Supriya Kumari

Marine bacteria: potential candidates for enhanced bioremediation

Bacteria are widespread in nature as they can adapt to any extreme environmental conditions and perform various physiological activities. Marine environments are one of the most adverse environments owing to their varying nature of temperature, pH, salinity, sea surface temperature, currents, precipitation regimes and wind patterns. Due to the constant variation of environmental conditions, the microorganisms present in that environment are more suitably adapted to the adverse conditions, hence, possessing complex characteristic features of adaptation. Therefore, the bacteria isolated from the marine environments are supposed to be better utilized in bioremediation of heavy metals, hydrocarbon and many other recalcitrant compounds and xenobiotics through biofilm formation and production of extracellular polymeric substances. Many marine bacteria have been reported to have bioremediation potential. The advantage of using marine bacteria for bioremediation in situ is the direct use of organisms in any adverse conditions without any genetic manipulation. This review emphasizes the utilization of marine bacteria in the field of bioremediation and understanding the mechanism behind acquiring the characteristic feature of adaptive responses.

Understanding molecular identification and polyphasic taxonomic approaches for genetic relatedness and phylogenetic relationships of microorganisms

The major proportion of earths biological diversity is inhabited by microorganisms and they play a useful role in diversified environments. However, taxonomy of microorganisms is progressing at a snails pace, thus less than 1% of the microbial population has been identified so far. The major problem associated with this is due to a lack of uniform, reliable, advanced, and common to all practices for microbial identification and systematic studies. However, recent advances have developed many useful techniques taking into account the house-keeping genes as well as targeting other gene catalogues (16S rRNA, rpoA, rpoB, gyrA, gyrB etc. in case of bacteria and 26S, 28S, β-tubulin gene in case of fungi). Some uncultivable approaches using much advanced techniques like flow cytometry and gel based techniques have also been used to decipher microbial diversity. However, all these techniques have their corresponding pros and cons. In this regard, a polyphasic taxonomic approach is advantageous because it exploits simultaneously both conventional as well as molecular identification techniques. In this review, certain aspects of the merits and limitations of different methods for molecular identification and systematics of microorganisms have been discussed. The major advantages of the polyphasic approach have also been described taking into account certain groups of bacteria as case studies to arrive at a consensus approach to microbial identification.

Bacterial biofilms and quorum sensing: fidelity in bioremediation technology

Increased contamination of the environment with toxic pollutants has paved the way for efficient strategies which can be implemented for environmental restoration. The major problem with conventional methods used for cleaning of pollutants is inefficiency and high economic costs. Bioremediation is a growing technology having advanced potential of cleaning pollutants. Biofilm formed by various micro-organisms potentially provide a suitable microenvironment for efficient bioremediation processes. High cell density and stress resistance properties of the biofilm environment provide opportunities for efficient metabolism of number of hydrophobic and toxic compounds. Bacterial biofilm formation is often regulated by quorum sensing (QS) which is a population density-based cell–cell communication process via signaling molecules. Numerous signaling molecules such as acyl homoserine lactones, peptides, autoinducer-2, diffusion signaling factors, and α-hydroxyketones have been studied in bacteria. Genetic alteration of QS machinery can be useful to modulate vital characters valuable for environmental applications such as biofilm formation, biosurfactant production, exopolysaccharide synthesis, horizontal gene transfer, catabolic gene expression, motility, and chemotaxis. These qualities are imperative for bacteria during degradation or detoxification of any pollutant. QS signals can be used for the fabrication of engineered biofilms with enhanced degradation kinetics. This review discusses the connection between QS and biofilm formation by bacteria in relation to bioremediation technology.

Effect of biofilm parameters and extracellular polymeric substance composition on polycyclic aromatic hydrocarbon degradation

Marine bacterial biofilms were studied under different physicochemical conditions for enhanced bioremediation of polycyclic aromatic hydrocarbons (PAHs). Molecular characterization of ten environmental isolates was done by 16S rRNA gene sequencing. The effect of different physicochemical parameters, such as pH, salt concentration, temperature, carbon source on their biofilm production capability was monitored. Various topological parameters of the biofilms such as total biomass (EPS and cells content), thickness, roughness coefficient, diffusion distance and surface to biovolume ratio were studied using a confocal scanning laser microscope (CSLM). Among the various strains studied, the total biomass was maximum for P. aeruginosa N6P6 (106.64 μm3 μm−2) followed by S. acidaminiphila NCW702 (26.92 μm3 μm−2) indicating the formation of dense biofilm. Significant negative correlation (P < 0.05) was observed between the roughness coefficient of the biofilm and PAH degradation, whereas a significant positive correlation (P < 0.05) was observed between PAH (phenanthrene and pyrene) degradation and total biomass, thickness and diffusion distance of the biofilms. PAH degradation was studied both in planktonic and biofilm modes of growth. Biofilm facilitated degradation of the two PAHs was higher than the planktonic cells. This work demonstrates that the attached phenotypes of the marine bacteria showed noticeable variation in biofilm architecture and, in turn, biodegradation of PAHs.

