Study on Microbial Diversity of Soils for AOX Degrading ...shodhganga.inflibnet.ac.in/bitstream/10603/3275/12/12_chapter 3.pdffunctional background of this technique is very well understood

38

Study on Microbial Diversity of Soils for AOX Degrading

Bacteria

3.1 Introduction

Microorganisms are ubiquitous, i.e., they are present in all niches where life can

exist even if their environment is under constant stress. One of the major stresses

induced is the contamination of these niches by man made compounds i.e.

xenobiotics. Enumeration and unequivocal identification of microorganisms

existing in polluted environment is important for understanding microbial ecology

as well as for bioremediation of such polluted sites (Nogales et. al., 20001; Widada

et. al., 2002; Yoshida et. al., 2005).

It is accepted that microbial diversity of soil is vast and the classical

microbiological techniques alone are inefficient to enumerate the total diversity.

Amann et. al. (1995) reported enormous discrepancies when they compared total

cell counts from different habitats with percentage of culturable bacteria. This

difference could be due to interdependence of microorganisms on each other or

lack of knowledge of specific growth requirements otherwise available to

microorganisms in their natural habitat (Muyzer and Smalla, 1998). Culture

independent or molecular techniques are widely used nowadays, especially for

contaminated environment, along with conventional culture dependent techniques

to determine the microbial diversity. Molecular techniques do not necessarily

correlate the presence of microorganisms with their ability to degrade pollutant

present in the same ecosystem. However these techniques have several advantages

over culture dependent techniques. Dominant species of microorganisms that best

adapts to the ecosystem can be more prominently detected using these techniques

as metabolically active microorganisms contain more DNA and RNA. In other

words ecologically important microorganisms are assessed with molecular

techniques and not the inactive ones which do not contribute to ecosystem

functions. Another major advantage of these techniques is that microbial

39

communities can be studied without actually cultivating the microorganisms

thereby preserving the in situ metabolic status and composition.

Polymerase chain reaction (PCR) and Denaturing Gradient Gel Electrophoresis

(DGGE) of 16S rRNA genes is widely used technique for studying microbial

diversity and community structure. DGGE has several advantages over other

fingerprinting techniques. It can give substantial information about species

composition from complex microbial communities. Sequencing of bands can

reveal phylogenetic information. The technique is easy, reliable, reproducible,

rapid and inexpensive. Community changes over time can be studied as the

technique allows analyses of multiple samples simultaneously. Theoretical and

functional background of this technique is very well understood (Muyzer and

Smalla, 1998; Muyzer, 1999).

In the present chapter bacterial diversity of seven soil samples collected from three

regions, viz., northern-, western- and southern- India. The sites investigated were

irrigated with effluent of pulp and paper industry. Total genomic DNA from

samples was extracted and 16S rDNA region was PCR amplified. Simultaneously

each sample was individually enriched in Davis Mingiolis Medium (DMM)

supplemented with AOX. Total genomic DNA from these enrichments was

extracted and 16S rDNA region was PCR amplified. The PCR amplicons were

then resolved by DGGE and separated bands were excised from the gel. These

bands were subsequently PCR amplified and cloned in to suitable vector. After

getting recombinant colonies on Luria Agar with appropriate antibiotic, plasmid

was isolated from the clones. 16S rRNA gene insert from recombinant plasmid was

PCR amplified and sequenced. The AOX contaminated soil samples were

characterized for their physico-chemical properties.

3.2 Materials and methods

3.2.1 Soil sampling

Soil samples of fields irrigated with PAP industry effluent, from seven different

regions of Indian subcontinent, were collected according to standard methods

40

described by Alef and Nanniperri (1995). The samples were analyzed for their %

C, N, mg% P, pH, % water holding capacity (WHC) and AOX content.

For organic C determination 1 g dried soil samples were taken in 100 ml

Erlenmeyer flasks. To these flasks 10 ml of potassium dichromate solution (5%

w/v) was added and shaken gently to dissolve soil. 20 ml concentrated sulfuric acid

was added slowly with gentle mixing. After cooling the mixture 50 ml barium

chloride solution (0.4% w/v) was added and the flasks were swirled to mix

contents thoroughly. The mixture was centrifuged at 10,000 rpm for 10 mins at

room temperature. Supernatant was directly used for organic C determination by

recording absorbance on spectrophotometer (UV- 1601, Shimadzu, Japan) at 600

nm. Organic C in the soil samples was determined by following formula:

(Ks - Ko) X 0.1/W X 0.74

Where Ks is sample concentration, Ko is blank, W is sample weight in g, 0.74 is

factor applied to correct for incomplete digestion and 0.1 is for conversion from g

per Kg to percent. Glucose was used as standard.

For N determination 0.2 g soil samples were digested with 4.4 ml digestion

mixture at 360oC for 2 h. Digestion mixture was prepared by adding 0.42 g

selenium powder and 14 g lithium sulfate to 350 ml 30% hydrogen peroxide

(H2O2). To this mixture 420 ml concentrated sulfuric acid was added while keeping

the flask in ice cold water. The digestion mixture was stored at 4oC. After digesting

soils the mixtures were transferred to 100 ml volumetric flask and volume was

made up. The solution was allowed to stand over night. For total N estimation 0.1

ml of digested solution was mixed with 5 ml of color reagent and was allowed to

stand for 15 mins Then 5 ml of alkaline hypochlorite solution was added. After 1 h

absorbance of the solution was recorded at 660 nm on spectrophotometer. Total N

was determined by the following formula:

C X 0.1/sample weight

41

Where C is sample minus blank concentration, sample weight in g and 0.1 factor

for conversion in percent. Ammonium sulfate was used as standard.

For total P estimation 0.5 g finely ground soil samples were added to 250 ml

beaker. To this 5 ml of concentrated sulfuric acid was added and after mixing 3 ml

of 30% H2O2 in 0.5 ml portions was added to the mixture. The mixture was shaken

vigorously and H2O2 reaction was allowed to subside. Then 1 ml of hydrofluoric

acid in 0.5 ml portions was added to this mixture. The beakers were kept at 150oC

for 10-12 mins. After cooling the mixture 15 ml water was added to the beaker and

the mixture was allowed to cool further. Mixture was then transferred to 50 ml

volumetric flasks and volume was made up. To this 1 ml digested mixture 1 ml of

working color reagent was added and volume was made up to 10 ml. Color reagent

was prepared by dissolving 20 g ammonium molybdate and 0.5 g ammonium

potassium tartarate in 600 ml deionized water. To this solution 250 ml

concentrated sulfuric acid was added and volume was made up to 1000 ml. This

reagent was stored at 4oC in dark. Working color reagent was prepared freshly by

adding 1.5 g ascorbic acid in 100 ml stock color reagent. The solution was allowed

to stand for 30 mins in dark. Absorbance of the solution was recorded at 882 nm on

spectrophotometer. Total P was determined by following formula:

Cs X 10 X Vo/Va X W

Where Cs is sample solution concentration, Va is volume of aliquot in ml, Vo is

volume of extract in ml and W is dry weight of soil sample. Final volume of

analyte is 10 ml. Potassium dihydrogen phosphate was used as standard.

pH of soil samples was determined by adding 25 ml of 0.01M CaCl2 solution to 10

g of air dried soil samples. The mixture was stirred continuously for 1 min. and

allowed to stand for 1 h. pH was then measured in supernatant after second short

stirring.

42

% WHC of soil samples was determined by placing 20 g field moist soil samples

on funnel with filter paper. The funnel is then mounted on preweighed Erlenmeyer

flask. Then 100 g distilled water was added in small portions to the soil samples

and allowed to stand overnight. The funnel was covered with aluminum foil to

prevent evaporation. Next day the funnel was tapped gently to move adhering

water drops into flask and the flask was weighed. % WHC was determined by

following formula:

[(100 – Wp) + Wi/dwt] X 100

Where Wp is weight of percolated water in g, Wi is initial amount of water in g

contained in sample and dwt is soil dry weight in g.

AOX content was analyzed using AOX analyzer (MultiX 2000, Analytik Jena,

Germany) as per DIN Standard 38414/18. 25 mg of dried soil samples were mixed

with 50 mg AOX carbon and 25 ml nitrate stock solution (17.0 g NaNO3; 1.4 ml

concentrated nitric acid – volume made up to 1000 ml) in Erlenmeyer flasks. The

flasks were then kept on shaker for 1 h and the mixture was filtered through Cl-

free polycarbonate filter (25 mm diameter, 0.45 µm pore size). The filter cake

portion was washed with nitrate wash solution (prepared by diluting 50 ml nitrate

stock solution up to 1000 ml) to remove inorganic Cl-. The cake was then packed

in AOX sample tube between ceramic wool. The cake was then combusted at

950oC and gaseous halides passed through concentrated sulfuric acid to remove

moisture. Dried gaseous halides were titrated with electrolyte solution in

coulometric cell. Silver ions needed for halide precipitation were generated

electrochemically at silver anode in the titration cell. Concentration of halides was

determined by amount of charge consumed until analyte conversion was complete

which is directly proportional to the concentration of halides.

