Genomic characterization of denitrifying methylotrophic Pseudomonas aeruginosa strain AAK/M5 isolated from municipal solid waste landfill soil.

Municipal landfills are known for methane production and a source of nitrate pollution leading to various environmental issues. Therefore, this niche was selected for the isolation of one-carbon (C1) utilizing bacteria with denitrifying capacities using anaerobic enrichment on nitrate mineral salt medium supplemented with methanol as carbon source. Eight axenic cultures were isolated of which, isolate AAK/M5 demonstrated the highest methanol removal (73.28%) in terms of soluble chemical oxygen demand and methane removal (41.27%) at the expense of total nitrate removal of 100% and 33% respectively. The whole genome characterization with phylogenomic approach suggested that the strain AAK/M5 could be assigned to Pseudomonas aeruginosa with close neighbours as type strains DVT779, AES1M, W60856, and LES400. The circular genome annotation showed the presence of complete set of genes essential for methanol utilization and complete denitrification process. The study demonstrates the potential of P. aeruginosa strain AAK/M5 in catalysing methane oxidation thus serving as a methane sink vis-à-vis utilization of nitrate. Considering the existence of such bacteria at landfill site, the study highlights the need to develop strategies for their enrichment and designing of efficient catabolic activity for such environments.

Effect of Fagonia arabica on thrombin induced release of t-PA and complex of PAI-1 tPA in cultured HUVE cells.

Fagonia arabica (FA) possesses a thrombolytic property which has been earlier reported in our laboratory. Current study was undertaken to investigate the effect of aqueous extract of FA on thrombin-induced tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) release from cultured human umbilical vein endothelial cell line (HUVE) for studying its clot lytic activity. For this, establishment of cell line model has been done by isolating the cells from human umbilical cord. Cell toxicity was evaluated using XTT assay. Estimation of t-PA and PAI-1 t-PA complex were done using ELISA technique. Thrombin treatment induces the t-PA and PAI-1 release from HUVE cell line, and FA treatment was found to antagonize the thrombin induced t-PA and PAI-1 release. Our preliminary results suggest that FA may be used as an alternative to thrombolytic drug. However, study demands further experiments using animal model of thrombosis to establish the role of FA as a novel thrombolytic drug.

Effect of aqueous extract and fractions of Fagonia arabica on in vitro anticoagulant activity.

Fagonia arabica (FA) is a deobstruent and blood purifier, which possesses thrombolytic and antioxidant activities. In this study, the anticoagulant effects of FA and its derived fractions were evaluated. Plasma recalcification was performed with multisolvent extracts of FA and then with extracts prepared successively with increasing polarity of the solvents. Aqueous extract was the most potent anticoagulant extract, which was fractionated by thin-layer chromatography and column chromatography. Five fractions collected were checked for their anticoagulation effect. The most potent fraction was screened for phytoconstituents. Aqueous extract of FA is the most active anticoagulant (31 minutes). Results were statistically significant when compared to heparin (38 minutes) and saline (4.04 minutes; P > .001). The Fifth fraction (FA5), the most potent fraction (27 minutes), was found positive for flavonoids, saponin, tannin, triterpenoids, carbohydrates, reducing sugar, and monosaccharides. Aqueous FA and fraction FA5 were most active in in vitro anticoagulation, and any of the phytochemicals identified could be considered the active component.

Characterization of antibacterial activity of bikaverin from Fusarium sp. HKF15.

Redeemed interest in discovery and characterization of naturally occurring antimicrobials arises from the fact that these compounds possess diverse biological activities useful in the development of new therapeutics. In this study, 35 fungi from extreme environments of effluent treatment plants were screened for antimicrobial activity. An isolate HKF15 produced antimicrobial compound that showed activities against pathogenic and multidrug resistant bacteria; and was identified as Fusarium sp. The active principle was separated through activity-guided HPLC fractionation of 1:1 ethyl acetate crude extract that was identified as bikaverin using HRMS and (1)H NMR. Bikaverin is known for antitumor and antifungal activity and in this study; antibacterial activity of bikaverin was assessed, emphasizing the importance of repurposing of antibiotics.

In-vitro clot lytic potential of Fagonia arabica: a comparative study of two methods.

The tube method developed in our laboratory is a simple, inexpensive and a classical whole blood clot lytic procedure through which clot lytic potential of Fagonia arabica was found to be significant. Microtiter plate clot lysis (MPCL) assay is a rapid and precise turbidimetric clot lysis method which includes measurements of maximum absorbance (Max Abs), area under the curve (AUC) along with the standard clot lysis time. In the present study we have compared and validated clot lytic potential of F. arabica extract by tube method and MPCL assay. Percentage of clot lysis was calculated by measuring the difference of the absorbance taken at 0 and 240 min in the case of MPCL assay, whereas with the tube method according to the weight difference. Fagonia arabica (50 ug/ml) was capable of clot lysis by MPCL assay and showed clot lysis pattern similar to 60 U/ml streptokinase (positive control). The clot lysis times were significantly different from one another (P value ≤0.001). When Max Abs and AUC were compared to the clot lysis time the correlation coefficient (r value) was significant too (P value ≤0.001). Moreover, we have also found that both the methods showed almost the same clot lysis percentage by streptokinase as well as F. arabica. The correlation coefficient between streptokinase, and F. arabica done by tube method and MPCL assay was found to be statistically significant (P < 0.05). Fagonia arabica had the clot lytic potential checked by in-vitro methods, namely MPCL assay and the method.

