Bioremediation of phenolic compounds in

contaminated soil using white rot fungus

Supervisor - Submitted by-
Dr. Anand Mohan Pritpal Singh
Reg. no- 3440070045
( Asst. professor ) Roll no- RB1R09A26
Lovely Professional
University
Date- 20/05/2012

INTRODUCTION
Bioremediation can be defined as any process that uses
microorganisms or their enzymes to destroy or render
harmful contaminants that are altering the environment.
Both fungi and bacteria are capable of doing
bioremediation
It can be used to degrade wide range of compounds like
Poly aromatic hydrocarbons, chlorinated phenols, explosives,
dyes, etc.
Mycoremediation
Process of using fungi in order to degrade various harmful
contaminants in environment
Can be used for treatement of contaminated soils and industrial
effluents
Most commonly used fungi are white rot fungus capable of
degrading lignin (ligninolytic fungi) Eg. Phanerochaete
chrysosporium , Trametes versicolor , Agaricus bisporus , etc
Ligninolytic enzymes of white rot fungus are extracellular and
the use of these enzymes generally forms the basis of our
bioremediation process

S.NO. FINDINGS REFERENCES
1










2.

Lignin is a three-dimensional, naturally occurring
polymer and it constitutes one of the most structurally
complex and therefore resistant materials to microbial
degradation. The ability of white rot fungi to mineralize
lignin is generally attributed to the secretion of
extracellular ligninolytic enzymes, mostly laccase

The degradation of lignin and other recalcitrant
compounds by basidiomycetes is mediated by the
coordinated action of an enzymatic system .
The expression of the enzymatic system involved in
the degradation of xenobiotics has been shown to
mainly depend on the culture conditions



Naresh Magan ,
Silvia Fragoeiro
and Catarina Bastos
. Mycobiology 38(4)
: 238-248 (2010)
The Korean
Society of Mycology





Renata Yamanaka;
Clarissa F. Soares;
Dcio R.
Matheusand and
Ktia M.G.
Machado
(2008)

Literature review
S.NO. FINDINGS REFERENCES
3.










4.






A main feature of laccase is its highly non-specific
nature with regard to the breakdown of substrates.
Many xenobiotics share at least one of many sub-
structures (e.g.,functional groups) present in the
lignin molecule. This explains the ability of white rot
fungi to tolerate and degrade such a wide range of
environmental organic pollutants

The key step in lignin degradation by laccase or
the ligninolytic peroxidases involves the
formation of free radical intermediates, which are
formed when one electron is removed or added to
the ground state of a chemical


Naresh Magan , Silvia
Fragoeiro and Catarina
Bastos . Mycobiology
38(4) : 238-248 (2010)
The Korean Society of
Mycology


Reddy C.A. and
Mathew, Z. 2001.
Fungi in
bioremediation. G. M.
Gadd Cambridge, U.K.:
Cambridge
University Press
S.NO. FINDINGS REFERENCES

5.











6.
Laccases oxidize aromatic pollutants, such as
phenols, in the presence of oxygen (1, 2, 8). In this
reaction, the substrates are oxidized by one electron to
generate the corresponding phenoxy radicals, which
either polymerize to yield a phenolic polymer or are
further oxidized by laccase to produce a quinone

The substrate nonspecificity of laccases has led to
them being examined as agents for the biodegradation
of xenobiotic compounds, and their ability to oxidize
compounds such as chlorinated phenols and
polyphenols as well as aromatic amines
MURALIKRISHNA
CHIVUKULA AND V.
RE
NGANATHAN(1995).




PATRICK J.
COLLINS, MICHIEL J.
J. KOTTERMAN, JIM
A. FIELD AND ALAN
D. W. DOBSON(1996)
S.NO. FINDINGS REFERENCE



7.





8.

Chlorophenols constitute a significant category of
pollutants and are major components of paper pulp
bleach plant effluents and its nearby soils.

The large-scale use of chlorophenols has led to the
contamination of terrestrial and aquatic ecosystems,
resulting in the classification of chlorophenols as
priority pollutants

White-rot species, particularly Phanerochaete
chrysosporium, have been used for decontamination of
PCP-polluted soils and aqueous effluents.

