagcta acgcattaag cactccgcct
901 ggggagtacg accgcaaggt tgaaactcaa aggaattgac ggggacccgc acaagcggtg
961 gagcatgtgg tttaattcga agcaacgcga agaaccttac caaatcttga catcctttga
1021 ccgctctaga gatagagtct tccccttcgg gggacaaagt gacaggtggt gcatggttgt
1081 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttaagctt
1141 agttgccatc attaagttgg gcactctagg ttgactgccg gtgacaaacc ggaggaaggt
1201 ggggatgacg tcaaatcatc atgcccctta tgatttgggc tacacacgtg ctacaatgga
1261 taatacaaag ggcagcgaat ccgcgaggcc aagcaaatcc cataaaatta ttctcagttc 1321 ggattgtagt ctgcaactcg actacatgaa gctggaatcg ctagtaatcg tagatcagca
1381 tgctacggtg aatacgttcc cggtcttgta cacacccccg tcacacccga gatttgtaac
1441 acccgaaccg gtggaataac cttt
Data Sheet 5.5: 16S rRNA nucleotide sequence of isolate E9-3
135
LOCUS FJ573174 1510 bp DNA linear BCT 24-MAR-2009
DEFINITION Staphylococcus sciuri strain E9-4 16S ribosomal RNA gene, partial
sequence.
ACCESSION FJ573174
VERSION FJ573174.1 GI:225353983
KEYWORDS SOURCE Staphylococcus sciuri
ORGANISM Staphylococcus sciuri
Bacteria; Firmicutes; Bacillales; Staphylococcus.
REFERENCE 1 (bases 1 to 1510)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Isolation, identification, and characterization of bacterial
isolates degrading chlorophenols and adsorbable organic halides
(AOX)
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1510)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Direct Submission JOURNAL Submitted (17-DEC-2008) Microbial Sciences Division, Agharkar Research
Institute, G G Agarkar Road, Pune, Maharashtra 411004, India
FEATURES Location/Qualifiers
source 1..1510
/organism="Staphylococcus sciuri"
/mol_type="genomic DNA"
/strain="E9-4"
/isolation_source="soil irrigated with effluent of pulp and paper industry"
/db_xref="taxon:1296"
rRNA <1..>1510
/product="16S ribosomal RNA" ORIGIN
1 tccggatccg ttcgacagag tttgatctgg ctcaggatga acgctggcgg cgtgcctaat
61 acatgcaagt cgagcgaaca gatgagaagc ttgcttctct gatgttagcg gcggacgggt
121 gagtaacacg tgggtaacct acctataaga ctgggataac tccgggaaac cggggctaat
181 accggataat attttgaacc gcatggttca atagtgaaag acggtttcgg ctgtcactta
241 tagatggacc cgcgccgtat tagctagttg gtaaggtaac ggcttaccaa ggcgacgata
301 cgtagccgac ctgagagggt gatcggccac actggaactg agacacggtc cagactccta
361 cgggaggcag cagtagggaa tcttccgcaa tgggcgaaag cctgacggag caacgccgcg
421 tgagtgatga aggtcttcgg atcgtaaaac tctgttgtta gggaagaaca aatttgttag
481 taactgaaca agtcttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag
541 ccgcggtaat acgtaggtgg caagcgttat ccggaattat tgggcgtaaa gcgcgcgtag
601 gcggtttctt aagtctgatg tgaaagccca cggctcaacc gtggagggtc attggaaact 661 gggaaacttg agtgcagaag aggagagtgg aattccatgt gtagcggtga aatgcgcaga
721 gatatggagg aacaccagtg gcgaaggcgg ctctctggtc tgtaactgac gctgatgtgc
781 gaaagcgtgg ggatcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt
841 gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa gcactccccc
901 tggggagtac gaccgcaagg ttgaaactca aaggaattga cggggacccg cacaagcggt
961 ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaaatcttg acatcctttg
1021 accgctctag agatagagtc ttccccttcg ggggacaaag tgacaggtgg tgcatggttg
1081 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttaagct
1141 tagttgccat cattaagttg ggcactctag gttgactgcc ggtgacaaac cggaggaagg
1201 tggggatgac gtcaaatcat catgcccctt atgatttggg ctacacacgt gctacaatgg
1261 ataatacaaa gggcagcgaa tccgcgaggc caagcaaatc ccataaaatt attctcagtt 1321 cggattgtag tctgcaactc gactacatga agctggaatc gctagtaatc gtagatcagc
1381 atgctacggt gaatacgttc ccgggtcttg tacacaccgc ccgtcacacc acgagagttt
1441 gtaacacccg aagccggggg agtaaccttt tnggagctag ccggnaaaag ntgggacaaa
1501 tgattggggg
Data Sheet 5.6: 16S rRNA nucleotide sequence of isolate E9-4
136
LOCUS FJ573175 1401 bp DNA linear BCT 24-MAR-2009
DEFINITION Brevibacterium stationis strain E9-5 16S ribosomal RNA gene,
partial sequence.
