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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.
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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).
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• 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
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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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