Khadar valli and
Michael.H.Gold
Journal of
bacteriology, Jan.
1991, p. 345-352





Lamar, R.T. and
Dietrich, D.M. (1990).
Appl. Env. Microbiol.,
56, 3093-3100

OBJECTIVE
1. Collection of white rot fungi from wood samples and purification of
culture.
2. Mass culture of white rot fungus on spawn for adjusting against different
variable parameters.
3. Optimization of ligninolytic activity for pH, temperature, time, carbon
source, nitrogen source, and surfactants using one variable at a time
approach.
4. Response Surface Methodology Standardization for multiple parameters
for phenol degradation and laccase activity optimization and checking the
activity under real time environment.
5. To reduce the toxicity level of soil by reducing 2-4- dichlorophenol
concentration in soil using optimized conditions.

SIGNIFICANCE
Soil pollution is one of the major threats to environment and to
biodiversity.
Compounds causing pollution needs to be degraded or inactivated
The use of bioremediation to remove pollutants is typically less
expensive than the equivalent physical-chemical methods.
Use of white rot fungus to degrade xenobiotics such as
chlorophenols is an efficient method as these group of fungi can
withstand extreme conditions and concentration of contaminants.

MATERIAL AND METHODS

1. FUNGUS SAMPLE :- White rot fungus samples were
collected from various sources ( on trees and in soil)
2. CULTURING :- Potato Dextrose Agar (PDA) was used for
culturing fungi. Sample of white rot fungus was cultured on PDA petri
dishes and was incubated at 30C for 5 to 7 days.
Sub culturing was performed after 8-10 days continuously until pure
culture was obtained. Tetracycline (antibiotic) was used as an
antibacterial agent.





3. SPAWN PREPARATION:- Boiled wheat (200g) was added
with 1% (w/w) calcium carbonate and 2 % (w/w) calcium sulphate in a
polyethene bag and autoclaved.
Autoclaved spawn bags are inoculated with the pure culture with 5%
w/w i.e. 10 g of inoculum in 200 g spawn bag and incubated at 30C for
12 to 15 days.
4. ENZYME ACTIVITY ASSAY :- Laccase enzyme assay
was carried out using ABTS (2,2'-azino-bis 3-
ethylbenzothiazoline-6-sulphonic acid) as a substrate.
Stock solution of ABTS was prepared by adding 0.164g ABTS
in 30 ml distilled water (10mM).
Working solution was prepared by diluting 10 mM ABTS to
1mM ABTS with 0.1 M acetate buffer.
Assay was carried out by mixing 1 ml of 1 mM ABTS with 50l
of enzyme.
Activity = 700* ( A
i
A
0
)
Inc. time(t)

5. OPTIMIZATION (OVAT) :- The conditions for the excess
yield of laccase have to be optimized.
One variable at a time approach was as first step for optimization.
Each packet contained single variable with 10 g of wheat straw
and 30 ml czapadox medium.
Media constituents include KCl (Potassium chloride), MgSO
4
(
Magnesium sulphate), NaNO
3
( Sodium nitrate ) and the variable
selected.
List of variables that have to be selected are enlisted in the table.

S. No. Variables Sets
1. Carbon Source Glucose , Starch , Galactose , Cellulose
, Dextrose , Lactose , Sucrose , Fructose
, Control ( 0.6 g for each source )
2. Nitrogen Source Urea , Peptone , Yeast Extract ,
Ammonium chloride, Sodium nitrate ,
Control ( 0.6 g for each source)
3. Metals NaCl , KCl , MgCl2, FeCl3, CaCl2 ,
CuSo
4 ,
Control (0.15 g for each metal)
4. Temperature 27 C, 30 C, 32 C, 37 C
5. pH 4.0 , 4.5 , 5.0 , 5.5 , 6.0 , 6.5 , 7.5 , 8.0
, 8.5 , Control ( 7.0 )
6. PREPARATION OF SOIL SAMPLE :- Soil sample was
collected and mixed with 2-4-DCP (200 mg/l) in ratio 1:1 i.e. 50 ml
phenol solution (2mg 2-4 dichlorophenol in 1litre distilled water) in
50 g soil. Allowed the samples to stay for 48 hrs