ACCESSION FJ573175
VERSION FJ573175.1 GI:225353984
KEYWORDS SOURCE Brevibacterium stationis
ORGANISM Brevibacterium stationis
Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales;
Micrococcineae; Brevibacteriaceae; Brevibacterium.
REFERENCE 1 (bases 1 to 1401)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R.
TITLE Isolation, identification, and characterization of bacterial
isolates degrading chlorophenols and adsorbable organic halides
(AOX)
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1401)
AUTHORS Dhakephalkar,P.K., Lapsiya,K.L., Savant,D.V. and Ranade,D.R. TITLE Direct Submission
JOURNAL Submitted (17-DEC-2008) Microbial Sciences Division, Agharkar Research
Institute, G G Agarkar Road, Pune, Maharashtra 411004, India
FEATURES Location/Qualifiers
source 1..1401
/organism="Brevibacterium stationis"
/mol_type="genomic DNA"
/strain="E9-5"
/isolation_source="soil irrigated with effluent of pulp and paper industry"
/db_xref="taxon:1705"
rRNA <1..>1401 /product="16S ribosomal RNA"
ORIGIN
1 gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac ggaaaggcct tgtgcttgca
61 caaggtactc gagtggcgaa cgggtgagta acacgtgggt gatctgccct gcactgtggg
121 ataagcctgg gaaactgggt ctaataccat ataggaccgc atcttggatg gtgtggtgga
181 aagcttttgc ggtgtgggat gagcctgcgg cctatcagct tgttggtggg gtaatggcct
241 accaaggcgg cgacgggtat ccggcctgag agggtgtacg gacacattgg gactgagata
301 cggcccagac tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga
361 tgcagcgacg ccgcgtgggg gatgaaggcc ttcgggttgt aaactccttt cgctatcgac
421 gaagccactt ggtgacggta ggtagataag aagcaccggc taactacgtg ccagcagccg
481 cggtaatacg tagggtgcaa gcgttgtccg gaattactgg gcgtaaagag ctcgtaggtg
541 gtttgtcgcg tcgtctgtga aatcccgggg cttaacttcg ggcgtgcagg cgatacgggc 601 ataacttgag tgctgtaggg gagactggaa ttcctggtgt agcggtgaaa tgcgcagata
661 tcaggaggaa caccgatggc gaaggcaggt ctctgggcag ttactgacgc tgaggagcga
721 aagcatgggt agcgaacagg attagatacc ctggtagtcc atgccgtaaa cggtgggcgc
781 taggtgtagg gggcttccac gtcttctgtg ccgtagctaa cgcattaagc gccccgcctg
841 gggagtacgg ccgcaaggct aaaactcaaa ggaattgacg ggggcccgca caagcggcgg
901 agcatgtgga ttaattcgat gcaacgcgaa gaaccttacc tgggcttgac atatacagga
961 tcgggctaga gatagtcttt cccttgtggt ctgtatacag gtggtgcatg gttgtcgtca
1021 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg tcttatgttg
1081 ccagcacgtt atggtgggaa ctcatgagag actgccgggg ttaactcgga ggaaggtggg
1141 gatgacgtca aatcatcatg ccccttatgt ccagggcttc acacatgcta caatggtcga
1201 tacagtgggc agcgacatcg taaggtggag cgaatccctg aaagtcggcc ttagttcgga 1261 ttggggtctg caactcgacc ccatgaagtc ggagtcgcta gtaatcgcag atcagcaatg
1321 ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacgtcatg aaagttggta
1381 acacccgaag ccggtggact a
Data Sheet 5.7: 16S rRNA nucleotide sequence of isolate E9-5
137
A
B
C
Fig. 5.12 Phylogenetic dendrogram based on 16S rDNA sequence showing
the relationship of isolates E1-1, E1-2 and E9-1 (A), E9-2 and E9-5
(B) and E9-3 and E9-4(C) with the most closely related strains and
with each other. Bootstrap values (percentages of 1000 replications)
are shown at the nodes. Isolates obtained in this study are marked in
bold.
