Abdulsalam, Z. B. MCB 406 NOTE: BIODEGRADATION OF PESTICIDES, JULY, 2023.

MCB 406 NOTE: BIODEGRADATION OF PESTICIDES AND RECALCITRANT

COMPOUNDS

DEFINITION OF SOME TERMS

Biodegradation: Biodegradation is a natural process, where the degradation of a xenobiotic

chemical or pesticide by an organism is primarily a strategy for their own survival.
Bioremediation: Bioremediation can be defined as any process that uses microorganisms, plants
or their enzymes to return the environment altered by contaminants to its original condition. It
can also be described as “a treatability technology that uses biological activity to reduce or lessen
the concentration or toxicity of a pollutant.
Pollutants: Pollutant is any chemical (radionuclide, organo phosphorus compound or trace
gases) or geochemical substances (dust, sediment, grit etc.), biotic component or product or
physical agent (heat, sound etc.), that is released by man in to the atmosphere in such quantities
that will have adverse effects.

Xenobiotics: The expression “xenobiotic” is derived from the Greek word for xeno (foreign) and
biotics (of or related to life). These are synthetic compounds not formed by natural biosynthetic
processes, and can be recalcitrant. Therefore, xenobiotic compounds are foreign substrates in our
ecosystem and may have toxic effects when accumulated above threshold value.

Mineralization: This is the total removal or conversion of organic pollutants into CO2, H2O and
other inorganic ions which readily absorbed by plants.

Co-metabolism:This is defined as the metabolism of an organic compound in the presence of a

growth substrate that is used as the primary carbon and energy source. Compounds that supports
microbial growth are known as primary substrates, while cometabolic substrates are called
secondary substrates because they do not support growth.

Transformation: This is the metabolic modification of the molecular structure of a compound,

resulting in the loss/alteration of some characteristics properties of the original compound, with
no/low loss of molecular complexity. Transformation may affect the toxicity, solubility and
mobility of the organic compound.

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 Abdulsalam, Z. B. MCB 406 NOTE: BIODEGRADATION OF PESTICIDES, JULY, 2023.

CLASSIFICATION OF ORGANIC COMPOUNDS

Organic compounds are classified on the basis of their behaviour in the environment into:
 Biodegradable: Those organic compounds which undergo biological transformations
are termed as biodegradable.
 Persistent: Those organic compounds which do not undergo biodegradation in certain
environment, are termed as persistent.
 Recalcitrant: Those which resist biodegradation in a wide range of environments are
termed as recalcitrant. These compounds constitute a broad class of chemicals that
appear as persistent contaminants in soils and sediments, including petroleum and fuel
residues, tars, and creosotes. Such compounds have low solubility, low volatility, low
intrinsic reactivity, and they typically exhibit very slow release rates from soil or
sediment.

BIODEGRADATION AND BIOREMEDIATION

Biodegradation and bioremediation are matching processes to an extent that both of these are
based on the conversion or metabolism of pesticides by microorganisms. The difference between
these two is that, the biodegradation is a science whereas bioremediation is a technology process.
BIOREMEDIATION TECHNOLOGY

Bioremediation works either by transforming or degrading the contaminant or by both

ways in the field. Bioremediation process uses mainly microorganisms, plants or their enzymes

to detoxify contaminants in the environment. The goal of the technology process is to create

most favourable growth condition for the microorganisms. Bioremediation treatment includes:

land farming, bioreactors, biologically enhanced soil washing, composting and solid – phase

bioremediation. In situ bioremediation requires manipulation of aqueous constituents and

bioventing. Genetically modified microbes are used to enhance the capability of degradation.

However, the use of genetically – engineering technology for the use in environment is still

controversial because an adverse genotype can be readily mobilized in the environment.

In a development of technology following points should be taken into consideration:

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(i) heterogeneity of contaminant.

(ii) concentration of contaminant and its effect on biodegradative microbe

(iii) persistence and toxicity of contaminant

(iv) behaviour of contaminant in soil environment

(v) conditions favourable for biodegradative microbe or microbial population.