3.2.2 DNA extraction

Soil DNA was extracted using Ultra Clean Soil DNA Isolation kit according to the

manufacturer specifications (MoBio Laboratories, Carlsbad, CA, USA). 0.25 g

43

soil sample was added to 2 ml beads solution tubes and the tubes were vortexed for

~1 min. Then 60 µl of solution S1 was added and the content of the tubes was

mixed by gently inverting tubes several times. 200 µl of solution IRS (Inhibitor

Removal Solution) was added and the tubes were secured horizontally on a flat bed

and vortexed at top speed for 10 mins. The tubes were then centrifuged at 10,000

x g for 30 secs. and the supernatant was transferred to a clean microcentrifuge

tube. 250 µl of solution S2 was added to the tubes, vortexed for 5 sec. and

incubated at 4oC for 5 mins. After incubation the tubes were centrifuged for 1 min.

at 10,000 x g and the supernatant was transferred to fresh microcentrifuge tubes.

1.3 ml of solution S3 was added to the tubes and vortexed for 5 sec. The mixture

was then loaded on spin filter and

centrifuged at 10,000 x g for 1 min. while

discarding the flow through. Then 300 µl of

solution S4 was added to the tubes and

centrifuged at 10,000 x g for 30 secs. Flow

through was discarded and the tubes were

again centrifuged for 1 min. Spin filter was

then placed in fresh microcentrifuge tubes

and 50 µl of solution S5 was added in the center of the filter membrane and

centrifuged for 1 min. at 10,000 x g. This step was repeated and the spin filter was

discarded. Presence of DNA was checked by agarose gel electrophoresis (0.7%)

with ethidium bromide was incorporated in the gel (Fig. 3.1).

A second set of soil samples (10 g) was enriched in DMM having composition (gL-

1): K2HPO4, 7.0; KH2PO4, 3.0; MgSO4.7H2O, 0.1; (NH4)2SO4, 1.0; yeast extract,

1.0 and trace element solution 1 mlL-1

spiked with AOX (30 mgL-1

), separately.

Trace element solution contained (gL-1

) FeSO4.7H2O, 0.5; ZnSO4.7H2O, 0.5;

MnSO4.3H2O, 0.5 and 0.1N H2SO4 10mlL-1

. DNA from this set was extracted by

Sigma GenEluteTM

Bacterial Genomic DNA kit (Sigma Aldrich, USA). After 48 h

AOX was analyzed and a second transfer was given in fresh DMM with AOX by

using 10% enrichment as inoculum. AOX analysis was carried out by AOX

analyzer as per DIN standard 38409/14. To 50 ml sample 2.5 ml of nitrate stock

1 2 3 4 5 6 7

Fig. 3.1: Genomic DNA of soil

samples. Lane 1-7: SS1-SS7.

44

solution was added to set pH to 2-3. The sample

was kept at 4oC for overnight. The sample was

then adsorbed on AOX sample tube, containing

activated carbon packed between ceramic wool,

by using two channel injection pump. 25 ml

nitrate wash solution was passed through the

sample tubes to remove inorganic Cl-. The tubes

were partially air dried and then combusted at

950oC for AOX estimation. AOX reduction in

second set was analyzed after 48 h and genomic

DNA was extracted. 2 ml of enrichment was used

to pellet cells by centrifuging at 16,000 x g for 2 mins at room temperature. For

Gram negative bacterial preparation the cell pellet was resuspended thoroughly in

180 µl of Lysis Solution T/Buffer STL. For Gram positive bacterial preparation

pellet from second set (duplicate) was resuspended in 200 µl of Lysozyme Solution

and incubated for 30 mins at 37oC. 20 µl of RNase A Solution was added to both

the sets and incubated for 2 mins at room temperature. Cells were prepared for

lysis by adding 20 µl Proteinase K solution and incubated at 55oC for 30 mins.

After incubation 200 µl of Solution C was added to the tubes, vortexed and

incubated at 55oC for 10 mins. After incubation 200 µl of ethanol was added to the

tubes and contents were mixed thoroughly by vortexing for 5-10 secs. GenElute

Miniprep Binding Column was prepared by adding 500 µl of Column Preparation

Solution and centrifuging at 12,000 x g for 1 min. Eluate was discarded. The

column was then loaded with cell lysate and centrifuged at 6500 x g for 1 min.

Eluate was discarded and 500 µl of Wash Solution I was added to the tubes. The

tubes were centrifuged at 6500 x g for 1 min. and eluate was discarded. A second

wash was given and the column was dried to remove traces of ethanol by

centrifuging at 16,000 x g for 3 mins Elution of DNA was done by adding 200 µl

of Elution Solution at the center of the column, incubating for 5 mins at room

temperature and centrifuging at 6500 x g for 1 min. Presence of DNA was

confirmed by agarose gel electrophoresis (0.7%) with ethidium bromide

incorporated in the gel (Fig. 3.2).

1 2 3 4 5 6 7

Fig. 3.2: Genomic DNA of soil

samples enriched in DMM with

AOX. Lane 1-7: SS1-SS7.

45

3.2.3 Polymerase chain reaction (PCR)

PCR was carried out in Master cycler gradient thermalcycler (Eppendorf,

Germany). Universal primer pair FDD2 (5‘ CCG GAT CCG TCG ACA GAG TTT

GAT CIT GGC TCA G 3‘) and RPP2 (5‘ CCA AGC TTC TAG ACG GIT ACC

TTG TTA CGA CTT 3‘) was used to amplify the bacterial 1.5 kb rDNA region

from soil DNA (Fig. 3.3). Nested PCR was set up using amplicon of first PCR

reaction as template. For nested PCR primer pairs Com1 with GC clamped Primer

1 (5‘ CGG CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG

GCC TAC GGG AGG CAG CAG 3‘) and Com2 (5‘ CCG TCA ATT CCT TTG

AGT TT 3‘) to amplify V3-V5 variable region of 16S rDNA (Fig. 3.4) and GC

clamped Primer 1 and SRV3-2 (5‘ TTA CCG CGG CTG CTG GCA C 3‘) to

amplify V3 variable region of 16S rDNA (Fig. 3.5), were used. Amplicons of the

second PCR were used for DGGE. The PCR reaction system contained: 2 µl of

DNA for 1.5 kb PCR and 5 µl for nested PCR, 0.2 µM of each primer, 200 µM

dNTP (dATP, dTTP, dGTP and dCTP), 2 µl of 10X PCR buffer, 0.5 U of Taq

DNA polymerase (Sigma Aldrich, USA) and sterile deionized water to a final

volume of 20 µl. PCR amplification was performed under the following

conditions: initial denaturation at 94oC for 5 mins; 30 cycles of denaturation at

94oC for 1 min, annealing at 56

oC for 1 min and extension at 72

oC for 1 min; and a

final extension step at 72oC for 20 mins Presence of PCR products was confirmed

by agarose gel electrophoresis (1.5%) and staining with ethidium bromide solution.

1 2 3 4 5 6 7 M

Fig. 3.3: PCR

amplification of 16S

rDNA region. Lane 1-7:

SS1-SS7, Lane M: 100

bp ladder

1 2 3 4 5 6 7

Fig. 3.5: 200 bp

nested PCR of 16S

rDNA region. Lane

1-7: SS1-SS7

600 bp 500 bp

1 2 3 4 5 6 7 M

Fig. 3.4: 600 bp nested PCR of 16S

rDNA region. Lane 1-7: SS1-SS7,

Lane M: 100 bp ladder

1000 bp 900 bp

800 bp 700 bp

400 bp 300 bp

200 bp

100 bp

46

3.2.4 DGGE

A Dcode Universal Mutation Detection System (Bio-Rad, USA) was used to

perform DGGE analysis. Methodology for casting gel was followed as described

by the manufacturer. The polyacrylamide gel was made from acrylamide stock

solutions containing 0% and 100% denaturants [100% denaturant is defined as a

mixture of 7 M urea and 40% deionized formamide (Sigma)]. Analysis of the PCR

products was done using 8% polyacrylamide gel. Electrophoresis was performed in

1X TAE (20mM Tris, 10mM sodium acetate, 0.5mM sodium salt of EDTA) at

60oC. For perpendicular DGGE gel was cast using glass plates of the size 7.5 cm X

10.0 cm and for parallel DGGE gel was cast using glass plates of size 16 cm X 16

cm with 1 mm spacers. Gels were stained with ethidium bromide solution and

image was captured using Alphaimager 2200 gel documentation system (Alpha

Innotech, USA).

3.2.4.1 Perpendicular DGGE

Perpendicular DGGE was carried out to determine the appropriate denaturant

range to be used in parallel DGGE. Two gels were cast with denaturant range 0%

to 100%. In one tube PCR products of soil samples 1, 2, 3 and 4 were mixed in

equal proportion and in second tube PCR products of soil samples 5, 6 and 7 were

mixed. Sample from tube 1 was applied directly on gel 1 as a continuous line

across the gel. Similarly sample from tube 2 was applied on gel 2. Electrophoresis

was performed for 2 h at 200 V.

