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1


Bioremediation of Hazardous Di(2
-
ethylhexyl)phthalate

In Situ

and
Ex Situ


Sailas Benjamin*
, S. Pradeep and M. K. Sarath Josh

Enzyme Technology Laboratory, Biotechnology Division,

Department of Botany, University of Calicut, Kerala


673 635, India.



*Author for correspondence

Fax: +91
-
494
-
2400269

E
-
mail: sailasben@yahoo.co.in















2


1.
Introduction

Plastic materials have found wide applications in every aspect of modern human life
characterized
with technological advancement
and the
increasing
global population

(
Al
-
Salem
et.
al.
,

2009
).

Plastics are typically polymers of high molecular mass, and may contain modifying
substances to improve performance and/or reduce costs

(Simpson,
1995)
. Plastics can be
classified by chemical structur
e,
viz
., the molecular units that make up the polymer's backbone
and side chains. Some important groups in these classifications are the acrylics, polyesters,
silicones, polyurethanes, and halogenated plastics like

poly vinyl c
hloride (
PVC
)
. Plastics can
also be classified by the chemical process used in their synthesis, such as condensation,
polyaddition, and cross
-
linking. Other classifications are based on qualities that are relevant for
manufacturing or product design; e
xamples of such c
lasses are the thermoplastic
s

and
thermoset
s
, elastomer
s
, structural, biodegradable, and electrically conductive. Plastics can also
be classified by various physical properties, such as density, tensile strength, glass transition
temperature, and resistanc
e to various chemical products. Due to their relatively low cost, ease of
manufacture, versatility, and imperviousness to water, plastics are used in an enormous and
expanding range of products

(Simpson,
1995)
.


1.1.
Phthalates

Phthalic acid esters (PAE
s
)
are a major group of industria
lly
-
significant organic chemicals found
its application in various chemical industries as plasticizer for plastic products

(
Liang
et. al.
,

2008
).

PAE
s

p
lasticizer is
the predominant

additive for PVC to soften it thereby manufa
cturing
different products.

Most negative effects associated with
these plasticized PVC
(pPVC)
products
are the
leaching

of

phthalates
from the
polymer

into the environment
.
D
ue to the absence of
chemical bonding
of plasticizer with
the polymer
, the plasticizer
gradual
ly

migrat
es in to the
surrounding environment
from
the
plastic products (Fig.
1).

Many factors like t
emperature, pH,
organic solvents
,
etc
. can

accelerate the rate of
migration. Some

of the commonly used
phthalates are given in Tab
le1.


1.2.
Di(2
-
ethylhexyl)phthalate

Di(2
-
ethylhexyl)phthalate (DEHP)

is one of the

high molecular weight
PAE
s
, and is the
most
commonly used plasticizer

(Fig
.

2).

They are blended in varying proportions with
PVC

(
Jenke
,
3


2006)
.

Flexible PVC formulations are
widely

used for the production of
various
articles being
used
in the medical field; such as
urine bags, transfusion tubing,
etc
., and are further used for a
wide variety of
consumer products, flooring and wall coverings, food
contact applications
,
personal care products like lubricants, waxes, cleaning materials

and

toys

(
Hauser & Calafat
,


2005)
.

The

surrounding environment (air, oil, water, blood,
etc
.) has a very high affinity for the
DEHP;

the migration rate is dependent upon its ability to diffuse through the resin in to the
surrounding attracting media

(
Sarath Josh
et.

al.
,
20
12;
Graham,
1973)
.


1.3.
Phthalate pollution


Due to rapid urbanization and economic development

happened

globally
, over usage of PVC and
other polymer products and
subsequent
dispersal of polymer waste increased. The search for safe
alternatives for PAE
s

resulted with too little success. Generally
,

ingestion
via

food, inhalation of
perfumes and dermal contact are con
sidered to be the ro
utes of PAE
s

exposure in humans

(Florence, 2009;
Heudorf

et. al.
,
2007)
.

Release of DEHP into the ecosystem or wastewater
effluent occurs during the production phase and also
via

leaching and volatilization from plastic
products during
their usage
and after disposal
(
Sarath Josh
et.

al.
,
20
12
).

