Xenobiotics- Microbial Biotechnological approach .

1. UNIVERSITY OF AGRICULTURAL SCIENCES, BANGLORE
Topic: xenobiotics and biotechnological approaches
Department of biotechnology
Team : Kings boys

2. Xenobiotics definition
A xenobiotic is a chemical substance found within
an organism that is not naturally produced.
Actual meaning xenobiotic are the compound that
are foreign to an organism for its actual nature.
Environmental xenobiotic
 Environmental xenobiotics are xenobiotic
compounds with a biological activity that are
found as pollutants in the natural environment.

3. INTRODUCTION
• Environmental xenobiotics is an exciting new topic for
the end of the 20th century. We are now all dependent
on synthetic substances in agriculture,pharmaceuticals,
petrochemicals, colorants, adhesives, preservatives,
etc.
• Xenobiotics such as halogenated solvents undergo
photochemical reactions which have been shown to
cause depletion of the ozone layer
• Pesticides, when used for both agricultural and non-
agricultural purposes, are transformed to persistent
metabolites by soil and water microorganisms.

4. Recalcitrant Xenobiotic
The compounds that resist biodegradation and persist in the
environment for long period of time are called Recalcitrant
Xenobiotic.
Reasons for recalcitrant Xenobiotic
1. They are not recognized as substrate by
degradative microorganisms.
2. Highly stable in nature.
3. Insoluble in water.
4. They are highly toxic or release toxic products due to
microbial activity.
5. They have large molecular weight which prevents entry to
microbial cells.

5. TYPES of Recalcitrant Xenobiotic
1. Halocarbons
2. Poly chlorinated biphenyls(PCBs)
3. Synthetic Polymers
4. Alkylhenzyl sulphonates
5. Oil mixtures

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

8. 1) Pharmaceuticals
• Pharmaceutically active compounds (PhACs) can
enter the environment by one route or another as
the parent compound or as pharmacologically
active metabolites.
• PhACs can be entered into the environment in two
main ways; direct and indirect.
• PhAC's can be discharged directly by manufacturers
of the pharmaceuticals or effluents from hospitals.
• Indirect source of PhACs into the environment is
the passing of antibiotics, anesthetics and Growth
promoting hormones by domesticated animals in
urine and manure.

9. Fate in environment
Once PhACs are entered into the environment they
suffer one of three fates:
Biodegradation into carbon dioxide and water.
Undergo some form of degradation and form
metabolites.
Persist in the environment unmodified.

10. 2) Petrochemicals
• Petrochemicals (also known as petroleum
distillates) are chemical products derived from
petroleum.
• A hydrocarbon is an organic compound consisting
entirely of hydrogen and carbon.
• The majority of hydrocarbons found on earth
naturally occur in crude oil.
• Aromatic hydrocarbons (arenes), alkanes, alkenes,
cycloalkanes and alkyne-based compounds are
different types of hydrocarbons.

11. Biodegradation of Petroleum compounds
• Petroleum compounds are categorized into 2
groups
• Aliphatic hydrocarbon e.g. alkane, alcohol,
aldehyde
• Aromatic hydrocarbon e.g. benzene, phenol,
toluene, catechol
• H.C. (substrate) + O2 H.C.-OH + H2O
• H.C. (substrate) + O2 H.C. 29 O -H (O H)
monooxygenase
dioxygenase

12. 3) BIODEGRADATION OF PESTICIDES
• Pesticides are substances meant for destroying or
mitigating any pest.
• They are a class of biocide.
• The most common use of pesticides is as plant
protection products (also known as crop protection
products).
• It includes: herbicide, insecticide, nematicide,
termiticide, molluscicide, piscicide, avicide,
rodenticide, insect repellent, animal repellent,
antimicrobial, fungicide, disinfectant, and sanitizer.