FJ573173
FJ573174
EU855191.1
AB188210.1
AB009942.1
45
73
0.001
Staphyloccous sciuri strain E9-3
Staphyloccous sciuri strain E9-4
Staphyloccous sciuri strain CTSP9
Staphyloccous sp TUT1203
Staphyloccous pulvereri
Brevibacterium stationis strain E9-2
Brevibacterium stationis strain E9-5
Brevibacterium stationis strain LMG 21670T Corynebacterium ammoniagenes strain ATCC 6872
Corynebacterium sp strain B-5121
Brevibacterium sp strain B-5131
Corynebacterium casei strain 1MA
Corynebacterium thomssenii isolate 97-0130
Corynebacterium pseudogenitalium Corynebacterium tuberculostearicum strain Medalle X
Corynebacterium flavescens strain NCDO 1320 Corynebacterium falsenii isolate 97-0205
Corynebacterium diphtheriae strain CD450
AY971527.2
FJ573171
EU221345.1
AB188212.1
FJ573169
FJ573170100
45
74
0.002
Bacillus licheniformis
Bacillus subtilis strain E9-1
Bacillus subtilis strain PAB1C8
Bacillus sp strain TUT1206
Bacillus subtilis strain E1-1
Bacillus subtilis strain E1-2
FJ573172
AJ620367.1
FJ648509.1
FJ573175
DQ399759.1
DQ399760.1
DQ36113.1
AF537607.1
AJ43948.1
NR028975.1
X84441.1
AF537594.1
FJ409575.1
100
97
90
52
10065
100
66
0.005
138
Even though both the isolates obtained from enrichment E1 were B. subtilis, they
were different in their degradation capability and substrate specificities which was
evident from the results of biodegradation spectrum of chlorophenols. Kim et. al.
(2004) in their study on polychlorinated biphenyls (PCBs) isolated four stains of
the genus Bacillus from hexachlorocyclohexane contaminated soil. They further
observed that three strains utilized PCBs as sole carbon and energy source at
different rates. Even isolate E9-1 from enrichment E9 was identified as B. subtilis
but again its degradation capability and substrate specification were very different
from the other two Bacillus. Different species of the genus Bacillus have been
reported in the literature to degrade various environmental pollutants. Tondo et. al.
(1998) have reported Bacillus sp isolated from cellulose pulp mill effluent, which
could degrade 4,5,6-trichloroguaiacol (4,5,6TCG) at a concentration of 50 mgL-1
.
They attributed the reduction in level of 4,5,6TCG to bacterial metabolism and not
due to absorption or adsorption by the bacterial cells. Wang et. al. (2000) studied
removal of 2,4DCP by B. insolitus isolated from a mixed culture acclimated to
chlorophenols. They found that removal efficiency for 2,4DCP remained almost
same for suspended and immobilized culture. They also reported that at higher
initial concentration of 2,4DCP, degradation capability of immobilized pure culture
of B. insolitus was superior to immobilized mixed culture. Hirose et. al. (2003)
studied degradation of various substituted phenols by thermostable laccase bound
to B. subtilis spores. They reported 32% to 90% degradation of different
substituted phenolic compounds when incubated with the spore suspension.