Protection of PC12 cells from chemical ischemia induced oxidative stress by Fagonia arabica.

Fagonia arabica (Zygophyllaceae) is an important Ayurvedic herb, grows throughout arid regions of India, has been widely used as a folk remedy by the indigenous people for its anti-inflammatory, analgesic and antipyretic effects. In the present study, antioxidant potential of F. arabica and the associated mechanism of antioxidant defence in rat pheochromocytoma (PC12) cells subjected to chemical ischemia was studied. Effect of total extract of F. arabica was studied for its antioxidant potential on the chemical ischemia induced PC12 cells. Alterations in the activities of cellular antioxidant enzymes (SOD, CAT, GSH-Px and GSH-R) were measured. Antioxidant potential of herb (ABTS), extent of lipid peroxidation (MDA and 4-HAE), total antioxidant status (TAS) and total glutathione (reduced, oxidized and their ratio) were evaluated. F. arabica scavenges the free radicals (ABTS(.)+), and showed a concentration dependent antioxidant activity, highest being at 1000 microg/ml. Its treatment with ischemic cells ameliorates the GSH and TAS levels and also helps the cells to restore the activities of the cellular antioxidative enzymes and also reduced the degree of lipid peroxidation. F. arabica scavenges the free radicals and attenuates oxidative stress mediated cell injury during ischemia.

Identification of genes conferring arsenic resistance to Escherichia coli from an effluent treatment plant sludge metagenomic library.

The majority of bacteria elude culture in the laboratory. A metagenomic approach provides culture-independent access to the gene pool of the whole bacterial community. A metagenomic library was constructed from an industrial effluent treatment plant sludge containing about 1.25 Gb of microbial community DNA. Two arsenic-resistant clones were selected from the metagenomic library. Clones MT3 and MT6 had eight- and 18-fold higher resistance to sodium arsenate in comparison with the parent strain, respectively. The clones also showed increased resistance to arsenite but not to antimony. Sequence analysis of the clones revealed genes encoding for putative arsenate reductases and arsenite efflux pumps. A novel arsenate resistance gene (arsN) encoding a protein with similarity to acetyltransferases was identified from clone MT6. ArsN homologues were found to be closely associated with arsenic resistance genes in many bacterial genomes. ArsN homologues were found fused to putative arsenate reductases in Methylibium petroleiphilum PM1 and Anaeromyxobacter dehalogenans 2CP-C and with a putative arsenite chaperone in Burkholderia vietnamiensis G4. ArsN alone resulted in an approximately sixfold higher resistance to sodium arsenate in wild-type Escherichia coli W3110.

Isolation and characterization of bacterial strains Paenibacillus sp. and Bacillus sp. for kraft lignin decolorization from pulp paper mill waste.

Eight aerobic bacterial strains were isolated from pulp paper mill waste and screened for tolerance of kraft lignin (KL) using the nutrient enrichment technique in mineral salt media (MSM) agar plate (15 g/L) amended with different concentrations of KL (100, 200, 300, 400, 500, 600 ppm) along with 1% glucose and 0.5% peptone (w/v) as additional carbon and nitrogen sources. The strains ITRC S6 and ITRC S8 were found to have the most potential for tolerance of the highest concentration of KL. These organisms were characterized by biochemical tests and further 16S rRNA gene (rDNA) sequencing, which showed 96.5% and 95% sequence similarity of ITRC S(6) and ITRC S(8) and confirmed them as Paenibacillus sp. and Bacillus sp., respectively. KL decolorization was routinely monitored with a spectrophotometer and further confirmed by HPLC analysis. Among eight strains, ITRC S(6) and ITRC S(8) were found to degrade 500 mg/L of KL up to 47.97% and 65.58%, respectively, within 144 h of incubation in the presence of 1% glucose and 0.5% (w/v) peptone as a supplementary source of carbon and nitrogen. In the absence of glucose and peptone, these bacteria were unable to utilize KL. The analysis of lignin degradation products by GC-MS analysis revealed the formation of various acids as lignin monomers which resulted in a decrease in pH and a major change in the chromatographic profile of the bacterial degraded sample as compared to the control clear indications of biochemical modification of KL due to the bacterial ligninolytic system by ITRC S(6), namely, acetic acid, propanoic acid, butanoic acid, guaiacol, hexanoic acid, and ITRC S(8), namely acetic acid, propanoic acid, ethanedioic acid, furan carboxylic acid, 2-propanoic acid, butanoic acid, 3-acetoxybutyric acid, propanedioic acid, acetoguiacone, 1,2,3-thiadiazole, 5-carboxaldixime, 4-hydroxy-3,5-dimethoxyphenol, and dibutyl phthalate, indicating the bacterium characteristic to degrade G and S units of lignin polymer.

