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Introduction

The human populations of eastern India (West Bengal mainly) and Bangladesh are severely suffered by arsenic (As) contamination of water and food^{1, 2}。The concurrent application of As enriched irrigation seems to be the major reason for soil build-up^{3, 4}and subsequent accumulation in standing crops⁵。Generally, As residues are found in the top layer of soil or surface soil and enter the plants easily because of their low volatility and low solubility⁶。The As has both organic and inorganic forms⁷that found as an oxyanion in the environment⁸。The high-risk involvement and high cost of remediation⁹emphasize the scope of bioremediation through novel As-tolerant microbes¹⁰。

In As polluted area, there are several soil-based microbes¹¹with adaptive strategies for surviving in a contaminated atmosphere; mostly byutilizing the toxic arsenic as a nutrient for their metabolisms and proliferation⁶。Some bacteria such asBurkholderia¹²,Agrobacterium, Bacillus, Rhizobiumare best suitable candidate for arsenic bioremediation and plant growth promotion (PGP)¹³。These microorganisms can have a dual purpose environmental beneficial impacts by metal decontamination and plant growth promoters^{14, 15}。These PGP bacteria are able to enrich soil nutrient and able to mitigate soil arsenic also¹³。The physical and chemical process for arsenic mitigation are very costly so these rhizobium-legume symbiosis may a alternative new tool for As mitigation¹⁶。

阻力和植物g的独特属性rowth promotion (PGP) in rhizospheric microbes is quite noteworthy. These adopted indigenous soil microbes tend to manifest several PGP traits through secretion of 1-aminocyclopropane-1-carboxylate (ACC), deaminase, indole-3-acetic acid (IAA), phosphate solubilizers, producing siderophores that reduce metal toxicity and encourage plant-assisted bioremediation^{17, 18, 19}。Most remarkably, such PGP characters remain active even under intense arsenic stress conditions^{20, 21}。

In this backdrop, we have categorized the study and identified efficient As-resistant bacterium from contaminated rhizosphericsoil, and investigated the PGP attributes of identified bacteria with a view to harvest their capacity to develop plant’s resistance to stress conditions, encourage plant growth, and give a path to contribute accelerated remediation of arsenic polluted soils.

Materials and Methods

Soil Sample Analysis

Soil samples (2 cm diameter, 10 cm depth) were collected in sterilized zipper bags to maintain an aseptic condition from arsenic (As) contaminated rhizospheric zone of lentil in Chakdaha, West Bengal (23º05' N latitude and 88º54' E longitude), India, that noted for As concentrations in the groundwater above World Health Organization (WHO)-defined safe limit²²。Atomic AbsorptionaSpectrophotometera (AAS, Model-Perkin Elmer A Analyst 200) was used to measure total²³and available As²⁴of the soil samples.

Isolation;of;As-Tolerant;Bacteria

2 g of soil;(taken from lentil rhizosphere separately) was; suspended in 2 mL;sterile;distilled water, 1mL of each was re-suspended in Yeast Extract Mannitol (YEM) liquid medium separately in two conical flasks, spiked with 1 mM arsenate and 1 mM arsenate.The total experimental set up was incubated for 48 hours at 30°C²⁵。After incubation, 2 ml bacterial culture was further inoculated in a YEM liquid medium.The procedure was repeated twice. Around 0.1;mL;of As spiked culture was spread on a YEMA (Yeast;Extract;Mannitol Agar) solid medium plate to isolate the As tolerant bacteria. To test theRhizobiumstrain, the congo red YEMA test was categorized²⁶。

MIC (Minimum Inhibitory Concentration) Value of the Bacteria

The MIC is the lowest concentration of arsenate or arsenite which entirely inhibits microbial growth and activity^{27, 28}。1.0 mL of overnight bacterial culture was inoculated in two conical flasks containing 99.0 mL of YEM liquid medium, containing either arsenite (NaAsO₂; 1-50 mM) or arsenate (Na₂HAsO₄·7H₂O; 1-500 mM) separately and;incubated;at 30ºC with 48 hrs of shaking. The OD (optical density, measurement of microbial growth) of the bacterial cultures was detected using a UV-Vis spectrophotometer (Model: Varian CARY-50) at 600 nm wavelength.

