CHAPTER-IV METHODOLOGY - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/4514/12/12_chapter...

Chapter – IV Ch. Rajani Kumari, Ph.D., Thesis 2010 Methodology

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CHAPTER-IV

METHODOLOGY

The materials and methods of the rhamnolipid production(under

section 4A),bioremediation of contaminated soil, sediment and sludges

using rhamnolipids( under section 4B) and the biodegradation of soil

contaminated with Anthracene using rhamnolipids(under Section 4C)

were presented in this chapter.


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SECTION- 4A

4.1 MATERIALS AND METHODS USED IN THE PRODUCTION OF

RHAMNOLIPIDS USING VARIOUS ECONOMICAL CARBON

SUBSTRATES.

4.1.1 Isolation of pseudomonas aeruginosa

Pseudomonas aeruginosa from contaminated soil was isolated by

taking ten grams of contaminated soil and suspended in 100ml of NaCl

0.9% following serial dilution technique and 100µL aliquots of the

suspensions was spread on Pseudomonas agar plates and incubated the

plates at 35oC for 48 h.

4.1.2 Identification of isolate

According to the protocol followed in Bergy’s manual of

determinative microbiology, the isolates of similar morphology and colony

forming units were sub cultured on Pseudomonas agar (75) separately

and then characterized according to cell morphology, Physiological and

biochemical tests. The species of Pseudomonas are mainly identified by

their fluorescent pigment formation viz Pyocyanin when grown on

Pseudomonas agar, its capability of lysis of casein, a milk protein and

blood heamolysis. A pure culture from Microbial Type Culture Collection

(MTCC), Chandigarh was also obtained and is used as reference culture.


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The identified isolate along with pure culture of Pseudomonas

aeruginosa were analyzed under FT-IR spectroscopy in which 2 mg of

cell biomass was mixed with 300mg KBr and disc was prepared at 20000

lbs pressure and were examined in FT-IR spectrophotometer with a wave

range of 4000-400 cm-1and the resolution of 2 cm-1(220).

4.1.3 Maintenance of the stock cultures

Cultures of Pseudomonas aeruginosa were maintained in

Pseudomonas Agar slants (Table 4.1). The culture was streaked on

Pseudomonas agar with a sterile inoculation loop and sealed with a

cotton plug. After 24 hours of growth, the slant cultures were preserved

under refrigeration (4oC) until further use cultures were revived for every

15 days.

Table 4.1.1: COMPOSITION OF PSEUDOMONAS AGAR

Ingredient used Amount added

Gelatin peptone 16 g

Casein hydrolysate 10 g

Potassium sulphate 10 g

Magnesium chloride 1.4 g

Glycerol 10 ml

Cetyl trimethyl ammonium bromide 200mg

Nalidixic acid, sodium salt 15 mg

Agar 11g

Distilled water 1 liter


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4.1.4 Inoculum and medium

The culture from the slants was inoculated into a conical flasks

containing mineral medium (54) of the composition: NaNo3-1.5, KCl -

1.1, 1.1-NaCl, 0.00028-FeSo4.7H2o, 3.4-KH2Po4, 4.4-K2HPO4, 0.5-

MgSO4.-7H2O, 0.5- Yeast Extract, and Trace element solution of 0.05 ml

whose composition (g/L) is 0.29 -ZnSO4.7H2O, 0.24-CaCl2.4H2O, 0.25-

CuSO4.5H2O, 0.17-MnSO4. 7H2O, 1%v/v glycerol and pH 7.2.the flasks

were incubated in a rotatory shaker at 250 rpm at 30 ºC for 5days.

4.1.5 Biomass estimation

The sample aliquot of 10ml was collected from the culture flasks at

24h interval till the end of the experiment and centrifuged at 4000rpm

for 10 min to remove cells. The pellet containing cells were washed with

sterile water and centrifuged again. The cells were then dried at 110oC in

an oven, and the dry weight of the cells was measured until constant

weight was attained.

4.1.6 Detection of rhamnolipid biosurfactants

Sigmund and Wagner (9) technique was used for the detection of

glycolipid–type biosurfactant synthesis. To detect biosurfactant

production, blue agar plates were prepared adding cetyltrimethyl-

ammonium bromide (CTAB) (0.2 mg/ml) and methylene blue (5µg/ml).