7 RESPONSE SURFACE METHODOLOGY:-Response
surface methodology (RSM) is a collection of mathematical
and statistical techniques for empirical model building.
The objective was to optimize a response (output variable)
which is influenced by several independent variables (input
variables)
Box-Behnken taking glucose as a carbon source and Tween
-80 as a surfactant along with pH, temperature, inoculum
amount and incubation time was designed having 54 runs.
DESIGN SUMMARY(Design Expert 8.0.7.1)
BOX-BEHNKEN DESIGN RUNS
After getting the design order, different run order experiments were
performed to find out phenol degradation and laccase activitys
experimental results.
White rot fungus was grown in each packet containing wheat straw
and czapexdox medium.
2-4-dichlorophenol (0.02%w/v) was added in czapexdox medium to
check the degradation ability of white rot fungus during solid state
fermentation.
Each packet (run order) contained 10g wheat straw, 30 ml
contaminated czapexdox medium and the corresponding variables
from DOE.

Growth was observed in each packet with respect to the
conditions. One set of control was kept without inoculum.
White rot fungus growth observed
8. PHENOL DEGRADATION ANALYSIS:-Phenol
degradation analysis was carried out using 4-aminoantipyrine
method. Phenolic materials react with 4- amino antipyrine in the
presence of potassium ferricyanide to form a stable antipyrine dye.
Added 1ml of sample in 5 M NH
4
OH +25 M of 4 -
aminoantipyrine and 1% potassium ferricyanide.
Centrifuged at 2000 rpm for 2 minutes followed by measuring
absorbance at 510 nm.
Degradation (%) = 100 [Absorbance
(control)
- Absorbance
(r.o.)
]
Absorbance
(control)

9. DEGRADATION OF 2-4-DCP:- Two approaches; Whole
cell culture approach and Treatment with crude laccase were used for
degradation of 2-4-DCP. Both approaches were followed using
optimized conditions.
In whole cell culture approach white rot fungus was grown on
czapekdox media containing 2-4 dichlorophenol and wheat straw.
2-4- dichlorophenol containing water was extracted from phenol mixed
soil. It was done by ultrasonication of soil samples followed by
vortexing and centrifugation at 10000 rpm for 10 minutes. Water
collected from phenol containing soil was subjected to biological
treatment with white rot fungus

Crude laccase treatment approach involves direct treatment of phenol
contaminated water extracted from contaminated soil with crude
laccase enzyme
Crude laccase was the supernatant left after centrifugation of cell
extract of white rot fungus grown on czapekdox media and wheat
straw.
Conditions optimized for obtaining maximum laccase activity using
response surface methodology were used to grow white rot fungus.
pH Temp Inoculum
Amount
Carbon
Source
Surfactant I.P.
6.4 30
0
C 10.00 2.00% (w/v) 0.22% (w/v) 27

RESULTS AND DISCUSSIONS

Pure culture was obtained and grown on spawn to get enough
culture so as to go for one variable at a time approach.
Maximum enzyme activity was observed at pH=6 (53.97
IU/ml) ,glucose as a carbon source( 48.3 IU/ml ), urea as a
nitrogen source (33.46 IU/ml) , CuSo
4
as metal source ( 46.62
IU/ml) and optimum temprature 32 degree celcius (24.85
IU/ml) during one variable at a time approach.
Enzyme activities during one variable at a time approach
analysis were given in the table.


1. Carbon Source was the first variable and in this glucose,
starch, cellulose and galactose had shown better activity
S. No. Carbon Source Absorbance at
t= 0
Absorbance
at t= t
1

Enzyme
Activity( IU/ml )
1. Control 0.761 0.892 9.17
2. Glucose 0.411 1.101 48.3
3. Fructose 0.203 0.294 6.37
4. Starch 0.798 1.319 36.4
5. Lactose 0.796 0.966 11.9
6. Cellulose 0.343 0.632 20.23
7. Galactose 1.133 1.456 22.61
S.No. pH Absorbance at
t= 0
Absorbance at
t= t
1

Enzyme
Activity(IU/ml)
1. Control (7) 0.394 0.440 3.2
2. 4.5 0.140 0.189 3.43
3. 5.5 0.499 0.763 18.48
4. 6.0 0.264 1.035 53.97
5. 6.5 0.411 0.908 34.79
6. 7.5 0.208 0.310 7.14
7. 8.0 0.348 0.466 8.26