Identification of the As;Tolerant;Bacteria

The As;tolerant;bacterial;genomic DNA was;isolated and PCR amplification for molecular identification by 16S rRNA sequence analysis²⁷。The bacterial isolate was studied for Gram reaction, colony morphology and characterized for catalase, urease, and oxidase activities by standard protocols²⁹。

Scanning Electron Microscopic (SEM) Study

For the SEM study,First the bacterial cells were collected andallowed to wash properly with sodium;phosphate;buffer;(pH 7.4).Then a thin smear of bacterial cells were kept on a glass cover slip and prepared a smear, after that it was allowed to heat-fixover a flame;for;1–2 sec followed;by;fixation;with;2.5%;glutaraldehyde;for;45 min³⁰。The slides;were;then;dehydrated with;50–90%;of;alcohol;solutions;and;finally;through;absolute;alcohol;for;5;min;each;and observed;under;a;15kV;scanning;electron;microscope;(HITACHI,;S-530,;SEM,;and;ELKO Engineering).

Arsenite Transformation; and Species Detection

The arenite; transformation; ability of the bacterial; isolate; was determined by modified laboratory-based standard protocol²⁷。As concentrations were determined by using a spectrophotometric method^31.The recoveries of the As species fromthe liquid culture medium by the efficient bacterial strains were determined by HPLC-ICP-MS³²。所有化学物质用于物种形成研究意图gent grade. All of the experimental solutions were prepared with Mili-Q (Millipore, Bedford, MA, USA) water. A Perkin Elmer Series 200 Micro Pump was used instead of the quaternary pump for the isocratic method. The isocratic mobile phase was 30 mM NH₄H₂PO₄at pH 5.6. The flow rate was 1.0 mL min^-1with 100 μL sample injections. The LC column effluent was directly attached to the nebulizer with PEEK tubing (1.59 mm OD) and a low dead volume PEEK connector (Part No.:WE024375).The isocratic mobile phase was 30 mM NH₄H₂PO₄at pH 5.6. The flow rate was 1.0 mLmin^-1with 100 μL sample injections. The LC column effluent was directly attached to the nebulizer with PEEK tubing (1.59 mm OD) and a low dead volume PEEK connector (Part No.: WE024375).

Plant Growth Promoting (PGP) Characteristics of the As Tolerant Bacterium

The isolated arsenic resistant bacterial PGP attributes (IAA-production,;ACC;Deaminase activity,;phosphate;solubilization,;nodulation,;and;siderophore;production) were determinedin vitroin culture medium under arsenic stress conditions (spiking levels being 0 mg/L,;15 mg/L,;and 30 mg/L;As(III)/As(V)).

Ability to Solubilize Phosphate

This encompassesthe growth of the bacterial strain in Pikovskaya’s medium³³(containing 0.5%;of;tricalcium;phosphate;(TCP);spiked with three levels of arsenate and arsenite 0, 15, 30 mg/kg) at 30°C for 5-6 days and 170 rev/min. Then the culture was allowed to centrifuge at 6500 times gravity (×g) and supernatant was collected. The solubilized phosphate in supernatant of culture medium was estimated³⁴。

Screening of Indole Acetic Acid (IAA)

The As resistant isolates were determined by growing them in an L-tryptophan (0.5 mg/ml) supplemented minimal medium;in;presence;of;a different;concentration;of As (0, 15, 30 mg/L) and incubated for 5 days in the dark at 30⁰C. 2 ml bacterial suspension was transferred in 100 µl 10 mM orthophosphoric acid and 4 ml Salkowski’s reagent (2% solution of 0.5 M FeCl₃in 35% perchloric acid) in a test tube. The entire mixture was shaken vigorouslybefore incubation for 45 min until a pink colour develops. Absorbance of the resultant solution was determined at 530 nm for obtaining the content of IAA-like molecules in liquid culture medium³⁵。

ACC Deaminase Activity

The ACC Deaminase enzymeactivity is the quantity of α-ketobutyric acid production by the breakdown of ACC³⁶by the bacterial strain.To determine this, a minimal medium was prepared using1-aminocyclopropane-1-carboxylic acid orACC (3 gL⁻¹) as a source of nitrogen, spiked with three different concentrations of As(V) and As(III) (0, 15, 30 mg/L) separately and the bacterial cells were grown. The quantity of ketobutyrate (KB) formed per mg of protein per hour is the total value of the specific enzyme activity.

