Phytoremediation of xenobiotics

Introduction
• Xenobiotics (greek “xenos” = strange, foreign, foreigner) are chemically
synthesized compounds found within an organism/Biosphere that is not naturally
produced by or expected to be present within and thus are 'foreign to the
biosphere'.
• Xenobiotics compound have been produced artificially by chemical synthesis for
industrial or agricultural purposes, e.g. Halogenated H.C., aromatics, pesticides,
PAH.
• Natural substances can also become xenobiotic if they are taken by other organisms
such as natural human hormones by fish found downstream of sewage treatment, plant
outfalls or chemical defences produced by some organisms against predators
• Xenobiotics (greek “xenos” = strange, foreign, foreigner) are chemically
synthesized compounds found within an organism/Biosphere that is not naturally
produced by or expected to be present within and thus are 'foreign to the
biosphere'.
• Xenobiotics compound have been produced artificially by chemical synthesis for
industrial or agricultural purposes, e.g. Halogenated H.C., aromatics, pesticides,
PAH.
• Natural substances can also become xenobiotic if they are taken by other organisms
such as natural human hormones by fish found downstream of sewage treatment, plant
outfalls or chemical defences produced by some organisms against predators

1. Petrochemical industry oil/gas industry - refineries and the production of basic
chemicals e.g. vinyl chloride and benzenes
2. Plastic industry - closely related to the petrochemical industry uses a number of
complex organic compounds such as anti-oxidants, plasticizers, cross-linking
agents
3. Pesticide industry most commonly found central structures are benzene and
benzene derivatives, often chlorinated and often heterocyclic
4. Paint industry major ingredient are solvents, xylene, toluene, methyl
ethyl ketone, methyl isobutyl ketone and preservatives
5. Others Electronic industry, Textile industry, Pulp and Paper industry,
Cosmetics and Pharmaceutical industry, Wood preservation

ORIGIN OF DIFFERENT TYPES OF XENOBIOTIC
CHEMICAL COMPOUNDS IN THE ENVIRONMENT

Hazards of xenobiotics in
environment
Hazards of xenobiotics in
environment
• Xenobiotics pose a serious issue in sewage treatment plants, since they are
many in number and each will present its own problems as how to remove
them.
• Some xenobiotics are resistant to degradation for example synthetic
organochlorides, PAH, crude oil and coal.
• Many xenobiotics produce variety of biological effects such as
carcinogenic, toxic to humans, ecotoxicity and persistence in environment.

Possible environmental fate of a xenobiotic compound

Persistence of xenobiotic compounds
• These new compounds tend to resist biodegradation, with potential
consequences such as persistence in the environment or
bioaccumulation in food chains.
• The presence of artificial groups such as chloro-, nitro- or sulfonate-
in many synthetic chemicals makes them resistant to decomposition.
• The compounds are highly resistant to biodegradation is known as
recalcitrant compounds.
.

Persistence of xenobiotic compounds
in soil
Xenobiotic compound Persistence duration
Heptachlor 9 years
Aldrin, Dieldrin 9 years
DDT 10 years
BHC 11 years
Chlorodane 12 years
Diuron 19 months
Simazine 17 months
Atrazine 18 months
Monuron 36 months
2, 4- D 1 to 2 months

Xenobiotics remediation methods

Photo degradation
• Photo degradation involves in conversion of xenobiotics mainly
by photo oxidation and photolysis
• photolysis breakdown of toxic xenobiotics by sunlight
• Oxidation in presence of sunlight is called as photo oxidation.

Photo-degradation of Ethylene di
bromide

Bioremediation
 Microorganisms have the ability to act upon xenobiotics and
convert them into simpler non-toxic compounds.
 This process of degradation of xenobiotics and conversion into
non-toxic compounds by microorganisms is known as
“biodegradation”.

Phyto-remediation
• Phytoremediation can be defined as “the efficient use of plants
to remove, detoxify or immobilise environmental
contaminants in a growth matrix (soil, water or sediments)
through the natural biological, chemical or physical activities
and processes of the plants”.
• Phytoremediation is the term used to describe those
methodologies that employ living higher organisms, which
include green vegetation, plants, aquatic plants, trees and
grasses, to remove toxic compounds.

