DISCUSSION
Increased industrialization raised the level of arsenic and other heavy
metals in the groundwater (Mazumder et al. 2020; Shaji et al. 2021),
agricultural land (Shrivastava et al. 2017) that ultimately affected
plant and human life. Various studies demonstrated wide spread
occurrence of arsenic in different regions. Shukla et al. (2019)
revealed high concentration of arsenic of up to 300ppb in the ground
water of West Bengal, India. Many districts location of Bihar, India
were also found contaminated with arsenic, out of which half were highly
effective (50-100ppb) and even five districts were severely affected
with >100ppb arsenic (Kumar et al. 2021). Present study also
reported high concentration of arsenic in agricultural soil, and water
samples collected from different regions in India, and results are in
accordance with previous reports. Microbes are well known for stress
tolerance in environment. In order to access indigenous bacteria having
arsenic tolerance we isolated a total of 16 potential arsenic
tolerant bacterial isolates from samples collected from various
contaminated sites. Importance of microbes in combating stress has been
discussed in different studies. High pollution level in an environment
corresponds to high microbial diversity has been reported earlier (Lang
and Cai et al. 2009). Previous studies demonstrated that arsenic and
other heavy metal contaminated soil possess a diverse range of microbial
population that has been reported as heavy metal tolerant microbes
( Turpeinen et al. 2004; Sanyal et al. 2016; Suhadolnik et al. 2017;
Cavalca et al. 2019; Zhai et al. 2020). In addition to this,Bacillus mycoides NR5 exhibited resistance to multiple heavy
metals. Bacteria can possess multiple metal resistance. In our studyB. mycoides NR5 also exhibited resistance to again 9 heavy metal
tested. Following studies also indicated multiple metal resistance in
various potential microorganisms. Keramati et al. (2011) reported
multiple metal resistance in 6 bacteria against Cd, Zn, Ni, Pb, Cu and
Ag. Vallalar et al (2019) evaluated 20 bacteria against five metals Cu,
Cr, Co, Ni, and Zn and found resistance among them in the range of 25 to
400 mgL-1. Multiple mechanism of metal resistance in
bacteria was reported by Nanda et al. (2019). The metal transport across
cell membrane, accumulation of metal in cell membrane, metal entrapment
and redox reaction are the basic mechanisms of multiple heavy metal
tolerance in bacteria. In addition to this metallothioneins are also a
key player in arsenic resistance.
Arsenic removal and adsorption behaviour by B.mycoidesNR5 was investigated using adsorption isotherm by Langmuir (Langmuir
1918) and Freundlich (Freundlich 1906) isotherm model, that describes
the monolayer/multilayer adsorption of molecule on cell surface. It was
found that both models supported the adsorption behaviours of arsenic byB. mycoides NR5, but Langmuir was most fit in present study. Our
data is also in accordance with previous studies where in monolayer
sorption was recorded in bacteria mediated heavy metal and other
hazardous molecules (Andjelkovic et al. 2017; Altowayti et a. 2022;
Glatstein et al 2018; Sarim et al. 2019). Bacillus mycoides is a member of Bacillus cereus group and is easily distinguishable
from other closely related microbes ( Di Franco et al. 2005).
Bacillus is well known species to possess tolerance against various
kind of stresses, and multiple species of Bacillus have been
reported as arsenic tolerant species. Shivaji et al (2020) isolated an
arsenic tolerant novel species. Few other studies are as follow;Bacillus subtilis (Pepi et al. 2011); B. safensis (Raja
and Omine, 2012); B. flexus (Marwa et al. 2019); B.
megaterium (Islam et al. 2019) B. cereus (Ayangbenro and
Babalola 2020), B. firmus (Bagade et al. 2020). Heavy metal
resistance and antibiotic resistance are two aspect and their gene
generally located on plasmid, extra cellular DNA material. Co-occurrence
of Heavy metal (HM) and antibiotics resistance (AR) have been reported
in various studies (Chen et al. 2015; Chen et al. 2019; Glibota et al.
2020). In a study by Li et al. (2017) explained that genetic distance
linkage between HM and AR genes is closer and potential of co-transfer
in genomes is also high, and thus play a key role in shaping resistance
against both.
In order to strengthen the candidature of potential bacterium for
arsenic tolerance It should also support in plant growth and development
if applied in field condition. Hence, NR5 was also tested for any plant
growth promoting, and found that along with multiple heavy metal and
antibiotic tolerance bacteria also exhibited plant growth promoting
attributes such as phosphate solubilization, IAA production,
siderophore, ammonia production and nitrate reduction. The presence of
heavy metal tolerance along with the plant growth promoting traits in
microorganisms has been documented in some previous reports (Yu et al.
2014; Tirry et al. 2018; El-Meihy et al. 2019; Jain et al. 2020; Renu et
al. 2020). As the plant growth promoting bacteria are residing in the
close vicinity of plant roots and the soils contaminated with arsenic or
any other heavy metal, they are supposed to develop partial resistance
against heavy metals, and thus undoubtedly play an important role in
plant metal toxicity remediation.