Humans exposed to
DEHP showed carcinogenic, endocrine disrup
tion and reproductive problems

(Florence,
2009
;

Latini
et. al.
, 2004;

Matsumoto

et. al.
,
2008)
. In humans, PAE
s

are bio
-
transformed and
conjugated to glucuronides and excreted or stored in the body

(
Koch
et. al.
,
2004
)
.

Recent

report
showed

that
DEHP exposure may result in gender birth defect
s and the
feminization

of boys

(Sampson & De Korte, 2011)
.
Even though m
ig
ration of DEHP from the DEHP
-
PVC blood
storage bags (BB) into the stored blood and blood compone
nts has much been studied
, DEHP
-

p
PVC
is widely used in
medical devices
due

to their flexibility, convenience and cost
effectiveness.
Because of
their

potential
s

for human exposure and demonstrated
developmental
and reproductive
toxicity in animals, PAE
s

have
raised

conce
rn in the field
of public

health
,

(
Heudorf

et. al.
,
2007
;

Hill
et.

al.
,
2001
;

Shaz

et. al.
,
2011).


1.4.
Biodegradation

of
PAE
s

by fungi


4


Biodegradation is a critical process affecting the environmental fate of phthalate and phthalate
esters.
Microorganisms play the major role in the biodegradation process in various
environmental conditions;

it can be either by aerobic or anaerobic routes.
Many bacterial strains

and a

few fungi have been reported to degrade phthalates

(
Pradeep

et. al.
,
20
12
;

Pradeep &
Benjamin,
2012)
. We have tried to explore the potential of fungi in remediating PAE. There are
few studies previously done in this aspect
, in
1977

Engelhardt
et. al.

reported

the
fungal
degradation of diiso
butyl phthalate and related dialkyl phthalates by
Penicillium lilacinum
.

The
fungus
Sclerotium rolfsii

has been shown to catalyse the hydrolytic conversion of dimethyl

terephthalate to
terephthalate through the formation of monomethyl

terephthalate and the
presence of phthalate esterase was detected in the mycelia of
Sclerotium rolfsii

(
Sivamurthy

et.
al.
,
1991
)
.

Aspergillus niger

(AG
-
1) metaboli
z
ed dimethyl

terephthalate through monomet
hyl

terephthalate, terephthalate and protocatechuate. The quantitative UV analysis showed that 58%
of the dimethyl

terephthalate su
pplement was taken up in 144 h

(
Ganji
et. al.
,

1995
)
. The
application of two lypolytic enzymes (fungal cutinase
, yeast esterase)
for

the degradation of
phthalates was investigated. Compared to yeast esterase,

the degradation rate of fungal cutinase
produced by
F
usarium

oxysporum
for different phthalates was surprisingly hi
gh and
showed
high preference to

butylbenzy
l phthalate (BBP)

(
Kim
et. al.
,
2002
)
, DEHP

(
Kim
et. al.
,
2003
)
;
dibutyl phthalate (
DB
P)

(
Kim
&

Lee
,
200
5),
dipropyl phthalate (
DPrP
)

(
Kim
et. al.
,
2005
)
,
dipentyl phthalate

(DPP)
(
Ahn
et. al.
,
2006
)
, and
diheptyl phthalate (
DHP
)

(
Kim
et. al.
,
2007
)
.


In all these studies either free PAE
s

or PAE
s

blended PVC sheets were used.

However, no study
is seen in literature which describes the efficiency of microbial remediation of DEHP from the
PVC medical products including
BB
. In the l
ight of the aforesaid background,
we have isolated
a novel fung
us

form heavily plastics
-
contaminated soil

with the focus
to remediate the PAE
s

in
intact BB
by static submerged growth (28
o
C) in
a
simple basal salt medium (BSM). A two
-
stage
cultivation
strategy was adopted for the complete removal of DEHP from BB.


2. Materials and Methods


2.1. Isolation of fungal cultures

Soil samples were collected from 10 cm depth in 1M
2
chosen areas
,
heavily contaminated with
plastics. Soil samples were collected fr
om Municipal waste treatment centre at Puliyettummal,
Malappuram, Kerala,
India (
11.0548 N and 76.0616 E
).

After appropriate serial dilutions, the
5


isolates
were initially grown on BSM containing (g/L) K
2
HPO
4
, 1.0; NaCl, 1.0; NH
4
Cl, 0.5 and
MgSO
4,
0.4. BSM with 1.5% agar plates were supplemented with
1mM

DEHP. The initial pH of
the medium was adjusted at 7.2. The plates were incubated at
37
o
C in an incubator.
Pure cultures
were obtained by repeated sub
-
culturing and then maintained in
the

BSM
-
DEHP
medium.


