ISSN: 2320-5407 Int. J. Adv. Res.

12(04), 509-517

Journal Homepage: - www.journalijar.com

Article DOI: 10.21474/IJAR01/18576

DOI URL: http://dx.doi.org/10.21474/IJAR01/18576

RESEARCH ARTICLE
APPLICATION OF BIOREMEDIATION IN MITIGATION OF HEAVY METAL STRESS ON PLANTS

Dr. S. Padmavathi1 and G. Jayalakshmi2

1. Lecturer in Botany, Dr. V. S. Krishna Government PG College (A), Visakhapatnam Dt.
2. Lecturer in Botany, Government Degree College, V. Madugula, Anakapalli Dt.
……………………………………………………………………………………………………....
Manuscript Info Abstract
……………………. ………………………………………………………………
Manuscript History Heavy metals are toxic metals which are persistent in nature due to
Received: 24 February 2024 their nondegradable nature.Due to increase in industrialization and
Final Accepted: 27 March 2024 urbanization heavy metals are being added to theenvironment and
Published: April 2024 consequently causing heavy metal pollution of water and soil. These
heavy metals cause significant stress on crop plants and effect various
Key words:-
Bioremediationand Heavy Metal parameters such as growth and yield. Manymethods like physical,
chemical and biological methods are available to degrade these
heavymetals. Recently, bioremediation is being widely used because
of its cost-effective andenvironment friendly approach.
Bioremediation is a biological process which makes use
ofmicroorganisms to remove hazardous substances or environmental
pollutants from air, water,soil, industrial effluents etc. Bacteria and
fungi are mostly employed for bioremediation.These organisms are
genetically engineered for effective use in bioremediation. This not
onlydiscussed about the importance of bacteria for bioremediation of
heavy metals but alsodiscussed about the challenges and limitations of
bacteria for bioremediation. In this chapter, a variety of mechanisms
responsible for adaptation of microorganisms to high heavy metal
concentrations, e.g. metal sorption, uptake and accumulation,
extracellular precipitation and enzymatic oxidation or reduction, will
be reported. Moreover, molecular mechanisms responsible for their
metal tolerance will be described.

Copy Right, IJAR, 2024,. All rights reserved.

……………………………………………………………………………………………………....
Introduction:-
The rapid growth of industrialization is always coupled with the persistent problem of environmental pollution.
Accumulation of toxic metals released by the industries entering the ecosystem may lead to biomagnification. Very
few heavy metals function as trace elements but, many of them are of great concern in human biochemical
processes because of their toxicity. In this respect, the important point that needs to be taken care of for the
wellbeing of the ecosystem is the treatment of the processed wastes released into the environment from these
industries. “Heavy metals” are a group of metals and metalloids with a specific gravity that is at least four to five
times the specific gravity of water at the same temperature and pressure (1). High levels of heavy metals
causedegradation of soil quality, leading to reduction of yield and quality of agricultural products, posing
significant hazards to human, animaland ecosystem health. The crop plants growing in HM contaminated areas
usually show low growth, substantial oxidative stress and genetic changes. These HMs also enter the crop produce
and ultimately harm the human health. Heavy metal pollution of agricultural soils is a widespread environmental

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Corresponding Author:- Dr. S. Padmavathi
Address:- Lecturer in Botany, Dr. V. S. Krishna Government PG College (A),
Visakhapatnam Dt.
ISSN: 2320-5407 Int. J. Adv. Res. 12(04), 509-517

threat. Heavy metals induce deleterious effects on plants. In view of all these ill effects caused by HMs such as
inhibition of germination, retardation of growth and metabolism, we should prioritize the need for sustainable
techniques to mitigate its hazardous effects. So, it is an urgent necessity to remediate the plants.

The methods of HM remediation are broadly divided into three types physical, chemical and biological. The
physical processes of HM remediation are leaching, precipitation, ion exchange, reverse osmosis, etc. Chemical
methods are chemical oxidation, reduction chemical precipitation, etc. These technologies typically result in wastes
that have high metal concentrations, which are a major cause of pollution in the environment. Moreover, the
previously mentioned techniques could not be efficient or cost-effective, particularly in cases when the
concentrations of heavy metals are below 100 mg/l (2).

