Integrative chemical and omics analyses reveal copper biosorption and tolerance mechanisms of Bacillus cereus strain T6.

A comprehensive understanding of the cellular response of microbes to metal stress is necessary for the rational development of microbe-based biosorbents for metal removal. The present study investigated the copper (Cu) sorption and resistance mechanism of Bacillus cereus strain T6, a newly isolated Cu-resistant bacterium, by integrative analyses of physiochemistry, genomics, transcriptomics, and metabolomics. The growth inhibition assay and biosorption determination showed that this bacterium exhibited high tolerance to Cu, with a minimum inhibitory concentration of 4.0 mM, and accumulated Cu by both extracellular adsorption and intracellular binding. SEM microscopic images and FTIR spectra showed significant cellular surface changes at the high Cu level but not at low, and the involvement of surface functional groups in the biosorption of Cu, respectively. Transcriptomic and untargeted metabolomic analyses detected 362 differentially expressed genes and 60 significantly altered metabolites, respectively. Integrative omics analyses revealed that Cu exposure dramatically induced a broad spectrum of genes involved in Cu transport and iron homeostasis, and suppressed the denitrification pathway, leading to significant accumulation of metabolites for metal transporter synthesis, membrane remolding, and antioxidant activities. The results presented here provide a new perspective on the intricate regulatory network of Cu homeostasis in bacteria.

Molecular insights into plant-microbe interactions for sustainable remediation of contaminated environment.

The widespread distribution of organic and inorganic pollutants in water resources have increased due to rapid industrialization. Rhizospheric zone-associated bacteria along with endophytic bacteria show a significant role in remediation of various pollutants. Metaomics technologies are gaining an advantage over traditional methods because of their capability to obtain detailed information on exclusive microbial communities in rhizosphere of the plant including the unculturable microorganisms. Transcriptomics, proteomics, and metabolomics are functional methodologies that help to reveal the mechanisms of plant-microbe interactions and their synergistic roles in remediation of pollutants. Intensive analysis of metaomics data can be useful to understand the interrelationships of various metabolic activities between plants and microbes. This review comprehensively discusses recent advances in omics applications made hitherto to understand the mechanisms of plant-microbe interactions during phytoremediation. It extends the delivery of the insightful information on plant-microbiomes communications with an emphasis on their genetic, biochemical, physical, metabolic, and environmental interactions.

Insights on the role of periphytic biofilm in synergism with Iris pseudacorus for removing mixture of pharmaceutical contaminants from wastewater.

The potential of Iris pseudacorus and the associated periphytic biofilm for biodegradation of two common pharmaceutical contaminants (PCs) in urban wastewater was assessed individually and in consortium. An enhanced removal for sulfamethoxazole (SMX) was achieved in consortium (59%) compared to individual sets of I. pseudacorus (50%) and periphytic biofilm (7%) at concentration of 5 mg L-1. Conversely, individual sets of periphytic biofilm (77%) outperformed removal of doxylamine succinate (DOX) compared to individual sets of I. pseudacorus (59%) and consortium (67%) at concentration of 1 mg L-1. Enhanced relative abundance of microflora containing microalgae (Sellaphora, Achnanthidium), rhizobacteria (Acidibacter, Azoarcus, Thioalkalivibrio), and fungi (Serendipita) in periphytic biofilm was observed after treatment. SMX treatment for five days elevated cytochrome P450 enzymes' expressions, including aniline hydroxylase (48%) and aminopyrine N-demethylase (54%) in the periphytic biofilm. Nevertheless, I. pseudacorus showed 175% elevation of aniline hydroxylase along with other biotransformation enzymes, such as peroxidase (629%), glutathione S-transferase (514%), and dichloroindophenol reductase (840%). A floating bed phytoreactor planted with I. pseudacorus and the periphytic biofilm consortium removed 67% SMX and 72% DOX in secondary wastewater effluent. Thus, the implementation of this strategy in constructed wetland-based treatment could be beneficial for managing effluents containing PCs.

Characterization of a Bowman-Birk type trypsin inhibitor purified from seeds of Solanum surattense.

A Bowman-Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purified and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that differentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman-Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1-10) and temperatures (10-100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains ß sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) values (16.6 × 10-8 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high affinity towards trypsin with dissociation constant (Kd) 2.28 µM.