Andretta et. al. (2004) in their study on 4,5,6TCG degradation by B. subtilis strain
IS13 showed that the culture was able to degrade 4,5,6TCG only when the
inoculum was composed of cells in stationary phase of growth. They reported that
4,5,6TCG was partially degraded at a concentration of 100 mgL-1
. The
concentration >100 mgL-1
was found toxic to the bacteria. They also reported that
the rate of degradation of 4,5,6TCG by the isolate could be accelerated by addition
of other carbon sources such as glucose, molasses, etc. Matafonova et. al. (2006)
isolated a 2,4DCP degrading Bacillus sp from an aeration pond in the Baikalsk
PAP mill. They reported degradation of 2,4DCP by this strain at concentration up
to 400 μM however high concentration was inhibitory to growth. Al-Thani et. al.
139
(2007) isolated six strains of genus Bacillus from two different soil samples. They
reported that all the strains were capable of degrading 2CP at concentrations up to
1.5 mM and higher concentrations (2.5 mM) were inhibitory to growth. Singh et.
al. (2008) investigated biotransformation of pentachlorophenol (PCP) and PAP
mill effluent decolorization by bacterial strains in mixed culture. Using a mixed
inoculum consisting of two bacterial strains namely, Bacillus sp and Serratia
marcescens these authors have reported 94% degradation of PCP present in PAP
mill effluent. They also reported decline in pH, AOX, color, DO, BOD and COD
levels in the PAP mill effluent. Bacillus species have also been shown to possess
ability to synthesize plant growth promoting substances and have been used as
biocontrol bacteria for crops. Reva et. al. (2004) studied plant colonizing ability of
B. amyloliquefaciens and B. subtilis. Therefore it can be said that the use of this
genus in bioaugmentation trials can serve two purposes, one it will degrade AOX
from contaminated soil and second it will protect crops from pathogens and toxic
effects of AOX.
Isolates E9-2 and E9-5 both were closely related to Brevibacterium stationis. They
also had different degradation capability and substrate specificity among
themselves. This is the first time, to the best of our knowledge that Brevibacterium
stationis has been reported for organochlorine degradation. Though isolates E9-3
and E9-4, both identified as S. sciuri, had no difference in substrate specification,
their degradation capability was different. Kumar and Philip (2006) have reported
bioremediation of endosulfan contaminated soil and water using a mixed bacterial
culture consisting of Staphylococcus sp and Bacillus circulans I and II. They found
that the mixed bacterial culture was able to degrade 71.58 % and 75.88% of
endosulfan in aerobic and facultative anaerobic conditions, respectively. Their
study, on soil reactor, showed maximum endosulfan degradation efficiency of
95.48% by the mixed bacterial culture.
Out of seven isolates which were studied herein, three were identified as B.
subtilis, two as Brevibacterium stationis and remaining two as S. sciuri. There are
reports on degradation of different organochlorines by B. subtilis. The data
140
presented herein confirm ability of B. subtilis to degrade organochlorines. One
finds in the literature hardly any report on degradation of organochlorine by
Brevibacterium stationis. There is only one report on degradation of
organochlorine by Staphylococcus sp. and that too as a mixed culture with Bacillus
circulans.
5.3.7 Spectrum of chlorophenol biodegradation
Results of chlorophenol biodegradation (Fig. 5.13) showed that isolate E1-2 (B.
subtilis) was the most versatile organism and could degrade four different
chlorophenols, 3CP (25%), 2,3DCP (75%), 2,4DCP (72%) and 2,4,6TCP (32%)
apart from 2CP which is used as the substrate for its maintenance. Its range of
degradation was very wide as it could degrade mono-, di- and trichlorophenols.
Position of chlorine did not affect the degradation ability of isolate E1-2, i.e., it
could degrade ortho, meta as well as para substituted compounds. Next to this was
isolate E1-1 (B. subtilis) which could degrade three different chlorophenols, 4CP
(56%), 2,4DCP (42%) and 3,4DCP (34%) apart from 2CP which is used as
substrate for its maintenance. From the chlorophenol degradation pattern it was
seen that isolate E1-1 mostly favored ortho and para substituted chlorophenols.