2.2. Morphological characteri
z
ation

Fungal isolates were stained with lactophenol

cotton blue for studying the morphological
characteristics. The slides were observed unde
r the binocular microscope (100
X). The
photographs were taken using Image Analyser (Nikon Eclipse E 400, Towa Opticals, Japan)
fitted with Nikon digital camera (DXM 1
200F, Japan).


2.3. Molecular Characteriz
ation

The pure cultures were subjected to PCR amplification of
D1/D2 region of the large sub unit of
the 28S rDNA
and its subsequent sequencing employing standard protocols. The DNA
sequencing service of Xcelris La
bs, Ahmedabad, India was hired for the purpose.


2.4. Blood bag used

Though various brands of commercially available PVC BB were used, in the present study, HL
haemopack BB of Hindustan Latex Ltd, Thiruvananthapuram
, India

(Batch No. HO 30419B,
June 2009)

was used in this study
.
The anticoagulant in the sealed BB was drained off and
washed several times with ddH
2
O. Then it was cut into s
mall pieces of square size (~10
mm
2
) for
use in static submerged culture.


2.5. Solvent extraction of
DEHP
from BB

10
g of

BB pieces (~
10 mm
2
)
were
transferred in to a s
oxhlet apparatus containing 100 ml of
n
-
hexane, then boiled (69
o
C) for 2
h. To estimate the total weight (%) of
DEHP
in BB, the PAE
s

migrated in to
n
-
hexane was concentrated by reflex condensation.
DEHP in
n
-
hexane were

also
calculated from the standard graph
constructed from
commercial DEHP at 275
nm using UV
-
Vis
spectrophotometer (Shimadzu UV
-
1601, Japan).


2.6. Gas chromatography


Mass spectrometry (GC
-
MS)

6


The
n
-
hexane

extract of BB was used for GC
-
MS anal
ysis.
Agilent 6890N GC with 5975 inert
MSD with the
column HP5
-
MS (30m
× 0.25mm ×

0.25

m) was used. The carrier gas was
helium at a flow rate of 0.7 ml
/min. The m/z scan range was 40
-
450 m/z.

Initial column
temperature: 80
o
C

(hold 4min), increased to 300
o
C

(hold 4
min)
at 10
o
C/min.

Injection
temperature: 250
o
C.

Sample analysis was done at I
nterfield Laboratories, Kochi, Kerala, India.


2.7.
Fungal growth patterns
on BB

2.7.1. Growth profile

The fungi were cultured in
5 mL of BSM supplemented with 1
g BB
pieces as sole carbon source.
The spores were counted in Neubauer counting chamber and 100µl of inoculum

(~ 1×10
7
CFU)
were added to each 5
mL and incubated at static condition at 28
o
C. BSM without any carbon
source
was maintained as control. At 5
days inter
vals, the cultures were withdrawn for various
assays. The fungal mycelia bound to BB pieces were separated and pooled by repeated stirring
and washing on a magnetic stirrer (Rotek Magnetic Stirrer, India
) for 5
min. in each step. The
fungal gro
wth profile w
as measured at 600
nm in a Shimadzu UV
-
Vis (UV
-
1601, Japan)
spectrophotometer after proper dilution of the mycelia with ddH
2
O.




2.7.2. Calculation of biomass

The fungal suspension as harveste
d above was centrifuged at 9000g at 4
o
C for 10min

in a
refrigerated centrifuge (Rota 4R
-

V/ FM, Plasto Crafts). Then, supernatant was discarded and the
fungal pellet
(
biomass) was weighed out.


2.7.3. Change in pH

The pH of medium (whole flask) was measured using a digital pH meter (Systronics 335, Indi
a
)
at 5
days intervals.


2.8. Percentage utilization of DEHP in BB

7


At 5day intervals, the fungus
-
free BB was taken after repeated wash with ddH
2
O. The air
-
dried
BB pieces were transferred in to the Soxhlet apparatus for extraction with
n
-
hexane for
estimati
ng exact weight (%) loss.