USE OF TECHNOLOGY AT ACTUAL SITE (INSITU)

The use of technology at actual site requires (i) the knowledge of natural bioprocess at

contaminated site, (ii) detail and valid data of microbial biodegradation developed in

laboratory, (iii) monitoring onsite biodegradation process. Studies on microbial population,

activities and soil enzymes will provide the mirror image about the functional status of the

soil. Most of the bioremediation technologies for the field are designed to remove the

pollutant once it is generated or released into environment. Usually, these technologies

includes:

i. Bioaugmentation: This involves the addition of organism or enzyme to the

contaminant.

ii. Biostimulation: This involves the use of nutrients to stimulate the naturally occurring

organisms.

iii. Biofilters: This is the removal of organic gasses by passing air through compost or

soil containing microorganism,

iv. Bioreactors: This is the treatment of contaminant in a large tank containing organism

or enzymes.

v. Bioventing: This involves the venting of oxygen through soil to stimulate the growth

of natural microorganisms capable of degrading contaminant.

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vi. Composting: This involves mixing of contaminant with compost containing bioreme

diation organisms.

vii. Landfarming: This the use of farming, tilling and soil amendment techniques to

encourage the growth of bioremediation organism at contaminated site.

PESTICIDES

PESTICIDES: Pesticide can be defined as any substance or mixture of substances intended for

preventing, destroying, repelling, or mitigating pests. Any substance or mixture of substances

intended for use as a plant regulator, defoliant, or desiccant can also be included in the definition.

Pesticides are biologically active compounds, and, in time, the mechanism of action has shifted

from directly killing the pests to interfering with metabolic processes. Thus, they could interfere

with metabolic processes also in humans and other living organisms. The increase in the use of

pesticides, which is no doubt negatively affecting the environment since only 1 % of the applied

chemicals reached their targets while the remaining 99 % escaped in to the environment.

Example of pesticides include: organochlorides, organophosphorus, carbamates, synthetic

pyrethroids etc. Residues of all these pesticides have been found in soil, water, sediments and in

human body fluids such as breast milk, tears and semens. A lot of study have shown that these

chemicals are immune distruptors, neurotoxins, and have threatened reproduction in mammals.

For instance, SPs residues have been discovered in solid prepared food, indicating that these

substances can also reach humans through the food chains.

BIODEGRADATION OF PESTICIDES

In order to reduce the risk in environmental and public health associated with the use of

pesticides, it is necessary to develop rapid and effective methods to remove or minimize the

concentration of these insecticides in the environment. Pesticides in the soil could be degraded in

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different ways; the traditional methods included physical degradation, chemical degradation and

physical-chemical degradation, which basically caused secondary pollution. One promising

treatment method is to exploit the ability of microorganisms to remove pollutants from

contaminated sites. It is an alternative treatment strategy that is effective, minimally hazardous,

economical, versatile and environment-friendly.

The ability of native microorganisms to degrade pesticides have been used to clean up

soil polluted by pesticide. The use of bacteria is highlighted as the main microorganisms

evaluated for degradation of pesticides. Among the main microorganisms studied for this

purpose are bacteria belonging to the genera Arthrobacter, Bacillus, Flavobacterium,

Micrococcus, Pseudomonas, Klebsiella, Sphingomonas etc, which have all demonstrated the

capacity to consume pesticides as the only source of carbon and nitrogen. On the other hand, the

use of fungi for these purposes is scarce. Among the fungi recently evaluated for the degradation

of pesticides are Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and

Penicillium that uses reactive oxygen species as the primary mechanism.

MECHANISMS OF BIODEGRADATION PROCESS

The whole degradation mechanism is divided into three parts:

i. Adsorption of target, which occurs on the surface of the cell membrane and was a

dynamic equilibrium process that was also critical.

ii. Secondly, the target gets into the cell through the surface of the cell membrane, and

the penetrated rate and efficiency are related to the molecular structure of the target

isomers.

