3.1. Characterization of experimental site
The assessment of the level of As contamination of the selected soil under study were chemically attributed in terms of total (tri-acid extracted) and Olsen-extractable available As. Results revealed a considerable load of the heavy metal of 17.2 ± 1.72 and 1.50 ± 0.27 mg kg-1 respectively. The total microbial count from the soil was 6.4 ± 0.07 log CFU/g. The As resistant microbial count was 3.6 ± 0.09 log CFU/g soil (presented as mean of three observations ± SD). The considerably high proportion of As resistant microbial count can be a cue to address the problem of As contamination more efficiently through a low cost microbial remediation technique.
3.2. Assessment of As resistant bacteria from enrichment culture:
Employing the enrichment culture techniques for possible isolation of As resistant bacterial isolates in Yeast Extract Mannitol (YEMA) solid medium spiked with different As(V) and As(III) concentration, few colonies were observed. Eleven distinct colonies, namely, BcAL-1, JN 73, LAR-2, AR-30, GAR-1, GAR-2, LAR-7, GAR-11, LAR-20, LAR-3 and SAR-05, were picked from the plates and allowed for further growth and study.
3.3. Arsenic accumulation and oxidation-reduction potential of bacterial isolates:
The isolated eleven bacterial strains were investigated for their potential of As accumulation and oxidation/reduction. Initially, a qualitative analysis of ability of the bacterial strains to form Ag3AsO3 or Ag3AsO4 from the AgNO3 solution as visualized by the intensity of colour change to bright yellow and brownish silver was categorized. Four isolates were observed to have distinguishably brighter colour change. To confirm the test, the As accumulation and oxidation/reduction potential of the isolates were addressed through an quantitative estimation by incubating for 12 hr in a liquid culture medium spiked with 5 mM As(V) and As(III). The As content in cell pellet, liquid medium and impregnated filter paper were separately analyzed with the purpose of attaining the quantity of As oxidized or reduced (filter paper) and accumulated (in cell pellet). The results interestingly revealed the similar pattern of microbial alteration of As as in the qualitative test. The strains BcAL-1, JN 73, LAR-2 and AR-30 had shown significant ability to oxidize/reduce and accumulate As and thus enunciated the reduction of highest quantity of As from the initial concentration. As evident from the Table-1, the As recovery from the filter paper (a measure of As oxidization-reduction potential) followed the trend of BcAL-1 (1.63 ±0.43) > JN 73 (1.60±0.69) > LAR-2 (1.59±0.66)> AR-30 (1.56±0.71) for As (V) and BcAL-1 (1.08 ±0.61) > JN 73 (1.07±0.80) > LAR-2 (1.03±0.50)> AR-30 (1.01±0.63) for As (III). The bacterial strains BcAL-1 and JN 73 have shown maximum cellular absorption (40/39% for As(V) and 36% for As(III)), oxidation/reduction of As(III) (21/22%) and As(V) (33/32%) while left least residues (25/26% for As(V) and 40% for As(III)); (Fig-1) in solution followed by LAR-2 > AR-30.
3.4. MIC and arsenite oxidase activity of the selected bacterial isolates:
The four most efficient strains as obtained from the previous section were tested for their minimum inhibitory concentration of As. The results in Table-2 revealed BcAL-1 had highest MIC value (408 mM for As(V) and 46.2 mM for As(III)) followed by JN 73 (390 mM for As(V) and 41.2 mM for As(III)). The MIC values of the remaining two strains were considerably lower, as LAR-2 (300 mM, As(V) and 31.3 mM, As(III))> AR-30 (275 mM, As(V) and 28.1 mM, As(III)). The strain, SAR-05, as evident from both Table-1 and 2, showing no resistance to As, was taken as control.
The highest MIC value carrying bacteria also had the highest arsenite enzyme activity. As in Table 2, the specific As(III) oxidase activity of these four bacterial isolates (BcAL-1, JN 73, LAR-2 and AR-30) were 5.82, 5.30, 4.97, 4.60 nM min-1 mg-1 of protein respectively. Concurrently, the result brought about the synchrony that the bacterial strains having higher MIC value will have the higher As (III) oxidase activity.
3.5. Biochemical characterization and identification of the As-resistant PGP Bacteria
All the selected strains of bacteria were found to be gram negative and rod shaped. The strains (BcAL-1, JN 73, LAR-2 and AR-30) screened on the basis of phenotypic and biochemical tests have been represented in Table-3. All these strains were found to be oxidase, catalase and urease positive. Further employing 16S rRNA gene sequencing a phylogenetic tree was prepared (Fig-2). These identified bacterial isolates thus assumed to be Burkholderia cepacia (BcAL-1, accession number KJ461686), Burkholderia metallica (JN 73, accession number KJ507654), Burkholderia sp. (LAR-2, accession number MK634685 and Burkholderia cenocepacia (AR-30, accession number KY992359), as in Table-4. Further to confirm the results, the SEM study of the bacterial isolates (Fig- 3) was categorized.
3.6. Identification of arsenite oxidase gene:
The As(III) oxidase gene was detected in our selected As(III) oxidizing bacteria. A fragment of 1200bp was amplified via polymerase chain reaction (PCR) obtained from the genomic DNA of BcAL-1, JN 73, LAR-2 and AR-30 (Fig-4). Based on the gene sequencing, the sequences were submitted to gene bankit and the accession number was obtained. A phylogenetic tree of As(III) oxidase gene sequences of the As oxidizing bacteria were drawn (Fig-5) through Maximum likelihood algorithms and bootstrapping procedure.
3.7. Potential plant growth promoting attributes in screened As tolerant bacteria
The plant growth promoting traits of the four most efficient As tolerant isolates and one control (SAR-05) isolate were categorized. All of these strains were able to solubilize phosphate, produce IAA and ACC Deaminase under As(V) and As (III) stressed condition. BcAL-1, JN 73, LAR-2 and AR-30 were observed to solubilize highest amount of phosphate (570, 563, 553, 560 μg/L) under As free condition and even solubilized significant amount of phosphate when the culture medium are spiked with 15 and 30 mg/L of As(V) and As(III) (Table 5 and 6).
BcAL-1 was the best performer in nodulation, IAA production and ACC production both in As free and As stressed condition. Stresses imposed by As(V) spiking failed to effect phosphate solubilization, IAA production, ACC Deminase activity and nodulation significantly while under As(III) stress, phosphate solubilization, IAA production, siderophore production and ACC Deminase production were significantly impacted (Table-5 and 6). Comparing all the aspects of PGP in the selected bacterial isolates, two isolates, namely BcAL-1 and JN 73 were able to produce siderophore under all conditions, which the other two strains failed at higher As stress condition.