3. Scanning Electron Microscopy (SEM)

The surface patterns of the PVC BB pieces before and after treating with fungal consortium were
analyzed using the SEM (Hitachi, SU 6600, Japan) facility available at the National Institute
of
Technology (NIT), Calicut, India
. After 15
days of growth (first phase), only the liquid in the
flask was carefully drained off and fresh BSM was added for the complet
e utilization of DEHP.
After 15
days of this second phase of growth, the
fungal mycelia
bound to BB pieces were
harvested completely by repeated stirring and washing with ddH
2
O, then the BB pieces were air
dried for SEM analysis.


4. Statistics

All experiments were repeated 3 times. Mean values were given with
±

SD. Microsoft Excel was
used f
or the purpose.


5. Results

5.1. Confirmation of DEHP by GC
-
MS analysis

Extraction of
the
various commercial brands of BB with
n
-
hexane and further concentration
revealed that BB contained about 33.5% (w/w) phthalate.
The presence of DEHP in BB was
initia
lly confirmed by UV spectrometry, which showed a single characteristic peak at λ 275
nm
.
Analysis with
GC
-
MS revealed that DEHP was used as the plasticizer in the BB
GC profile

shows a sharp peak at RT 24.54
min with a corresponding m/z, 149
.

The MS fragmen
tation
spectrum of t
he GC peak obtained at RT 24.54
min revealed by molecular mass and standard
DEHP analyses that the spectrum is of DEHP
.


5.2. Culture

used

The fungal strain

specifically utilized D
EHP was

employed in this study

was

Aspergillus
parasiticus

BP10 (G
en bank accession No. JN968368).


8


5.3. Morphological characteriz
ation

A
. parasiticus

produced light green colonies with frilled margins on the BSM
-
agar plate
supplemented with extracted DEHP (Fig.
3
), and its mycelia grew profusely and p
roduced
globose sporangia
.



5.4. Fungal growth on BB

F
ungal growth (afte
r 5
days) on the BB pieces was presented with the help of digital cam
era and
stereomicroscope (Fig.

4
). Figure
4
A
shows the
digital image of
A. parasiticus

growing on the
surface of BB

under static condition, and Figure
4
B is its stereo image showing mycelia
penetrated or adhered on to the BB with fruiting bodies.


5.5. Growth profile

The growth profile of
A. parasiticus

was recorded from the absorbance of myc
elia suspension
measured
at 600
nm (Fig.
5
).
The fungal culture attained maximum
growth in

15
days

and
showed
maximum OD
(~
5.5
).



5.
6
. Biomass

Growth pattern of
A. parasiticus

on BB pieces showed that there was a sharp increase in the
biomass of mycelium (Fig.
6
). It attained maxim
um biomass in 15 days of growth. Pellet weight
of
A. parasiticus

(per g BB pieces) was about 0.
24g.


5.7. Change in pH during growth

It was observed that the pH of the medium decreased (from initial 7.2) to acidic pH within the
incubation period. In an ave
rage, the pH was declined to 3.3 by fungal growth after 3 weeks.

Even at 15
days of growth

of
A. parasiticus
, pH in the growth medium declined drastically

(Fig.

7)
.



5.8. Percentage loss of DEHP from
BB

At the decli
ning phase of fungal growth (15
days), the concentration of remaining
DEHP
in BB
pieces was determined by repeated stirring, washing, drying and
extraction with
n
-
hexane
. Then
9


the percentage loss of DEHP from BB pieces by the activity of fungal culture or consortium was
calculated (Fig.
8
). About two
-
third parts of DEHP (total 33.5
%, w/w BB) bound to BB pieces
were consumed in about 15 days of growth,
i.e
.,
A. parasiticus

consumed 67% of DEHP.


6. SEM pattern of BB

A
fter the complete consumption of DEHP by
the fungal growth

on the BB sur
face
, it became
rough and stiff. Figure
9

shows the SEM profile of the outer surface of the BB after the complete
consumption of DEHP by
A. parasiticus
. Before fungal treatment, the surface and PVC mesh
spaces were seen tightly packed

with DEHP and its agg
regates,
the complete removal of DEHP
made the BB a micro
-
porous network.