3.2.4.2 Parallel DGGE

On the basis of results obtained from perpendicular DGGE, polyacrylamide gel for

parallel DGGE was cast with denaturant range from 30% to 70%. PCR products

from individual soil samples were loaded in separate wells. Electrophoresis was

performed for 6 h at 130 V.

47

3.2.5 Cloning of DGGE fragments

3.2.5.1 Excision of DGGE bands and PCR

Samples of individual DGGE bands of interest were obtained by excising small

cores of the gel with sterile 1000-µl pipette tips. These gel cores were added to

sterile 1.5 ml microcentrifuge tubes with 20 µl of sterile double-distilled water for

elution of the DNA from the gel (diffusion of DNA into the water overnight at

4oC). Five microliter of the DNA extract was used as PCR template for Com1-GC

Clamp – Com2 and Com1-GC Clamp – SRV3-2 primer sets using same PCR

program as described above. The PCR products were purified by PEG-NaCl (20%

Polyethylene Glycol 8‘000, 2.5M Sodium Chloride) to remove unincorporated

primers. The volume of PCR reaction mixture was made up to 100 µl with sterile

deionized water and equal volume of PEG-NaCl was added. The mixture was

mixed properly and incubated at 37oC for 1.5 h. DNA was precipitated by

centrifugation at 12,000 rpm for 20 mins, followed by two washes of 70% ethanol

by centrifugation at 12,000 rpm for 10 mins, each. DNA was dried at room

temperature to remove any traces of ethanol and then resuspended in 20 µl sterile

deionized water.

3.2.5.2 Generation of T overhangs

Vector DNA, pBluescript

(pBS) was linearized by

restriction digestion with

EcoRV (Bangalore Genei,

India). The reaction system

contained 1 µg of DNA, 0.2 µl

of 100X BSA, 40 units of

EcoRV, 2 µl of 10X buffer and

sterile deionized water to a

final volume of 20 µl.

Restriction digestion was carried out overnight at 37oC and the reaction was

stopped by incubating the system at 65oC for 20 mins Linearization was confirmed

by agarose gel electrophoresis (Fig. 3.6). Linearized vector DNA was purified by

1 2 3

Fig. 3.6: Linearized pBS vector using

EcoRV. Lane 1: Linearized pBS, Lane 2:

Supercoiled pBS, Lane 3: Supercoiled DNA

ladder

16,210 14,174 12,138 10,102 8,066

7,045 6,030 5,012 3,990 2,972

2,067

48

GeNeiTM

Quick PCR Purification Kit (Bangalore Genei, India). To 1 volume of

sample 5 volumes of binding buffer was added and loaded on spin column. The

tubes were centrifuged at 10,000 rpm for 1 min. Flow through was discarded and

500 µl of wash buffer I was added to the tubes. The tubes were centrifuged at

10,000 rpm for 1 min. and flow through was discarded. Wash buffer II was

prepared by diluting 1 volume of concentrated wash buffer II with 4 volumes of

95% ethanol. 700 µl of dilute wash buffer II was added to the tubes and

centrifuged for 1 min. at 10,000 rpm. Flow through was discarded and the tubes

were centrifuged for 5 mins to remove any traces of ethanol. Spin column was then

placed in fresh microcentrifuge tubes and 50 µl elution buffer was added at the

center of the spin column membrane. The tubes were centrifuged at 10,000 rpm for

1 min. This step was repeated and the spin column was discarded. Final volume of

purified product was reduced to 20 µl by concentration using vacuum dryer.

For addition of T overhangs the linearized vector DNA was dispensed in four

tubes, each tube containing 250 ng of DNA. The reaction system contained along

with DNA, 1 µl of 10X PCR buffer, 0.2 µM of dTTP, 5 U of Taq DNA polymerase

(Sigma Aldrich, USA) and sterile deionized water to a final volume of 10 µl. The

reaction was carried out at 72oC for 15 mins Reaction mixture was purified by

PEG-NaCl. Aliquots of vector DNA with T overhangs were prepared with 50 ng

DNA in each tube. The tubes were stored at -20oC and were used as and when

required.

3.2.5.3 Ligation of DGGE bands and pBS vector

Ligation was carried out at vector : insert of 1 : 2.5. The 10 µl reaction system

contained 50 ng of vector DNA, 125 ng of insert DNA, 10 U of T4 DNA ligase, 1

µl of 10X ligation buffer and sterile deionized water. The reaction was carried out

at 16oC for 21 h. After incubation whole reaction mixture was used to transform

competent cells.

49

3.2.5.4 Preparation of competent cells

E. coli strain JM109 was selected for transformation. A single colony was

inoculated in 50 ml Luria Bertani (LB) broth and incubated overnight at 37oC with

vigorous shaking at 300 rpm. After incubation the broth was transferred in 2 L

flask containing 50 ml sterile LB broth. The flask was incubated at 37oC with

shaking at 250 rpm till the OD600 reached ~0.9. To this 400 ml sterile LB broth was

added and further incubated till OD600 reached ~0.8. The cells were harvested by

centrifugation at 3,600 rpm for 15 mins at 4oC. Supernatant was decanted and cells

were resuspended in 100 ml ice cold TFB I buffer having composition:

CH3COOK, 30 mM; CaCl2.2H2O, 10 mM; MnCl2.4H2O, 50 mM; RbCl, 100 mM

and 15% glycerol, pH was adjusted 5.8 with 1M acetic acid and filter sterilized.

The cells were mixed by gentle shaking and harvested by centrifugation at 3,600

rpm for 15 mins at 4oC. Cells were then resuspended in TFB II buffer having

composition: PIPES disodium salt, 100 mM; CaCl2.2H2O, 75 mM; RbCl, 10 mM

and 15% glycerol, pH was adjusted to 6.5 with 1M potassium hydrooxide and filter

sterilized. Cells were mixed by gentle shaking and 100 µl aliquots were prepared in

1.5 ml sterile microcentrifuge tubes. The tubes were stored at -70oC.

Transformation efficiency of the competent cells was determined by using pUC19

as described by Sambrook et. al. (1989). Transformation of the competent cells

was done as described below wherein 1 ng of pUC19 was used instead of ligation

mixture. Transformation efficiency was calculated by the following formula:

No. of CFU/ng of DNA X 1000 = Y CFU/μg of DNA

3.2.5.5 Transformation

Total ligation mixture was used for transformation. Competent cells were thawed

on ice. The mixture was added to the tube containing competent cells, mixed by

swirling gently with pipette tip and was incubated on ice for 30 mins Heat shock

was given to the cells by incubating in water bath at 42oC for 2 mins followed by

cold shock by incubating on ice for 1 min. 900 µl of prewarmed (37oC) LB broth

was added to the tubes and incubated at 37oC by shaking at 250 rpm for 1.5 h.

Cells were harvested by centrifugation at 10,000 rpm for 10 mins at room

50

temperature. Supernatant was discarded and cell pellet was resuspended in 100 µl

fresh LB broth. LB agar plates containing 100 µg/ml ampicillin were spread with

X-Gal (X-Galactose) (10 µl of 100 mg/ml stock) and IPTG (Isopropyl β-D-1-

thiogalactopyranoside) (100 µl of 100 mM stock) and incubated at 37oC for 30

mins 100 µl of cell suspension was spread on these preincubated plates and the

plates were kept for incubation at 37oC. Clones were selected by blue white

screening procedure. They were subcultured on fresh LB agar plates containing

100 µg/ml ampicillin and simultaneously inoculated in 2 ml LB broth containing

100 µg/ml ampicillin for plasmid DNA isolation.

3.2.6 Isolation of plasmid DNA

Recombinant plasmids were isolated

from overnight grown cultures by

alkaline lysis method described by

Sambrook et. al. (1989). Cells from

overnight grown cultures were

harvested by centrifugation at

12,000 rpm for 5 mins at 4oC. Cell

pellet was washed with 1 ml

phosphate buffered saline (PBS),

twice, to remove debris. Then 200

µl of solution I (50mM Glucose;

25mM Tris.Cl, pH 8.0; 10mM EDTA, pH 8.0) was added to the cell pellet and the

mixture was vortexed for ~30 secs. 10 µl of RNase (1 mg/ml) was added to the cell

suspension and the mixture was incubated for 2-3 mins at room temperature. To

the cell suspension 400 µl of freshly prepared solution II (0.2N NaOH, 1% SDS)

was added. The mixture was mixed properly by gently tapping the tubes and

incubated for 2-3 mins at room temperature. Then 300 µl of solution III (5M

Potassium acetate, 60 ml; Glacial acetic acid, 11.5 ml; Deionized water, 28.5 ml)

was added to the tubes and the content of the tubes was mixed by gently inverting

the tubes for 3-4 times and were kept for incubation at -20oC for 5 mins The tubes

were then centrifuged at 12,000 rpm for 10 mins at 4oC and the supernatant (~800

1 2 3 4

Fig. 3.7: Plasmid DNA preparation by

alkaline lysis method. Lane 1-4:

Linearized and supercoiled plasmid DNA

from clones

51

µl) was collected in fresh tubes. To the supernatant 0.8 volumes, i.e., ~600 µl of

isopropanol was added and mixed by gently inverting the tubes for 3-4 times. The

tubes were centrifuged at 14,000 rpm for 10 mins at room temperature.