2. Next variable is the different pH conditions and best
activity was observed at 6.0, 6.5 as shown below:-

3. Nitrogen Source is the next variable and it was clear
that best activity is achieved using urea and yeast extract
as nitrogen source as shown in the table below :-
S.No. Nitrogen Absorbance at
t= 0
Absorbance
at t= t
1

Enzyme
Activity (IU/ml)
1. NaNO3 0.349 0.549 14.00
2. Urea 0.456 0.934 33.46
3. Peptone 0.287 0.287 0
4. Yeast extract 0.551 0.825 19.18
4. Next variable was concentration of metals and the
best variables for which there was maximum activity are
copper sulphate and potassium chloride (KCl) as shown
in table below:-

S. No. Metals Absorbance at
t= 0
Absorbance
at t= t
1

Enzyme
Activiy(IU/ml )
1. Control 0.457 0.520 4.41
2. KCl 0.299 0.625 22.82
3. CuSo
4
0.421 1.087 46.62
4. FeCl3 0.226 0.227 0
5. CaCl2 0.200 0.668 32.76
5. Next variable was the incubation temperature and
optimum temperature was found to be in between 30
32 degree celcius.
S. No. Temperature
(degree
celcius)
Absorbance
at t= 0
Absorbance
at t= t
1

Enzyme
Activity
( IU/ ml)
1. 27 0.133 0.166 2.31
2. 30 0.149 0.251 7.14
3. 32 0.706 1.061 24.85
4. 37 0.259 0.265 0.42
9.17
48.3
6.37
36.4
11.9
20.23
22.61
0
10
20
30
40
50
60
L
a
c
c
a
s
e

a
c
t
i
v
i
t
y

(
I
U
/
m
l
)

Carbon source
Effect of carbon source on
laccase activity
14
33.46
0
19.18
0
5
10
15
20
25
30
35
40
NaNO3 Urea Peptone Yeast
extract
L
a
c
c
a
s
e

a
c
t
i
v
i
t
y

(
I
U
/
m
l
)

Nitrogen source
Effect of Nitrogen source on
laccase activity
laccase activity
3.2 3.43
18.48
53.97
34.79
7.14
8.26
0
10
20
30
40
50
60
Control
(7.0)
4.5 5.5 6 6.5 7.5 8
L
a
c
c
a
s
e

a
c
t
i
v
i
t
y

(
I
U
/
m
l
)

p H
Effect of pH on laccase activity
4.41
22.82
46.62
0
32.76
0
5
10
15
20
25
30
35
40
45
50
Control KCl CuSo4 FeCl3 CaCl2
L
a
c
c
a
s
e

a
c
t
i
v
i
t
y

(
I
U
/
m
l

)