7. Discussion

In this

study, we used commercial
BB as a model plastic

and describe how
Aspergillus
parasiticus
,
completely utilized
the
DEHP
in BB. We enriched
A. parasiticus

by repeatedly
growing on BSM
-
DEHP
-
agar plates, wherein DEHP acted as the sole carbon source. To our
information, no report is available yet in literature describing bioremediation of
DEHP
bound to
a medical device like BB directly. Having obtained the pur
e fungal culture utilizing various
PAEs, we proceeded further to assess the type of PAE
s

present in the BB. By GC
-
MS analysis
we confirmed that the PAE
s

present in the BB was only DEHP, which in comparison a much
larger PAE
s

molecule.
Only a little informa
tion is available in literature on the type and extent of
PAEs
used in medical devices. Hence, we initially tested the type and quantity (by solvent
extraction) of plasticizer used in three BBs commercially available in Kerala, India. They
contained an ave
rage of 35% w/w DEHP

(
Sarath Josh
et. al
.
,
2012).

Most of the studies related
to the plasticizer used in PVC medical products reported DEHP as the principal plasticizer bein
g
used
(Florence, 2009;
Latini
et.

al.
,
2010)

t
hus, we concerned how the xenobiotic

DEHP in
medical devices (debris and waste

after use) would be
bioremediated before reaching the
environment
. Hence, we used BB as a model with
in situ
DEHP.

Webb
et
.

al
.

(
1999)

studied

the
physicochemical nature of the adhesion of deteriogenic fungus
Aureobasidium pullulans

on to
both
pPVC
and unplasticized PVC

(uPVC)
, which showed that the myco
-
adherence to
pPVC
was much higher with apparently nil on

uPVC
.
Sabev
et
.

al
.

(2006)

also investigated the fungal
colonization on
dioctyl phthalate (
DOP
) or di
octyl adipate (DOA)
-
pPVC
buried in grassland and
10


forest soil for up to 10

months, and found that
Penicillium janthinellum
was the principal
colonizer, but
Doratomyces
sp. being much efficient in adherence
.

This adherence should be the
initial
stage
of
biofilm formation (Reynolds and

Fink,
2001)

and this primary anchoring enables
the fungus to better utilize the DEHP bound to BB as we described.
We observed

sharp decline
of pH of the growth me
dium to acidic state in about 2
weeks of initial growth, which
leveled off
the growth and thereby limited the complete removal of DEHP from the BB during the first
phas
e of growth. However, almost 70
% DEHP bound to BB consumed during this period.
Hence, we adopted second phase of growth by replenishing the BSM and cou
pled elevation of
pH to 7.2


keeping the mycelia bound to BB intact and undisturbed
-

which accelerated further
growth and complete removal of DEHP from BB in another couple of weeks. This restored pH
(7.2)
enhanced

the fungus to completely extract and co
nsume the DEHP on BB for its sustenance
in the second phase. Reports show that apart from other intermediates; phthalic acid, benzene
carboxylic acid, orthohydroxy benzoic acid, muconic acid,
etc
., were formed during

the
biodegradation of DEHP
(
Chen
et. al
.
,
2007
;

Liang
et.

al.
,
2008).

In fact, all these intermediates
ultimately metabolized in to CO
2

and H
2
O
(
Liang
et.

al.
,

2008)
. Such acids formed during the
DEHP metabolism (also carbonic acid from CO
2
) would have lowered the pH of the medium as
found in o
ur results. Lee
et
. al.

(2004)
showed declining of pH up to 3.4 (initial pH 5.4) during
the growth of the fungus
Daldinia concentrica

in
DBP
containing medium, which was not
dependent on the increased biomass.
Interestingly, as revealed by the SEM images, this
metabolic activity of fungi created micropores on the PVC BB sheet making it naked with lost
plasticity. This

myco
-
remediated DEHP
-
free

PVC sheets can be recycled for further use.


8. Conclusions

and p
erspective

DEHP
pPVC
waste
are often dumped in to the soil or buried. During this process, the DEHP will
be leached in to the soil, which ultimately reaches the human system.
To our knowledge, no
other report describes bio
-
remediation of phthalate present
in a commercial product like
PAEs
blended PVC medical device.

We showed that DEHP present in commercial BB can be bio
-
remediated efficiently,

using mycelial fungus
. In the light of our ongoing studies, we also
suggest that apart from BB, other DEHP
-

(or an
y PAE
s
)
-
p
PVC medical and allied products
could als
o be used as substrate for the fungus
.