13. DIFFERENT METHODS
a) Detoxification: Conversion of the pesticide
molecule to a non-toxic compound. A single moiety
in the side chain of a complex molecule is
disturbed(removed), rendering the chemical non-
toxic.
b) Degradation: Breakdown or transformation of a
complex substrate into simpler products leading to
mineralization.
E.g. Thirum (fungicide) is degraded by a strain of
Pseudomonas and the degradation products are
dimethylamine, proteins, sulpholipids, etc

14. c) Conjugation (complex formation or addition reaction):
An organism makes the substrate more complex or
combines the pesticide with cell metabolites.
Conjugation or the formation of addition product is
accomplished by those organisms catalyzing the reaction
of addition of an amino acid, organic acid or methyl
crown to the substrate thereby inactivating the
pesticides
d) Changing the spectrum of toxicity: Some pesticides
are designed to control one particular group of pests,
but are metabolized to yield products inhibitory to
entirely dissimilar groups of organisms, for e.g. the
fungicide PCNB is converted in soil to chlorinated
benzoic acids that kill plants.

15. 4) BIODEGRADTION OF PLASTICS
• Plastic is a broad name given to different
polymers with high molecular weight, which
can be degraded by various processes.
• The biodegradation of plastics by
microorganisms and enzymes seems to be the
most effective process.
• It consist of two steps- fragmentation and
mineralization. But at the core, reaction
occurring at molecular level are oxidation and
hydrolysis.

16. other HAZARDS OF XENOBIOTICS
• Highly toxic in nature.
• Causing skin problems and various types of cancers.
• They are easily not degraded so they remain in
environment for many years.
Hazards of xenobiotics in environment
Xenobiotics pose a serious issue in sewage treatment
plants
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.

18. Biotechnological approaches in xenobiotics
The limitations of conventional remediation technologies
include poor environmental compatibility, high cost of
implementation and poor public acceptability.
Efficiency and performance of bio and phytoremediation
approaches can be enhanced by genetically modified
microbes and plants.
Phytoremediation can also be stimulated by suitable plant-
microbe partnerships, i.e. plant-endophytic or plant-
rhizospheric associations.
Synergistic interactions between recombinant bacteria and
genetically modified plants can further enhance the
restoration of environments impacted by organic
pollutants.

20. Genetic engineering
• Genetic engineered new strains of microbes that have
the unique characteristics compare to the wild type
and broad spectrum of catabolic potential for
bioremediation of xenobiotics.
• GEMs have been developed by the identification and
manipulation of certain genetic sequences. GEMs
exhibit enhanced degradability for a wide range of
xenobiotics and have potential for bioremediation
from various environmental sources.
• In 1970, the first GEMs called “superbug” was
constructed to degrade oil by the transfer of plasmids
which could utilize a number of toxic organic chemicals
like octane, hexane, xylene, toluene, camphor and
naphthalene.

21. Approaches for the construction of GEM’s
1. The identification of organisms suitable for
modification with the relevant genes
• Microorganisms are well adapted to survive in soil
environment may not be able to survive in aquatic
environment and hence cannot be used successfully.
• Therefore, aquatic microbes can be used to develop
GEMs for bioremediation of aquatic sources.
• Scientists have developed Anabaena sp. and Nostoc
sp.by the insertion of linA (from P. paucimobilus) and
fcbABC (from Arthrobacter globiformis) respectively.
• The gene linA controls the biodegradation of lindane,
and fcbABC confers the ability to biodegrade
halobenzoates and can be used to remediate these
pollutants from water sources.

22. 2. The pathway construction, extension and
regulation.
• GEMs have developed by improving existing
catabolic pathways to degrade some more
compounds which are not possible to degrade by
using wild strain.
• The complete catabolic pathway may be encoded
by a single microorganism, or by a consortium of
microorganisms, each performing one or more of
the stages of bioremediation of xenobiotics.

23. 3. Modification of enzyme specificity and affinity.
• GEMs have been developed by hybrid gene clusters
which alter their enzymatic activity .
• These gene clusters encode the enzyme possessing
improved transforming capability.
• E. coli strain is genetically modified to express a
hybrid gene cluster for the degradation of
trichloroethylene (TCE).

24. 4. bioprocess development, monitoring, control, and
bioaffinity, bioreporter, sensor applications for
chemical sensing, toxicity reduction, and end point
analysis.
• The use of lux gene-based system has been
developed that offers several advantages for
monitoring bioremediation processes.
• Bioluminescence can be easily detected and do not
require expensive devices, exogenous addition of
chemicals or co-factors.
• Bioluminescence producing GEMs also help us to
understand the spread of microbes in the polluted
area and end point of the bioremediation.
• Further, GEMs possess chemical sensors that allow
the monitoring of contaminant bioavailability.