Doxorubicin-Conjugated Innovative 16-mer DNA Aptamer-Based Annexin A1 Targeted Anti-Cancer Drug Delivery.

Aptamers are small, functional single-stranded DNA or RNA oligonucleotides that bind to their targets with high affinity and specificity. Experimentally, aptamers are selected by the systematic evolution of ligands by exponential enrichment (SELEX) method. Here, we have used rational drug designing and bioinformatics methods to design the aptamers, which involves three different steps. First, finding a probable aptamer-binding site, and second, designing the recognition and structural parts of the aptamers by generating a virtual library of sequences, selection of specific sequence via molecular docking, molecular dynamics (MD) simulation, binding energy calculations, and finally evaluating the experimental affinity. Following this strategy, a 16-mer DNA aptamer was designed for Annexin A1 (ANXA1). In a direct binding assay, DNA1 aptamer bound to the ANXA1 with dissociation constants value of 83 nM. Flow cytometry and fluorescence microscopy results also showed that DNA1 aptamer binds specifically to A549, HepG2, U-87 MG cancer cells that overexpress ANXA1 protein, but not to MCF7 and L-02, which are ANXA1 negative cells. We further developed a novel system by conjugating DNA1 aptamer with doxorubicin and its efficacy was studied by cellular uptake and cell viability assay. Also, anti-tumor analysis showed that conjugation of doxorubicin with aptamer significantly enhances targeted therapy against tumors while minimizing overall adverse effects on mice health.

In situ textile wastewater treatment in high rate transpiration system furrows planted with aquatic macrophytes and floating phytobeds.

Plants are known to remediate dyes, metals and emerging contaminants from wastewaters. Vetiveria zizanioides, a perennial bunchgrass showed removal of Remazol Red (RR, 100 mg/L) up to 93% within 40 h. Root and shoot tissues of V. zizanioides revealed induction in dye degrading enzymes viz. lignin peroxidase by 2.28 and 1.43, veratryl alcohol oxidase 2.72 and 1.60, laccase 6.15 and 3.55, and azo reductase 2.17 and 2.65-fold, respectively, during RR decolorization. Substantial increase was observed in the contents of chlorophyll a, chlorophyll b, and carotenoids in the plant leaves during treatment. Anatomical studies of roots, HPLC and GC-MS analysis of metabolites, and phytotoxicity assessment confirmed phytotransformation of RR into nontoxic metabolites. Floating phytobed with V. zizanioides treated textile wastewater (400 L) effectively and reduced ADMI, COD, BOD, TDS, and TSS by 74, 74, 81, 66 and 47%, respectively within 72 h. In-situ treatment of textile wastewater for 5 days in constructed furrows planted with semiaquatic plants, V. zizanioides, Ipomoea aquatica and its consortium-VI decreased ADMI by 68, 61 and 76%, COD by 75, 74 and 79%, BOD by 73, 71 and 84%, TDS by 77, 75 and 83%, and TSS by 34, 31 and 51%, respectively. This treatment was also useful to remove arsenic, cadmium, chromium and lead from wastewater. Overall observation suggests wise strategy to use this plantation in the furrows of high rate transpiration system and phytobeds in deep water for textile wastewater treatment.

Decolorization and detoxification of dye mixture and textile effluent by lichen Dermatocarpon vellereceum in fixed bed upflow bioreactor with subsequent oxidative stress study.

Navy Blue HE22 (NBHE22), dye mixture and real textile effluent were decolorized and degraded by lichen Dermatocarpon vellereceum. Up-flow bioreactor showed about 80%, 70%, 80% and 65% removal of American dye manufacturer index (ADMI), biological oxygen demand (BOD), total suspended solids (TSS) and total dissolved solids (TDS), respectively of dye mixture at flow rate of 25mlh-1. The removal of ADMI, BOD, TSS and TDS of real textile effluent were 75%, 65%, 82% and 70%, respectively at flow rate of 30mlh-1. Significant induction of extracellular enzymes such as manganese peroxidase and lignin peroxidase was observed up to 46% and 36% during decolorization of dye mixture, while 43% and 24% during effluent treatment, respectively. Exponential enhancement in the activities of stress enzymes such as catalase (CAT) and guaiacol peroxidase (GPX) was observed after exposure to NBHE22 (116% and 125%, respectively), dye mixture (150% and 300%, respectively) and effluent (400% and 350%, respectively) endorsing the stress tolerance ability of model lichen. Phytotoxicity and genotoxicity studies demonstrated less toxic nature of metabolites resulted from biodegradation.