Chlorophenol degradation spectrum for isolates E9-2, E9-3 and E9-4 was paltry as
they could degrade only one chlorophenol each, 2,4DCP (34%), 2CP (66%) and
2CP (40%), respectively, apart from 4CP used as their substrate for maintenance.
All these three isolates favored ortho and para substituted chlorophenols. Isolates
E9-1 and E9-5 could not degrade any other chlorophenol apart from 3CP and 4CP,
respectively which were used as their substrates for maintenance. Abiotic loss of
different chlorophenols was, on an average, below 5% and the reduction in
chlorophenol levels reported here are in addition to the abiotic chlorophenol losses.
141
Fig. 5.13 Spectrum of chlorophenol biodegradation by isolates: (A) %
chlorophenol degradation and (B) growth of isolates measured in
terms of increase in absorbance
5.3.8 Biodegradation of extracted AOX by isolates
Contrary to the results of spectrum of chlorophenol biodegradation isolate E1-1
was found superior to all the isolates in degrading AOX and could degrade 34.62%
AOX (Fig. 5.14). Isolate E1-2 could degrade 19.24% AOX whereas isolate E9-2
could degrade 15.38% AOX. Isolate E9-5 could degrade 9.17% AOX. There was
abiotic loss of AOX in the control flasks over the course of incubation which was
averaged ~7%. The reduction in AOX levels reported here are in addition to the
abiotic AOX losses. Rest of the isolates showed no degradation of AOX.
A B
142
0
5
10
15
20
25
30
35
40
E1-1
E1-2
E9-1
E9-2
E9-3
E9-4
E9-5
Con
trol
Isolates
AO
X D
eg
ra
da
tio
n (
%)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
E1-1 E1-2 E9-1 E9-2 E9-3 E9-4 E9-5
Isolates
Op
tic
al
De
ns
ity
(6
00
nm
)
Fig. 5.14 Biodegradation of AOX by isolates (A) and growth of isolates
measured in terms of increase in absorbance (B) [error bars
represents standard deviation of the mean of triplicate samples, not
shown when no larger than the symbols]
Of the seven isolates studied isolate E1-1 was found to be superior to other isolates
with respect to AOX degradation efficiency. The spectrum of chlorophenol
biodegradation study was not sufficient to predict the performance of these isolates
in a complex mixture of AOX which is known to contain more than 300 different
types of organochlorines. From the chlorophenol degradation study isolate E1-2
was found to be versatile however its action on different chlorophenols alone was
not relevant for its ability to remove AOX from BCWW though chlorophenols are
one of the major portions of AOX. On the other hand isolate E1-1, which appeared
to be less versatile than E1-2, was able to degrade other organochlorines which is
relevant to its ability to remove AOX along with chlorophenols. Isolate E9-2 was
also selected for further studies because spectrum study showed that it was capable
of degrading 2,4DCP, which was found in BCWW during GC-MS analysis. But it
was clear from AOX degradation study that it was not efficient at removing AOX.
Fulthorpe and Allen (1995) had reported similar results in their study on AOX
degradation by three bacterial species. In their preliminary studies on degradation
of simple chlorinated compounds Ancylobacter aquaticus A7 and Pseudomonas P1
were found to be more versatile with broader substrate range as compared to
A B
143
Methylobacterium CP13. But when the three isolates were tested for their ability to
degrade AOX from BKME, strain CP13 was found to be superior with respect to
the other two species.
5.3.9 AOX biodegradation from BCWW by isolates and their
consortium
Comparable results were obtained with respect to AOX degradation ability of the
three isolates when compared with the results of previous experiment (Fig. 5.15).
Isolate E1-1 was found superior to the other two isolates, with AOX degradation of
15.25%. Isolate E1-2 could degrade 5.76% of AOX whereas isolate E9-1 could
degrade 13.04% of AOX. The consortium developed using the isolates could
degrade 20.37% of AOX. Reduction of AOX was same for flasks containing dead
bacterial consortium and negative control flasks. Abiotic loss of AOX in the
control flasks over the course of incubation which was averaged ~2%. The
reduction in AOX levels reported here are inclusive of the abiotic AOX losses.