Fates of organic xenobiotics on plant

Phyto-remediation in field studies

Green liver concept
• The metabolic processes involved in phytodegradation have strong
similarities to those used by animals for modification and degradation of
drugs and other toxins.
• This has given rise to a conceptual model for phytodegradation known as
the “green liver” model (Sanderman, 1994).
Three main biochemical processes;
1. Conversion or transformation (phase I)
2. conjugation (phase II)
3. compartmentalization (phase III)

Contaminant metabolism in plants

Enzyme involvement in
phytoremediation

•Cytochrome P450 is a super family of enzymes similar
to hemoproteins which are critically important in
xenobiotic metabolism
•The name P450 because it gives an absorption peak at
450 nm in spectrophotometer
•Cytochrome P450 in encoded in human genome 57
(Guengerich, 2003). Which is involved in xenobiotic
metabolism
Cytochrome P450: Terminal oxidase in
Xenobiotic metabolism

Phytoremediation by genetically
engineered plants
Phytoremediation by genetically
engineered plants

Anarelly et al., 2017
Objective
To evaluate the effect of Pennisetum purpureum cultivation time
on the reduction of the concentrations of 1,4-CB and 1,3,5-CB
in sewage sludge.
Phytoremediation of chlorobenzenes in sewage sludge
cultivated with Pennisetum purpureum at different
stages.
Phytoremediation of chlorobenzenes in sewage sludge
cultivated with Pennisetum purpureum at different
stages.

Experimental Details
 Location: The experiment was carried out in a greenhouse, at
the experimental farm ‘Professor Hamilton de Abreu
Navarro’, at the Institute of Agricultural Sciences, Montes
claros
 Design : CRD
 Treatment : 6
 Replication : 3
 Six treatments, which corresponded to five evaluation
periods (30, 60, 90, 120 and 150 days, from the planting of
cuttings) during the cultivation of P. purpureum and one
control

 CBs are extracting using solid liquid extraction technique (Pinho
et al., 2014) and analyzed in GC ECD. 1,4-CB had retention
time of 7.004 min and 1,3,5- CB had retention time of 10.400
min.
 Means relative to the CBs concentrations in the sewage sludge
cultivated with P. purpureum collected at different times was
compared using ANOVA, treatment means are compared with
control using Dunnett’s test.
 Treatment means were fitted to regression models, testing the
coefficients up to 0.10 probability level by t-test.
Experimental Details

Initial properties of Sewage and
Sludge
pH = 6.2
P2O5 (total) = 25 g dm-3
K2O (total) = 2.9 g dm-3
Ca (total) = 75 g dm-3
Mg (total) = 26 g dm-3
S = 10.1 g dm-3
Si (soluble) = 14.2 mg dm-3
1,4-CB = 0.004 mg kg-1
1,3,5-CB = 0.023 mg kg-1

Table 1. Concentration of 1,3,5-CB in sewage sludge with
and without P. purpureum cultivation in five evaluation
periods
Period/
Depth (cm)
30CSS 60CSS 90CSS 120CSS150CSS 150C CV(%)
0-10 0.065b 0.058b 0.025b 0.022b 0.018b 0.041a 137.19
10-20 0.091b 0.045b 0.033b 0.031b 0.024b 0.0067a 62.758
20-30 0.115b 0.063b 0.031b 0.046b 0.034b 0.0023a 74.22
30-40 0.0074a 0.0082a0.0056a0.0097a 0.0034a 0.0029a 56.4
>40 0.00391a0.0074a 0.013a 0.0068a 0.0023a 0.0028a 37.02
CSS- Cultivated with Sewage and Sludge , C- Control (uncultivated)

Fig 1. Trend line of concentration of 1,3,5-CB mg kg -1
from initial depths

Fig 2. Trend line of concentration of 1,3,5-CB
mg kg -1
from deeper depths

period
depth
(cm)
30CSS 60CSS 90CSS 120CSS 150CSS 150C CV(%)
0-10 0.161b 0.115b 0.048b 0.032b 0.0125b 0.431a 31.85
10-20 0.288b 0.152b 0.095b 0.083b 0.078b 0.483a 34.56
20-30 0.268b 0.155b 0.114b 0.172b 0.102b 0.313a 85.39
30-40 0.175b 0.384a 0.189a 0.505a 0.159a 0.201a 38.68
>40 0.07b 0.315a 0.335a 0.573a 0.106a 0.217a 36.16
Table 2. Concentration of 1,4-CB in sewage sludge with
and without P. purpureum cultivation in five evaluation
periods

Fig 3. Trend line of concentration of 1,4-CB mg kg -1
from initial
depths

Fig 4. Trend line of concentration of 1,3,5-CB mg kg -1
from
deeper depths

Conclusion
 P. purpureum cultivation in sewage sludge for 150
days promotes reductions in the concentrations of
1,3,5-CB and 1,4-CB in the layers with greater
concentration of roots, compared with uncultivated
sewage sludge.
 Cultivation for at least 150 days is advisable to
maintain the levels of the contaminants within the
safest limits for the agricultural use of the waste.

