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.