Supernatant was discarded and the plasmid DNA pellet was washed with ice cold

70 % ethanol by centrifuging at 14,000 rpm for 10 mins at room temperature. The

plasmid DNA pellet was dried to remove any traces of ethanol and resuspended in

40 µl sterile deionized water. Presence of plasmid was confirmed by agarose gel

electrophoresis (0.7%) and staining with ethidium bromide solution (Fig. 3.7).

Plasmid DNA band from the gel was cut using surgical blade and the gel piece was

added to sterile 1.5 ml microcentrifuge tubes with 50 µl of sterile double-distilled

water. The gel pieces were crushed with sterile 200 µl tips and the tubes were kept

overnight at 4oC for elution of the DNA from the gel (diffusion of DNA into the

water overnight at 4oC). Inserts in plasmid DNA were detected by PCR

amplification with GC clamped Primer 1 and SRV3-2 primer pair and purified by

PEG-NaCl. Quality of PCR product was checked by agarose gel electrophoresis

(1.5%) with ethidium bromide incorporated in gel.

3.2.7 DNA sequencing and phylogenetic tree

16S rDNA insert was sequenced using GC clamped Primer 1 in sequencing

reaction using Big Dye Terminator Kit (Applied Biosystems Inc., Foster City, CA).

The reaction system contained 5 μl of template DNA, 3 μl of sequencing buffer, 2

μl of premix, 0.2 μM of primer and sterile deionized water to a final volume of 20

μl. Cycle sequencing PCR was carried out in Master cycler gradient thermalcycler

(Eppendorf, Germany) under the following conditions: initial denaturation at 96oC

for 1 min; 25 cycles of denaturation at 96oC for 10 secs, annealing at 50

oC for 5

secs and extension at 60oC for 4 min. After the reaction was over, 2 μl of 125 mM

EDTA and 2 μl of 3M sodium acetate (pH 4.8) were added in the tube. The content

was mixed properly and transferred in 1.5 mL microcentrifuge tube. 50 μl of 95%

ice chilled ethanol was added to the tube and content was mixed properly. The tube

was incubated at room temperature in dark for 15 mins After incubation DNA was

pelleted by centrifuging at 12,000 rpm for 25 mins at room temperature.

Supernatant was discarded and DNA pellet was washed twice with 70% ice chilled

52

ethanol by centrifuging at 12,000 rpm for 10 mins at room temperature. DNA

pellet was air dried completely to remove any traces of ethanol.

To the DNA pellet 12 μl of Hi-Di Formamide was added and the tube was

vortexed briefly to resuspend DNA pellet. A brief spin was given and DNA was

loaded on the plate. Before actual run DNA was denatured by heating the plate at

94oC for 3 mins and then cooling to 4

oC. The sequencing reaction was run on ABI-

PRISM automated sequencer (3100 Avant DNA Analyzer).

ClustalW and DAMBE programs were used to edit, assemble and align all the 16S

rDNA gene sequences including the reference sequences obtained from GenBank

database. The sequence distance matrix for all pair wise sequence combinations

was analyzed by the use of MEGA 4.1 program with neighbor-joining method of

phylogenetic tree construction with 1000 bootstrap replicates.

3.3 Results and discussion

3.3.1 Soil properties:

Each soil sample was unique in terms of its physic-chemical properties (Table 3.1).

The pH of the soils ranged from 6.02 – 7.72. Soil Sample 1 (SS1) had highest C

content, which was evident from its dark color whereas SS3 had lowest C content,

evident from its red color. Overall C (%) content was in the range of 0.74 – 5.8.

Total N (%) and P (mg %) were in the range of 0.01 – 0.38 and 11.71 – 31.53.

Water holding capacity was in the range of 25.52 – 87. 15 %. AOX content

(mg/Kg) in soil ranged from 0.13 – 27.41. The results clearly indicate that the field

soil is contaminated with AOX.

53

Table 3.1: Soil sample properties

Sample C (%) N (%) P (mg %) pH Water

Holding

Capacity

(%)

AOX

(mg/Kg)

SS1 5.8 0.38 31.53 7.26 62.65 7.87

SS2 2.14 0.08 14.41 6.06 40.94 15.04

SS3 0.74 0.05 11.71 7.65 25.52 0.13

SS4 0.83 0.01 18.01 7.56 31.36 0.29

SS5 1.44 0.05 21.84 7.72 41.16 7.60

SS6 2.18 0.01 22.97 7.60 72.04 18.50

SS7 3.41 0.26 30.63 6.02 87.15 27.41

3.3.2 DGGE

PCR amplicons on perpendicular DGGE traveled at right angle to the denaturing

gradient forming a sigmoidal curve (Fig. 3.8). Minimum mobility of DNA was

observed at denaturant concentration ~80% and higher. There was significant

transition in the mobility of DNA over the denaturant gradient of 55 – 82%. The

same gradient was used for resolution of PCR amplified 16S rRNA gene fragments

by parallel DGGE.

54

The denaturant gradient was subsequently narrowed to improve the resolution of

PCR amplicons. Optimum resolution of the amplicons was finally obtained over a

gradient of 30 – 70% denaturant, at 130 V for 6 h. Figures 3.9 and 3.10 shows well

resolved DGGE profile representing microbial community of soil samples from

seven fields irrigated with PAP industry wastewater and soil samples enriched in

DMM along with AOX, respectively. SS1 showed maximum diversity as

compared to other soil samples. This could be due to high C content in the soil and

lower contents of toxic AOX. Soil samples 7, 6 and 2 had C content slightly less

than SS1 but AOX content was higher which can be the reason for lower diversity

of microbial community in these soil samples. Soil samples 4 and 5 had

comparable diversity with respect to soil samples 7, 6 and 2 though there was a

strong difference in the C and AOX contents of both the soils when compared with

other soil samples and amongst themselves. SS3 showed lowest diversity amongst

the seven soil samples tested. It also had lowest C content and AOX content. In

0 20 40 60 80 100 0 20 40 60 80 100

Fig. 3.8: Melting behavior of PCR amplicons of soil samples 1-4 (A) and 5-7 (B)

A B

55

case of enrichment diversity SS1 and 6 showed maximum diversity. SS2, 3 and 5

showed comparable diversity whereas SS4 and 7 showed the lowest. The

difference in the banding patterns for soil samples and their enrichment could be

due to higher AOX concentration. As DNA was extracted from enrichment after 48

h there is a possibility that only bacteria that can use parent AOX compounds were

present in high number whereas bacteria that used degradation products as C

source might be present in very low numbers. During total genomic DNA

extraction DNA copy number of such bacteria was less and due to biasness of PCR

technique their DNA did not get amplified.

1 2 3 4 5 6 7

Fig. 3.9: DGGE pattern of seven soil samples from fields irrigated with PAP industry

effluent. Lane 1-7: SS1-SS7.

11

12

13

116

115

114 113

111

17*

14*

13* 12* 11*

15*

21

22

23 24

25 26

27

28

31

32

33

72

74

75

76

61

62

63

65

66

64

51

52 53

54

56

57

58

41

42

43

44

45 46

48

47

18*

71

73 55 112

56

The diversity of soil samples from different regions can be grouped into ten major

groups viz. α-, β-, γ- and δ-Proteobacteria, Firmicutes with High GC content ,

Firmicutes with Low GC content, Bacteroides/Cytophaga, Actinobacteria,

Chloroflexaceae, Dictyoglomi whereas culturable diversity can be grouped into

five major groups viz. α-, β- and γ-Proteobacteria, Firmicutes with Low GC

content and Bacteroides/Cytophaga. The soil samples were composed of different

bacterial strains both unculturable and culturable, though there were some

overlaps. These overlaps were more prominent in case of culturable diversity. The

fact that there were overlaps for some of the genera, in spite of geographic

separation in the samples, underscores the potential importance of these genera.

1 2 3 4 5 6 7

Fig. 3.10: DGGE pattern of seven soil samples enriched with AOX. Lane 1-7: SS1-SS7.

13A

22A

61A

14A

11A 12A

23A

32A

31A

33A

51A

52A

21A 71A 72A

53A

63A

41A

42A

62A

64A

57

Table 3.2: Identification of clones

Soil

Sample

No.

Band

No.

