Sudhir K. Shukla

Calcium-mediated modulation of Pseudomonas mendocina NR802 biofilm influences the phenanthrene degradation.

A potential biofilm forming and phenanthrene utilizing marine bacterium Pseudomonas mendocina NR802 was isolated from Rushukulya, Odisha, East Coast of India. The effect of Ca(2+) and Mg(2+) on biofilm growth and phenanthrene degradation was evaluated. Among the various tested concentrations, 20 mM of Ca(2+) and Mg(2+) showed a significant enhancement in biofilm production by the bacterium. The SEM-EDAX study showed that the elemental composition of the biofilm varied significantly when grown in the presence of Ca(2+) and Mg(2+). The CSLM analysis of biofilms grown in the presence of 20 mM Ca(2+) and Mg(2+) reveal the critical role of these ions on biofilm architectural parameters such as total biomass, biofilm thickness, roughness coefficient and surface to biovolume ratio. Ca(2+) was found to enhance the extracellular polymeric substances (EPS) production and phenanthrene degradation. Ca(2+) enhanced the biofilm growth in a dose dependent manner, whereas Mg(2+) significantly increased the cell growth in biofilm. More than 15% increase in phenanthrene degradation was observed when biofilm was grown in the presence of an additional 20 mM Ca(2+). This study also supports the fundamental role of Ca(2+) in biofilm growth, architecture as well as biofilm-mediated pollutant degradation.

Effect of calcium on Staphylococcus aureus biofilm architecture: a confocal laser scanning microscopic study.

Bacterial adhesion is a threshold event in the formation of biofilms. Several studies on molecular and biochemical aspects have highlighted that the protein matrix of the biofilm is of interest in developing strategies to combat biofouling. The prevalent role of biofilm associated protein (Bap) of Staphylococcus aureus in early adhesion and the putative presence of Ca(2+) binding EF hand motif in Bap was the motivation for this study. Biofilm assays (S. aureus strains V329 and M556) were done in micro-titer plates and confocal laser scanning microscopy (CLSM) was used to study the biofilm architecture. The results showed that Ca(2+) did not influence planktonic growth of the cultures; however, it modulated the biofilm architecture of S. aureus V329 in a dose dependent manner. Strain M556 was found to be a weak biofilm former and showed no significant change in the presence of Ca(2+). When tested with increasing NaCl concentration, there was no reversal of the Bap-dependent Ca(2+) inhibition of S. aureus V329 biofilm. This indicates that the interaction of Bap and Ca(2+) is not mere electrostatic. CLSM images of V329 biofilm showed reduction in biofilm thickness as well as altered biofilm topography with varying Ca(2+) concentrations. The inhibition effect of Ca(2+) on strain V329 biofilm disappeared in the presence of chelating agent EDTA at a non-inhibiting concentration (0.15 mM). The paper elaborates the role of Ca(2+) in biofilm architecture of S. aureus.

Characterization of Stenotrophomonas acidaminiphila NCW-702 biofilm for implication in the degradation of polycyclic aromatic hydrocarbons

Biofilm formation and polycyclic aromatic hydrocarbons (PAHs) degradation by a marine bacterium Stenotrophomonas acidaminihila NCW‐702 was investigated.

Biofilm-Mediated Bioremediation of Polycyclic Aromatic Hydrocarbons

Contamination of the environment with noxious polycyclic aromatic hydrocarbons (PAHs) has increased in recent times. They are of key concern because of their toxic and mutagenic effect on living organisms. Among the various methodologies available for cleanup of contaminants, the impending bioremediation technologies are promising. Biological processes are preferred over chemical treatment as they are environmentally safe and techno-economically viable. Planktonic cultures grown under controlled laboratory conditions are extensively studied for bioremediation. However, in nature, microbiota are mostly found in the form of biofilms, whose development is supported on solid surfaces where nutrient gradients are available bacterial. Bacterial biofilms can be the potential tools for bioremediation of PAHs. They are very common in environmental and clinical settings. Biofilm is a highly rigid structure resistant to a variety of environmental problems. Therefore, optimization of bioremediation processes as per the field conditions requires a thorough knowledge of biofilm structure, dynamics, and the microbiota interaction with pollutants and other environmental factors. This chapter deals with the prospects of using biofilms for bioremediation of PAHs.

Removal of toxic Co-EDTA complex by a halophilic solar-salt-pan isolate Pseudomonas aeruginosa SPB-1.

In this study, a promising bioremediation approach was developed to remove [Co(III)-EDTA](-) complex that is generated during the waste management process. Though several studies have been reported on bioremediation of cobalt, the removal of [Co(III)-EDTA](-) complex has not been tested. A [Co(III)-EDTA](-) resistant bacterium, Pseudomonas aeruginosa SPB-1 was isolated from the solar-salt-pan and physical parameters were optimized for its growth. The various studies showed that the removal of [Co(III)-EDTA](-) from the bulk liquid was due to the adsorption of the complex by the biomass. Using absorption/desorption isotherm over a range of pH (1-8), the maximum adsorption of [Co(III)-EDTA](-) was found to be at pH 7.0 and maximum desorption from the biomass occurred at pH 1.0, thus rendering an ion exchange property to P. aeruginosa SPB-1 biomass. P. aeruginosa SPB-1 biomass could be used as bio-resin that showed 80.4±3.27% adsorption capacity up to fourth cycle and the biomass was viable till the ninth cycle with 10.5±7.3% adsorption. Radiation tolerance potential i.e. D10 value for the strain was found to be ~300 Gy, which suggests the potential use of the bacterium in bioremediation of moderately active nuclear waste.