On these plates, a drop of microbial culture grown in the mineral

medium for rhamnolipid production was placed and incubated at 45°C


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for 24 h and observed for the formation of a dark blue halo around the

drop of culture.

i) Drop collapse method

To test the biosurfactant production Drop collapse method was one

of the easy, sensitive and qualitative methods (96). This technique is

based on the principle that a drop of a liquid containing a biosurfactant

will collapse and spread completely over the surface of oil. In this

method, each well of a micro titre plate was added with 2 µl of mineral

oil, equilibrated for 1 h at room temperature, and later 5 µl of the culture

was added to the surface of the oil. After 1 min the shape of the drop on

the surface of the oil was observed. The cultures that gives flat drops

with scoring system ranging from ‘+’ to ‘+ + + +’ corresponding to partial

to complete spreading on oil surfaces indicates the ability of

biosurfactant production of the cultures. Those cultures that give ‘-’

indicates the inability of biosurfactant production by the culture.

ii) The oil spreading technique

The oil spreading technique measures the diameter of clear zones

caused when a drop of a biosurfactant- containing solution is placed on

an oil water surface (136) 50 ml of distilled water was added to a large

Petri dish ,followed by the addition of 20 µl of crude oil to the surface of

the water. Ten micro liters of the culture were then added to the surface


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The height of emulsion layer E24(%) = --------------------------------------------X100 The height of total solution

of oil. The diameter of the clear zone on the oil surface was measured

and related to concentration of biosurfactant by using a standard curve

prepared with commercially available biosurfactant, surfactin at

concentrations ranging from 50 to 2000 mg/l. The diameter of triplicate

samples from the same culture of each strain were determined

iii) Emulsification assay

The emulsifying activity of biosurfactant was determined according

to Cooper and Goldenberg (49). Five milliliters of 0.5% (w/v) rhamnolipid

(crude extract) solution was mixed with 5 ml of hydrophobic substrates

like Kerosene in the test tubes (120x15 ml), vortexed to homogeneity and

left to stand for 24 h at 4 oC. Emulsifying activity was expressed as the

percentage of the total height occupied by the emulsion in the test tube.

Emulsification index was determined as follows:

A mixture of 2 ml supernatant and 3 ml kerosene (or diesel) was

vortically stirred for 2 min and the height of emulsion layer was

measured after 24 h to determine the emulsification index (49). The

equation used to determine the emulsification index (E24(%)) is as follows:


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4.1.7 LC-MS analysis of rhamnolipids

Mass spectral analysis of rhamnolipid was done in Shimadzu LC-

MS (LCMS-2010A). Analysis was performed at 400C (LC column oven)

and 850C (MS ionization chamber) with Luna 5µ C18 (2) 100A column

(250 x 4.6 mm). Acetonitrile and HPLC water (1:1) was used as solvent at

0.2 ml min-1. The column effluent from the LC was nebulized into an

Electron Spray Ionization (ESI) region under N2 gas for generating

molecular masses, ranging from m/z 200 to 800, which were detected in

negative mode.

4.1.8 Experimental protocol for the production of rhamnolipids by

pseudomonas aeruginosa using different carbon and nitrogen

sources

The experiment was conducted by using two carbon sources and

four Nitrogen sources. The production of rhamnolipids was observed

from initial day to 8th day. For each flask duplicate flasks were kept. The

two Carbon sources used were fried Groundnut Oil and Glycerol. The

Nitrogen sources used are Sodium Nitrate (NaNO3), Ammonium Chloride

(NH4Cl), Ammonium Nitrate (NH4NO3) and yeast extract. The rate of

production of rhamnolipids and growth of microorganisms were noted by

using UV-Visible Spectrophotometer.

The experiments were conducted in 250 ml Erlenmyer flasks. Each

flask contained 100 ml of Mineral medium, 10ml of culture and 2 ml /l


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of carbon source. A replicate for each carbon source was kept. A flask

with only mineral media and culture was also kept for comparision. A

control was kept with only mineral media.

Prior to sterilization the pH of the medium was adjusted to 7.2.

Then the flasks were sterilized in an autoclave at 200atm pressure for 15

minutes. After sterilization the culture was inoculated and flasks were

kept in the incubatory rotatory shaker. The carbon source in the medium

(i.e., glucose) was replaced by glycerol (40 g/L), groundnut oil (40 g/L) for

the experiments exploring the effect of carbon substrates on rhamnolipid

production. To investigate the effect of nitrogen source on rhamnolipid

production, addition of the nitrogen source in medium (i.e.NaNO3), NH

4Cl (50 mM), NH 4NO3 (50 mM), and yeast extract (10 g/L) were replaced.