Closest Phylogenetic Affiliation %

Homology

Total Diversity

1 11 Uncultured Burkholderia sp. clone DB3 16S ribosomal

RNA gene, partial sequence

94

12 Uncultured Klebsiella sp. clone S1-5 16S ribosomal


94

13 Uncultured Desulfonatronospira sp. clone 87 16S

ribosomal RNA gene, partial sequence

94

11* Uncultured bacterium isolate DGGE gel band W14 16S


98

12* No sequence similarity found in database



15 Bacillus subtilis strain nE2 16S ribosomal RNA gene,

partial sequence

98

16 Pectobacterium sp. R13 16S ribosomal RNA gene,

partial sequence

93

17 Uncultured Bacilli bacterium clone GASP-

KC2W1_F11 16S ribosomal RNA gene, partial

sequence

96

18 Uncultured Streptomyces sp. clone JAB SMS 34 16S


99

111 Leptothrix sp. MBIC3364 gene for 16S rRNA, partial

sequence

97

112 Bacillus sp. KSL5401-267 16S ribosomal RNA gene,

partial sequence

97

113 Bacillus chandigarhensis strain SK 47 16S ribosomal


98

58

114 Bacillus niacini strain NBPP53 16S ribosomal RNA

gene, partial sequence

98

116 Bacillus firmus strain NQ11 16S ribosomal RNA gene,

partial sequence

98

2 21 Bacillus sp. JC32 partial 16S rRNA gene, isolated from

Toluene

98

22 Uncultured Gemella sp. clone 12BA42 16S ribosomal


86

23 Brachybacterium sp. 5-1 16S ribosomal RNA gene,

partial sequence

98


partial sequence

98

25 Uncultured Bifidobacterium sp. clone FS29_G01 16S


99

26 Uncultured Actinobacterium clone 01QGJ 16S


99

27 Rhizobium sp. P146 16S rRNA gene, isolate P146 87

28 Brachybacterium conglomeratum strain G134a 98

3 31 Uncultured Raoultella sp. clone QRSYY3 16S


91

32 Desulfovibrio sp. enrichment culture clone Jdgsrb034

16S ribosomal RNA gene, partial sequence

93

33 Uncultured Acidobacteriaceae bacterium partial 16S

rRNA gene, clone AMBB2

97

4 41 Uncultured Burkholderiaceae bacterium clone

Amb_16S_887 16S ribosomal RNA gene, partial

sequence

97

42 Uncultured Rhizobiales bacterium clone

A10I2_INITIAL

99

43 Uncultured Haliscomenobacter sp. partial 16S rRNA

gene, clone CL3.D51

95

44 Bacillus pumilus partial 16S rRNA gene, strain 1Sm46

84

59


partial sequence

98

46 Uncultured Chloroflexi bacterium clone MSB-5D9 16S


90

47 Uncultured Actinobacterium clone 01QGJ 16S


100

48 Uncultured Syntrophobacterales bacterium partial 16S

rRNA gene, clone JML-70

87

5 51 Uncultured Flavobacteria bacterium clone GASP-

WC1W3_E06 16S ribosomal RNA gene, partial

sequence

98

52 Uncultured Chloroflexi bacterium clone 2627 16S


98

53 Uncultured Gemmatimonadetes bacterium clone

AKYH491 16S ribosomal RNA gene, partial sequence

97

54 Uncultured bacterium clone nbw33a01c1 16S


83

55 Uncultured Actinobacterium clone GASP-

KC3W3_B12 16S ribosomal RNA gene, partial

sequence

98

56 Bacillus badius strain B2S2 16S ribosomal RNA gene,

partial sequence

97

57 Uncultured Paenibacillaceae bacterium clone MFC87

16S ribosomal RNA gene, partial sequence

81

58 Balneimonas sp. S4c-b9 16S ribosomal RNA gene,

partial sequence

98

6 61 Arthrobacter sp. PN6 partial 16S rRNA gene, strain

PN6

93

62 Uncultured candidate division TM7 bacterium partial

16S rRNA gene, clone CM3H12

98


partial sequence

98


partial sequence

99

60

65 Janthinobacterium sp. Acj 119 gene for 16S ribosomal

RNA, partial sequence

98

66 Pseudomonas fragi strain R30 16S ribosomal RNA


92

7 71 Pseudomonas sp. R-35714 partial 16S rRNA gene,

strain W5a

94

72 Uncultured Acidobacteriaceae bacterium partial 16S

rRNA gene, clone AMBB2

92


partial sequence

96

74 Uncultured bacterium clone PCD-27 16S ribosomal


84

75 Paenibacillus zanthoxyli strain JH31 16S ribosomal


91

76 Massilia timonae strain HNL19 16S ribosomal RNA


97

Diversity with AOX enrichment

1 11A Acidovorax sp. XJ-L63 16S ribosomal RNA gene,

partial sequence

94

12A Comamonas sp. TS32 16S ribosomal RNA gene,

partial sequence

97

13A Aquincola tertiaricarbonis L10 16S ribosomal RNA


84

14A Sphingobacterium multivorum strain DW-1 16S


83

2 21A Brevundimonas diminuta strain 207 16S ribosomal


97

22A Comamonas sp. TS32 16S ribosomal RNA gene,

partial sequence

98

23A Chryseobacterium indoltheticum strain LMG 4025 84

3 31A Flavobacterium saliperosum strain AS 1.3801 16S


77

61

32A Alcaligenes sp. STC1 gene for 16S rRNA, partial

sequence

88

33A Flavobacterium saliperosum strain AS 1.3801 16S


77

4 41A Comamonas sp. TS32 16S ribosomal RNA gene,

partial sequence

98

42A Flavobacterium columnare strain Z13 16S ribosomal


87

5 51A Comamonas sp. AM13 16S ribosomal RNA gene,

partial sequence

84

52A Comamonas aquatica strain 530 16S ribosomal RNA


97

53A Bacillus firmus strain HU75 16S ribosomal RNA

(rrnE) gene, partial sequence

80

6 61A Comamonas sp. AM12 16S ribosomal RNA gene,

partial sequence

84

62A Mycoplana bullata strain 3P04AC 16S ribosomal RNA


95

63A Comamonas aquatica strain 530 16S ribosomal RNA


94

64A Sphingobacterium multivorum gene for 16S rRNA,

partial sequence

95

7 71A Comamonas aquatica strain st18f 16S ribosomal RNA


98

72A Sphingobacterium multivorum gene for 16S rRNA,

partial sequence

95

Sequence based identification (phylogenetic affiliation) of eluted DGGE bands is

shown in Table 2. Phylogenetic analysis (based on sequence homology) each of the

54 cloned bands revealed the genus Bacillus as the major phylotype in the AOX

contaminated soil samples. It was detected in four out of seven soil samples from

different regions. Less than 97% 16S rRNA gene sequence homology of clones

15*, 17*, 112, 113, 114, 116, 21, 44 and 56 with the closest match i.e. the genus

62

Bacillus indicated possible new/yet unreported species. Bacillus species are present

worldwide in soil samples and are known to be one of the most metabolically

active bacteria. Gilbride and Fulthorpe (2004) reported presence of two Bacillus

spp from secondary treatment systems of kraft pulp mill wastewater. They also

reported Bacillus in their studies on effect of chemical and physical parameters on

a pulp mill biotreatment bacterial community (Gilbride et. al., 2006). There are

reports on this genus as a well known degrader of environmental pollutants such as

4,5,6 Trichloroguaiacol by Bacillus sp (Tondo et. al., 1998), 2,4 DCP by B.

insolitus (Wang et. al., 2000) which coincides well with isolation of this genus

from more than one soil samples.

Brachybacterium was the second most frequent genus present in majority of

samples. Clones 23, 24, 28, 45, 63, 64 and 73 displayed similarities with

Brachybacterium with sequence homology from 96% to 99%. Sabdono and

Radjasa (2008) investigated the phylogenetic diversity of bacterial community

associated with organophosphorous degradation in corals of mid west coast of

Indonesia. Their analysis of 16S rDNA showed presence of Brachybacterium sp

alongwith Brevibacterium sp, Kyotococcus sp, Bacillus sp, Chromohalobacter sp

and Oceanobacillus sp.

The rest of the genera were found in one or more than one samples. Clones 11*,

54, 62 and 74 showed sequence similarity with uncultured bacterium clone while

for clones 12*, 13* and 14* no sequence similarity was found in database.

63

FJ897771.1

112

GQ497298.1

GQ150478.1

15

GQ475512.1

AY603657.2

GQ495047.1

AB518985.1

EU919206.1

FJ897757.1

116

FJ973528.1

DQ013307.1

113

114

GQ199756.1

17

FN394541.1

AB015048.1

18

AY694691.1

GQ410442.1

FM177043.1

111

16

GU097456.1

FJ982845.1

GU066861.1

13*

EU919218.1

14*

11*

EU876657.1

12*

GQ471864.1

13

11

12

Fig. 3.11 Phylogenetic dendrogram based on 16S rRNA sequence showing

the relationship of bands 11, 12, 13, 11*, 12*, 13*, 14*, 15, 16, 17,

18. 111, 112, 113, 114, 116 with the most closely related strains and

with each other. Bootstrap values (percentages of 1000 replications)

are shown at the nodes.

Bacillus sp. HU29

Bacillus sp. KSL5401-267

Bacillus subtilis strain nE2

Bacillus subtilis strain R372

Bacillus axarquiensis

Bacillus sp. M_22

Bacillus sp. MB73

Bacillus firmus strain NQ11

Bacillus firmus strain RKS160

Bacillus niacini strain NBPP53

Bacillus chandigarhensis strain SK 47

Bacillus sp. 210_54

Uncultured Bacillus sp. clone K18

Leptothrix sp. MBIC3364

Uncultured Streptomyces sp. clone JAB SMS 34

Uncultured Desulfonatronospira sp. clone 87 Uncultured Dictyoglomus sp.