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iii. Finally, xenobiotic target conducts a rapid enzymatic reaction in the membrane. The

microorganisms then convert organic macromolecules into small non-toxic

molecules, thus avoiding the secondary pollution.

Studies have shown that mineralization and co-metabolism were the main mechanisms for the

further degradation of pesticides and their intermediate products.

FACTORS INFLUENCING BIODEGRADATION

There are a lot of factors affecting the biodegradation but broadly can be grouped into

two: intrinsic and extrinsic factors.

INTRINSIC FACTORS: They are also known as the inherent/natural factors. They the factors

relating to only the microorganisms and the chemicals. For the microorganisms, these include:

the number and diversity of the microorganisms, the capacity and capability of the

microorganisms, and the physiological state of the microorganisms. While for the chemicals: the

type and concentration of the chemical compounds, the physical states of the chemical

compounds and the nature and chemical structure of the chemical compounds.

EXTRINSIC FACTORS: These are the factors that are external to the microorganisms. They

include; pH, temperature, oxygen concentration, humidity, soil texture, vegetation cover, rainfall,

topography, availability of nutrients, presence of other contaminants, presence of other

competitors/predators.

There are three possible regions where pesticide contamination can occur in terrestrial

ecosystem:

(i) surface soil

(ii) vadose zone

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(iii) ground water or saturated zone.

The biodegradation in surface is primarily aerobic and rapid because the surface soils have large

number of aerobic microorganisms and their number usually decreases with the depth.

Biodegradation is slow in other two zones i.e. vadose and ground water. Therefore, different

bioremediation technologies are required to deal with different regions of terrestrial ecosystem

METHODS OF BIODEGRADATION OF PESTICIDES

SOIL SAMPLING: To study, the microbial degradation of pesticide, random soil sampling with

utmost precision is required. Depending on experimental design or analysis, the soil sampling

requires a precision to reduce the uncertainty and variability in results.

STORAGE: Soil samples should be processed and analysed immediately or stored only for a

very limited period. Microbial analysis must be performed as soon as possible to minimize the

effects of storage on microbial population and their biochemical activities. Reduced microbial

activity has been reported when soil samples were stored in field moist condition at 4 °C for

three months.

RESIDUE ANALYSIS: Similarly, pesticide residue analysis needs to be performed in a short

time to avoid the changes during the storage. Sampling, sample processing, sampling constant,

matrix effect, extraction and cleanup are very important steps in pesticide residue analysis.

Residue analyses are influenced by uncertainty and variability during sample processing.

Residues can be quantified by using LC, HPLC, GC-ECD and GC-MS. Validation of pesticide

residue method is required to quantify the biodegradation of pesticide by microbes.

An alternative method for analysis with high precision and accuracy requires the use of

radiolabelled pesticide. The use of 14C-radiolabelled/specific radiolabelled pesticide allows very

sensitive and specific measurements i.e. the rate of degradation of pesticide, metabolites or

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mineralization as 14CO2 evolution. The technique provides the actual image of microbial

degradation of pesticide.

DIFFERENT APPROACHES FOR BIODEGRADATION

 Complete biodegradation of a pesticide involves the oxidation of parent compound to

form carbon dioxide and water. This process provides both carbon and energy for the

growth and reproduction of microbes. Each degradation step is catalyzed by specific

enzyme produced by a degrading cell or enzyme found external to the cell. Degradation

of pesticide by either external or internal enzyme will stop at any step if an appropriate

enzyme is not present. Absence of an appropriate enzyme is one of the common reason

for persistence of any pesticide. If an appropriate microorganism is absent in soil or if

biodegrading microbial population has been reduced due to toxicity of pesticide in that

case a specific microorganism can be added or introduced in soil to enhance the activity

of the existing population. The microorganisms could be either natural or genetically

engineered.

 "Super strains" that can degrade the pesticide at a fast rate can also be developed. The

problem is that the introduction of microorganism to contaminated site may fail because

they are unable to survive in a new environment more than a few days or weeks.