For the experiments examining the effect of carbon to nitrogen (C/N)

ratio, the concentration of carbon source (groundnut oil) was fixed at 40

g/L, while the concentration of nitrogen source (NaNO3) was adjusted to

0.85, 2.125, 4.25, 8.5,and 17 g/L, resulting in a C/N ratio of 130, 52,

26,13, and6.5, respectively. After centrifugation of the culture medium at

8000rpm for 20 minutes, the supernatant was collected and analyzed for

rhamnolipid production and emulsification index. The centrifuged cells

were used to monitor biomass. The Rhamnolipid estimation was noted at

0, 48, 96,192 hrs.


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4.1.9 Rhamnolipid estimation

Total Rhamnolipid concentration was determined by measuring the

concentration of hydrolysis released rhamnose by the Whistler and

Wolfram method after ethyl ether extraction and acid hydrolysis of the

sample. The culture supernatant was obtained to analyze by

centrifugation at 6,000 rpm. Then 5 ml of this supernatant was

transferred into small borosilicate tubes. To these tubes one drop of 2N

HCl was added for rhamnolipid separation from the medium. If a lot of

rhamnolipids are present, the medium will become cloudy. It is then

extracted three times with 500-700 µl of diethyl ether and combined the

fractions in another small borosilicate tubes. The ether was evaporated

from all the tubes by putting the rack containing the tubes in a hot water

bath. The rediue at high concentration looks like a brown oily

substance. This residue was resuspended in 50mM Sodium bicarbonate.

Depending on the concentration, the rhamnolipids were diluted to 2-20

times. To 1 ml of this sample, 4.5 ml of Reagent A (90ml H2SO4 + 15ml

dist. H2O) was added. Heated in water bath for 10 minutes at 100 oC and

then cooled with cold water for some time. Then 0.1ml of Reagent B

(0.1ml of Thioglycolic acid + 2.9ml of distilled water) was added and

mixed well. The samples were kept in darkness for 3 hours. The

concentration of rhamnolipids was estimated using U.V-Visible

Spectrophotometer at 400 and 430nm. The concentrations of


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rhamnolipids were obtained by comparing with the standard graph

prepared for L-rhamnose (Hi media). Rhamnolipids were quantified as

Rhamnose content by a Colorimetric method by using standards of

Rhamnose. The Rhamnolipid content was calculated by comparing with

the standard graph of Rhamnose of various concentrations.

Standardization of Rhamnose was done according to Whistler and

Wolfram method, 1962 (40). Rhamnolipid concentration (RL) was

calculated using the formula

RL= [54.18(A400 –A430) –1.49] F

Where A400 and A430 are absorbances at 400 and 430 nm, respectively,

and F is the dilution factor.

Table 4.1.2 :Rhamnose standards showing absorbance at 400 and

430 in UV-Visible spectophotometer

Concentration of

Rhamnose (ppm)

A400 A430

0 0.00 0.00

8 0.285 0.080

16 0.436 0.115

24 0.542 0.076

32 0.652 0.085

40 0.738 0.064


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4.1.10 Separation and purification of rhamnolipids

After the growth of the cells for 192Hrs (8 days), cells were removed

by centrifugation and the pH of the supernatant was lowered to 2.0-3.0

using 2N HCL after overnight incubation at 40C to precipitate the

rhamnolipids. The precipitate formed thereafter was collected by

centrifugation. This precipitate was dissolved in 50mM sodium

bicarbonate buffer (pH 8.6) and acidified to pH 2.0-3.0. The final

precipitate was recovered by centrifugation and the rhamnolipid was

extracted with a mixture of chloroform and methanol (2:1). The solvent

was evaporated in vacuum. The residue was dissolved in methanol and

filtered through a 0.22µm filter (Millipore) and a honey colored

rhamnolipid was obtained.

SECTION – 4B

4.2 Materials and methods used in bioremediation of

contaminated soil, sediment and sludges using rhamnolipids.