Phytobeds with Fimbristylis dichotoma and Ammannia baccifera for treatment of real textile effluent: An in situ treatment, anatomical studies and toxicity evaluation.

Fimbristylis dichotoma, Ammannia baccifera and their co-plantation consortium FA independently degraded Methyl Orange, simulated dye mixture and real textile effluent. Wild plants of F. dichotoma and A. baccifera with equal biomass showed 91% and 89% decolorization of Methyl Orange within 60h at a concentration of 50ppm, while 95% dye removal was achieved by consortium FA within 48h. Floating phyto-beds with co-plantation (F. dichotoma and A. baccifera) for the treatment of real textile effluent in a constructed wetland was observed to be more efficient and achieved 79%, 72%, 77%, 66% and 56% reductions in ADMI color value, COD, BOD, TDS and TSS of textile effluent, respectively. HPTLC, GC-MS, FTIR, UV-vis spectroscopy and activated oxido-reductive enzyme activities confirmed the phytotrasformation of parent dye in to new metabolites. T-RFLP analysis of rhizospheric bacteria of F. dichotoma, A. baccifera and consortium FA revealed the presence of 88, 98 and 223 genera which could have been involved in dye removal. Toxicity evaluation of products formed after phytotransformation of Methyl Orange by consortium FA on bivalves Lamellidens marginalis revealed less damage of the gills architecture when analyzed histologically. Toxicity measurement by Random Amplification of Polymorphic DNA (RAPD) technique revealed bivalve DNA banding pattern in treated Methyl Orange sample suggesting less toxic nature of phytotransformed dye products.

Co-plantation of aquatic macrophytes Typha angustifolia and Paspalum scrobiculatum for effective treatment of textile industry effluent.

Field treatment of textile industry effluent was carried out in constructed drenches (91.4m×1.2m×0.6m; 65.8m3) planted independently with Typha angustifolia, Paspalum scrobiculatum and their co-plantation (consortium-TP). The in situ treatment of effluent by T. angustifolia, P. scrobiculatum and consortium-TP was found to decrease ADMI color value by 62, 59 and 76%, COD by 65, 63 and 70%, BOD by 68, 63 and 75%, TDS by 45, 39 and 57%, and TSS by 35, 31 and 47%, respectively within 96h. Heavy metals such as arsenic, cadmium, chromium and lead were also removed up to 28-77% after phytoremediation. T. angustifolia and P. scrobiculatum showed removal of Congo Red (100mg/L) up to 80 and 73%, respectively within 48h while consortium-TP achieved 94% decolorization. Root tissues of T. angustifolia and P. scrobiculatum revealed inductions in the activities of oxido-reductive enzymes such as lignin peroxidase (193 and 32%), veratryl alcohol oxidase (823 and 460%), laccase (492 and 182%) and azo reductase (248 and 83%), respectively during decolorization of Congo Red. Anatomical studies of roots, FTIR, HPLC, UV-vis Spectroscopy and GC-MS analysis verified the phytotransformation. Phytotoxicity studies confirmed reduced toxicity of the metabolites of Congo Red.

Efficient decolorization and detoxification of textile industry effluent by Salvinia molesta in lagoon treatment.

Salvinia molesta, an aquatic fern was observed to have a potential of degrading azo dye Rubine GFL up to 97% at a concentration of 100mg/L within 72h using 60±2g of root biomass. Both root as well as stem tissues showed induction in activities of the enzymes such as lignin peroxidase, veratryl alcohol oxidase, laccase, tyrosinase, catalase, DCIP reductase and superoxide dismutase during decolorization of Rubine GFL. FTIR, GC-MS, HPLC and UV-visible spectrophotometric analysis confirmed phytotransformation of the model dye into smaller molecules. Analysis of metabolites revealed breakdown of an azo bond of Rubine GFL by the action of lignin peroxidase and laccase and formation of 2-methyl-4-nitroaniline and N-methylbenzene-1, 4-diamine. Anatomical tracing of dye in the stem of S. molesta confirmed the presence of dye in tissues and subsequent removal after 48h of treatment. The concentration of chlorophyll pigments like chlorophyll a, chlorophyll b and carotenoid was observed during the treatment. Toxicity analysis on seeds of Triticum aestivum and Phaseolus mungo revealed the decreased toxicity of dye metabolites. In situ treatment of a real textile effluent was further monitored in a constructed lagoon of the dimensions of 7m×5m×2m (total surface area 35m(2)) using S. molesta for 192h. This large scale treatment was found to significantly reduce the values of COD, BOD5 and ADMI by 76%, 82% and 81% considering initial values 1185, 1440mg/L and 950 units, respectively.