25. Advantages
• Microbes are confined to aerobic catabolic and co-
metabolic pathways and therefore cannot be applied
to anaerobic environment.
• GEMs are developed by inserting genes for oxygenases
make it possible to use them in anaerobic
environmental conditions.
• Microbes or GEMs which use xenobiotics as a source
of carbon, energy or nitrogen, can get nutrients and
grow.

26. Cont,,
• Scientists have been developing a novel strategy to
construct “Suicidal Genetically Engineered
Microorganisms (SGEMs)" by exploring the
antisense RNA-regulated plasmid addiction.
• To design a novel S-GEM is based on the knowledge
of killer-anti-killer genes that makes the microbes
susceptible to programmed cell death after
degradation of xenobiotics.
• This technology helps in the removal of microbes
after bioremediation by their autolysis and
therefore, reducing the risks to the human beings
and environment.

27. Limitations of GEM’s
• The information on the genes is very limited which
limits the development of GEMs.
• Second main obstacle in the application of GEM is
the regulatory affairs and hazards associated with
them.

28. WHITE-ROT FUNGI AND THEIR
ENZYMES AS A
BIOTECHNOLOGICAL TOOL
FOR
XENOBIOTIC BIOREMEDIATION
MARIEM ELLOUZE AND SAMI SAYADI
HTTP://DX.DOI.ORG/10.5772/64145

29. • Biological methods, being eco-friendly and cost cheap
techniques, were proposed for xenobiotic
degradation purposes in order to overcome problems
of xenobiotics.
• Compared to bacteria, most of the fungi are robust
organisms and generally more tolerant to high
concentrations of pollutants.
• White-rot fungi (WRF) constitute an eco-physiological
group comprising mostly of basi‐diomycetes and
litter-decomposing fungi. Recently, there has been a
great interest in white-rot fungi and their ligninolytic
enzymes, including laccase, manganese peroxidase
(MnP) and lignin peroxidase (LiP), for the degradation
of a wide range of xenobiotics.

30. • The expression of these enzymes depends on the
strain itself: some white-rot fungi produce LiP and
MnP, but not laccase, while others produce MnP and
laccase, but not LiP, acting simultaneously or
separately on xenobiotics released from the
environment.
• The potential of white-rot fungi can be harnessed
thanks to emerging knowledge of the physiology and
the morphology of these organisms.

31. • The importance of high extracellular levels of these
enzymes to enable the efficient degradation of
recalcitrant compounds under in vivo conditions
relates to the sorption and complexation of enzymes
in soil and the probable loss of their activity once
externalized.
• Many studies reported the effective degradation of
pesticides by fungal strains, including P.
chrysosporium and T. versicolor, and involving two
different enzyme systems: laccase and peroxidases

32. • It is known that white-rot fungi can degrade lignin in
the way that the mycelia of the organisms penetrate
the cell cavity and release ligninolytic enzymes to
decompose materials to a white sponge-like mass.
• Enzymatic treatment, involving mainly peroxidases
and/or laccases, is currently considered as an
alternative method for the removal of toxic
xenobiotics from the environment.

33. Peroxidase system
• The lignin degradation system consists on
peroxidases, H2O2 -producing enzymes, veratryl
alcohol, oxalate, and manganese. All of these
enzymes are glycosylated heme proteins that
couple the reduction of hydrogen peroxide to water
with the oxidation of a variety of sub‐ strates.
• Lignin peroxidases (LiPs) belong to the family of
oxidoreductases and were firstly described in the
basidiomycete P. chrysosporium in 1983. This
enzyme has been recorded for several species of
white-rot basidiomycetes.

34. • LiP is dependent of H2O2 , with an unusually high
redox potential and low optimum pH. This enzyme is
able to oxidize a variety of substrates including
polymeric ones and has consequently a great
potential for application in various industrial
treatment processes.
• MnP catalyzes the oxidation of phenolic structures to
phenoxyl radicals. The product Mn3+, being highly
reactive, complex with chelating organic acids, such
as oxalate, lactate, or malonate.
• On the other hand, it was reported that MnP may
oxidize Mn(II) without H2O2 and with decomposi‐
tion of acids, and concomitant production of peroxyl
radicals.