Microbial reduction of (Co(III)-EDTA) − by Bacillus licheniformis SPB-2 strain isolated from a solar salt pan

Naturally stressed habitats are known to be repositories for novel microorganisms with potential bioremediation applications. In this study, we isolated a [Co(III)-EDTA](-) reducing bacterium Bacillus licheniformis SPB-2 from a solar salt pan that is exposed to constant cycles of hydration and desiccation in nature. [Co(III)-EDTA](-) generated during nuclear waste management process is difficult to remove from the waste due to its high stability and solubility. It is reduced form i.e. [Co(II)-EDTA](2-) is less stable though it is toxic. This study showed that B. licheniformis SPB-2 reduced 1mM [Co(III)-EDTA](-) in 14 days when grown in a batch mode. However, subsequent cycles showed an increase in the reduction activity, which was observed up to four cycles. Interestingly, the present study also showed that [Co(III)-EDTA](-) acted as an inducer for B. licheniformis SPB-2 spore germination. Vegetative cells germinated from the spores were found to be involved in [Co(III)-EDTA](-) reduction. More detailed investigations showed that after [Co(III)-EDTA](-) reduction, i.e. [Co(II)-EDTA](2-) complex was removed by B. licheniformis SPB-2 from the bulk liquid by adsorption phenomenon. The bacterium showed a D10 value (radiation dose required to kill 90% cells) of ∼250 Gray (Gy), which signifies the potential use of B. licheniformis SPB-2 for bioremediation of moderately active nuclear waste.

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.

Phenotypic Switching in Biofilm-Forming Marine Bacterium Paenibacillus lautus NE3B01

Biofilm-forming marine bacterium Paenibacillus lautus NE3B01 was isolated from a mangrove ecosystem, Odisha, India. This isolate formed a swarming type of colony pattern on the solid culture medium with 0.5–2xa0% agar. Phase contrast microscopy study of a growing colony of P. lautus on solid media and swarming pattern revealed the existence of two phenotypically distinct cells (i.e. cocci and rods) across the colonies. However, in actively growing planktonic culture, only rod-shaped cells were observed. Biofilm growth studies (crystal violet assay) with the isolate showed significant biofilm formation by 6xa0h, and the detachment phase was observed after 18xa0h. Biofilm parameters (such as total biomass, roughness coefficient, biofilm thickness, etc.) of 24-h-old P. lautus biofilm were studied by confocal scanning laser microscopy (CSLM). The CSLM study showed that P. lautus formed a biofilm with an average thickness of 14.8xa0±xa02.6xa0μm, a high roughness coefficient (0.379xa0±xa00.103) and surface to bio-volume ratio (4.59xa0±xa01.12xa0μm2/μm3), indicating a highly uneven topography of the biofilm. This also indicates that the 24-h-old biofilm is in dispersal phase. Scanning electron microphotographs of P. lautus also supported the existence of two distinct phenotypes of P. lautus. The current findings suggest that P. lautus has two vegetative phenotypes and to decongest the overcrowded biofilm the bacterium can switch over to motile rods from nonmotile cocci and vice versa.

Differential Radio-Tolerance of Nutrition-Induced Morphotypes of Deinococcus radiodurans R1

Deinococcus radiodurans R1 is a highly radio-tolerant bacterium. Depending on the nutrient availability D. radiodurans R1 exists in three morphologies viz. monococcal, diplococcal and tetracoccal. In this study, we examined whether nutrition-induced morphotypes of D. radiodurans showed similar DNA damage upon gamma radiation exposure. Total DNA damage after radiation exposure was estimated by comparing percent double-strand breaks (DSBs) in genomic DNA. It was found that all three morphotypes exhibited different radiation tolerances which were also dependent on the radiation dose given. Monococcal forms were found to be most radio-tolerant at most of the tested radiation doses. Results showed that these nutrient-starved-condition induced morphotypes show lesser DNA DSBs upon irradiation, hence show higher radio-tolerance.

Isolation and characterization of culturable bacteria present in the spent nuclear fuel pool water

A spent nuclear fuel (SNF) pool is a key facility for safe management of nuclear waste, where spent nuclear fuel rods are stored in a water pool. The spent fuel rods carry a significant amount of radioactivity; they are either recycled or stored for further processing. Pool water acts as a heat sink as well as a shield against the radiation present in spent/burned fuel rods. The water used in these pools is filtered by an ultra-filtration process which makes certain the purity of water. As the life span of these pools is approximately 20 to 40xa0years, the maintenance of pure water is a big challenge. A number of researchers have shown the presence of bacterial communities in this ultrapure water. The bacterial types present in SNF pool water is of increasing interest for their potential bioremediation applications for radioactive waste. The present study showed the isolation of six bacterial species in the SNF pool water samples, which had significant radio-tolerance (D10 value 248xa0Gy to 2xa0kGy) and also biofilm-forming capabilities. These strains were also investigated for their heavy metal removal capacity. Maximum biofilm-mediated heavy metal (Co and Ni) removal (up to 3.8xa0μg/mg of biomass) was observed by three isolates (FPB1, FPB4, and FPB6). The ability of these bacterial isolates to survive in radioactive environments can be of great interest for remediation of radioactive contaminants.