Metals
Effect of metal on laccase activity
RSM EXPERIMENTAL RESULTS (BOX-BEHNKEN)
S.
No pH
Temper
ature
Inoculum
Amount Carbon Surfactant I.P %degradation
Laccase
Activity
1 6 25 6 2 0.3 20 70.05 24.01
2 6 25 6 2 0.1 20 61.13 16.17
3 5 30 8 3 0.1 20 50.34 9.8
4 7 30 8 1 0.1 20 65.32 14.35
5 5 30 6 2 0.2 10 40.56 6.16
6 6 35 10 2 0.1 20 66.47 19.39
7 6 30 8 2 0.2 20 77.73 33.88
8 6 30 10 1 0.2 10 60.81 22.75
9 6 35 8 2 0.3 30 81.21 23.17
10 7 35 8 1 0.2 20 59.8 12.95
11 6 25 8 2 0.3 10 47.32 11.9
12 5 25 8 3 0.2 20 38.47 5.32
S.No. pH
Temp
erature
Inoculum
Amount Carbon Surfactant I .P
%
degradation
Laccase
activity
13 6 35 8 2 0.3 10 46.71 16.87
14 5 35 8 3 0.2 20 43.54 7.42
15 7 30 10 2 0.2 30 80.88 29.26
16 5 30 10 2 0.2 30 60.67 12.39
17 6 25 10 2 0.3 20 70.27 27.93
18 6 30 8 2 0.2 20 79.88 34.93
19 6 30 8 2 0.2 20 74.32 33.81
20 6 35 8 2 0.1 10 40.37 11.76
21 7 35 8 3 0.2 20 62.99 13.58
22 6 35 6 2 0.3 20 67.26 17.57
23 6 35 10 2 0.3 20 68.08 18.13
24 6 30 10 3 0.2 10 62.17 23.73
S.No. pH Temperature
Inoculum
Amount Carbon Surfactant I .P
%
degradation
Laccase
Activity
25 6 25 10 2 0.1 20 69.9 28.07
26 6 30 10 1 0.2 30 82.5 36.98
27 6 30 10 3 0.2 30 83.79 37.1
28 5 30 10 2 0.2 10 44.38 8.12
29 6 30 8 2 0.2 20 76.94 35.91
30 6 35 6 2 0.1 20 65.18 17
31 5 30 8 1 0.3 20 49.94 9.17
32 7 30 8 3 0.1 20 68.7 18.83
33 6 30 8 2 0.2 20 79.23 35.49
34 5 30 6 2 0.2 30 59.51 13.93
35 6 30 8 2 0.2 20 81.42 37.54
36 6 30 6 1 0.2 30 77.16 24.08
S.No. pH Temperature
Inoculum
Amount Carbon Surfactant I .P
%
degradation
Laccase
Activity
37 6 35 8 2 0.1 30 70.84 19.67
38 7 30 6 2 0.2 30 76.08 22.19
39 6 30 6 1 0.2 10 54.14 10.22
40 6 30 6 3 0.2 10 63.21 11.06
41 7 30 8 3 0.3 20 70.88 20.58
42 7 30 10 2 0.2 10 67.74 22.75
43 5 35 8 1 0.2 20 36.89 5.81
44 6 30 6 3 0.2 30 78.91 18.76
45 7 25 8 1 0.2 20 59.54 14.28
46 6 25 8 2 0.1 30 71.28 17.15
47 6 25 8 2 0.1 10 53.81 10.99
48 6 25 8 2 0.3 30 73.75 18.76
S.No. pH Temperature
Inoculum
Amount Carbon Surfactant I .P
%
degradation
Laccase
Activity
49 7 30 6 2 0.2 10 64.96 13.72
50 7 30 8 1 0.3 20 74.57 19.18
51 5 30 8 3 0.3 20 56.79 10.08
52 7 25 8 3 0.2 20 63.85 16.59
53 5 30 8 1 0.1 20 49.3 8.89
54 5 25 8 1 0.2 20 40.62 4.83
ANALYSIS OF RESULTS
Experimental data obtained from experimental runs
was analysed using Design Expert 8.0.7.1.
Fit summary program looked for highest order model
that was significant for our data without any lack of
fit.
This program calculated the effects for all model
terms. Statistically significant model was detected
and suggested model was underlined. This became
the default model on model screen
Diagnostics Program determines the predicted value
of experimental data for each particular run.
MODEL DESIGN SUMMARY
Quadratic model was suggested for my design.
OPTIMIZATION OF PHENOL DEGRADATION
Optimization was done using the statistical model
generated during analysis process.
Optimization program searches the design space, using
the models created during analysis to find factor settings
that meet the defined goals.
This program needs to define the criteria required for
optimization of the response. To maximize the
degradation, goal would be "maximize" and the upper
limit would be 100%.

Lower limits were kept at 70%. After determining the
optimization criteria, this program looked through all the given
solutions to see which ones best meet our requirements as
specified in maximization criteria.