 Another strategy is to add a specific genes that can confer specific degradation capability

to indigenous microorganism. The addition of degradative genes relies on delivery and

uptake of genetic material by a indigenous microorganism. There are two possible

approaches that can be taken i.e. (i) the use of microbial cells to deliver gene via

conjugation, (ii) to add naked gene in soil and allow its uptake via transformation.

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BIOAVAILABILITY: contaminants can be degraded only when they exist in the aqueous phase

and come into contact with the cell membrane of a microorganism. In this way, the contaminant

serves as a substrate for the microorganism and is incorporated through membrane transport into

the cell and utilized as an energy source in the cell’s metabolic pathways. However, physical or

chemical phenomena can limit the bulk solution concentration of the contaminant and thus

significantly reduce the ability of the microorganisms to assimilate the contaminant. Therefore,

the bioavailability of the contaminant can control the overall biodegradation of these compounds.

RECENT DEVELOPMENTS IN PESTICIDE BIODEGRADATION

Microbes, their catabolic gene and respective enzymes capable of degrading the pesticides

have been isolated and identified by several researchers. Several bacteria competent of degrading

either organochlorides, organophosphate, carbamates, Pyrethroids and other pesticides have been

isolated and characterized.

Genetic studies of microbial degradation indicates that the plasmids are the main place for the

gene of interest usually spread throughout the microbial community.

After, understanding the gene of interest and enzyme involved, the Superbugs can be created to

achieve the desired result at fast rate in short time frame.

LIMITATIONS OF BIOREMEDIATION

There are several limitations to bioremediation technology:

 One major limitation is the nature of the organisms. The removal of pollutants by

organisms is not a benevolent gesture. Rather, it is a strategy for survival. Most

bioremediation organisms do their job under environmental conditions that suit their

needs. Consequently, some type of environmental modification is needed to

encourage the organisms to degrade or take up the pollutant at an acceptable rate. In

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many instances the organism must be presented with low levels of the pollutant over a

period of time. This induces the organism to produce the metabolic pathways needed

to digest the pollutant. When using bacteria and fungi, it is usually necessary to add

fertilizer or oxygen to the material containing the pollutant. This can be disruptive to

other organisms when done in situ. In situations where simple compounds and metals

are being taken up it is likely that these pollutants are at toxic levels for the

organisms. Overall, the organisms do not always live as well on the pollutant diet as

on other nutrients found more commonly in their environment. This causes problem

when doing in situ remediation.

 Other limitations of immediate concern are cost/benefit ratios i.e. cost versus overall

environmental impact. Neither the government nor industry wants to spend large

amounts of money to clean up pollution. Industry in particular always likes to keep

their costs of products and services down for gain in the market. The petroleum

industries are embroiled currently in a battle with the EPA about the added costs of

maintaining new Clean Air Act standards.

 Bioremediation is generally very costly, labor intensive, and can take several months

for the remediation to achieve acceptable levels. Air bioremediation in particular is

very inefficient, considering the volume of polluted air generated by industry.

 Another problem is that both ex situ and in situ technologies can cause environmental

disruption beyond the damage done by the pollution.

 The long-term effects of introducing naturally occurring non-native bioremediation

organisms into an area are not fully understood.

 The impact of genetically altered bioremediation organisms is even less understood.

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 Bioremediation has been proven to work effectively under laboratory conditions.

 Short-term studies show that it also works under several field conditions.

 Like many technologies with good scientific foundations its merits are marred by

over-optimistic speculations and fraudulent claims.

 In spite of its limitations, bioremediation is benefiting from the rush to use

biotechnology to solve public health problems.

 Bioremediation’s popularity is further enhanced because it is perceived as being more

“green” than other remediation technologies. Companies and individuals are investing

in biotechnology future in spite of the high risks. As a result, bioremediation

companies have a viable future regardless of its long-term effectiveness.

 In field, microbial consortia through enrichment technique can be used for

mineralization of specific pesticide at faster rate.

 In comparison to other remediation processes i.e. incineration, thermal disposition,

use of chemicals etc., the bioremediation has a better future in development of

technology for removal of contaminants from actual site.

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