Bio Remediation
Biological methods offer promising solution to HM remediation as they are known to be faster, cheaper and safer
methods. In this context phytoremediation and the use of rhizosphere microbes have emerged as an important
alternative to ensure high efficiency and better performance. While it has been known that plants provided with
some strategies to avoid, tolerate and reduce the accumulation of heavy metals in their tissues (3), these
mechanisms, however, have certain limitations and plants have to depend on rhizospheric bacteria for
detoxification or heavy metal transformation mechanisms (4). It was experimentally proved by them in rice plants
by killing bacteria with chloramphenicol that plants require rhizospheric bacteria for methylation of Arsenic (As)
into a less toxic form. Several studies have shown that microorganisms have unique capacities to absorb and
transform toxic heavy metals into less toxic forms (5).

Research on PGPB suggests that the usage of plant growth-promoting bacteria has begun to be a promising
alternative for the alleviation of plant stress caused by heavy metals Scientists have developed several strategies to
minimise the adverse effects of HMs on plant growth, including the application of metal accumulator plants, the
use of nanoparticles, the use of biofilms and the recent application of Plant Growth Promoting Bacteria (PGPB)
(6). Therefore, it is important to continually assess and monitor the levels of heavy metals in an environment and
evaluate the effects of human exposure and should come up with probable solutions for sustainable environment.

Many studies suggest the minimization of heavy metal stress of plants by the application of PGPB. The main
objective of this study is to assess the effect and mechanisms of PGPB inoculants of on improving growth,
managing oxidative stress and improving the yield.

Bioaugmentation, bioventing and bioreactor are the possible biological methods of HM remediation. In this article
bioremediation process is explained.

Microbial remediation:
Microbial organisms are very important and natural recyclers. Many bacteria growing in heavy metal contaminated
areas possess mechanisms operates by them to survive in hostile environments and resistant genes acquired through
horizontal gene transfer methods.

Various microorganisms are used for heavy metals biosorption.Saccharomyces cerevisiae (yeast), Streptococcus
(bacteria) and Aspergillus niger (fungi) is widespread. Genetically engineered organisms can be generated which
can likely to reduce different types of polycyclichydrocarbons (PAHs). Flavobacterium, Pseudomonas, Bacillus,
Arthrobacter, Corynebacterium, Mycobacterium, Stereumhirsutum, Pleurotus ostreatus, Azotobacter,
Phormidiumvalderium, Ganoderma applantus are some microbial species that help to remediate heavy metals
more efficiently.

Different microbes have been proposed to be efficient and economical alternatives for the removal and
transformation of heavy metals from soil and water. There were quite a few studies carried on with regard to
bacterial application in remediation of phytotoxicity induced by heavy metals. Further exploration of bacteria is
needed for more efficient methods under field conditions, as well as the understanding of resistance mechanisms
are still required.

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Mechanisms operated by bacteria

Microbial bioremediation is mainly about converting harmful substances into substances that are less toxic to
human health and the environment. Bacteria found to have several distinct mechanisms including physiological,
biochemical and genetic to deal with extreme environmental conditions.

It has been reported that some of heavy metal resistant-PGPRs can also reduce both uptake and transportation of H
Ms to aerial parts of plant by decreasing metal bioavailability in soil. (7). Rhizospheric bacteria initiate mutualistic
relationships with plants and minimize the stress. By improving nitrogen fixation, phytohormone synthesis, and
mineral uptake, PGPRs also support plant growth. Since one of the most well-known effects of auxins is
stimulation of rhizogenesis, the most common way that rhizobacteria are thought to stimulate root growth is
through their ability to synthesize indole acetic acid (IAA), the most common, naturally occurring plant hormone of
the auxin class (8). Additionally, studies demonstrating that Azospirillum mutants weak in auxin synthesis did not
improve wheat root development further supported the significance of bacterial auxins in boosting plant root
proliferation (9). Thus, mutational analyses of the significance of auxin in plant/microbe interactions gave
consistent results across two bacterial genera and different plant species. PGPR can increase the tolerance of plants
to salinity, acting primarily through the accumulation of osmolytes, increasing the absorption of nutrients, the
nitrogen fixation, the solubilization of P and other essential elements, but similarly with the activity of ACC
deaminase, the production of auxins, siderophores, and exopolysaccharides (10).

