isolation, screening and selection of dechlorinating...

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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


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


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.

http://www.ncbi.nlm.nih.gov/BLAST

http://www.ebi.ac.uk/

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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 E9-5

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



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

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=1423


http://www.ncbi.nlm.nih.gov/nuccore/225353978?from=1&to=1488&report=gbwithparts

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







isolates degrading chlorophenols and adsorbable organic halides

(AOX)

JOURNAL Unpublished



TITLE Direct Submission JOURNAL Submitted (17-DEC-2008) Microbial Sciences Division, Agharkar Research

Institute, G G Agarkar Road, Pune, Maharashtra 411004, India


source 1..1488



/strain="E1-2"



rRNA <1..>1488

/product="16S ribosomal RNA" ORIGIN

1 tcccggattc ccttttnggc agagtttgat ctggctcagg acgaacgctg gcggcgtgcc










601 caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa 661 actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt

721 agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg



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





132


DEFINITION Bacillus subtilis strain E9-1 16S ribosomal RNA gene, partial

sequence.

ACCESSION FJ573171

VERSION FJ573171.1 GI:225353980








(AOX)

JOURNAL Unpublished






source 1..1513



/strain="E9-1"



rRNA <1..>1513


1 ttcccggatt cccttttngg caagtttgat ctggctcagg acgaacgctg gcggcgtgcc










601 caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa 661 actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt

721 agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg



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





133


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.





(AOX)

JOURNAL Unpublished


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



source 1..1457

/organism="Brevibacterium stationis"


/strain="E9-2"



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





134


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.





(AOX)

JOURNAL Unpublished






source 1..1464

/organism="Staphylococcus sciuri"


/strain="E9-3"



rRNA <1..>1464


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





135


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.





(AOX)

JOURNAL Unpublished






source 1..1510

/organism="Staphylococcus sciuri"


/strain="E9-4"



rRNA <1..>1510


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





136


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.





(AOX)

JOURNAL Unpublished


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



source 1..1401

/organism="Brevibacterium stationis"


/strain="E9-5"



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





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



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



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



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

isolation, screening and selection of dechlorinating...

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