Heavy metal interaction with cell surface make many conformational
changes that can have effect on cell surface topology. To Investigate
changes on cell surface of NR5 scanning electron microscopy (SEM) and
transmission electron microscopy (TEM) was used and observed that metal
interaction causes swollen surface with deposition of metal. Alteration
in cell surface topology due to exposure of arsenic has also been
observed in following studies (Pandey and Bhatt, 2015; Felestrino et al.
2018; Mujawar et al. 2019). Surface chemistry probably play an active
role in the interaction of metal to cell surface. Sinha and, Mukherjee
(2009) and El-Helow (2001) showed that, Bacillus sp. containing
cell wall components such as polysaccharides, teichoic and teichuronic
acids or phospholipid layers and other functional groups might be
responsible for the heavy metal interaction through extracellular
substance secretion. The cell surface morphological changes inCryptococcus sp. and Ochrobactrum intermedium BB12 after
heavy metals exposure appeared as shrunken and distorted cell wall in
the presence of Cd and depressions in the presence of Pb and Zn (Singh
et al. 2013, Renu et al. 2022). The results of the present study also
confirmed morphological and topological changes of bacteria exposed to
heavy metals. In addition to this FTIR spectra also indicated as cell
surface amine group involvement in arsenic interaction and advocated
biosorption as principal mechanism of arsenic removal opted by NR5.
Beside cell surface adsorption bacteria can also transform metal in to a
different state so that either its impact gets reduced or it can
eliminate from cell environment using porins. Presently study through
silver nitrate assay where left yellow precipitate of silver
ortho-arsenite (Ag3AsO3) in the medium indicate arsenate reduction might
be a secondary mechanism for arsenic removal by NR5. Ability of microbes
to reduce arsenic was evaluated by arsenic transformation assay using
silver nitrate (AgNO3) methods. Test is based on the
reaction between arsenic ion and silver nitrate qualitatively (Krumova
et al. 2008). Silver nitrate mediated arsenite oxidation potential
(Rehman et al. 2010; Dey et al. 2016; Selvankumar et al. 2017) and
arsenate reduction (Das and Barooah, 2018) have been determined in
multiple studies. Bacterial cell after transforming As(V) to As (III)
efflux the arsenite As(III) out of cell system through arsenite
transporter proteins (Sarkar et al. 2013). Afterward the PGP trait
evaluation, tentative confirmation of mechanisms of arsenic removal by
NR5, bacterium was subjected to test in in planta. Results showed
that bacterium amended plant were able to reduce arsenic toxicity and
increased plant growth and development in comparison to plants without
bacterium. Multiple studies have indicated that due to accumulation of
heavy metal in plant parts leading to several cell function disruption
there is decrease in shoot and root length (Sandil et al. 2019; Jamil et
al. 2024). Metal resistant bacteria can reduce heavy metal uptake in
plant through various defence mechanisms such as precipitation,
volatilization, methylation, oxidation, reduction, complex formation,
alkalization or biosorption (Etesami, 2018). Bacteria have developed
mechanisms of extrusion of As to out of cell. A special resistance
mechanism of As in plant transform arsenate to arsenate phosphoglycerate
and transport this complex out of cell using efflux transporter (Zhang
et al. 2022). In addition to this glutathion, thioredoxin, kinase and
s-transferase are also responsible for oxidative stress tolerance
(Gautam et al. 2020). Although there was a little increase in root
length in As stress plants till 60 DAS, but it declined in 75 DAS. It
could be due to As accumulation in roots over the period. Studies have
shown that there was no significant changes in root length in case of
cadmium stress (Lu et al. 2023). Overall bacteria amended plant showed
improved growth close to control plants. Estimation of defence related
biomolecule proline is an active indicator of stress level in plants;
thus, all the plants were tested for their proline level at different
time intervals. Proline, an imino acid, is well known for accumulation
under abiotic stress. Study demonstrated that proline accumulation
helpful to adjust osmotic pressure at cellular level followed by
protection of intracellular organelles from disintegration. In addition
to this, proline also serves as nitrogen storage which can be a nutrient
source to resume the cell growth after releasing stress (Chandrashekhar
and Sandhyarani 1996). Increase in the osmoprotectant proline content
was directly proportional to the heavy metal concentration. In
comparison to control plant at 45 DAS the proline content in As treated
plants was increased 2.390 µmole/g fresh weight. On the other hand, As
and NR5 inoculated plants exhibited reduced intracellular proline
concentration 1.726 µmole/g. Stress induced rapid enhancement in proline
accumulation in plants is reported in various studies (Ghosh et al.
2017; Awasthi et al. 2018; Renu et al. 2020), and the possible mechanism
of defence might be chelating ability, scavenging of free radical and
increased activity of electron transport chain.