Solutions pH Temp
Inoculum
Amount
Carbon
Source Surfactant
I.P.
(days) %degradation
1. 6.39 30.43 10.00 1.92 0.30 30.00 89.8336
2. 6.29 30.10 10.00 1.68 0.22 27.45 88.0542
3. 6.38 30.22 10.00 1.82 0.30 30.00 88.9742
4. 6.19 31.17 6.00 2.43 0.30 29.59 87.7163
78 solutions were given for maximizing the degradation.
Solution 1 was selected as most desirable solution.
OPTIMIZED SOLUTIONS- PHENOL DEGRADATION
RESPONSE SURFACE PLOTS: SOLUTION 1
This plot represents the interaction between pH and temperature
towards phenol degradation.
Shape of response surface plot indicates that there was very high
interaction between pH and temperature Changes in pH and
temperature has very high impact on degradation efficiency.
In this plot, there was not much interaction being observed between
pH and carbon source.
Response surface lied between pH 6.00 to 7.00 and carbon source of
1.5 to 2.5%. Both of the factors were approximately contributing
equally to degradation efficiency.
In this response surface plot, no interaction was observed
between inoculum amount and surfactant
It indicates that these factors did not contribute for much
variation in degradation efficiency.
Laccase Activity Optimization
The criterias were specified for optimization of
laccase activity.
Lower limit was kept at 15 IU and upper limit was
kept at 40 IU.
Sixty three solutions were given for maximization of
laccase activity.
Most desirable solution for maximizing laccase
activity is given below:-


pH Temp
Inoculum
Amount
Carbon
Source Surfactant
Incubation
Period

Laccase
Activity
6.35 29.83 10.00 1.99 0.22 27.01 38.767
RESPONSE SURFACE PLOTS: LACCASE ACTIVITY
In this plot, interaction between pH and temperature towards
laccase enzyme activity was shown.
Optimized surface lied between pH range 6.0 to 7.0 and temperature
of 27
0
c

to 30
0
c.
In this plot, no interaction was observed between two factors
(inoculum amount and surfactant) which means that these factors
did not show much variation for the response (laccase activity).
In this plot, effect of pH and carbon source on response was shown.
Shape of graph indicted that both the factors are equally contributing
toward response


DEGRADATION OF 2-4-DCP USING OPTIMIZED
CONDITIONS
Whole cell treatment approach was found to be more
prominent than treatment of 2-4- DCP with crude laccase.
The optimal degradation of 2-4-DCP was found at pH 6.4,
temperature of 31
0
C, incubation period of 30 days and using
1.9% carbon source (glucose) and 0.3% surfactant (Tween 80).
Approximately 85% removal of 2-4 DCP from contaminated
soil was observed using whole cell treatment process.
Degradation of phenol was analysed using 4-aminoantipyrine
method.
Crude laccase enzyme treatment had shown 72% removal of 2-4
dichlorophenol.
Colour difference was observed between control and treated sample.
Spectrophotometric analysis of sample indicated the degradation of
phenol from sample.
Colour difference during phenol degradation analysis
References
Naresh Magan ,Silvia Fragoeiro and Catarina Bastos ;
Environmental Factors and Bioremediation of
Xenobiotics Using White Rot Fungi ; Mycobiology 38(4) :
238-248 (2010) The Korean Society of Mycology
Renata Yamanaka1; Clarissa F. Soares1; Dcio R.
Matheus2; Ktia M.G. Machado ; Ligninolytic enzymes
produced by trametes villosa ccb 176 under different
culture conditions ; Brazilian Journal of Microbiology
(2008) 39:78-84.
Muralikrishna Chivukula and V. Renganathan ; Phenolic
Azo Dye Oxidation by Laccase from Pyricularia oryzae ;
Applied and Environmental biology, Dec. 1995, p. 4374
4377 0099-2240/95/$04.0010 Copyright q 1995,
American Society for Microbiology

Patrick J. Collins , Michiel J.J.Kotterman,Jim A.Field, Alan
D.W.Dobson ; Oxidation of Anthracene and Benzo[a]pyrene
by Laccases from Trametes versicolor ; Applied and
environmental biology, Dec. 1996, p. 45634567 Copyright
q 1996, American Society for Microbiology
Khadar Valli and Michael H. Gold ; Degradation of 2,4-
Dichlorophenol by the Lignin-Degrading Fungus
Phanerochaete chrysosporium ; Journal of bacteriology ,
Jan. 1991, p. 345-352 Copyright 1991, American Society
for Microbiology
Richard T. Lamar and Diane M. Dietrich ; In Situ Depletion
of Pentachlorophenol from Contaminated Soil by
Phanerochaete spp.; Applied and environmental biology,
Oct. 1990, p. 3093-3100