0
5
10
15
20
25
E1-1
E1-2
E9-2
Live
Con
sortium
Dea
d Con
sortium
Con
trol
AO
X D
eg
ra
da
tio
n (
%)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
E1-1
E1-2
E9-2
Live
Con
ceortiu
m
Dea
d Con
sortium
Op
tic
al
De
ns
ity
(6
00
nm
)
Fig. 5.15 Biodegradation of AOX by selected isolates and their consortium
(A) and growth of isolates and consortium measured in terms of
increase in absorbance (B) [error bars represents standard deviation
of the mean of triplicate samples, not shown when no larger than the
symbols]
A B
144
The isolates were to be used for bioaugmentation trials in soil which is irrigated
with effluent of PAP industry; they were tested for their ability to degrade AOX
from actual wastewater. In the previous experiment it was found that all the three
isolates degraded AOX at different rates. It was thought worth testing AOX
degradation by mixing all the three isolates and forming a consortium so as to
increase the rate of degradation. Results showed that there was not much difference
in the rate of degradation between isolate E1-1 and consortium. Also a consortium
of dead cells of all the isolates was tested to prove that the reduction in AOX level
was due to metabolism by the bacteria and not due to adsorption on the cells. The
result of the dead consortium was comparable with that of results described by
Tondo et. al. (1998). Tiku et. al. (2010) studied holistic bioremediation of pulp
mill effluents using autochthonous bacteria. Their consortium of three bacterial
strains: Pseudomonas aeruginosa (DSMZ 03504), P. aeruginosa (DSMZ 03505)
and Bacillus megaterium (MTCC 6544), was capable of reducing not only COD
and BOD levels in the effluent but was also capable of reducing total dissolved
solid (TDS), AOX and color.
5.3.10 Plasmid DNA isolation
Both the techniques showed the same results. The O‘Sullivan and Klaenhammer
method based on alkaline lysis indicated that all the isolates were free of low
molecular weight plasmid DNA and the Tolmasky et. al. method based on hot
triton X-100 lysis indicated that all the isolates were free of high molecular weight
plasmid DNA in comparison with the positive control (E. coli V157) under similar
experimental conditions. The results suggest that the organochlorine degradation
activity is not plasmid borne and required genes are located on the bacterial
chromosome.
This observation is very important. As the degradation ability is chromosomal
based the use of these cultures becomes more dependable in bioremediation. The
results were comparable with that of Tondo et. al. (1998) and Andretta et. al.
(2004). Tondo et. al. (1998) attributed the presence of degradative genes on
chromosome to the necessity of bacterial cells to detoxify its surrounding
145
environment in order to survive. They reasoned that the toxic compounds were
always present in nature exerting a selective pressure on microorganisms during
evolution, justifying the presence of stable biodegrading routes in the bacterial
metabolism.
5.3.11 Biodegradation of 2,4DCP at different pH
The results of 2,4DCP biodegradation by the isolates at different pH were
comparable to that of spectrum of chlorophenol biodegradation (Fig. 5.16). Again
isolate E1-2 was found superior to other isolates in terms of percent efficiency for
2,4DCP removal and activity over wide pH range. It could degrade 2,4DCP at
three different pH. The rate of degradation at pH 5 after 120 h of incubation was
49.15% and 3.50% under shake and static culture conditions, respectively; 53.53%
and 10.39% under shake and static culture conditions, respectively at pH 6 and
71.67% and 19.37% under shake and static culture conditions, respectively at pH
7. Reduction of 2,4DCP at pH 8 was 14.70% under shake culture conditions.
However similar degradation was observed for flasks at shake culture without
bacterial inoculation. Isolate E1-1 could degrade 2,4DCP at two different pH and
the rate of degradation under shake and static culture conditions was 46.70% and
7.05%, respectively at pH 7 and 65.64% and 12.10%, respectively at pH 8.