11


Acknowledgements

The authors would like to thank the Ministry of Environment and Forests, Government of India
for a research Grant No. 19/62/2005
-
RE.


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14


Table & Figures


Table 1
: Most common phthalates

Name

Acronym

Structural formula

Di
-
methyl phthalate

DMP

C
6
H
4
(COOCH
3
)
2

Di
-
ethyl phthalate

DEP

C
6
H
4
(COOC
2
H
5
)
2

Di
-
allyl

phthalate

DAP

C
6
H
4
(COOCH
2
CH=CH
2
)
2

Di
-
n
-
propyl phthalate

DPP

C
6
H
4
[COO(CH
2
)
2
CH
3
]
2

Di
-

n
-

butyl phthalate

DBP

C
6
H
4
[COO(CH
2
)
3
CH
3
]
2

Di
-

isobutyl phthalate

DIBP

C
6
H
4
[COOCH
2
CH(CH
3
)
2
]
2

Butyl cyclohexyl phthalate

BCP

CH
3
(CH
2
)
3
OOCC
6
H
4
COOC
6
H
11

Di
-
n
-
pentyl

phthalate

DNPP

C
6
H
4
[COO(CH
2
)
4
CH
3
]
2

Dicyclohexylphthalate

DCP

C
6
H
4
[COOC
6
H
11
]
2

Butyl benzyl phthalate

BBP

CH
3
(CH
2
)
3
OOCC
6
H
4
COOCH
2
C
6
H
5

Di
-
n
-

hexyl phthalate

DNHP

C
6
H
4
[COO(CH
2
)
5
CH
3
]
2

Di
-
isohexyl phthalate

DIHxP

C
6
H
4
[COO(CH
2
)
3
CH(CH
3
)
2
]
2

Di
-
isoheptyl

phthalate

DIHpP

C
6
H
4
[COO(CH
2
)
4
CH(CH
3
)
2
]
2

Butyl decyl phthalate

BDP

CH
3
(CH
2
)
3
OOCC
6
H
4
COO(CH
2
)
9
CH
3

Di
-

(2
-
ethyl hexyl) phthalate

DEHP

C
6
H
4
[COOCH
2
CH(C
2
H
5
)(CH
2
)
3
CH
3
]
2

Di
-
n
-
octyl phthalate

DNOP

C
6
H
4
[COO(CH
2
)
7
CH
3
]
2

Di
-
iso octyl phthalate

DIOP

C
6
H
4
[COO(CH
2
)
5
CH(CH
3
)
2
]
2

n
-
octyl decylphthalate

ODP

CH
3
(CH
2
)
7
OOCC
6
H
4
COO(CH
2
)
9
CH
3

Di
-
isononyl phthalate

DINP

C
6
H
4
[COO(CH
2
)
6
CH(CH
3
)
2
]
2

Di
-
isodecyl phthalate

DIDP

C
6
H
4
[COO(CH
2
)
7
CH(CH
3
)
2
]
2

Diundecyl phthalate

DUP

C
6
H
4
[COO(CH
2
)
10
CH
3
]
2

Di
-
isodecyl phthalate

DIUP

C
6
H
4
[COO(CH
2
)
8
CH(CH
3
)
2
]
2

Di
-
tridecyl phthalate

DTDP

C
6
H
4
[COO(CH
2
)
12
CH
3
]
2


















15


Fig.

1
: Diagrammatic representation of PAE
s

in PVC plastics



Fig.
2
: Chemical
structure of DEHP










16


Fig.

3
:
Fungal
growth

on BSM
-
DEHP agar plate
.
Ex situ

utilization of DEHP




Fig.

4
A
:
Fungal growth

on
the surface of BB
.

In

situ

utilization of DEHP







17


Fig.
4B
:

Steriomicroscopic image showing mycelial adherence on the surface of BB




Fig.

5
:
Growth profile.

Opti
cal density was measured at 600nm at
5days interval up to 25
days.









18


Fig.

6
:
Biomass profile.
Measurements were tak
en at 5days interval, up to 25
days of cultivation.






Fig.

7
:
pH profile,
measured at 5days interval up to 25days.








19


Fig.
8
:
DEHP consumption
profile.

Measurements were taken at 5days interval, up to 25
days of
cultivation.









Fig.

9
:
SEM profi
le shows of BB,
after complete
utiliz
ation

of DEHP by
the fungus

in two stages

of treatment