Bioremediation studies were carried out for the industrially

polluted soils, sediments and sludges in microcosms to evaluate the

efficiency of bioremediation using sterile samples as controls. The

treatments were natural attenuation (ability of degradation of the

polluted sample naturally), biostimulation (adding nutrients to improve

the natural biodegradation rate) and bioaugmentation (addition of a


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microbial consortium previously isolated from contaminated soil and

acclimated to contaminated samples along with crude rhamnolipids and

nutrients)

4.2.1 MATERIALS

i) Soils

Soil samples were collected at IOC oil station where the crude oil is

stored and supplied to various stations. There is a regular spillage of oil

during transfer of oil from many years. Soil samples were collected from

the depth of 10-15 cm from various spilled locations and pooled up to

form a representative soil sample. The soil sample was grounded and

sieved through a 2mm sieve for chemical analysis according to standard

method (APHA, 1998) and reported in table 5.2.1.

ii) Sediments

Sediment samples were collected from Khazipally Lake from a

depth of 10-15cms of the lakebed at points of potential contamination

with industrial effluents, and passed through a 2mm to get uniform

sample.

iii) Sludge

Primary Sludge sample was collected from a treatment plant where

the effluents from different pharma and bulk drug industries were get

treated and discharged. The physico chemical analyses of the samples


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were carried out using standard methods (APHA, 1998). All the analysis

values were expressed as mean of triplicates and discussed in the

chapter results and discussion.

Plate 4.1: Contamination of Khazipally Lake with industrial

Plate 4.2 : Contaminated lakebed of Khazipally lake


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4.2.2 COMPOSITION OF NUTRIENT SOLUTION

The composition of nutrient solution was given in the table 4.2.1

4.2.3 EQUIPMENT

The instrumentation used for carrying out the present study

included Centrifuge (R24 Remi, India), UV-VIS Spectrophotometer,

Rotatory shakers, HPLC (Agillent), GC,

4.2.4 Acclimatization of pseudomonas aeruginosa for

bioaugmentation studies

Composite samples of contaminated sites were collected from

various points of potential contamination. These samples were sterilized

in an autoclave. Briefly, 5 g of above three samples were added to 100 ml

Table 4.2.1 : Composition of nutrient

Solution

S.No Ingredient used Amount added g/lit

1 NaNO3 2.0

2 K2HPO4 2.0

3 NH4SO4 0.8

4 MgSO4 0.8

5 Yeast Extract 0.1


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of nutrient broth and isolated Pseudomonas culture in three different

flasks aseptically and incubated for 48 h at 300 C in rotary shaker 50

rpm. After 48 h 5 ml supernatant of above culture is again transferred

into 3 different flasks containing 100ml nutrient broth and incubated for

48 h at 300 C. The carbon source was derived from the sample organic

matter added during acclimatization process. The cultures were placed in

an orbital shaker at 35oC and 250 rpm. The culture solution provided the

microorganisms with the mineral nutrients necessary for survival and

cell growth. 0.1 ml of the culture was plated on nutrient agar plates and

incubated at 37 oC for 24 h. This acclimatized culture was maintained on

nutrient agar slants and sub cultured every two weeks for further use.

Sub cultures were prepared every 5-7 days by transferring 2ml of full-

grown culture into125ml of fresh medium in a 250ml Erlenmeyer flask.

4.2.5 Experimental protocol for bioremediation studies

Three samples (1. Soil 2. Sediment 3. Sludge) were taken for

bioremediation studies. For each sample, four types of bioremediation

studies viz Natural attenuation, biostimulation, bioaugmentation and

combination of biostimulation and bioaugmentation were carried out in

duplicates (Plates 4.3, 4.4 & 4.5 )

• For each sample, one pair was sterilized and kept as controls. They

are CSO (Soil); CSE (sediment); CSL (sludge) .


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• In natural attenuation or biodegradation studies, the sample does not

receive any amendments and monitored for biodegradation. They are

NASO (soil); NASE (sediment) and NASL (sludge).

• In biostimulation studies, the samples receiving nutrients alone are

designated as BSSO1 (Soil); BSSE1 (Sediment); BSSL1 (sludge).

• In biostimulation studies, the samples receiving rhamnolipids alone

are named as BSSO2 (Soil); BSSE2 (Sediment); BSSL2 (sludge).

• In Biostimulation with both rhamnolipids and nutrients, the samples

were known as BSSO3 (Soil); BSSE3 (Sediment); BSSL3 (sludge).

• In the bioaugmentation treatment, the sterile samples are added with

Pseudomonas aeruginosa and nutrients are named as BASO1 (Soil);

BASE1 (sediment) and BASL1 (sludge).