The mechanisms operated by bacteria to alleviate toxic effects on plants of important HMs are discussed below.

Arsenic (As)
Arsenic is known as one of the most important toxic metalloids. Anthropogenic sources of arsenic are mainly
mining industries. Arsenic is available in both inorganic and organic forms. Inorganic forms primarily elemental
form (As 0), arsenite (As III) and arsenate (As V). As (III) is much more toxic and mobile than As (V), and hence,
microbial arsenic redox transformation has a major impact on arsenic toxicity and mobility which can greatly
influence the human health. The organic form of arsenic is methyl arsenic acid (CH 3 ASH2). Usually As enters food
chain of humans through water and crop plants cultivated soils.

Alleviation of As stress of plants by inoculation with As resistant bacteria

Bioremediation with special reference to bacteria hold promise to alleviate HM induced phytotoxicity. Among
microbial approaches, oxidation, reduction and biomethylation are the major detoxification processes which have
direct implications on amelioration of arsenic contaminated soils.

Bioremediation by microbes (bacteria, fungi& yeast) are quite effective and relies on deliberate action of natural or
engineered microbial activity to reduce, mobilize, or immobilize, volatilize As through sorption, bio-methylation
and redox reactions.

Molecular mechanisms of As bacterial remediation mainly catalize various metabolic reaction oxidation, reduction,
methylation and volatization(11). The bioremediation processes carried by bacteria are the oxidation of more toxic
As (III) to less toxic form As (V) with the help of an enzyme arsenic oxidase (AioBA). Thermus thermophiles, Th.
Aquaticus and A. faecalis and Bacillus arsenic oselenatis are the effective bacteria employed in arsenic
remediation. In furthering detoxification processes there are bacteria which convert inorganic arsenate to organic
form of As. Bacteria convert more toxic form of methylated arsenate to less toxic methylated forms. This process is
catalysed by an enzyme S. adenosyl methionine transferase. The final form of sequential methylation of arsenic is
the trimethyl arsenic which was least toxic. The bacterial genes arsM genes present in bacteria are responsible for
synthesizing the S. adenosyl methionine transferase enzyme.

The genome of several microorganisms metabolising As has been characterised from various ecosystems(12).
Previous studies documented the arsenic resistant bacterium, Herminiimonasarsenicoxydans which is isolated from
an industrial water treatment plant. It is resistant to high As concentrations and able to oxidise As(III) to As(V)
(13). In another research, a Rhizobium strain isolated from a gold mine in Australia carries the genes involved in
the resistance and detoxification of As on a plasmid. Such a genetic tool could be interesting from a
phytoremediation perspective by transferring the As detoxification capacity to related plant-associated bacteria
(14). In series of studies, arsenic tolerant strains are isolated which can bring about the oxidation ofarsenite to

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arsenate named as Halomonas A3H3 and Pseudomonas xanthomarina S11, respectively isolated from the
Mediterranean Sea-contaminated sediments (15) and a French old gold mine (16).

Arsenic resistant bacteria could be utilized for promoting plant growth. By increasing the IAA production,
siderophore production, potassium solubilization and P solubilization, the chromium resistant bacterial species
Bacillus thuringiensis, B. ceres and B. Subtilis and Aspergillus niger PMI -118 in Cicer arietinunm plant. They
improved growth of root and shoot and dry weight. Several experiments proved that As resistant bacteria regulate
the arsenic induced oxidation stress and significant reduction of activity of antioxidant enzymes of SOD, CAT,
APX and GPX. Inoculation of bacterial species such as Pseudomonas, Bacillus and Bruvindimonas(17)

Chromium (Cr)
Among the heavy metals, chromium is considered as one of the most toxic heavy metals, exists in Cr (III) and Cr
(VI). being the most stable states. Cr (III)is less toxic and poorly mobile and is less toxic. however, Cr (VI) is
highly soluble and mobile, is more toxic than Cr (III) and reported to be mutagenic, carcinogenic, and teratogenic
(18).