Ipomoea hederifolia rooted soil bed and Ipomoea aquatica rhizofiltration coupled phytoreactors for efficient treatment of textile wastewater.

Ipomoea aquatica, a macrophyte was found to degrade a highly sulfonated and diazo textile dye Brown 5R up to 94% within 72 h at a concentration of 200 mg L(-1). Induction in the activities of enzymes such as azoreductase, lignin peroxidase, laccase, DCIP reductase, tyrosinase, veratryl alcohol oxidase, catalase and superoxide dismutase was observed in leaf and root tissue in response to Brown 5R exposure. There was significant reduction in contents of chlorophyll a (25%), chlorophyll b (17%) and carotenoids (30%) in the leaves of plants. HPLC, FTIR, UV-vis spectrophotometric and HPTLC analyses confirmed the biotransformation and removal of parent dye from solution. Enzymes activities and GC-MS analysis of degradation products lead to the proposal of a possible pathway of phytotransformation of dye. The proposed pathway of dye metabolism revealed the formation of Napthalene-1,2-diamine and methylbenzene. Toxicity study on HepG2 cell lines showed a 3 fold decrease in toxicity of Brown 5R after phytoremediation by I. aquatica. Hydrophytic nature of I. aquatica leads to its exploration in a combinatorial phytoreactor with Ipomoea hederifolia soil bed system. Rhizofiltration with I. aquatica and soil bed treatment by I. hederifolia treated 510 L of effluent effectively within 72 h. I. aquatica along with I. hederifolia could decolorize textile industry effluent within 72 h of treatment as evident from the significant reductions in the values of COD, BOD, solids and ADMI. Further on field trials of treatment of textile wastewater was successfully carried out in a constructed lagoon.

Bioreactor with Ipomoea hederifolia adventitious roots and its endophyte Cladosporium cladosporioides for textile dye degradation.

In vitro grown untransformed adventitious roots (AR) culture of Ipomoea hederifolia and its endophytic fungus (EF) Cladosporium cladosporioides decolorized Navy Blue HE2R (NB-HE2R) at a concentration of 20 ppm up to 83.3 and 65%, respectively within 96h. Whereas the AR-EF consortium decolorized the dye more efficiently and gave 97% removal within 36h. Significant inductions in the enzyme activities of lignin peroxidase, tyrosinase and laccase were observed in roots, while enzymes like tyrosinase, laccase and riboflavin reductase activities were induced in EF. Metabolites of dye were analyzed using UV-vis spectroscopy, FTIR and gas chromatography-mass spectrometry. Possible metabolic pathways of NB-HE2R were proposed with AR, EF and AR-EF systems independently. Looking at the superior efficacy of AR-EF system, a rhizoreactor was developed for the treatment of NB-HE2R at a concentration of 1000 ppm. Control reactor systems with independently grown AR and EF gave 94 and 85% NB-HE2R removal, respectively within 36h. The AR-EF rhizoreactor, however, gave 97% decolorization. The endophyte colonization additionally increased root and shoot lengths of candidate plants through mutualism. Combined bioreactor strategies can be effectively used for future eco-friendly remediation purposes.

Phytoremediation of sulfonated Remazol Red dye and textile effluents by Alternanthera philoxeroides: An anatomical, enzymatic and pilot scale study.