• The sterilized samples bioaugmented with Pseudomonas aeruginosa

and rhamnolipids are BASO2 (Soil); BASE2 (sediment) and BASL2

(sludge).

• For the sterilized samples where Pseudomonas aeruginosa along with

both nutrients and rhamnolipids were added, they are known as

BASO3 (Soil); BASE3 (sediment) and BASL3 (sludge).

• In the treatment receiving both biostimulation and bioaugmentation

with the nutrients, rhamnolipids and Pseudomonas aeruginosa to the

non sterile samples were designated as BASSO (Soil); BASSE

(sediment); and BASSL (sludge).


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In each tray of surface area of 20 cm2 and volume of 150 cm3, the

corresponding sample of 200 gms was taken in duplicates for their

respective treatments. 50 ml of nutrient solution per kg of

soil/sediment/sludge was added for biostimulation and bioaugmentation

studies. The inoculum size was set to adjust the final CFUs between 106

and 107 per gram soil for each bacterium for biostimulation studies. The

biosurfactant solution that was obtained in the rhamnolipid production

experiment was applied at a concentration of 100ml kg-1of sample

(soil/sediment/sludge) for biostimulation and bioaugmentation

experiments. The samples were mixed regularly with sterile spatula for

through aeration. Deionized water was then added every day to the trays

to maintain moisture content of approximately 60% of the water holding

capacity. These conditions were applied to all treatments.

The samples in tray 1(control) was sterilized 3 times by autoclaving

at 121oC for 30 min. Natural attenuation with simple aeration was

evaluated in tray 2 which did not receive any nutrients, rhamnolipid or

culture supplementation.

Biostimulation with aeration, nutrients and rhamnolipid addition

was evaluated in trays 3, 4 & 5 individually and combinely.


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Plate 4.3: Experimental setup for biodegradation of soil

Plate 4.4: Experimental setup for Biodegradation of sediment


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4.2.6 Analytical procedures

Samples in the treatment units were sampled at 0, 2, 4, 6, 8, 12

and 16 weeks for chemical and microbiological analysis. The

performance of each treatment was examined by monitoring such as

total organic carbon (TOC), basal respiration, microbial biomass carbon

(Cmic), metabolic quotient (qCO2), dehydrogenase activity, and phyto

toxicity.

Composite samples were collected from different areas of the

microcosm for monitoring above-mentioned parameters and the

analytical procedures are given below.

Plate 4.5: Experimental setup for biodegradation of Sludge


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4.2.6.1 Total organic matter (TOM) and total organic carbon (TOC).

Determination of TOC values gives a gross measure of all forms of

organic carbon including organic contaminants and natural matter (189).

The total organic matter in sediments was determined using wet

oxidation method of Walkley and Black (1934) as described by Jackson

(95) which involves oxidation with dichromate and back titration of

excess dichromate with ferrous ammonium sulphate. 0.5 g of sample

(soil/sediment/sludge) concentrated H2SO4 was added and allowed to

stand for ½ hour. Then after 200ml of water was added along with 10ml

of Orthophosphoric acid, 0.2gms of Sodium Fluoride and 1ml of di

phenyl amine indicator. The contents of the flask were titrated against

0.5N ferrous procedure is followed for blank.

TOC was determined using the formula given by Navarro (180):

TOC= (TOM-9.33/1.745) where TOM is the total organic matter of the

sample.

4.2.6.2 Respiratory measurements and soil microbial biomass

Respiratory measurements were conducted in triplicate without

(basal respiration) and after addition of a growth substrate to the

samples (substrate induced respiration, SIR).

i) Basal Respiration

CO2 monitoring was performed by transferring 2g of sample from

different treatment units into a plastic vial. The vials were placed in


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closed 1liter glass jars. A glass vial containing 10ml 0.2N NaOH was

placed in each jar to trap CO2 resulting from substrate mineralization.

The NaOH trap was periodically replaced. BaCl2 (10ml) was added to

NAOH trap and the amount of CO2 produced by each microcosm was

determined by titrating with 0.1N HCl.

ii) Substrate induced Respiration (SIR)

Microbial biomass carbon (Cmic) was estimated by the SIR method.