Pectobacterium sp. R13

Uncultured Klebsiella sp. clone OHW7

Klebsiella sp. GSK

Uncultured Raoultella sp. clone QRSYY3

Uncultured Burkholderia sp. clone DB3

Uncultured Klebsiella sp. clone S1-5

64

FJ795660.1Brachybacterium

28

23

EF204388.1Brachybacterium

24

EU925627.1Brachybacterium

26

25

FJ518656.1Uncultured

27

22



21

AB518983.1Bacillus

FN293160.1Bacillus

AB518985.1Bacillus

AJ784178.1Rhizobium


the relationship of bands 21, 22, 23, 24, 25, 26, 27, 28 with the most

closely related strains and with each other. Bootstrap values

(percentages of 1000 replications) are shown at the nodes.

Brachybacterium sp 5-1

Brachybacterium conglomeratum strain G134a

Brachybacterium sp JSM 073009

Uncultured Bifidobacterium sp clone

FS29G01

Uncultured Gemella sp clone 12BA42 Uncultured Gemella sp clone 22B334

Bacillus sp MB71

Bacillus sp JC32

Bacillus sp MB73

Rhizobium sp P146

65

32

EU919218.1Uncultured

GQ503787.1Desulfovibrio


33

AM935718.1Uncultured

AM935622.1Uncultured

31

FM957865.1Aeromonas

FM957859.1Aeromonas


the relationship of bands 31, 32, 33 with the most closely related

strains and with each other. Bootstrap values (percentages of 1000

replications) are shown at the nodes.

Uncultured Raoultella sp clone QRSYY3 Desulfovibrio sp enrichment culture clone

Uncultured Shewanella sp clone QRSYY2

Uncultured Acidobacteriaceae clone AMBB2 Uncultured Acidobacteriaceae clone AMDG11

Aeromonas punctata isolate 08015 Aeromonas hydrophila isolate 08030

66

42

GQ242372.1Uncultured

43

FM175791.1Uncultured

EF018609.1Uncultured

41


FN423944.1Uncultured

48

44

DQ416787.1Bacillus

GQ487547.1Bacillus

45



47




46

DQ811880.1Uncultured

FM162975.1Bacillaceae

AM268213.1Bacillus





Uncultured Rhizobiales bacterium clone A10I2

Uncultured Haliscomenobacter sp

Uncultured Burkholderiaceae bacterium clone

Uncultured Nitrosomonadaceae bacterium clone

Uncultured Syntrophobacterales bacterium

Bacillus sp G2DM-52 Bacillus nealsonii strain Hb-0704

Brachybacterium sp JSM 073009 Brachybacterium sp 5-1

Brachybacterium conglomeratum strain G134a Uncultured Bifidobacterium sp clone FS29_G01

Uncultured Chloroflexi bacterium clone GASP-KB1S2-D03

Bacillaceae bacterium ACEMC 21-1 Bacillus pumilus

Uncultured Chloroflexi bacterium clone MSB5D9

67

56

EU221363.1Bacillus

GQ927170.1Bacillus

FN293160.1Bacillus


57

AB073206.1Paenibacillus


54

52






51



58

GQ344408.1Balneimonas

53


AY921664.1Uncultured

AY921665.1Uncultured




55





Bacillus badius strain B2S2

Bacillus firmus strain Z1-7

Bacillus sp JC32 Uncultured Bacillus sp clone GASP-WB2S3

Paenibacillus chondroitinus Uncultured Paenibacillus sp clone GASP-WB1S1_C04

Uncultured Chloroflexi bacterium clone 2627 Uncultured Chloroflexi bacterium clone 1969 Uncultured Chloroflexi bacterium clone g2

Uncultured Flavobacteria bacterium clone GASP-WC1W3_E06 Uncultured Flavobacteria bacterium clone GASP-WC2W2_G09

Uncultured Sphingobacteria bacterium clone GASP-MB1W3_C06 Uncultured Sphingobacteria bacterium clone GASP-WB1S2_H09

Balneimonas sp S4c-b9

Uncultured Gemmatimonnadets bacterium clone

Uncultured Gemmatimonnadets bacterium clone AKYH950

Uncultured Gemmatimonnadets bacterium clone AKYH491

Uncultured Actinobacterium clone



68

GQ339896.1Massilia

65

AB480763.1Janthinibacterium


GQ339887.1Massilia


66

AM886079.1Pseudomonas

62

FN397645.1Arthrobacter

63

AY275514.1Arthrobacter

64




61

AB490594.1Uncultured

AB490624.1Uncultured

AY972273.1Pseudomonas


the relationship of bands 61, 62, 63, 64, 65, 66 with the most closely

related strains and with each other. Bootstrap values (percentages of

1000 replications) are shown at the nodes.

Massilia sp PB248

Janthinibacterium sp Acj.119

Uncultured Janthinibacterium sp Massilia sp MK02

Uncultured Janthinibacterium sp

Pseudomonas sp R-35714

Arthrobacter sp PN6

Arthrobacter sp MSB2040

Brachybacterium sp JSM 073009 Brachybacterium conglomeratum strain G134a

Brachybacterium sp 5-1

Uncultured Bifidobacterium sp

Uncultured Bifidobacterium sp

Pseudomonas fragi strain R30

69

71

AM886079.1Pseudomonas

76

AY159797.1Massilia

74

EU373360.1Massilia




75

73

EF212892.1Paenibacillus

AM162327.1Paenibacillus

DQ358725.1Paenibacillus

AY972273.1Pseudomonas

72

EU143314.1Metschnikowia


the relationship of bands 71, 72, 73, 74, 75, 76 with the most closely



Pseudomonas sp R-35714

Massilia sp. B36

Massilia timonae strain HNL19

Brachybacterium sp. 5-1 Brachybacterium conglomeratum strain G134a Uncultured Massilia sp. clone U000107354

Paenibacillus urinalis strain 5402403 Paenibacillus sp. J16-10 Paenibacillus zanthoxyli strain JH31

Pseudomonas fragi strain R30

Metschnikowia sp. UWOPS04-218.3

70

EU841527.1Commamonas

12A

FJ976655.1Commamonas


DQ884343.1Pseudomonas

DQ656489.1Aquincola

DQ436455.1Aquincola

157841137Acidovorax

13A

11A

14A

FJ459994.1Sphingobacterium



the relationship of bands 11A, 12A, 13A, 14A with the most closely



GQ250440.1Brevundimonas

GQ891673.1Brevundimonas

EU730914.1Brevundimonas

EU434543.1Brevundimonas

21A

22A




23A

AY468448.1Chryseobacterium

AY468444.1Chryseobacterium

DQ208662.1Chryseobacterium


the relationship of bands 21A, 22A, 23A with the most closely



Commamonas aquatica strain 530

Commamonas sp. SP1 Commamonas aquatica strain st18f

Pseudomonas sp JS-1

Aquincola tertiaricarbonis L10 Aquincola tertiaricarbonis L108

Acidovorax sp. Ca8-2J04

Sphingobacterium multivorum

Sphingobacterium multivorum strain C2-30-2

Brevundimonas sp. MJ15

Brevundimonas sp. ZF12

Brevundimonas diminuta strain 207

Brevundimonas diminuta strain b55

Commamonas aquatica strain st18f Commamonas sp. SP1 Commamonas aquatica strain 530

Chryseobacterium indoltheticum strain LMG 4025 Chryseobacterium indoltheticum strain LMG 13342 Chryseobacterium sp.KL2C1

71








41A

42A

EU521691.1Flavobacterium


the relationship of bands 41A, 42A with the most closely related



31A


Commamonas sp TS32

Commamonas aquatica strain st18f

Flavobacterium sp R2A-7

72


52A



51A

53A

AJ746159.1Bacillus

FJ897758.1Bacillus

FJ226761.1Bacillus

GQ279347.1Bacillus






63A



61A

62A

FM213397.2Mycoplasma

EU977700.1Mycoplasma

FN397633.1Brevudimonas

FN397632.1Brevudimonas


AB100739.1Sphingobacterium

AB100738.1Sphingobacterium


64A

65A


the relationship of bands 61A, 62A, 63A, 64A. 65A with the most




Commamonas aquatica strain DNPA9 Commamonas aquatica strain 634

Bacillus sp MG102

Bacillus firmus strain RKS161

Bacillus flexus strain OS1 Bacillus flexus strain EP23



Commamonas sp SP1

Mycoplasma bullata

Mycoplasma bullata strain 3P04AC Brevudimonas sp MCS35 Brevudimonas sp CM282

Sphingobacterium multivorum Sphingobacterium multivorum Sphingobacterium multivorum

Sphingobacterium multivorum strain

C2-30-2

73




71A



72A


the relationship of bands 71A, 72A with the most closely related



Some of the clones could not be classified even upto genus level because of their

low sequence homology with the reference sequences available in the gene bank

database. Clones 11 and 41 showed sequence similarity with uncultured

Burkholderiaceae bacterium clone and uncultured Burkholderia sp clone with 97%

and 94% homology, respectively. Aitken et. al. (1998) isolated 11 strains from a

variety of contaminated sites (oil, motor oil, wood treatment and refinery) with the

ability to degrade Benzo(a)Pyrene. The organisms were identified as at least three

species of Pseudomonas, as well as Agrobacterium, Bacillus, Burkholderia and

Sphingomonas species. Rapp and Timmis (1999) studied degradation of

chlorobenzenes in liquid cultures and soil microcosms using Burkholderia sp.