Nodulation Efficiency

The nodulation efficiency of bacterial strain³⁷was assessed through a pot study. Soils were sterilized and the seeds of lentil were sown in the sterilized soil spiked with As (0, 15, 30 mg/kg) and the nodule counts were taken after 30 days.

Screening for Siderophore Production

The siderophores production was qualitatively assayed by using the Chrome Azural S method of Schwyn and Neilands³⁵。The MM9 (Tris buffer, casamino acids (0.3%), L-glutamic acid (0.05%), (+)- biotin (0.5 ppm), and sucrose (0.2%)) liquid medium with the absence of Fe was used for bacterial growth. The bacterial culture medium was inoculated in this medium and allowed to incubate for 5 days at 30ºC temperature. For control 0.2 µM of iron (freshly prepared, filter-sterilized FeSO_4.7H₂O stock solution) was also inoculated. The stationary phase of bacterial culture wascollected and pelleted by centrifugation (6,500 x g for 15 minutes). In supernatant solution, the most important qualitative conformation of the presence of siderophore is simply the color change from blue to orange.

Statistical Analysis

Statistical computations were performed usingMicrosoft Excel 2016andSPSS version 23.0.

Results

Characterization of the Experimental Site

The total As concentration and available As concentration of experimental rhizospheric soil of lentil were measured. Results revealed a considerable load of the total and available arsenic i.e. 17.2 ± 1.72 and 1.50 ± 0.27 mg kg^-1respectively (presented as the mean of three observations ± SD).

Isolation and Identification of the As Tolerant Bacteria

For isolation of Rhizobium, the YEMA medium with congo red was used.A few distinct colonies were found (Fig 1). From these,a distinct colony was picked and allowed to further study.

Figure 1: Rhizobium Isolates in Congo Red YEMA Medium. Click here to view Figure

The isolated strains of rhizobacteria were found to be gram-negative and rod-shaped. Theseisolated strainswere studied for phenotypic and biochemical studieshave been represented in Table-1.The bacterial isolate waspositive for catalase, glucose, sucrose, sorbitol, lactose, mannitol, maltose, xylose, and fructose; while oxidase,indole, citrate, MR, VP, urease activity were found to be negative.

Table 1: Morphological and Biochemical Properties of the Bacteria.

Further employing 16S rRNA gene sequencing a phylogenetic tree was prepared (Fig-2). These identified bacterial isolates are thus assumed to beRhizobium leguminosarum(LAR-7, accession numberMK696942).

Figure 2: Click here to view Figure

Further to confirm the results, the SEM study of the bacterial isolates (Fig- 3) was also categorized.

Figure 3: SEM Image of the Bacterial Strain. Click here to view Figure

MIC of the Arsenic Resistant Bacterial Isolates

The arsenic tolerant strains were tested for their minimum inhibitory concentration of As.The result showed that LAR-7(Rhizobium leguminosarum) hada high MIC value.Bacterial isolate had emerged with a high MIC towards arsenate (260 mM) and arsenite (27.5 mM).

Arsenite’transformation’and’species’detection

The As accumulation and volatilization potential of the isolate wasexperimented with through a quantitative estimation by incubating for 24 hr in a liquid culture medium spiked with 30mM As(III). The As content in liquid medium wasanalyzedto attain the quantity of Asdecontamination. The bacterial strain LAR-7 has shown a significantlevel of arsenic decontamination ability. 35% of total arsenic was decreased from the liquid culture medium containing 30 mMof arsenite.The bacterial transformation of arsenite to arsenate was 437.5μM h^-1, which is considered 35%of the total arsenite of media (Table 2). LAR-7 could oxidize 35% of 30 mM arsenite within 24 hours.