Glucose (80g), (NH4)2SO4 (13g), K2HPO4(2g) are used as growth substrate

for the SIR test (150).Approximately 10 mg of the ground mixture of

growth substrate was mixed with 1g(dry weight) of the sample taken

from each unit of treatment. The microbial biomass (Cmic) was calculated

as mg microbial biomass-C/kg dry soil using the SIR method and using a

conversion factor of 30 (101).

iii) Metabolic quotient

The metabolic quotient (q CO2) is an important parameter for the

functioning of the nutrient cycle in the ecosystem (6). It is calculated as

ratio of basal respiration to microbial biomass carbon. The ratio of

microbial biomass carbon to organic carbon (Cmic/Corg) serves as a long-

term indicator of the efficiency of microbes to decompose organic matter.

iv) Dehydrogenase activity

The efficiency of the microbial community to utilize the organic

matter was investigated through enzymatic activity. Dehydrogenase


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activity in a sample is used to monitor microbial activity and as an index

of the total the by oxidative activity in a sample.

Biological oxidation of organic compounds can result in

dehydrogenation processes that are catalyzed by dehydrogenase

enzymes. These enzymes play an essential role in the oxidation of organic

matter by transferring hydrogen from microbial populations capable of

degrading organic substrates to the electron acceptor.

The metabolic activity of the microbial biomass was determined by

measuring dehydrogenase activity (DHA) based on the method optimized

by Mosher et al., (137). Analysis was initiated by adding 2.5ml deionized

water and 0.2 ml of 0.75% freshly prepared 2-p-iodophenyl-3-p-

nitrophenyl-5 p g phenyl tetrazolium chloride (INT) solution (pH 7.9) into

1gm of sediment/soil (dry weight equivalent). The sample was incubated

in the dark at 27oC for 22 h, and the INT-formazan (INTF) formed was

extracted by the addition of 5ml of methanol. The tube was then further

incubated in dark at 27oC for 2 h. The extracted INTF was filtered and

measured the absorbance at 428 nm on UV-VIS spectrophotometer.

Dehydrogenase activity was expressed as mg INTF formed Kg-1dry sample

h-1.

4.2.6.3 Phytotoxicity Assay and germination Index

The phytotoxicity of a sample was measured through

Germination Index. The germination index is inversely related to the


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presence of phototoxic substances in samples. Seeds of garden cress

(Lepidium sativum) were used to evaluate the phytotoxicity of samples,

Their germination potential was examined at 27+ 1oC in darkness

prior to the assays as a control for the (90% guaranteed) viability of the

seeds. About 5ml of each extract is pippetted into a sterilized Petri dish

lined with Whatman No 2 filter paper. Ten cress seeds are evenly placed

on the filter paper and incubated at 27+1oC in the dark for 48hrs and

triplicates were analyzed for each set of samples.

Germination Index is calculated as the percentage of seeds

germinated with 10 ml of extracts multiplied by the average length of

roots in mm expressed as percentage of a control with distilled water

(89).

The percentage of relative seed germination, relative root

elongation and germination indexes (GI) are calculated by the following

formula:

No of seeds germinated in extract Relative seed germination (%) = ------------------------------------------- X100

No of seeds germinated in contact

Mean root length in extract Relative root growth (%) = ----------------------------------------- X 100

Mean root length in control

(% Seed germination) x (% root growth) Germination index = -----------------------------------------------------

100


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4.2.7 Analysis of residual pollutants from the samples using FTIR

Corresponding samples (2 g) from each plot (collected from five

different sites and mixed) were collected, air dried and finely ground. The

samples were prepared by mixing the sample and KBr (FT-IR grade) in

the ratio of 1:100 and pressed into pellets using hydraulic press. The

pellets were analysed in Perkin Elmer spectrophotometer for FT-IR

analysis with the wave number ranging from4000– 400 cm-1. To compare

one spectrum to another, a linear baseline correction was applied.

4.2.8 Analysis of metallic pollutants from the samples.

To determine the metallic ions present in the samples,

approximately 3g of sample was digested with HCl and HNO3 (1:1) at

95oC until the mixture attained a constant colouration. The sample was

subsequently filtered and diluted with a 10% HCl solution for analysis

using AAS.