Their results demonstrated that Burkholderia sp had a very high affinity for

chlorobenzenes at nanomolar concentrations. Nogales et. al. (2001) carried out a

study on combined use of 16S rDNA and 16S rRNA techniques for bacterial

community analysis of polychlorinated biphenyl polluted soil. They found that

Burkholderia sp was the abundant bacterial genus found in the contaminated soil

alongwith other genera like Sphingomonas, Pseudomonas, Acidobacterium,

Variovorax. Study carried out by Mahmood et. al. (2005) on cultivation

independent technique to analyze bacteria involved in PCP degradation and

sequencing of bands showed that Burkholderia was prominent degrader amongst

Commamonas aquatica strain st18f Commamonas aquatica strain 530 Commamonas aquatica strain 525

Sphingobacterium multivorum strain C2-30-2 Sphingobacterium multivorum

74

other bacteria identified. De Los Cobos-Vasconcelos et. al. (2006) reported

isolation of a strain of Burkholderia tropicalis from the Santa Alejandrina marsh,

Minatitlán, Veracruz, México. They studied co-metabolic degradation of

chlorophenols with this isolate and found that it was able to degrade 2 CP, 4 CP,

2,4 DCP and 2,6 DCP but was unable to degrade 2,4,5 TCP, 2,4,6 TCP and PCP.

The authors reported that the isolate was also able to degrade benzene, toluene and

xylene.

Clones 26, 47 and 55 showed sequence similarity with uncultured Actinobacterium

clone with 99%, 100% and 98% homology, respectively. Various genera such as

Rhodococcus, Gordonia, Mycobacterium, Streptomyces from actinobacteria class

have been reported for organochlorine degradation. (Tsitko, 2007; Benimeli et. al.,

2007; Cuozzo et. al., 2009).

Clones 33 and 72 showed sequence similarity with uncultured Acidobacteriaceae

bacterium with 97% and 96% homology, respectively. Ho et. al. (2007) studied

biological sand filtration for the complete removal of 2-methylisoborneol (MIB)

and geosmin. Community profile analysis in their study using 16S rRNA-directed

PCR-DGGE identified four bacteria most likely involved in the biodegradation of

geosmin within the sand filters and bioreactors. These included an

Acidobacteriaceae member, a Pseudomonas sp, Alphaproteobacterium, and a

Sphingomonas sp.

Clones 46 and 52 showed sequence similarity with uncultured Chloroflexi

bacterium clone with 90% and 98% homology, respectively. Yoshida et. al. (2005)

constructed microcosms to study dechlorination of polychlorinated dibenzo-p-

dioxins/dibenzofurans by seeding with polluted river sediment. Phylogenetic

characterization of these microcosms detected a significant number of Chloroflexi

clones alongwith other members.

Clones 57 and 76 showed sequence similarity with uncultured Paenibacillaceae

bacterium clone and Paenibacillus zanthoxyli strain JH31 with 81% and 92%

75

homology, respectively. Singh et. al. (2009) isolated Paenibacillus sp. D1 from the

effluent treatment plant of a seafood processing industry. Their study showed that

this isolate had a broad spectrum tolerance towards a number of pesticides at very

high concentrations. The isolate was also tolerant to many different fungicides and

most commonly used insecticides such as organophosphate, carbamate and

cyclodiene organochloride classes. Daane et. al. (2002) established that bacteria

belonging to genus Paenibacillus, isolated from the petroleum-contaminated

sediment and salt marsh rhizosphere can use naphthalene or phenanthrene as a sole

carbon source and can degrade the PAHs. They also isolated and characterized

PAH-degrading bacteria associated with the rhizosphere of salt marsh plants. They

categorized the isolated bacteria into three main bacterial groups - the

grampositive, spore-forming group, Paenibacillus, gram-negative Pseudomonas

and gram-positive, non-spore forming Nocardioforms. They observed that

phenanthrene-enriched isolates were able to utilize a greater number of PAHs than

are the naphthalene-enriched isolates (Daane et. al., 2001).

Clones 66 and 71 showed sequence similarity with Pseudomonas fragi strain R30

and Pseudomonas sp R-35714 with 92% and 94% homology, respectively. Fava et.

al. (1995) reported that their bacterial culture identified as Pseudomonas pickettii

was able to completely degrade 2 CP, 3 CP and 4 CP. The culture had a broad

biodegradation spectrum and could degrade other compounds such as

chlorocatechols, phenol, benzoic acid, hydroxybenzoic acid and hydroquinone.

Romero et. al.. (1998) isolated Pseudomonas aeruginosa from a stream heavily

polluted by a petroleum refinery. The species was found to be actively growing

over high dosages of phenanthrene with complete removal of the pollutant in a

period of 30 days. Buitrón et. al. (1998) studied degradation of a mixture of

phenol, 4 CP, 2,4 DCP, 2,4,6 TCP by acclimated activated sludge and by isolated

bacteria. They reported that the bacteria isolated for acclimated activated sludge

were identified as Pseudomonas sp, Aeromonas sp, Flavomonas oryzihabitans and

Chryseomonas luteola. Lee et. al. (1998) isolated a PCP degrading bacteria from

PCP contaminated soil from Pusan, Korea which was identified as a member of the

genus Pseudomonas. There are various other reports on degradation of a variety of

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xenobiotic compounds and heavy metals by Pseudomonas (Pazlarová et. al., 1997;

Nogales et. al., 2001; Ellis et. al., 2003; Moharikar and Purohit, 2003; Junca and

Pieper, 2004; Mahmood et. al., 2005). Gilbride and Fulthorpe (2004) reported

Pseudomonas in more than one secondary treatment systems for kraft pulp-mill

wastewater. But in our study Pseudomonas was found in only two soil samples.

This could be due to presence of more recalcitrant AOX in other soil samples as

compared to soil samples 6 and 7, though concentration of AOX was highest in

these soil samples amongst all the samples tested. Fulthorpe and Allen (1995) in

their study on organochlorine removal from bleached kraft pulp and paper-mill

effluents reported that Pseudomonas was least effective in removing

organochlorine as compared to Methylobacterium and Ancylobacter aquaticus

which were also used in the study. They attributed this ineffectiveness to the ability

to remove recalcitrant AOX.

Clones 12, 13, 16*, 18* and 111 showed sequence similarity with uncultured

Klebsiella sp clone, uncultured Desulfonatronospira sp clone, Pectobacterium sp

R13, uncultured Streptomyces sp clone and Leptothrix sp MBIC3364 with 94%,

94%, 93%, 99% and 97% holmology, respectively. Ghanem et. al. (2007) isolated

chlorpyrifos degrading Klebsiella sp from Damascus wastewater treatment plant in

Syria. They reported that Klebsiella sp was able to utilize chlorpyrifos as a sole

carbon source in mineral medium. Yang et. al. (2010) studied atrazine degradation

by a consortium of Klebsiella sp A1 and Commamonas sp A2. They reported that

Klebsiella sp A1 could utilize atrazine as a sole carbon and nitrogen source. Their

study showed that the consortium had high atrazine mineralizing ability and was

insensitive to commonly used nitrogen fertilizers. Winter et. al. (1991) studied

organochlorine degradation from spent sulfite bleached plant effluents from a

paper mill using actinomycetes. Most of the isolates they isolated belonged to

Streptomyces and could degrade AOX from low and high molecular weight

fractions. Benimeli et. al. (2007) isolated five actinomycete strains from pesticide

contaminated sediment which were able to grow on lindane. The most promising

strain in their study which could utilize lindane as a sole carbon source was

identified as Streptomyces sp M7. Cuozzo et. al. (2009) reported dechlorinase

77

activity in Streptomyces sp M7 for lindane degradation. They found that cell

activity was four and a half times higher when grown with lindane compared to

glucose. Nakajima-Kambe et. al. (2009) investigated degradation activities of

bacteria that can degrade aliphatic polyesters on various aliphatic–aromatic

copolyesters (PBSTIL, PBST, and EcoflexTM

). They reported that strain TB-71,

Leptothrix sp showed the best degradation activity. Two other strains reported in

their study were Acidovorax delafieldii BS-3 and Paenibacillus amylolyticus TB-

13.

Clones 22, 25, and 27 showed sequence similarity with uncultured Gemella sp

clone, uncultured Bifidobacterium sp clone, Rhizobium sp P146 with homology

86%, 99%, and 87% homology, respectively. Hamdi and Tewfik (1970) studied

herbicide 3,5-dinitro-o-cresol (DNOC) degradation using 31 strains of Rhizobia

and 5 strains of Azotobacter. They reported that the rate of degradation varied from

slight to complete with varying tolerance to herbicide by different cultures. They

also reported that Rhizobia degraded herbicide via reductive pathway.

Clones 31 and 32 showed sequence similarity with uncultured Raoultella sp clone

and Desulfovibrio sp with 91% and 93% homology, respectively. Claus et. al.