Disruption of the quorum sensing regulated pathogenic traits of the biofilm-forming fish pathogen Aeromonas hydrophila by tannic acid, a potent quorum quencher

Abstract The quorum sensing (QS) phenomenon regulates a myriad of pathogenic traits in the biofilm forming fish pathogen, Aeromonas hydrophila. Blocking the QS mechanism of A. hydrophila is a novel strategy to prevent disease in fish. This study evaluated the effect of tannic acid, a QS inhibitor, on A. hydrophila-associated QS regulated phenomena. A streaking assay with Chromobacterium violaceum (CVO26) reported the presence of N-acyl homoserine lactone (AHL) in A. hydrophila, which was confirmed by HPLC and GC-MS analysis. Tannic acid-treated A. hydrophila showed a considerable reduction in violacein production, blood haemolysis activity and the pattern of swarming motility. Biofilm formation was significantly reduced (p < 0.001) (up to 95%), after tannic acid treatment for 48 h. Analysis by qRT-PCR revealed significant downregulation (p < 0.001) of AhyI and AhyR transcripts in A. hydrophila after tannic acid treatment. Co-stimulation of Catla catla with A. hydrophila and tannic acid attenuated pathogen-induced skin haemorrhages and increased the relative survival rate up to 86.6%. The study provides a mechanistic basis of tannic acid as a QS blocker and indicates its therapeutic potential against A. hydrophila-induced pathogenesis.

Synergistic effect of quorum sensing genes in biofilm development and PAHs degradation by a marine bacterium

ABSTRACT Quorum sensing (QS) is a prevalently found intercellular signaling system in bacteria. QS system bestows behavioral coordination ability in bacteria at high population density. QS via acylated homoserine lactone (AHL) is extensively conserved in Gram-negative bacteria and plays crucial role in regulating many biological processes. The role of QS genes coding for AHL synthase enzyme (lasI and rhlI) was established in bioremediation of polycyclic aromatic hydrocarbons (PAHs) viz. phenanthrene and pyrene. AHL producing biofilm forming marine bacterium Pseudomonas aeruginosa N6P6 was isolated by selective enrichment on PAHs. AHL production was confirmed using AHL bioreporters and GC-MS analysis. Biofilm development and its architecture was significantly (P < 0.05) affected by alterations in lasI/rhlI expression. The lasI/rhlI gene expression pattern significantly influences biofilm formation and subsequent degradation of PAHs. The integrated density of Pseudomonas aeruginosa N6P6 biofilm was highest for 48 h old biofilm and the PAHs (phenanthrene and pyrene) degradation was also found maximum (85.6 % and 47.56 %) with this biofilm. A significant positive correlation (P < 0.05) was observed between lasI expression and PAHs degradation. The role of QS genes in biofilm formation and degradation of PAHs was validated by blocking the transcription of lasI/rhlI by a QS inhibitor (QSI) tannic acid. Further, application of such QS positive isolates in PAHs contaminated sites could be a promising strategy to improve the PAHs bioremediation.

Interaction of Pb(II) and biofilm associated extracellular polymeric substances of a marine bacterium Pseudomonas pseudoalcaligenes NP103

Three-dimensional excitation-emission matrix (3D EEM) fluorescence spectroscopy and attenuated total reflectance fourier-transformed infrared spectroscopy (ATR-FTIR) was used to evaluate the interaction of biofilm associated extracellular polymeric substances (EPS) of a marine bacterium Pseudomonas pseudoalcaligenes NP103 with lead [Pb(II)]. EEM fluorescence spectroscopic analysis revealed the presence of one protein-like fluorophore in the EPS of P. pseudoalcaligenes NP103. Stern-Volmer equation indicated the existence of only one binding site (n=0.789) in the EPS of P. pseudoalcaligenes NP103. The interaction of Pb(II) with EPS was spontaneous at room temperature (∆G=-2.78kJ/K/mol) having binding constant (Kb) of 2.59M-1. ATR-FTIR analysis asserted the involvement of various functional groups such as sulphydryl, phosphate and hydroxyl and amide groups of protein in Pb(II) binding. Scanning electron microscopy (SEM) and fluorescence microscopy analysis displayed reduced growth of biofilm with altered surface topology in Pb(II) supplemented medium. Energy dispersive X-ray spectroscopy (EDX) analysis revealed the entrapment of Pb in the EPS. Uronic acid, a characteristic functional group of biofilm, was observed in 1H NMR spectroscopy. The findings suggest that biofilm associated EPS are perfect organic ligands for Pb(II) complexation and may significantly augment the bioavailability of Pb(II) in the metal contaminated environment for subsequent sequestration.

Marine Bacterial Biofilms in Bioremediation of Polycyclic Aromatic Hydrocarbons (PAHs) Under Terrestrial Condition in a Soil Microcosm

Polycyclic aromatic hydrocarbons (PAHs) in soil retain for a quite long period due to their hydrophobicity and aggregation properties. Biofilm-forming marine bacterial consortium (named as NCPR), composed of Stenotrophomonas acidaminiphila NCW702, Alcaligenes faecalis NCW402, Pseudomonas mendocina NR802, Pseudomonas aeruginosa N6P6, and Pseudomonas pseudoalcaligenes NP103, was used for the bioremediation of PAHs in a soil microcosm. Phenanthrene and pyrene were used as reference PAHs. Parameters that can affect PAH degradation, such as chemotaxis, solubility of PAHs in extracellular polymeric substances (EPS), and catechol 2,3-dioxygenase (C23O) activity, were evaluated. P. aeruginosa N6P6 and P. pseudoalcaligenes NP103 showed chemotactic movement towards both the reference PAHs. The solubility of both the PAHs was increased with an increase in EPS concentration (extracted from all the 5 selected isolates). Significantly (P < 0.001) high phenanthrene (70.29%) and pyrene (55.54%) degradation was observed in the bioaugmented soil microcosm. The C23O enzyme activity was significantly (P < 0.05) higher in the bioaugmented soil microcosm with phenanthrene added at 173.26 ± 2.06 nmol min−1 mg−1 protein than with pyrene added at 61.80 ± 2.20 nmol min−1 mg−1 protein. The C23O activity and gas chromatography-mass spectrometer analyses indicated catechol pathway of phenanthrene metabolism. However, the metabolites obtained from the soil microcosm added with pyrene revealed both the catechol and phthalate pathways for pyrene degradation.