Alleviation of Cr stress of plants by inoculation with Cr resistant bacteria

Recent studies evaluated the effect of application of bacteria in alleviating crop plants under chromium stress.
Stenotrophomonas maltophilia, Bacillus thuringiensis B. cereus, and B. subtilis are the chromium resistant strains
applied for this purpose. The treatment of Cicer seeds with these chromium resistant bacteria showed significant
improvement in all the growth parameters by increasing the IAA production, Siderophore production, increasing
the P and K solubility.

Chromium reductase enzyme in chromium resistant bacteria, such as Pseudomonas putida and Bacillus subtilis, can
reduce the toxic Cr(VI) to the less toxic and immobilized trivalent form of chromium Cr(III) through reductive
immobilization. (19).Plant growth is adversely affected by Cr due to its impairment of critical metabolic processes.
Reactive oxygen species (ROS) production is linked to the harmful effects of Cr and results in oxidative stress in
plants. Changes in many physiological and biochemical processes have been noted in Ocimumtenuiflorum (20),
Vallisneria spiralis (21) and Triticum aestivum (22), and where the production of MDA by Cr metal induced the
degradation of membrane permeability.

Inoculation of wheat seeds with a bacterium strain resistant to Cr, Staphylococcus aureus strain K1 has shown the
detoxification potential of chromium-induced stress by increasing growth parameters and the production of
carotenoid and chlorophyll. The mechanisms behind the reduction of bacterial Cr6+ are particularly important
because they convert harmful and mobile chromium derivatives into reduced species that are harmless and less
mobile (23). Microbial cell walls are mainly composed of polysaccharides, lipids and proteins, providing many
functional groups that can bind heavy metal ions, including carboxylate, hydroxyl, amino and phosphate (24).

Mercury (Hg)
Mercury (Hg) is the most poisonous because of its non-degradable nature.
Mercury is a highly toxic trace metal that is ubiquitous in nature (25) and is the main cause of severe pollution all
around the world (26).

Accumulation of heavy metals more particularly mercury in agriculture soils has become a major concern for
food crop production. Once they enter the plant body they start to show visible toxic symptoms.

Since autotrophic plants function as the primary and principal entry point of heavy metals, they get
accumulated once they enter the food chain. Hence, a significant amount of these heavy metals is found in the
animal body leading to biomagnification. Though reports related to the mechanism of accumulation of mercury
in plants and their tissue distribution are scanty, literature pertaining to the effect of mercury on the plant
systems are plentily available in comparison to animal systems. Some of the possible mechanisms through
which mercury can impair important biochemical and physiological processes in living organisms include
inhibition of germination (27), a decrease of biomass production (28), inhibition of photosynthesis (27),
hindrance of protein function and induction of oxidative stress (29) anddamaging effect on DNA (30).

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Alleviation of Hg stress of plants by inoculation with Hg resistant bacteria

There are many instances to isolate mercury resistant bacteria from contaminated areas. Mercury tolerant bacterial
strains were isolatedfrom mercury contaminated areas and identifiedthe bacterial strain showing high mercury
resistance which showed growth up to 600 ppm mercury chloride concentration in growth medium. Based on the
high tolerance, they identified the bacterial isolate named as HG 2 that showed tolerance to 600 ppm mercuric
chloride concentration. This bacterial isolate identified as Brevundimonas by phylogenetic analysis using the
MEGA 6.0 software using 16S r RNA analysis (31).