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SECTION 4-C

4.3 BIODEGRADATION OF ARTIFICIALLY CONTAMINATED SOIL

WITH ANTHRACENE USING RHAMNOLIPID BIOSURFACTANT

PRODUCED BY PSEUDOMONAS AERUGINOSA

In this study anthracene was taken as a model compound for

degradation studies. Anthracene was a basic substance for production of

anthraquinone, dyes, pigments, insecticides, wood preservatives and

coating materials. Anthracene was a nucleus for polymer soluble

pigments. Anthracene family compounds are base materials for

colorings. They have useful functions such as light and temperature

sensitivity, heat resistance, conductivity, emittability, and corrosion

resistance. Anthracene enters the ecosystem mainly through tobacco

smoke and ingestion of food contaminated with combustion products

that are generated during combustion processes.

Anthracene was a tricyclic aromatic hydrocarbon (three of benzene

like rings joined side by side –C14H10) derived from coal tar with melting

point of 218oc, and boiling point of 354 oC (figure 4.3.1). It was a

hydrophobic substance that can be soluble in most organic solvents

such as alcohols, benzene and chloroform.


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Figure 4.3.1: Structure of Anthracene

4.3.1 MATERIALS

4.3.1.1Glassware

All the glassware used in the experiments including test tubes,

pipettes, Petri plates, measuring cylinders, volumetric flasks, standard

flasks and culturing flasks were of Borosil branded glass.

4.3.1.2Deionized water

Deionized water used for rinsing the glassware, media preparation

and for chemical analysis was collected from model ss1-s deionizer from

Shiter scientific industries, Bombay.

4.3.1.3 Chemicals

All the chemicals used in the study were obtained from Hi-chem.,

Hyderabad.


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4.3.1.4 Instruments

The instruments used for carrying out the present study included

centrifuge (R 24 remi, India), spectrophotometer (ECIL, India), autoclave,

rotary evaporator, incubator, rotary shakers (NEOLAB, India), laminar

air flow (CLAS, India) etc

4.3.1.5 Organism

An Oxidase-positive, gram-negative, rod-shaped bacterium

Pseudomonas aeruginosa was used in the present study. The culture

was obtained from MTCC (Microbial Type Culture Collection)

Chandigarh.

4.3.2 METHODS

4.3.2.1 Determination of pH

pH in the experiments was determined by means of a digital pH

meter (model DPH 100) from Insta Instruments, India, and

4.3.2.2 Sterilization

Sterilization of the culture media and glassware was done by

autoclaving at 15 lbs for 15 minutes.

4.3.2.3 Estimation of Growth

Growth was determined in terms of increase in optical density at

A570 of the bacteria suspension which was directly measured in a

Systronics make Colorimeter at 570nm, taking uninoculated media as

blank.


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4.3.2.4 Preparation of mineral media

All the medium and chemicals required for cultivation and

isolation of mixed consortia of micro organisms were procured from Hi

media (74). The mineral medium (table 4.3.1 and 4.3.2) provides a

supportive for the growth of microorganisms. Media contains all the

nutrients in required amounts for the development of the cultures

present in the conical flask.

Table: 4.3.1 Composition of Mineral Medium

SL.No Ingredient used Amount added (g/lit)

1 NaHPO42H2O 7

2 KH2PO4 1

3 CaCl22H2O 0.1

4 Feric Citrate 0.02

5 MgSO4.7H2O 0.2

6 NH4Cl 1.07

7 Trace Element Solution 0.025

8 pH 7.2

Table: 4.3.2 Composition of Trace Element Solution

Sl.No Ingredient used Amount added (g/lit)

1 ZnSO4.7H2O 0.007g

2 CaCl2.4H2O 0.024g

3 CuSO4.5H2O 0.005g

4 MnSO4.7H2O 0.005g


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4.3.2.5 Preparation of soil samples

Garden soil from JNTU university campus, Hyderabad is collected,

air-dried, and sieved using a 2 mm sieve. It is later sterilized in an

autoclave. The soil was spiked with anthracene by shaking for 30s with a

vortex mixer at 15 minutes intervals to allow the acetone to volatilize and

to mix the soil at a concentration of 20 mg anthracene/ 1kg of garden

soil. Sterile inorganic salts solution (2.0 ml) was then added to bring the

soil moisture content to 80% The salts solution contained 0.8 g of

K2HPO4, 0.2 g of KH2PO4, 0.01 g of FeCl3 and 0.1 g each of NH4NO3,

MgSO4 7H2O andCaCl2 2H2O g/l of distilled water (pH 7.0).The wet

mixture was placed on a stainless steel plate and the acetone was

allowed to evaporate. The average total anthracene concentration in the

soil was 20mg kg-1. The mobilization experiments were carried out 12 h

after contamination since many studies have shown increasing HOC

sequestration as a function of the contact time with soil (86).

