Effect of rhizobacteria on arsenic uptake by macrophyte <i>Eichhornia crassipes</i> (Mart.) Solms

<p>Wastewater flowing in streams and <i>nallahs</i> across India carries several trace metals, including metalloid arsenic (As), which are considered serious environmental contaminants due to their toxicity, and recalcitrant nature. In this study, we determined the phytoremediation of As by <i>Eichhornia crassipes</i> (Mart.) Solms either alone or in association with plant growth-promoting rhizobacteria. <i>Pseudomonas</i> and <i>Azotobacter</i> inoculation to <i>E. crassipes</i> resulted in enhanced As removal compared to uninoculated control. Co-inoculation with a consortium of <i>Pseudomonas, Azotobacter, Azospirillum, Actinomyces</i>, and <i>Bacillus</i> resulted in a higher As (<i>p</i> < 0.05) phytoaccumulation efficiency. <i>P. aeruginosa</i> strain jogii was found particularly effective in augmenting As removal by <i>E. crassipes</i>. Our findings indicate that the synergistic association of <i>E. crassipes</i> and various rhizobacteria is an effective strategy to enhance removal of As and thus may be utilized as an efficient biological alternative for the removal of this metalloid from wastewaters.</p

Analysis of enhancement in gamma ray shielding proficiency by adding WO3 in Al2O3-PbO-B2O3 glasses using Phy-X/PSD

Gamma ray shielding ability of WO3 borate glasses is analyzed by using the Phy-X/PSD software. Mass attenuation coefficient of glasses gets enhanced with addition of tungsten oxide resulting in lowering of the values of MFP, HVL, TVL and EBF at 10 mol % of tungsten as compared to many commercial and radiation shielding glasses/concretes. The higher value of FNRCS (∑R) in studied glasses than concretes shows their neutron shielding ability. The sample with highest value of tungsten is most suitable for making radiation shielding materials. The OPD, OMV, n¯c, nb and dbb values indicate the rigid and stable glass structure due to the formations of stable BO4 groups. Poisson ratio confirms high cross density and stability of prepared glasses as its value decreases from 0.233 to 0.226. The increased values of elastic constants and VHN are also in the favour of higher stability and rigidity of the glasses due to addition of WO3. All of these parameters are in good relation with each other and support the shielding power of the studied glasses

Spot-assay correlation in Msm.

<p>To check suitability of the assay. <b>a</b>. <b>MBC correlation</b> showing inter-assay variability (n = 3) in the cfu-based assay in a 25-μl volume in Msm using RIOE. <b>b</b>. <b>Correlation of spot <i>vs</i>. cfu in Msm</b> for RIOE. MBC values were deduced from this finding (<b>a</b>.) and were compared with the data obtained by the conventional method. The spot-assay MBC of four RIOE reference drugs on Msm was compared with the standard cfu-based plating assay. Cfu-based MBC: Minimum concentration that yielded a ≥2 log₁₀ reduction from the starting cfu. Spot-based MBC: Minimum concentration that resulted in no growth (NG) or countable colonies (upto 30 colonies). The data for all four drugs correlated well for both methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117577#pone.0117577.g005" target="_blank">Fig. 5b</a>). <b>c</b>. <b>Correlation of spot <i>vs</i>. cfu of Mtb</b> for RIOE. This method was subsequently replicated in Mtb for all four RIOE reference drugs.</p

MBC₉₀ studies: MBC₉₀ testing on <i>M. tuberculosis</i> (sensitive and drug-resistant isolates) is possible.

<p>MBC₉₀ studies: MBC₉₀ testing on <i>M. tuberculosis</i> (sensitive and drug-resistant isolates) is possible.</p

Spot-assay correlation in Msm.