Objectives
To determine the phytotoxic effects on the plant Ludwigia
octovalvis in order to assess its applicability for phytoremediation
gasoline-contaminated soils.
Asia et al., 2015
Phytoremediation of contaminated soils containing
gasoline using Ludwigia octovalvis (Jacq.) in greenhouse.
Phytoremediation of contaminated soils containing
gasoline using Ludwigia octovalvis (Jacq.) in greenhouse.

Experimental details
 Location : The experiment was conducted in a
greenhouse located at University Kebangaan
Malaysia.
 Design : Factorial RCBD
 2 factors: Gasoline concentration, Period of
evaluation
 Gasoline concentrations: control, 1, 2, and 3 g
gasoline per kilogram
 Periods of evaluation : 0, 7, 14, 28, 42, and 72
Days of Planting

Fig 1.Experimental Layout for the phytotoxicity test (R1, R2, R3 three
replicates, CC control contaminant without plants, PC plant control
without the gasoline contaminant)

 Microbial plate count is done by serial dilution.
 Total Petroleum Hydrocarbons (TPH) was extracted
using ultrasonic solvent extraction method (Tang et al.
2012) and analyzed with GC-FID.
 Treatment means are analysed using SPSS version 16
with two-way ANOVA at a 95 % confidence level
(p≤0.05).

Fig 2. Effect of gasoline concentration on dry
weight

Fig 3. Total bacterial population for different gasoline
concentration

Fig 4. SEM image showing root cross sections at 500× of L. octovalvis
after 72-day exposure to a gasoline concentration of 3 g kg-1
and the
corresponding control

Fig 5. The percentage Plant degradation of TPH in soil mixture by
L. octovalvis exposed to gasoline contamination at 1, 2, and 3 g
kg-1

Fig 6. Percentage of gasoline in L. octovalvis after exposure to
1, 2, and 3 g kg-1
in plant between two parts (lower and upper
layers) in plant

Fig 7. The GC-FID chromatogram profile of gasoline degradation by
L. octovalvis after exposure to 2 g kg-1
gasoline for days 0 and 72

Conclusion
• Based on soil extraction, the highest TPH removal rate was 79.8
percent in treatment cultivated with L. octovalvis compared to
the removal rate by the corresponding unplanted controls of only
48.63 percent.
• L. octovalvis has the ability to survive, remove and also provide
suitable conditions for rhizobacteria to degrade hydrocarbons at
all investigated gasoline concentrations (1, 2, and 3 g/kg).

OBJECTIVE
To assess the potential of bacterial isolates and Brassica juncea to
degrade phorate in soil
Rani and Asha., 2012
Biodegradation Of Phorate In Soil And Rhizosphere Of Brassica
Juncea (L.) (Indian Mustard) By A Microbial Consortium

• Location: NEERI, Nagpur, India
• Design: factorial RCBD
• Factors: phorate concentration, cultures, periods of evaluation
• Phorate conc: 10 mg kg-1
, 20mg kg-1
• Crop treatments: Control (C), B juncea (B), Microbial consortium (M),
B juncea and microbial consortium (BM).

Contd..
• Phorate concentration was analyzed according to Singh et al. (2003) in
GC-ECD using a 1:10 split ratio.
• Soil dehydrogenase activity was estimated according to Casida et al.
(1964) and expressed as mg of triphenyl formazan produced g-1
soil 24
h-1
.
• Acid and alkaline phosphatase activities in the soil samples were
estimated as described by Tabatabai and Juma (1988) and expressed as
the amount of para-nitrophenol released g-1
of soil min-1
.

Physico-chemical characterization of the soil.

Fig1. The variations in microbial
counts in the soil with time in different treatments
at concentrations of 10 and 20 mg kg-1
phorate.

Fig 2. The variations in the dehydrogenase activity in the soil with
time in different treatments at concentrations of 10 and 20 mg kg-1
phorate.

Fig 3. The variations in the acid phosphatase activity in the soil with
time in different treatments at concentrations of 10 and 20 mg kg-1
phorate.

Fig 4. The variations in the alkaline phosphatase activity in the soil
with time in different treatments at concentrations of 10 and 20 mg kg-1
phorate.

Fig 5. The variations in concentration of phorate with
respect to time in different treatments at 10 and 20 mg kg-1
phorate in soil.

