Operational parameters optimization for remediation of crude oil-polluted water in floating treatment wetlands using response surface methodology.

The application of floating treatment wetlands (FTWs) is an innovative nature-based solution for the remediation of polluted water. The rational improvement of water treatment via FTWs is typically based on multifactorial experiments which are labor-intensive and time-consuming. Here, we used the response surface methodology (RSM) for the optimization of FTW's operational parameters for the remediation of water polluted by crude oil. The central composite design (CCD) of RSM was used to generate the experimental layout for testing the effect of the variables hydrocarbon, nutrient, and surfactant concentrations, aeration, and retention time on the hydrocarbon removal in 50 different FTW test systems planted with the common reed, Phragmites australis. The results from these FTW were used to formulate a mathematical model in which the computational data strongly correlated with the experimental results. The operational parameters were further optimized via modeling prediction plus experimental validation in test FTW systems. In the FTW with optimized parameters, there was a 95% attenuation of the hydrocarbon concentration, which was very close to the 98% attenuation predicted by the model. The cost-effectiveness ratio showed a reduction of the treatment cost up to $0.048/liter of wastewater. The approach showed that RSM is a useful strategy for designing FTW experiments and optimizing operational parameters.

First report of diazotrophic Brevundimonas spp. as growth enhancer and root colonizer of potato.

Rhizobacteria contain various plant-beneficial traits and their inoculation can sustainably increase crop yield and productivity. The present study describes the growth-promoting potential of Brevundimonas spp. isolated from rhizospheric soil of potato from Sahiwal, Pakistan. Four different putative strains TN37, TN39, TN40, and TN44 were isolated by enrichment on nitrogen-free malate medium and identified as Brevundimonas spp. based on their morphology, 16S rRNA gene sequence, and phylogenetic analyses. All strains contained nifH gene except TN39 and exhibited nitrogen fixation potential through acetylene reduction assay (ARA) except TN40. Among all, the Brevundimonas sp. TN37 showed maximum ARA and phosphate solubilization potential but none of them exhibited the ability to produce indole acetic acid. Root colonization studies using transmission electron microscopy and confocal laser scanning microscopy showed that Brevundimonas sp. TN37 was resident over the root surface of potato; forming sheets in the grooves in the rhizoplane. TN37, being the best among all was further evaluated in pot experiment using potato cultivar Kuroda in sterilized sand. Results showed that Brevundimonas sp. TN37 increased growth parameters and nitrogen uptake as compared to non-inoculated controls. Based on the results obtained in this study, it can be suggested that Brevundimonas spp. (especially TN37) possess the potential to improve potato growth and stimulate nitrogen uptake. This study is the first report of Brevundimonas spp. as an effective PGPR in potato.

Growth stimulatory effect of AHL producing Serratia spp. from potato on homologous and non-homologous host plants.

Plant growth promoting rhizobacteria are known to improve plant performance by developing healthy and productive interactions with the host plants. These associations may be symbiotic or asymbiotic depending upon the genetic potential of the resident microbe and promiscuity of the host. Present study describes the potential of two Serratia spp. strains for promotion of plant growth in homologous as well as non-homologous hosts. The strains KPS-10 and KPS-14; native to potato rhizosphere belong to genus Serratia based on 16S rRNA gene sequences (accession no. LN831934 and LN831937 respectively) and contain multiple plant growth promoting properties along-with the production of quorum sensing acyl homoserine lactone (AHL) molecules. Both Serratia spp. strains showed solubilization of inorganic tri-calcium phosphate while KPS-14 also exhibited phytase activity (1.98 10-10 kcat). KPS-10 showed higher P-solubilization activity (128.5 µg/mL), IAA production (8.84 µg/mL), antifungal activity and also showed the production of two organic acids i.e., gluconic acid and lactic acid. Both strains produced three common AHLs: C6-HSL, 3oxo-C10-HSL, 3oxo-C12-HSL while some strain-specific AHLs (3OH-C5-HSL, 3OH-C6-HSL, C10-HSL specific to KPS-10 and 3OH-C6-HSL, C8-HSL, 3oxo-C9-HSL, 3OH-C9-HSL specific to KPS-14). Strains showed roots and rhizosphere colonization of potato and other non-homologous hosts up to one month. In planta AHLs-detection confirmed a likely role of AHLs during seedling growth and development where both extracted AHLs or bacteria inoculated roots showed extensive root hair. A significant increase in root/shoot lengths, root/ shoot fresh weights, root/shoot dry weights was observed by inoculation in different hosts. PGP-characteristics along with the AHLs-production signify the potential of both strains as candidate for the development of bio-inoculum for potato crop in specific and other crops in general. This inoculum will not only reduce the input of chemical fertilizer to the environment but also improve soil quality and plant growth.

Pseudomonas sp. AF-54 containing multiple plant beneficial traits acts as growth enhancer of Helianthus annuus L. under reduced fertilizer input.