Alternanthera philoxeroides Griseb. a macrophyte was found to degrade a highly sulfonated textile dye Remazol Red (RR) completely within 72 h at a concentration of 70 mg L(-1). An induction in the activities of azoreductase and riboflavin reductase was observed in root and stem tissues; while the activities of lignin peroxidase, laccase and DCIP reductase were induced in leaf tissues. Some enzymes namely tyrosinase, veratryl alcohol oxidase, catalase and superoxide dismutase displayed an increase in their activity in all the tissues in response of 72 h exposure to Remazol Red. There was a marginal reduction in contents of chlorophyll a (20%), chlorophyll b (5%) and carotenoids (16%) in the leaves when compared to control plants. A detailed anatomical study of the stem during uptake and treatment revealed a stepwise mechanism of dye degradation. UV-vis spectrophotometric and high performance thin layer chromatographic analyses confirmed the removal of parent dye from solution. Based on the enzymes activities and gas chromatography-mass spectroscopic analysis of degradation products, a possible pathway of phytotransformation of RR was proposed which revealed the formation of 4-(phenylamino)-1,3,5-triazin-2-ol, naphthalene-1-ol and 3-(ethylsulfonyl)phenol. Toxicity study on Devario aequipinnatus fishes showed that the anatomy of gills of fishes exposed to A. philoxeroides treated RR was largely protected. The plants were further explored for rhizofiltration experiments in a pilot scale reactor. A. philoxeroides could decolorize textile industry effluent of varying pH within 96 h of treatment which was evident from the significant reductions in the values of American dye manufacturers' institute color, chemical oxygen demand, biological oxygen demand, total dissolved and total suspended solids.

Enhanced phytotransformation of Navy Blue RX dye by Petunia grandiflora Juss. with augmentation of rhizospheric Bacillus pumilus strain PgJ and subsequent toxicity analysis.

This study reveals the beneficial synergistic phytoremediation potential of Petunia grandiflora Juss. with its rhizospheric bacterial isolate Bacillus pumilus strain PgJ to decolorize reactive Navy Blue RX (NBRX) dye by their active enzymatic machinery. In vitro cultures of P. grandiflora and B. pumilus gave 80.01% and 76.80% while their consortium decolorized NBRX up to 96.86% within 36 h. Significant induction in the enzyme activities of lignin peroxidase (207%), tyrosinase (133%), laccase (161%), riboflavin reductase (78%) were seen in the roots of tissue cultured plants while enzymes tyrosinase (660%), laccase (689%), riboflavin reductase (528%) were induced significantly in the B. pumilus cells. Metabolites of treated NBRX were analyzed using UV-vis spectroscopy, gas chromatography and biotransformation was visualized using high performance thin layer chromatography profile. Metabolites of the dye exhibited reduced phytotoxicity Sorghum vulgare and Phaeseolus mungo and significant reduction in cytogenotoxicity on Allium cepa roots when compared to NBRX.

Bacterial assisted phytoremediation for enhanced degradation of highly sulfonated diazo reactive dye.

PURPOSE: Phytoremediation is the exploitation of plants and their rhizospheric microorganisms for pollutants treatment like textile dyes, which are toxic, carcinogenic and mutagenic from the effluent. The purpose of this work was to explore a naturally found plant and bacterial synergism to achieve an enhanced degradation of Remazol Black B dye (RBB). METHODS: In vitro cultures of Zinnia angustifolia were obtained by seed culture method. Enzymatic analysis of the plant roots and Exiguobacterium aestuarii strain ZaK cells was performed before and after decolorization of RBB. Metabolites of RBB formed after its degradation were analyzed using UV-Vis spectroscopy, high-performance liquid chromatography (HPLC), Fourier transform infrared (FTIR) and gas chromatography-mass spectrometry (GC-MS). Phytotoxicity studies were performed. RESULTS: The consortium ZE was found to be more efficient than individual plant and bacteria. Z. angustifolia roots showed significant induction in the activities of lignin peroxidase, laccase, DCIP reductase and tyrosinase during dye decolorization. E. aestuarii showed significant induction in the activities of veratryl alcohol oxidase, azo reductase and DCIP reductase. Analysis of metabolites revealed differential metabolism of RBB by plant, bacteria and consortium ZE. E. aestuarii and Z. angustifolia led to the formation of 3,6-diamino-4-hydroxynaphthalene-2-sulfonic acid, (ethylsulfonyl)benzene, and 3,4,6-trihydroxynaphthalene-2-sulfonic acid and propane-1-sulfonic acid, respectively, whereas consortium ZE produced 4-hydroxynaphthalene-2-sulfonic acid, naphthalene-2-sulfonic acid and 4-(methylsulfonyl)phenol. The phytotoxicity study revealed the nontoxic nature of the metabolites formed after dye degradation. CONCLUSION: Consortium ZE was found to be more efficient and faster in the degradation of RBB when compared to degradation by Z. angustifoila and E. aestuarii individually.