4.3.2.6 Acclimatization to anthracene by shake flask culture

method

In order to acclimatize the anthracene to the mixed cultures in

acclimatization phase by shake flask culture method, the acclimatization

studies were carried out to check the capacity of organisms to tolerate

the toxic compound and their capability to utilize the anthracene.


90

Initially the freshly prepared mixed culture solution were slowly

acclimatized to the lower concentrations of anthracene compound, by

taking the mineral media with anthracene compound in the range of 10-

50mg/L (10,20,30 &, 50 mg/L) in an Erlenmeyer conical flask and 10%

fresh culture were inoculated and incubated aerobically at 370C by

shake flask method on rotary shaker at 200rpm. The mineral media

provides a balance mixture of required nutrients that enhance growth of

microorganisms under laboratory conditions. Shaking was done on

rotary shaker. Constant shaking leads to the microorganisms for proper

aeration. The samples were collected and parameters like TOC, Microbial

Count and compound reduction were studied at regular intervals.

4.3.3 Experimental protocol

The amendments were prepared with each set receiving the

following treatments in duplicates.

• The first set of autoclaved spiked soil was treated as Control (C).

• The second set of non autoclaved treatment of spiked soil does not

receive any amendments and monitored for natural ability of

degradation. The samples were named as Natural Attenuation (NA).

• In the third set of treatment, the spiked soil was amended with

rhamnolipid and nutrient solution and the samples were named as

Biostimulation (BS).


91

• In the fourth set of treatment, the spiked soil was sterilized and

amended with acclimatized mixed consortia along with rhamnolipids

and nutrients. The treatment samples were named after

Bioaugmentation (BS).

In each microcosm, 100gms of spiked soil was taken in duplicates for

their respective treatments. The inoculum size was set to adjust the final

CFUs between 106 and 107 per gram soil. The soils are moistened by

adding deionised water for maintaining moisture content of 40% every 3

days until the end of the experiment. The biosurfactant solution was a

cell free broth obtained from the growth of pseudomonas aeruginosa

strain in mineral medium. This crude biosurfactant was applied at a

concentration of 10ml/100gm of soil .The inoculum for seeding spiked

soil was prepared by growing the consortium in nutrient broth for 24hrs

at 370 C .The nutrient solution described above (devised with the goal of

being a nitrogen source in the soil) was added to a concentration of 10ml

/100gm of soil for biostimulation and bioaugmentation studies.

Plate 4.6: Experimental Set up for Anthracene Biodegradation


92

4.3.4 Biodegradation study on spiked soil

The biodegradation study was carried out on the spiked soils of

autoclaved and non-autoclaved at regular intervals and sampling was

done at 1, 2, 4, 8, 12, weeks to evaluate the natural ability of the soil to

degrade the anthracene.

4.3.5 Total organic content

The TOC was calculated as per procedure given in Page 82.

4.3.6 Methodology for direct total microbial count

Soils usually are aerobic, mesophytic, heterotrophic, and occurs in

clusters or chains. The total number of calculatable bacterial

enumeration was done by using serial dilution and plating techniques.

Medium used for this is nutrient agar as it was the simple media for soil

sample. In this method, 1 gram of soil was added to 10 ml of saline in a

test tube, which is taken as original sample. From the original sample

1ml is transferred to the 2nd tube make the concentration of soil tube to

be 10-1 like that the dilution to be increased up to last test tube (10-7).

From this dilution, 0.1 to 0.5 ml inoculum was taken and plated on

nutrient agar using spread plate method. The colonies forming unit

(CFU) was used to count the number of bacteria in 1gm of soil and

estimated by using a formula.

CFU = no of colonies / volume of plates x dilution factor


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4.3.7 Analysis of the soil extract by gas chromatography

The analysis of extract was performed by gas chromatography

(Agilent technologies; model: 6890N). The GC was equipped with a split

injector (split ratio 50/1) and a flame ionization detector both set at

300oC; carrier gas was nitrogen 1.50 mL min-1; the column was fused

silica capillary column (30.0 m χ 0.32mm, film thickness 0.25 µm);

temperature programming was 60-320oC. Ramp 5OC min-1, injection

volume 1 µ L (139).

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