Reduction of 2,4DCP was almost same for flasks with pH 5 and 6 and for negative
control flasks. Isolate E9-2 could degrade 2,4DCP at two different pH and the rate
of degradation was 52.51% and 6.96% under shake and static culture conditions,
respectively at pH 6 and 62.00% and 14.83% under shake and static culture
conditions, respectively at pH 7. Reduction of 2,4DCP was same for flasks with
pH 5 and 8 and negative control flasks. The results indicated that aeration was
necessary for the isolates to degrade organochlorine effectively. There was abiotic
loss of 2,4DCP in the control flasks over the course of incubation which was
averaged ~2% for pH 5 and 6, ~7% for pH 7 and ~11% for pH 8. The reduction in
2,4DCP levels reported here are in addition to the abiotic losses.
146
Isolate E1-1
0
10
20
30
40
50
60
70
80
5 6 7 8pH
2,4
DC
P D
eg
rad
ati
on
(%
)
Shaking
Static
Isolate E1-2
0
10
20
30
40
50
60
70
80
5 6 7 8
pH
2,4
DC
P D
eg
rad
atio
n (
%)
Shaking
Static
A1 B1
A2 B2
A3 B3
147
Control
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
5 6 7 8pH
Op
tic
al
De
ns
ity
(6
00
nm
)
E1-1, Shaking
E1-1, Static
E1-2, Shaking
E1-2, Static
E9-2, Shaking
E9-2, Static
Fig. 5.16 2,4DCP biodegradation by selected isolates at different pH (A1-A3)
and growth of isolates measured in terms of increase in absorbance
(B1-B3), Negative control (A4) and positive control (B4) [error bars
represents standard deviation of the mean of triplicate samples, not
shown when no larger than the symbols]
5.3.12 Biodegradation of AOX at different pH
The results of AOX biodegradation by the three isolates at different pH were
comparable with that of AOX degradation by different isolates (Fig. 5.17). Isolate
E1-1 was found superior to all the three isolates with respect to AOX degradation.
It could degrade AOX at three different pH and the rate of degradation was 15.38%
at pH 6, 32.14% at pH 7 and 28.46% at pH 8. Reduction in AOX at pH 5 was same
as that in control flask. Isolate E1-2 could also degrade AOX at three different pH
and the rate of degradation was 7.69% at pH 5 and 6 and 19.23% at pH 7.
Reduction in AOX level at pH 8 was same as that in the control flask. Isolate E9-2
could degrade AOX at two different pH and the rate of degradation was 3.84% at
pH 6 and 30.76% at pH 7. Reduction in AOX level at pH 5 and 8 was equal and
moreover almost similar as in control flask. There was abiotic loss of AOX in the
control flasks over the course of incubation which was averaged ~1% for pH 5 and
6, ~7% for pH 7 and ~15% for pH 8. The reduction in AOX levels reported here
are in addition to the abiotic losses.
A4 B4
148
Isolate E1-1
0
5
10
15
20
25
30
35
5 6 7 8
pH
AO
X D
eg
rad
ati
on
(%
)
Isolate E1-1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
5 6 7 8pH
Op
tic
al D
en
sit
y (
60
0 n
m)
Isolate E9-2
0
5
10
15
20
25
30
35
5 6 7 8pH
AO
X D
eg
rad
ati
on
(%
)
A1 B1
A2 B2
A3 B3
149
Control
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
E1-1 E1-2 E9-2
Isolates
Op
tical
Den
sit
y (
600 n
m)
pH 5
pH 6
pH 7
pH 8
Fig. 5.17 AOX biodegradation by selected isolates at different pH (A1-A3)
and growth of isolates measured in terms of increase in absorbance
(B1-B3), Negative control (A4) and positive control (B4) [error bars
represents standard deviation of the mean of triplicate samples, not
shown when no larger than the symbols]
The ability of the selected isolates to degrade 2,4DCP and AOX was studied at
different pH because the isolates were to be used in bioaugmentation trials. It is a
common practice to alter the pH of the original soil during bioaugmentation trials
in order for the bacteria to work efficiently. But the isolates were found to be
versatile and retained their degradative ability at a broader pH range. This study
indicates the possibility of developing an economical bioaugmentation protocol
using these isolates.
Thus, the characterization of the isolates with respect degradation of chlorophenols
and AOX allows us to infer that they are promising for designing protocols for
bioaugmentation trials to remediate soil from AOX contamination.
A4 B4