Plant growth promoting rhizobacteria (PGPR) are capable to increase the growth and yield of crops in eco-friendly and sustainable manner. To evaluate the response of sunflower towards inoculation with PGPR, a sunflower root associated bacterium AF-54 isolated from Diyar Gali Himalayan Mountain region, Azad Jammu and Kashmir (AJK), identified as Pseudomonas sp. by 16S rRNA sequence analysis and was characterized using polyphasic approach. The bacterium produced 23.9 µgmL-1 indole-3-acetic acid in tryptophan-supplemented medium, showed 44.28 nmoles mg-1 protein h-1 nitrogenase activity through acetylene reduction assay and released 48.80 µg mL-1 insoluble phosphorus in Pikovskaya's broth. During P-solubilization, the pH of the Pikovskaya's medium decreased from 7 to 3.04 due to the production of acetic acid, malic acid and gluconic acid. Pseudomonas sp. AF-54 showed metabolic versatility by utilizing 79 carbon sources from BIOLOG GN2 plates and resistance to many antibiotics. Furthermore, it inhibited the growth of Fusarium oxysporum in dual culture assay. To evaluate the plant-inoculation response, series of experiments conducted in hydroponic, sterilized soil and fields at AJK, and Faisalabad where inoculated plants with reduced fertilizer showed a significant increase in growth, yield, oil contents and achene NP uptake as compared to non-inoculated control. AF-54 showed extensive root colonization in sterilized and non-sterile conditions documented through yfp-labeling and fluorescent in situ hybridization coupled with confocal laser scanning microscopy. This study concludes that the Pseudomonas sp. strain AF-54 containing multiple plant growth promoting traits can be a potential candidate for biofertilizer production to enhance sunflower crop yield with reduced application of chemical (NP) fertilizers.

Successful phytoremediation of crude-oil contaminated soil at an oil exploration and production company by plants-bacterial synergism.

Phytoremediation is a promising approach for the cleanup of soil contaminated with petroleum hydrocarbons. This study aimed to develop plant-bacterial synergism for the successful remediation of crude oil-contaminated soil. A consortia of three endophytic bacteria was augmented to two grasses, Leptochloa fusca and Brachiaria mutica, grown in oil-contaminated soil (46.8 g oil kg-1 soil) in the vicinity of an oil exploration and production company. Endophytes augmentation improved plant growth, crude oil degradation, and soil health. Maximum oil degradation (80%) was achieved with B. mutica plants augmented with the endophytes and it was significantly (P < 0.05) higher than the use of plants or bacteria individually. Moreover, endophytes showed more persistence, the abundance and expression of alkB gene in the rhizosphere as well as in the endosphere of the tested plants than in unvegetated soil. A positive relationship (r = 0.70) observed between gene expression and crude oil reduction indicates that catabolic gene expression is important for hydrocarbon mineralization. This investigation showed that the use of endophytes with appropriate plant is an effective strategy for the cleanup of oil-contaminated soil under field conditions.

Isolation and characterization of a ß-propeller gene containing phosphobacterium Bacillus subtilis strain KPS-11 for growth promotion of potato (Solanum tuberosum L.).

Phosphate-solubilizing and phytate-mineralizing bacteria collectively termed as phosphobacteria provide a sustainable approach for managing P-deficiency in agricultural soils by supplying inexpensive phosphate to plants. A phosphobacterium Bacillus subtilis strain KPS-11 (Genbank accession no. KP006655) was isolated from potato (Solanum tuberosum L.) rhizosphere and characterized for potato plant growth promoting potential. The strain utilized both Ca-phosphate and Na-phytate in vitro and produced 6.48 µg mL(-1) indole-3-acetic acid in tryptophan supplemented medium. P-solubilization after 240 h was 66.4 µg mL(-1) alongwith the production of 19.3 µg mL(-1) gluconic acid and 5.3 µg mL(-1) malic acid. The extracellular phytase activity was higher (4.3 × 10(-10) kat mg(-1) protein) than the cell-associated phytase activity (1.6 × 10(-10) kat mg(-1) protein). B. subtilis strain KPS-11 utilized 40 carbon sources and showed resistance against 20 chemicals in GENIII micro-plate system demonstrating its metabolic potential. Phytase-encoding gene ß-propeller (BPP) showed 92% amino acid similarity to BPP from B. subtilis (accession no.WP_014114128.1) and 83% structural similarity to BPP from B. subtilis (accession no 3AMR_A). Potato inoculation with B. subtilis strain KPS-11 increased the root/shoot length and root/shoot weight of potato as compared to non-inoculated control plants. Moreover, rifampicin-resistant derivative of KPS-11 were able to survive in the rhizosphere and on the roots of potato up to 60 days showing its colonization potential. The study indicates that B. subtilis strain KPS-11 can be a potential candidate for development of potato inoculum in P-deficient soils.

Bacterial rhizosphere and endosphere populations associated with grasses and trees to be used for phytoremediation of crude oil contaminated soil.