(2007) investigated the capacity of Raoultella terrigena to degrade a nitroaromatic

explosive TNT. They reported that with low concentrations of nutrient

supplements the isolate could completely degrade TNT. Liang et. al. (2009)

isolated Raoultella sp X1 capable of degrading an organophosphorous pesticide

dimethoate. They found that the isolate showed poor degradation ability when the

pesticide was used as a sole carbon source but via co-metabolism could degrade up

to 75% of the pesticide.

Clones 42, 43 and 48 showed sequence similarity with uncultured Rhizobiales

bacterium clone, uncultured Haliscomenobacter sp and uncultured

Syntrophobacterales bacterium clone with 99%, 95% and 87% homology,

respectively. Carvalho et. al. (2006) studied degradation of fluorobenzene by

Rhizobiales strain F11. They reported that the organism had enzymes for ortho

78

cleavage pathway and not for meta cleavage pathway. Their findings suggested

that degradation was via 4 fluorocatechol with subsequently ortho cleavage and

partially via catechol.

Clones 51, 53 and 58 showed sequence similarity with uncultured Flavobacteria

bacterium clone, uncultured Gemmatimonadetes bacterium clone and Balneimonas

sp. S4c-b9 with 98%, 97% and 98% homology, respectively. Crawford and Mohn

(1985) studied removal of PCP from contaminated soil. They showed that

inoculating such soils, including actual waste dump soils, with cells of PCP

degrading Flavobacterium resulted in PCP removal. Yu and Ward (1996)

investigated biodegradation of PCP. They reported presence of three predominant

strains Flavobacterium gleum, Agrobacterium radiobacter and Pseudomonas sp

out of which Flavobacterium showed highest PCP degradation ability. Their study

found that rates of PCP degradation by individual isolates were lower than for the

three isolates combined.

Clones 61 and 65 showed sequence similarity with Arthrobacter sp PN6 and

Janthinobacterium sp. Acj 119 with 93% and 98% homology, respectively. Marks

et. al. (1984) reported degradation of 4-Chlorobenzoic acid by a mixed population

which was established from sewage sludge. Arthrobacter sp was isolated by them

and was shown to be capable of utilizing 4-chlorobenzoate. Paris and Blondeau

(1999) investigated bacterial population from activated sludge samples of a PAP

mill. They found four main bacterial groups with Arthrobacter as the major one.

Elväng et. al. (2001) studied 4 CP degradation with Arthrobacter chlorophenolicus

in liquid cultures as well as soil microcosms and demonstrated that inoculation

with this culture was effective for cleaning-up soil containing high concentrations

of 4 CP.

Clone 77 showed sequence similarity with Massilia timonae strain HNL19 with

97% homology.

79

In case of sequences of bands from enriched samples Commamonas sp was

detected in all except one sample. Bands 12A, 22A, 41A, 51A, 52A, 61A, 63A,

and 71A showed similarity to Commamonas with sequence homology from 84% to

98%. Sylvestre (1995) had reviewed PCB catabolic pathways in Commamonas

testosteroni B-356. Fedi et. al. (2001) analyzed fifteen bacterial strains, which

were isolated form polychlorinated biphenyl contaminated area and were able to

utilize biphenyl as sole carbon and energy source, for the presence of bph operon.

The authors found that most of the isolates belonged to genera Commamonas,

Ralstonia, Alcaligenes and Pseudomonas. Dercová et. al. (2006) used

organomineral complex (OMC) for bioremediation of pentachlorophenol (PCP)

contaminated soil. They found that bioaugmenting soil with Commamonas

testosteroni CCM 7350 alongwith OMC resulted in biodegradation of PCP without

any initial toxicity to the organism. Marrón-Mantiel et. al. (2006) isolated five

bacterial strains degrading 2,4 D from soil samples of central region of Mexico.

The isolates were identified as Commamonas sp, Pseudomonas putida, Klebsiella

oxytoca, Acinetobacter sp, Acinetobacter lwoffii. The authors reported more than

97% 2,4 D removal efficiency with these cultures in chemostat. Dong et. al. (2008)

characterized phenol degrading bacterial strains isolated from soil which belonged

to genera Commamonas, Pseudomonas, Acinetobacter and Cupriavidus.

Rests of the genera were found in one or more than one samples. Bands 14A, 64A

and 72A showed sequence similarity with Sphingobacterium multivorum with

83%, 95% and 95% homology, respectively. Pesce and Wunderlin (2004) reported

lindane biodegradation aerobically by a consortium of acclimated bacteria from

sediment at a polluted site on the Suquia River, Cordoba, Argentina. They isolated

four different bacteria which were identifed as Sphingobacterium spiritivorum,

Ochrobactrum anthropi, Bosea thiooxidans and Sphingomonas paucimobilis.

Macbeth et. al. (2004) studied use of sodium lactate to biostimulate deep, fractured

basalt at a U.S. Department of Energy site to bioremediate it from trichloroethene

contamination. They found that dechlorinating indigenous microbial community

got established at that site due to biostimulation and characterization of this

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community showed presence of Sphingobacteria, Bacteroides, Proteobacteria

along with other dominant members.

Bands 31A and 33A showed sequence similarity with Flavobacterium saliperosum

strain AS whereas 42A showed sequence similarity with Flavobacterium

columnare strain Z13 with 87% homology.

Bands 11A and 13A showed sequence similarity with Acidovorax sp XJ-L63 and

Aquincola tertiaricarbonis L10 with 94% and 84% homology, respectively.

Monferrán et. al. (2005) acclimated a subsurface microbial community to 1,2-

dichlorobenzene (1,2-DCB) and isolated a strain of Acidovorax avenae from this

community. They reported that this organism was capable of completely degrading

chlorobenzene, 1,2-DCB, 1,3-DCB and 1,4-DCB. Lechner et. al. (2007) isolated

three strains namely L10T, L108 and CIP I-2052 from methyl tert-butyl ether

(MTBE)-contaminated groundwater and from a wastewater treatment plant. On the

basis of physiological properties, DNA–DNA relatedness values and the

phospholipid and cellular fatty acid profiles the authors proposed the name

Aquincola tertiaricarbonis gen. nov., sp. nov for these strains.

Bands 21A and 23A showed sequence similarity with Brevundimonas diminuta

strain 207 and Chryseobacterium indoltheticum strain LMG 4025 with 97% and

84% homology, respectively. Philips et. al. (2008) studied endophytic bacteria

associated with prairie plants for their hydrocarbon degradation potential and

activity. They reported that Brevundimonas and Pseudomonas dominated

endophytic communities and were associated with high hydrocarbon degradation

and activity.

Bands 32A, 53A and 62A showed sequence similarity with Alcaligenes sp STC1,

Bacillus firmus strain HU75 and Mycoplana bullata strain 3P04AC with 88%, 80%

and 95% homology, respectively. Valenzuela et. al. (1997) assessed the ability of

Alcaligenes eutrophus to degrade 2,4 D, 2,4,6 TCP and other chlorophenol

mixtures in bleached kraft mill effluent microcosms. They reported that the culture

81

was able to degrade these compounds when bleached kraft mill effluent was

amended with mixtures of these compounds. Andreoni et. al. (1998) studied

Alcaligenes eutrphus for its ability to degrade 2,4,6 TCP from soil versus

indigenous soil microflora. Their results indicated that when Alc. eutrophus was

used as inoculum it was effective in degrading 2,4,6 TCP in short time. A study on

biodegradation and detoxification of a mixture of phenolic compounds was carried

out by Gallego et. al. (2003). They used mixed and indigenous cultures which were

isolated from polluted Buenos Aires river and identified as Alcaligenes and

Acinetobacter. El-Sayed (2009) reported isolation and characterization of two 4 CP

degrading bacteria from petroleum contaminated soil. The bacteria were identified

as Alcaligenes sp and Bacillus subtilis.

Gilbride and Fulthorpe (2004) in their studies on composition and diversity of

bacterial populations from secondary treatment systems of bleached kraft pulp-mill

wastewater reported some of the genera of bacteria, which were also found in this

study such as Acidovorax, Commamonas, Klebsiella, Burkholderia,

Flavobacterium, Paenibacillus, Rhizobium, Acidobacterium. Therefore, their

presence in soil samples irrigated with PAP industry effluent should not be a

surprise.

Some clones (11*, 62) showed sequence similarity with unknown clones from the

database while for some clones (12*, 13*, 14*) no sequence similarity could be

found in the database. These organisms could be prevalent in the nature but due to

our inability to characterize them these clones remained uncharacterized.

Sequences of excised bands, found in this study, were most closely related to

cultured and/or uncultured organisms capable of degrading organochlorines and

other environmental contaminants from different ecological niches. This study

gives us a lead to selectively enrich and isolate microorganisms capable of AOX

degradation. These microorganisms could then be subsequently used to develop a

process to bioremediate soil from AOX contamination.

Documents

Study on Microbial Diversity of Soils for AOX Degrading ...shodhganga.inflibnet.ac.in/bitstream/10603/3275/12/12_chapter 3.pdffunctional background of this technique is very well understood