<p>To check suitability of the assay. <b>a</b>. <b>MBC correlation</b> showing inter-assay variability (n = 3) in the cfu-based assay in a 25-μl volume in Msm using RIOE. <b>b</b>. <b>Correlation of spot <i>vs</i>. cfu in Msm</b> for RIOE. MBC values were deduced from this finding (<b>a</b>.) and were compared with the data obtained by the conventional method. The spot-assay MBC of four RIOE reference drugs on Msm was compared with the standard cfu-based plating assay. Cfu-based MBC: Minimum concentration that yielded a ≥2 log₁₀ reduction from the starting cfu. Spot-based MBC: Minimum concentration that resulted in no growth (NG) or countable colonies (upto 30 colonies). The data for all four drugs correlated well for both methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117577#pone.0117577.g005" target="_blank">Fig. 5b</a>). <b>c</b>. <b>Correlation of spot <i>vs</i>. cfu of Mtb</b> for RIOE. This method was subsequently replicated in Mtb for all four RIOE reference drugs.</p

Optimisation of assay conditions.

<p>Initial assay optimization was done in Msm: <b>a. Minimum culturable volume in Msm</b>: Msm culture was dispensed in 1.56-μl aliquots (1.56μlX64) multiple times, up to 50μl twice (50μlX2), on 24-well agar beds and conventional plates. The sum of the cfu obtained for each volume was plotted. The parallel lines of the cfu data plotted against the plating volumes confirmed that there was no variation in the net bacterial counts obtained from plating different volumes. Hence, the 25-μl volume was selected. <b>b. Drug carry-over assay in Msm</b>: Untreated and isoniazid-treated samples yielded a similar number of cfus. Isoniazid exposure yielded similar bacterial counts from both the conventional and 24-well spot-assay over a range of concentrations up to 16 μg/ml.</p

The schematics of spotting from an MIC plate to a 24-well plate.

<p><b>a</b>. Culture aspiration from a MIC plate using filter-tips fixed at alternate positions of a 12-channel Pipetman. <b>b</b>. <b>Twenty-four-well plate</b>: Row-wise dispensing into a 24-well plate. Two compounds/row, with a 10c-DR each: e.g., Row A: Compound#1 = 2–11 wells and Compound#2 = 14–23 wells (1 & 13 = media control, 12 & 24 = culture control). One plate of 24 wells = data for 2 compounds (10c-DR) for 1 row of MIC plate: The left half of the 24-well plate contains the 1^st compound (#2 to 11), and the right half contains the 2^nd compound (#14 to 23). <b>c. A picture of a spot-assay in a 24-well plate</b>: colony morphology and countable colonies (e.g., rifampicin on the left half and isoniazid on the right half of the plate).</p

Kinetic screen-spot vs. conventional assay.

<p>The survival kinetics of all three Mtb-AS target genes (<i>ppk</i>, <i>pyrH</i> and <i>coaD</i>) demonstrated quantitative cidality in real time on different days. All findings are reproducible and exhibit comparable overlapping graphs (two-tailed P values <0.001 for all three AS data), with a strong positive correlation (ppk r² = 0.693, <i>pyrH</i> r² = 0.920 and <i>coaD</i> r² = 0.856).</p

A plate map for the MIC in a 384-well plate.

<p>A total of 30 test compounds/plate (2 compounds/row) could be tested as a 10-concentration dose response (10c-DR as A2–11 to P2–11 and A14–23 to N14–23). <b>Quality controls (QC)</b>: Columns 1 and 13 = media controls (MC), columns 12 and 24 = culture controls (CC). The 31^st and 32^nd rows = reference drug controls (e.g., isoniazid in rows O&P14 to O&P23) and rows MN14 to MN23 were used for additional reference drug QC, if required. Culture addition was performed with the Multidrop-Combi liquid handling system, and incubation was performed at 37°C for 3–4 days (Msm) and 3–4 weeks (Mtb). Post-incubation, the MIC data were recorded using a spectrophotometer (Spectramax^384Plus) at O.D._600nm.</p

Spot-assay correlation in Mtb and the Manhattan analysis.

<p>The spot-assay was replicated in Mtb for RIOE. <b>a. An MBC correlation between the spot-assay and the conventional method</b>: A strong positive correlation (r² = 0.808). <b>b & c: The Manhattan analysis</b> of isoniazid-treated reference controls exhibited variation within the 2-fold in the spot-assay (<b>b</b>) and conventional MBC methods (<b>c</b>).</p