Table 2: Arsenic Transformation and Species Detection of the;bacterial’isolates’under 10mg/L As(III);Application.

Each value is a mean ofthree replicates. mean ± standard deviation.

In this study, about 61% of the total As was recovered in As species. The transformation of arsenite to arsenate in liquid culture broth varied from 0 (uninoculated control) to 39% (medium inoculated with LAR-7) by the selected arsenic resistant microbial strains.

Potential Plant; growth;promoting;properties;of;As tolerant bacteria

The plant;growth;promoting;traits;of;the;As;tolerant;isolate was categorized. The strain LAR-7 was able to solubilize phosphate, produce IAA and ACC Deaminase under As(V) and As (III) stressed conditions (Table-3).

Table 3: Plant Growth Promoting Properties of the Bacterium LAR-7.

Each;value;is’a’mean’of’three’replicates. mean ±’standard;deviation.

LAR-7 was observed to solubilize a good amount of phosphate (440.8, 440.5, 337.5μg/L when the medium was spiked with 0,15,30 mg/kg arsenate respectively).Similarly, the same results were also found under arsenite stress conditions. This bacterial isolate also solubilizesphosphate 440.8, 140.0, 137.2 μg/L when the medium was spiked with 0,15,30 mg/kg arsenite respectively. Under As stress condition, theamount of phosphate solubilization was decreased but still the bacteria also able to solubilize phosphate.

LAR-7 also was’able’to’produce;Indole;acetic;acid;(IAA) and had a good ACC Deaminase activity. The quantity of IAA and ACC Deaminase activity were 16.4,12.8,12.8 μM IAA ml^-1(medium containing 0,15,30 mg/kg arsenate)and 1.99,1.99,1.87 nM α-ketobutyrate mg protein^-1h^-1(medium containing 0,15,30 mg/kg arsenite) respectively. Under arsenite (medium containing 0,15,30 mg/kg arsenite) stress conditionthe quantity of IAA and ACC Deaminase activity were 16.4,10.0,10.0 μM IAA ml^-1and 1.99,1.09,1.00 nM α-ketobutyrate mg protein^-1h^-1respectively. Under arsenite and arsenate stress condition arsenic resistant bacterial isolate LAR-7 was also able to produce the root nodule and also the ability to produce siderophore.The siderophore production was qualitatively tested. So LAR-7 (Rhizobium leguminosarum) is an arsenic resistant bacterium also able to carry a bunch of plant growth promoting properties (Fig- 4).

Figure 4: PGP Attributes of the Bacterial Strain LAR-7. Click here to view Figure

Discussion

Identification and isolation;of;As-resistant;bacterial;strains;from;contaminated;soils

In the course of identification and;characterization;of;arsenic;resistant;PGP;bacteria;from the As polluted area in the present investigation LAR-7 (Rhizobium leguminosarum) bacterial isolate had emerged with a high MIC towards arsenate (260 mM) and arsenite(27.5) mM which is higher than the previously reported MIC for arsenate (260 mM) and arsenite (27.5mM) forRhizobium radiobacterof 10 mM of As(V) in agricultural soils³⁸and also greater than other As-oxidizing, As-resistant bacteria in soil;(183 mM arsenate and 6 mM of arsenite)¹⁵, in ground water (200 mM;arsenate and 5 mM;arsenite)³⁹, in mines (10 mM As(V))⁴⁰。LAR-7 also showed a greater MIC value thanRhizobium radiobacterwhich showed the highest;MIC;of >1,500 mg/L;of arsenic⁴¹。The isolate LAR-7 in our present;investigation;was;identified;asRhizobium leguminosarum(based;on;16S;rRNA;gene;sequence;homology) which can oxidize at the rate of 437.5μM h⁻¹(35%) in presence of an arsenite concentration (30 mM) within 24 h under laboratory condition. The behaviour is somewhat similar toAlcaligenessp. completely oxidizes arsenite within 40 h when exposed to relatively reduced arsenite concentrations (1 mM)⁴²。The enhanced oxidation efficiencies of isolates in the present research have been quantitatively corroboratedviaspeciation analysis. It was previously reported that some beta and gamma proteobacteria are also able to oxidise arsenic⁴³。