35. LACCASE SYSTEM
• Laccases which are blue multicopper oxidases,
catalyze the monoelectronic oxidation of a large
spectrum of substrates, for example, ortho- and para-
diphenols, polyphenols, aminophenols, and aromatic
or aliphatic amines, coupled with a full, four electron
reduction of O2 to H2O.
• Laccases act on both phenolic and nonphenolic lignin-
related compounds as well as highly recalcitrant
environmental pollutants, and they can be effectively
used in paper and pulp industries, textile industries,
xenobiotic degradation, and bioremediation and can
act as biosensors.

36. • The importance of high extracellular levels of these
enzymes to enable the efficient degradation of
xenobiotic compounds under in vivo conditions
relates to the sorption and complexation of enzymes
in soil and the probable loss of much of their activity
once externalized.
• This group may be a useful and a powerful tool for
bioremediation purposes thanks to fungal capacities
to degrade many xenobiotic substances.

37. RECENT STUDY OF XENOBIOTIC IN FUNGI

38. Biodegradation of Toxic Organic Compounds in Environmental Soil
Endophytic Bacteria and Phytoremediation Reported cases of successful bioremediation using
endophytic bacteria

39. Rhizospheric Bacteria and Phytoremediation (Rhizoremediation) bacteria

42. INTRODUCTION
• Remediation processes based on plants and on
plant–microbe interactions have been proposed to
clean up sites contaminated with xenobiotics
including polychlorinated biphenyls (PCBs).
• The extensive root system of plants allows them to
pump large amounts of chemicals from soil.
• In addition, plant roots exude many chemicals that
promote rhizobacterial growth and metabolis.

43. Engineering of the biphenyl catabolic
enzymes
Bacterial biphenyl catabolic pathway

44. Transgenic plant expressing biphenyl-
degrading enzymes
• In recent years, few investigations have addressed
the feasibility of constructing transgenic plants
producing active PCB-degrading enzymes.
• As indicated above, BPDO is a three-component
enzyme requiring the participation of BphAE, BphF,
and BphG.
• Analyses of tobacco plants transiently expressing B.
xenovorans LB400 genes encoding the BPDO
components or transformed with them have shown
that each of the components can be produced
individually as active protein in plants.

45. Deciphering the molecular mechanisms
involved in plant–microbe interactions
• The choice of plants is likely to impact on the success of the
rhizoremediation technology.
• Some plants such as Cucurbita pepo (zucchini) accumulate
high level of hydrophobic chemicals, others, such as alfalfa
possess extensive root systems that exhibit high affinity
toward hydrophobic chemicals
• a critical criterion is the ability of plants to support the
metabolism and survival of the PCB-degrading rhizobacteria
• A clear example was provided by Narasimhan et al. who
showed that PCB removal by Pseudomonas putida PML2
which is a phenylpropanoid-utilizing and PCB-degrading
rhizobacteria was significantly lower in rhizosphere of
an Arabidopsis mutant exuding less flavonoids than in the
rhizosphere of the wild-type strain

46. CONCLUSION
• PCB-rhizoremediation process is a promising approach to
restore contaminated sites.
• However, challenging issues remain to be overcome. Efforts
are required to understand how plants will respond to the
presence of high levels of PCB metabolites.
• New approaches should be conceived that will allow co-
ordinate expression of several heterologous genes together
in same plant cells and same compartments.
• Finally we need to have better insight into the mechanisms
by which rhizosphere bacteria perceive and modify plant
small molecules such as flavonoids that to some extent
mimic some of the PCB structural features and how these
interactions impact on the catabolic pathways involved in
PCB degradation.