Effect of Fagonia Arabica (Dhamasa) on in vitro thrombolysis.

BACKGROUND: Atherothrombotic diseases such as myocardial or cerebral infarction are serious consequences of the thrombus formed in blood vessels. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs have certain limitations which cause serious and sometimes fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases. Herbs and their components possessing antithrombotic activity have been reported before; however, herbs that could be used for thrombolysis has not been reported so far. This study's aim was to investigate whether herbal preparations (aqueous extract) possess thrombolytic activity or not. METHODS: An in vitro thrombolytic model was used to check the clot lysis effect of six aqueous herbal extracts viz., Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn along with Streptokinase as a positive control and water as a negative control. RESULTS: Using an in vitro thrombolytic model, Tinospora cordifolia, Rubia cordifolia, Hemidesmus indicus, Glycyrrhiza glabra Linn, Fagonia Arabica and Bacopa monnieri Linn showed 19.3%, 14.5%, 20.3%, 17.8%, 75.6% and 41.8% clot lysis respectively . Among the herbs studied Fagonia arabica showed significant % of clot lysis (75.6%) with reference to Streptokinase (86.2%). CONCLUSION: Through our study it was found that Dhamasa possesses thrombolytic properties that could lyse blood clots in vitro; however, in vivo clot dissolving properties and active component(s) of Dhamasa for clot lysis are yet to be discovered. Once found Dhamasa could be incorporated as a thrombolytic agent for the improvement of patients suffering from Atherothrombotic diseases.

Impact of bioaugmentation with a consortium of bacteria on the remediation of wastewater-containing hydrocarbons.

GOALS, SCOPE AND BACKGROUND: It has been observed that hydrocarbon treated wastewaters still contain high COD and a number of intermediates. This suggests that the required catabolic gene pool for further degradation might be absent in the system or, that its titer value is not significant enough. By providing the desired catabolic potential, the overall efficiency of the treatment system can be improved. This study aims to demonstrate this concept by bioaugmentation of a lab-scale reactor treating refinery wastewater with a consortium having the capacity to complement the alkB genotype to the available microbial population. METHODS: Two reactors were set up using activated biomass collected from a refinery treatment plant and operated at a continuous mode for a period of 8 weeks. The feed to both reactors was kept constant. Crude oil was spiked regularly. One reactor was bioaugmented with a consortium previously described for crude oil spill remediation. The efficiency of the bioaugmented reactor was demonstrated by reduced COD. The changes in the microbial population over a period of time were analyzed by RAPD. Catabolic activity of the biomass in both reactors was monitored by PCR. The presence of the catabolic loci was confirmed by Southern Hybridization. RESULTS AND DISCUSSION: 52.2% removal of COD was observed in the bioaugmented reactor while only 15.1% reduction of COD was observed in the reactor without bioaugmentation. The change in microbial population can be seen from the 4th week, which also corresponds to improved catabolic activity. The presence of the bedA locus was seen in all samples, which indicates the presence of aromatic degraders, but the appearance of the alkB locus, from the 6th week onwards, which was observed only in the samples from the bioaugmented reactor. The results suggest that the gene pool of the bioaugmented reactor has catabolic loci that can degrade accumulated intermediates, thus improving the efficiency of the system. CONCLUSIONS: In this study, improvement of efficiency of bioremediation was demonstrated by addition of catabolic loci that are responsible for degradation. Bioaugmentation was carried out in biomass that was collected from an ETP (effluent treatment plant) treating hydrocarbon containing wastewater to study the strategies for improvement of the treatment system. Biostimulation, only marginally improved the efficiency, when compared to bioaugmentation. The improved efficiency was demonstrated by COD removal. The presence of the alkB locus suggests the importance of a catabolic gene pool that acts on accumulated intermediates. It is well documented that straight chain aliphatics and intermediates of aromatic compounds after ring cleavage, accumulate in refinery wastewater systems, thereby hindering further degradation of the wastewater. Supplementation of a catabolic gene pool that treats the lower pathway compounds and alkanes will improve the overall efficiency. In this study, results suggest that the alkB locus can also be used to monitor the degradative mode of the activated biomass. RECOMMENDATIONS AND PERSPECTIVES: Pollution from petroleum and petroleum products around the globe are known to have grave consequences on the environment. Bioremediation, using activated sludge, is one option for the treatment of such wastes. Effluent treatment plants are usually unable to completely degrade the wastewater being treated in the biological unit (the aerator chambers). The efficiency of degradation can be improved by biostimulation and bioaugmentation. This study demonstrates the improved efficiency of a treatment system for wastewater containing hydrocarbons by bioaugmentation of a consortium that supports degradation. Further experiments on a pilot scale are recommended to assess the use of bioaugmentation on a large scale. The use of molecular tools, like DNA probes for alkB, to monitor the system also needs to be explored.