T. Subba Rao

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.

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.

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.

Staphylococcus aureus biofilm removal by targeting biofilm-associated extracellular proteins

Background & objectives: Among cell surface proteins, biofilm-associated protein (Bap) promotes biofilm development in Staphylococcus aureus strains. The aim of this study was to investigate proteinase-mediated biofilm dispersion in different isolates of S. aureus. Methods: Biofilm assay was done in 96-well microtitre plate to evaluate the effect of proteinase K on biofilms of bovine mastitis S. Aureus isolates. Extracellular polymeric substances were extracted and evaluated for their composition (protein, polysaccharides and extracellular DNA), before and after the proteinase K treatment. Results: Biofilm assay showed that 2 µg/ml proteinase K significantly inhibited biofilm development in bap-positive S. aureus V329 as well as other S. aureus isolates (SA7, SA10, SA33, SA352), but not in bap-mutant M556 and SA392 (a weak biofilm-producing strain). Proteinase K treatment on S. aureus planktonic cells showed that there was no inhibition of planktonic growth up to 32 µg/ml of proteinase K. Proteinase K treatment on 24 h old preformed biofilms showed an enhanced dispersion of bap-positive V329 and SA7, SA10, SA33 and SA352 biofilms; however, proteinase K did not affect the bap-mutant S. aureus M556 and SA392 biofilms. Biofilm compositions study before and after proteinase K treatment indicated that Bap might also be involved in eDNA retention in the biofilm matrix that aids in biofilm stability. When proteinase K was used in combination with antibiotics, a synergistic effect in antibiotic efficacy was observed against all biofilm-forming S. aureus isolates. Interpretation & conclusions: Proteinase K inhibited biofilms growth in S. aureus bovine mastitis isolates but did not affect their planktonic growth. An enhanced dispersion of preformed S. aureus biofilms was observed on proteinase K treatment. Proteinase K treatment with antibiotics showed a synergistic effect against S. aureus biofilms. The study suggests that dispersing S. aureus by protease can be of use while devising strategies against S. aureus biofilms.

Reduction of [Co(III)–EDTA]− complex by a novel process using phototrophic granules: a step towards sustainable bioremediation

Microbial granules are more effective than monoculture bacteria in the bioremediation process of environmental contaminants. Among various types of microbial granules phototrophic granules are relatively advantageous in bioremediation than heterotrophic granules since they are self-sustaining. [Co(III)–EDTA]− generated during nuclear waste management is difficult to remove from the radioactive waste due to its high solubility and stability. In this study, phototrophic granules generated from a freshwater source were used for the reduction of [Co(III)–EDTA]− under anoxic conditions. The results of the study showed that the phototrophic granules efficiently reduced the highly soluble [Co(III)–EDTA]− to a lesser soluble form, [Co(II)–EDTA]2– up to a concentration of 20 mM. The rate of [Co(III)–EDTA]− reduction was found to be relatively high when the process was performed with acclimatized phototrophic granules when compared to non-acclimatized granules. Specific studies using antibiotics to kill the heterotrophic bacterial population of the phototrophic granules showed that [Co(III)–EDTA]− reduction was carried out predominantly by the phototrophs. Investigations were also carried out to test the robustness of phototrophic granules to perform [Co(III)–EDTA]− reduction after gamma irradiation. The phototrophic granules showed substantial radio-tolerance and could reduce [Co(III)–EDTA]− without any significant loss in the activity, when irradiated up to 2 kGy. The study suggests that phototrophic granules have potential application in bioremediation of moderate level nuclear waste.

Heterologous Expression and Purification of a 238 kDa Large BiofilmAssociated Surface Protein (Bap) in Escherichia coli

Biofilm-associated protein (Bap) is a large surface protein (~238 kDa) that plays a significant role in the development of Staphylococcus biofilms. Surface proteins in S. aureus are functionally redundant, which implies that a null mutant that affects one surface protein might only be partially defective in the studied function. Therefore, the objective of this study was to clone, overexpress and purify the full Bap protein in E. coli to enable us to characterize the protein in detail for future experiments. The challenging part of this study was to resolve the problem of plasmid instability of recombinant construct, which is speculated to be due the large size of the gene and the presence of 13 direct tandem repeats, when conventional E. coli strains such as DH5-α and XL1-Blue were used as a cloning host and optimizing the parameters for overexpression of the gene. The full bap gene (~6.8 kb) was amplified by long-range Taq polymerase and cloned in an expression vector pET21b in E. coli stbl2 and in BL21(DE3)-pLysS for over expression. DNA sequencing of the cloned gene confirms 100% identity with bap gene in-situ (S. aureus V329). Successful expression of the full length of Bap protein in E. coli BL21(DE3)-pLysS was confirmed by the SDS-PAGE and Western blotting using Anti-His tag antibody. To the best of our knowledge, it is first attempt to clone and overexpress full-length Bap protein in E. coli. The use of recombinant Bap gene will allow us to study and aid in its biophysical characterization.