Results of mercury stress mitigation experiments (32), showed improved growth of Cajanus plants under mercury
stress caused by mitigating effect of Brevundimonas bacterium coupled with increased production of IAA. The
seeds of the pigeon pea plant under Hg stress were inoculated with mercury resistant Bacillus sp. bacterium
observed to show enhanced rates of germination and growth in terms of length and biomass compared to Hg
exposed pigeon pea seeds (untreated).

Rhizospheric bacteria play a great role in alleviation of stress by means of accumulation and transform them into
less toxic forms. There are many instances of minimization of stress by using bacterium to improve plant growth
parameters (32). Enhanced production of oxidative enzymes is an important parameter in evaluation of heavy metal
stress. Oxidative stress is a direct mechanism of plants facing heavy metal stress. Oxidative stress is the increased
production of oxygen free radicles. Reactive oxygen species damage enzymes and cell membranes by binding with
them. An experiment conducted on pigeon pea(33) has proved that when seeds exposed to stress are treated with
Hg resistant Brevundimonas bacterium showed lowered production of antioxidant enzymes such as CAT, POX and
SOD and also reduced lipid peroxidation. In Cajanus seeds exposed to mercury stress. The plant antioxidant
response has been analyzed by quantifying the catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase
(APX) and glutathione reductase (GR) enzyme activity in pigeon pea seedlings exposed to mercury and different
PGPB treatments. These studies indicated that the bacteria acted as a shield and protect the Cajanus plants from
mercury toxicity and hence extent of lipid peroxidation and antioxidant enzyme activities, MDA content and
proline content were increased in the mercury exposed pigeon pea and a parallel reduction was observed in
Brevundimonasbacterium treated pigeon pea seedlings under mercury stress indicate that these plants faced less
stress in presence of Brevundimonas bacterium.

Studies conducted to know the mechanisms of mercury resistance showed that resistance is not due to genes
responsible for detoxification. In this research it was identified that two membrane proteins account for conferring
mercury resistance. These proteins make the membrane impermeable to mercury compound may be responsible for
the mercury resistance of the strain (34).

Enzymatic reduction of Hg2+ to Hg0.

The central enzyme in the microbial mercury detoxification system is the mercuric reductase protein encoded by
mer A genes, which catalyzes the reduction of Hg(II) to volatile Hg(0). In addition to merA, mer operons encode
for proteins involved in regulation, Hg binding and degradation of organomercury. The detoxification processes
operated by mercury resistant bacteria are mainly due to mer operon which consists mer A and the two
regulatorgenes (merR and merD) in Pseudomonas putida. In this no mer B gene is detected (35).

The possible mechanisms operated in bacteria for the mitigation of heavy metal stress in crop plants treated by
heavy metal resistant bacteria were represented diagrammatically for the quick understanding.

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Mercury pollution cycle

Biomagnification

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IAA
Phosphate
production
solubilization

Decreased Increased
damage to DNA percentages of
germination

Benefits of
Becterial
treatment of
crop plants
Increased in
growth of root
Decreased and shoot
levels of proline

Increased Levels
Decreased levels of of Chlorophyll
Anti-oxidant content
enzymes

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Conclusions:-
The growth and output of crop plants are significantly altered by heavy metal pollution in agricultural soils, which
eventually affects human health. Thus, it is necessary to look for mitigation techniques for the harmful effects that
heavy metals have on plants. As a potential treatment option, rhizospheric bacterial remediation shields plants from
heavy metal stress. In addition, intriguing insights can be gained from techniques for separating metal-resistant
microorganisms for bioremediation from contaminated areas. These bacteria use a variety of strategies and genetic
mechanisms to mitigate the harmful effects caused by heavy metals to plants.

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Rotterdamseweg, Netherlands (www.excelwater.com/thp/filters/Water-Purification.htm).
39. Image by <a href="https://www.freepik.com/free-vector/set-colorful-fishes-hand-drawn-
style_2112045.htm#query=small%20fish&position=10&from_view=keyword&track=ais&uuid=201f0bd4-
c06b-408c-b533-1f9bc1963875">Freepik</a>

517