48. HIGHLIGHTS
• Nine genes from different microorganisms were
synthesized and modified.
• Phenol was degraded completely and imported into
the tricarboxylic acid cycle.
• All genes were regulated by monocistronic
transcriptional pattern.
• The engineered E. coli could effectively degrade
phenol in coking wastewater

49. INTRODUCTION
• Phenol is one of the most frequently pollutants
found in industrial effluents, landfill runoff waters
and rivers and its concentration can reach 10 g/L in
some wastewaters.
• Phenol is potentially carcinogenic to humans, and
deaths in adults have resulted after ingestion of 1-
32g of phenol.
• Phenols are categorized as priority hazardous
substances due to their proven toxic, mutagenic,
carcinogenic, and teratogenic effects

50. MO’S REPORTEDLY UTILISED FOR PHENOL BIODEGRADATION
• Pseudomonas putida,
• Rhodococcus erythropolis,
• Bacillius sp.,
• Alcaligenes faecalis,
• Ralstonia taiwanensis,
• Nocardia hydrocarbonoxydans and
• Candida tropicalis

51. METHOD
• In this study, two metabolic modules were
introduced into Escherichia coli, to elucidate the
metabolic capacity of E. coli for phenol degradation.
• The first module catalyzed the conversion of phenol
to catechol.
• The second module cleaved catechol into the three
carboxylic acid circulating intermediates by the
ortho-cleavage pathway.

52. PHENOL DEGRADING
PATHWAY
• Phenol is first catalysed to
catechol by phenol hydroxylase,
which attaches a hydroxyl group
to the ortho-position of the
aromatic ring under aerobic
conditions, to facilitate
degradation.
• Then, catechol is cleaved by
dioxygenases, either between the
hydroxyl groups or adjacent to
one of the hydroxyl groups
through ortho- or meta-cleavage.
• The final products of catechol
degradation, namely, acetyl-CoA
and succinyl-CoA, were assumed
to be imported into the TCA cycle
of bacteria.

53. CONSTRUCTION OF PHENOL DEGRADATION STRAIN BL-
phe/cat
• All nine genes used in this study are from different
microorganisms, as follows:
• pheA1 and pheA2 from Rhodococcus erythropolis;
• catA, catB and catC from Rhodococcus species AN-
22;
• catD from Rhodococcus jostii; and
• pcaI, pcaJ and pcaF from Pseudomonas putida.

54. Scheme of gene clusters in different transformants (BL-phe, BL-cat and
BL-phe/cat).
pheA1, pheA2 (large and small subunits of phenol hydroxylase);
catA (catechol 1,2-dioxygenase); catB (cis,cis-muconate lactonizing
enzyme); catC (muconolactone isomerase); catD (β-ketoadipate enol-
lactone hydrolase);
pcaI, pcaJ (β-ketoadipate succinyl-CoA transferase);
pcaF (β-ketoadipyl-CoA thiolase)

55. • All synthesized genes were seamlessly connected with T7
promoter and terminator to construct gene expression cassette via
the modified overlap-extension PCR method.
• The PCR products were purified and cloned into pGEM-T easy
vector, and sequenced prior to cloning in the expression vector
• All gene expression cassettes were re-amplified with primer
containing different restriction enzyme cutting sites.
• The PCR fragment was then excised using restriction
endonucleases and inserted into pCAMBIA1301 vector in proper
order.
• All the cassettes were transformed to the host E. coli strain BL221-
AI after their assembly in the expression vector.
• The transformant was named BL-phe/cat.
• The strains BL-phe and BL-cat respectively containing the modules
for the conversion of phenol to catechol and catechol degradation.

56. RESULT
• The key intermediate metabolites and products in the
process of phenol degradation can be detected in the
chassis cell by stable carbon isotopic tracer(13C6-catechol)).
• Proteomic analyses showed that low phenol concentrations
can activate downstream signalling pathways and these
results indicated that the engineered E.coli could completely
and quickly degrade phenol.
• This analysis showed that all genes in the phenol
degradation pathway were over-expressed and affected cell
division and energy metabolism of the host cells.
• The engineered E. coli could effectively be used in the
remediation of phenol-polluted wastewater.

57. • Phenol was completely degraded and imported into
the tricarboxylic acid cycle by the engineered
bacteria.
• Phenol in coking wastewater was degraded
powerfully by BL-phe/cat.
• The engineered E. coli can improve the removal
rate and shorten the processing time for phenol
removal and has considerable potential in the
treatment of toxic and harmful pollutants.