Conclusion
• The presence of B. Juncea aided the bacterial degradation
of phorate in soil as the degradation percent of phorate
was found to increase by 14% in the presence of B.
Juncea in comparison with the bacterial consortium
alone.

Phytoremediation of methyl tert-butyl ether
(MTBE) with Hybrid Poplar Trees
Objective
To study the uptake and volatilization of methyl tert-butyl
ether (MTBE)in lab-scale hydroponic systems by hybrid
poplar trees.
Ma et al., 2004

LOCATION: University of Missouri—Rolla, Rolla,
Missouri, USA.
Four concentrations of MTBE: 148, 296, 593, and 1186 ppm
were dosed to the reactors, with two replicates for each
concentration.

Contd..
The Hybrid poplar cuttings rooted in one-fourth strength modified
Hoagland solution for about 1 week.
After the cuttings showed developing leaves and roots, they were
moved to 250-mL flasks filled with one-fourth strength Hoagland
solution in Bio reactor equipped with diffusion traps on lower and
upper regions.
MTBE extracted using activated carbon disulphide and analysed
in gas chromatograph.

Table 1.Transpiration rates of each tree dosed
in the study, showing percent decrease after
dosing initiated.

Fig 2. Total MTBE collection from the vacuum
diffusion traps over the 7-d experiment.

Fig 3. Relative fate of MTBE in
phytoremediation systems

Fig 1.MTBE concentrations in the transpiration
stream (X-axis) of Tree #6 along stem
height (Y-axis, independent variable).

Table 2. Average plant tissue MTBE
concentrations (μg g-1
) for individual trees. Two
replicates
were tested per concentration.

Fig 4. Distribution of MTBE in different plant
compartments.

Conclusion
The exponential decline of MTBE concentration in the
transpiration stream indicated that MTBE diffusion occurred and the
direct collection of MTBE mass from stems with diffusion traps
substantiated the finding.
A small amount of MTBE is stored in the plant biomass after
uptake, with the largest amount of mass located in the old-growth
stems, i.e., woody tissues.
This shows that Hybrid poplars can be extensively used for
remediation of soils contaminated with MTBE

To evaluate the degradation of metolachlor by
CYP2B6 rice plants to confirm the metabolic activity of the
introduced CYP2B6.
Kawahigashi et al., 2005
Objective
Phytoremediation of Metolachlor by Transgenic
Rice Plants Expressing Human CYP2B6

Location : fukuyama universty, fukuyama, hiroshima,
japan.
Statistical significance between treatment means are
compared using student t-test with 99 % confidence level
Treatments : 2
1. Indegenious variety : nippon bare
2. Trangenic rice: CYP2B6 rice

Small scale experiment
Ten 12-day-old plants were transferred to a plastic plant box
with 80 mL of MS culture medium (26)
 30 µM metolachlor was added along with the medium
It is then Incubated at 27 °C under 16 h of light daily for 1 or 6
days.
Plant samples was extracted with acetone and analysed using
GC-MS.

Fig 1. Herbicide tolerance of CYP2B6 rice plants to
alachlor and metolachlor in hydrophonics

Table 1. Small-scale Residual Metolachlor Analysis

Fig 2. The amount of metolachlor absorbed from the culture
medium.

Fig 3. Amount of residual herbicide in plants and culture
medium.

Conclusion
•CYP2B6-transgenic rice plants make it possible to
remove metolachlor faster from the culture medium and
soil than can nontransgenic Nipponbare.
•CYP2B6-transgenic rice plants can be used for
phytoremediation of metalochlor after conducting
appropriate field trails.

Summary
 Phytoremediation is an emerging technology that is certainly
on the verge of being a big part of the solution to the
contamination problem, which can be prescribed as
‘environmental medicine’.
 P. purpureum cultivation helps in remediation of sewage
sludge contaminated with Chloro-Benzenes mainly in
rhizosphere region.
B. Juncea along with microbial consortium is effective in
remediation of soils contaminated with phorate in green house
conditions

Contd…
L. octovalvis has the ability to survive, remove and also
provide suitable conditions for rhizobacteria to degrade
hydrocarbons at all investigated gasoline concentrations.
Hybrid poplars volatilizes and stores MTBE in plant
tissue , Thus can be extensively used for remediation of soils
and water bodies contaminated with MTBE.
CYP2B6-transgenic rice plants removed metolachlor faster
from the culture medium and soil than Non-transgenic
Nipponbare.

Phytoremediation of xenobiotics

Phytoremediation of xenobiotics

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