Different grasses and trees were tested for their growth in a crude oil contaminated soil. Three grasses, Lolium perenne, Leptochloa fusca, Brachiaria mutica, and two trees, Lecucaena leucocephala and Acacia ampliceps, were selected to investigate the diversity of hydrocarbon-degrading rhizospheric and endophytic bacteria. We found a higher number of hydrocarbon degrading bacteria associated with grasses than trees and that the endophytic bacteria were taxonomically different from rhizosphere associated bacteria showing their spatial distribution with reference to plant compartment as well as genotype. The rhizospheric soil yielded 22 (59.45 %), root interior yielded 9 (24.32 %) and shoot interior yielded 6 (16.21 %) hydrocarbon-degrading bacteria. These bacteria possessed genes encoding alkane hydroxylase and showed multiple plant growth-promoting activities. Bacillus (48.64 %) and Acinetobacter (18.91 %) were dominant genera found in this study. At 2 % crude oil concentration, all bacterial isolates exhibited 25 %-78 % oil degradation and Acinetobacter sp. strain BRSI56 degraded maximum. Our study suggests that for practical application, support of potential bacteria combined with the grasses is more effective approach than trees to remediate oil contaminated soils.

Genetic, physiological and biochemical characterization of Bacillus sp. strain RMB7 exhibiting plant growth promoting and broad spectrum antifungal activities.

BACKGROUND: Plant growth promoting rhizobacteria (PGPR) are functionally diverse group of bacteria having immense potential as biofertilizers and biopesticides. Depending upon their function, they may serve as partial replacements for chemical fertilizer or pesticides as an eco-friendly and cost-effective alternatives as compared to their synthetic counterparts. Therefore, isolation, characterization and practical evaluation of PGPRs having the aforementioned multifaceted beneficial characteristics, are essentially required. This study describes the detailed polyphasic characterization of Bacillus sp. strain RMB7 having profound broad spectrum antifungal activity and plant growth promoting potential. RESULTS: Based on 16S rRNA gene sequencing, strain RMB7 was identified as Bacillus specie. This strain exhibited the production of 8 mg. L(-1)of indole-3-acetic acid (IAA) in tryptophan-supplemented medium. It was able to solubilize 50.6 mg. L(-1) tri-calcium phosphate, reduced 601ηmol acetylene h(-1)/vial and inhibited >70% growth of nine fungal phytopathogens tested in vitro. Under natural pathogen pressure, inoculation with strain RMB7 and RMB7-supernatant conferred resistance by arugula plant against Pythium irregulare with a concurrent growth improvement over non-inoculated plants. The T-RFLP analysis based on 16S rRNA gene showed that inoculation with RMB7 or its supernatant have a major impact on the indigenous rhizosphere bacterial population. Mass spectrometric analysis revealed the production of lipopeptide surfactins as well as iturin A presence in crude extract of RMB7. PCR-amplification further confirmed the presence of genes involved in the biosynthesis of these two bioactive lipopeptide compounds. CONCLUSIONS: The data show that Bacillus sp. strain RMB7 has multifaceted beneficial characteristics. It may be an ideal plant growth promoting as well as biocontrol agent, for its integrated use in disease and nutrient management strategies.

Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11.

An Enterobacter sp. Fs-11 was isolated from sunflower rhizosphere, identified on the basis of 16S rRNA gene sequence analysis (GeneBank accession no. GQ179978) and studied for its root colonization and growth promotion ability in sunflower. Morphologically, it was rod shaped Gram-negative, motile bacterium, producing 4.5 µg mL(-1) indole acetic acid in tryptophan-supplemented medium. It utilized 27 out of 95 substrates in BIOLOG GN2 micro plate system. It was able to convert insoluble tri-calcium phosphate to soluble phosphorus up to 43.5 µg mL(-1) with decrease in pH of the medium up to 4.5 after 10 days incubation at 28 ± 2 °C in the Pikovskaya's broth. High performance liquid chromatography of cell free supernatant showed that Fs-11 produced malic acid and gluconic acid (2.43 and 16.64 µg mL(-1), respectively) in Pikovskaya's broth. Analysis of 900 bp fragment of pyrroloquinoline quinine pqqE gene sequence showed 98 % homology with that of E. cloacae pqqE gene. Confocal laser scanning microscope revealed strong colonization of fluorescently labeled Fs-11 with sunflower roots. Sunflower inoculation with Fs-11 and its rifampicin resistant derivative in sterile sand and natural soil showed that Fs-11 colonized sunflower roots up to 30 days after transplanting in both sterile sand as well as natural soil. Moreover, Fs-11 inoculation resulted in increased plant height, fresh weight, dry weight and total phosphorus contents as compared to un-inoculated plants. The data showed that Enterobacter sp. Fs-11 is an efficient phosphate solubilizing and plant growth promoting rhizobacterium and has great potential to be used as bio-inoculant for sunflower under phosphorus deficient conditions.