Plant Growth Promoting Attributes in As Resistant, Arsenite Oxidizing Bacterial Strains

Recent studies have thrown some light on the PGP traits shown by the As oxidizing bacteria. The isolated bacterial strains ofAcinetobacter sp., Klebsiella sp., Pseudomonas sp., Enterobacter sp.andComamonas sp.from As- polluted tannery wastes under agricultural lands in Thailand³⁵possess both As tolerance and siderophore production capacity¹⁰。Arthrobacterglobiformis,:Bacillus:megaterium,:Bacillus:cereus,:Bacillus;pumilus,:Staphylococcus:lentus,Enterobacter;asburiae,;Sphingomonas;paucimobilis,:Pantoea:spp.,”Rhizobium:rhizogenes,and:Rhizobium:radiobacter:are:some:arsenic:resistant:bacteria.Rhizobium:rhizogenes:has:ahigh:MIC:Value¹³。Rhizobium;leguminosarum;are;also;capable;to;mitigate;As⁴⁴。

Rhizobium community can survive the heavy metal pollutant soil and effective to enrich the soil;nutrient;and;save;the;enhance;the;growth;of;the;plant;in;contaminated;field¹⁶。Bradyrhizobium japonicum:has:potential:as:a:plant:growth-promoting:rhizobacterium also carries a bunch of PGPR attributes.Rhizobium melilotialso produce IAA-like molecules and able to solubilize a significant amount of phosphate⁴⁵under stress conditions.Rhizobium japonicum, Rhizobium spp,Rhizobium leguminosarumare the three plant growth promoting bacteria which are showed their PGPR activities under heavy metal stress conditions. The LAR-7 also could solubilize a significant amount of phosphate and produce IAA. Most bacteria under:Pseudomonas:sp.,:Acinetobacter sp.:and:Paenibacillus sp.;were reported to be potential plant growth promoters³⁵along withBacillus aryabhattaiwhich behaves similar to our studied strain forbeing an As resistant plant growth promoter²⁰。

Similar observations were also obtained with As-resistant bacteria of Alpha proteobacteria, Beta proteobacteria, and Gamma proteobacteria manifesting potential PGP attributes^{46, 10}。Pseudomonas sp。has been reported to possess high As(III) oxidizing capacity while at the same time found to solubilize a significant amount of phosphate, indulge in siderophores,:IAA-like molecules,:and:ACC:Deaminase:production³⁵。

The:present investigation has indisputably established the manifestation of:PGP:traits:ofAs:tolerant, As;oxidizing;bacterial;isolate;Rhizobium leguminosarum,insolubilizing phosphate;, producing siderophores, root nodule, IAA-like molecules, and ACC deaminase under As stress.Rhizobium leguminosarum(LAR-7) had emerged as best performing candidate isolates with regard to As resistance and PGP traits.

Conclusion

In pursuit of providing environmental safeguard to restore food safety and sustaining food security to the burgeoning population and combat abiotic pollution, a low-cost alternative to exorbitant pollution control strategies remained an absolute priority. The outcome of the present investigation revealed the candidate bacterial isolateRhizobium leguminosarumas a potent, novel bacterial strain for As mitigation and may be useful, through fulfillment of mass production and field validation protocols, in As decontamination and plant growth promotion.

Acknowledgement

The authors are grateful to the ICAR-Niche area of Excellence-As Research Laboratory, Bidhan Chandra Krishi Viswavidyalaya (BCKV), Kalyani, Nadia as well as Department of Genetics & Plant Breeding and Department of Agronomy, Bidhan Chandra Krishi Viswavidyalaya (BCKV), Mohanpur, Nadia, West Bengal, India for providing technical and all other necessary assistance during the study.

Funding Source

这项研究没有收到任何特定的格兰特from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work.

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