Hormetic effect of ionic liquid 1-ethyl-3-methylimidazolium acetate on bacteria

h i g h l i g h t s Among three ILs tested, only [EMIM

Compatibility of the green fluorescent proteinnext term and a general nucleic acid stain for quantitative description of a Pseudomonas putida biofilm

Better understanding of biofilm development is essential for making optimal use of beneficial biofilms as well as for devising effective control strategies for detrimental biofilms. Analysis of biofilm structure and quantification of biofilm parameters using optical (including confocal) microscopy and digital image analysis techniques are becoming routine in many laboratories. The purpose of this study was to evaluate a dual labeling technique based on fluorescence signals from the green fluorescent protein (GFP) and those resulting from staining with the general nucleic acid stain SYTO 60 for the quantitative description of a model biofilm. For this purpose, a Pseudomonas putida KT2442 derivative was genetically tagged with the green fluorescent protein gene. Biofilm formation by this strain was investigated using flow cells and confocal laser scanning microscopy (CLSM). Percentage surface coverage as well as microcolony size quantified using GFP and SYTO 60 signals showed significant correlation (R=0.99). The results indicated that intrinsic labelling of this model biofilm using constitutively expressed proteins such as GFP can be used for real-time biofilm observation and generation of reliable quantitative data, comparable to those obtained using conventional methods such as nucleic acid staining. Non-destructive time series observation of GFP-expressing biofilms in flow-cells can thus be confidently used for four-dimensional (x, y, z, t) analysis and quantification of biofilm development. The results also point to the possibility of using GFP and SYTO 60 to study dual species biofilms, as quantitative data generated using both fluorophore signals are comparable

Microbial transformation of Se oxyanions in cultures of Delftia lacustris grown under aerobic conditions

Delftia lacustris is reported for the first time as a selenate and selenite reducing bacterium, capable of tolerating and growing in the presence of ≥ 100 mM selenate and 25 mM selenite. The selenate reduction profiles of D. lacustris were investigated by varying selenate concentration, inoculum size, concentration and source of organic electron donor in minimal salt medium. Interestingly, the bacterium was able to reduce both selenate and selenite under aerobic conditions. Although considerable removal of selenate was observed at all concentrations investigated, D. lacustris was able to completely reduce 0.1 mM selenate within 96 h using lactate as the carbon source. Around 62.2% unaccounted selenium (unidentified organo-selenium compounds), 10.9% elemental selenium and 26.9% selenite were determined in the medium after complete reduction of selenate. Studies of the enzymatic activity of the cell fractions show that the selenite/selenate reducing enzymes were intracellular and independent of NADPH availability. D. lacustris shows an unique metabolism of selenium oxyanions to form elemental selenium and possibly also selenium ester compounds, thus a potential candidate for the remediation of selenium-contaminated wastewaters in aerobic environments. This novel finding will advance the field of bioremediation of selenium-contaminated sites and selenium bio-recovery and the production of potentially beneficial organic and inorganic reactive selenium species

The ionic liquid 1-alkyl-3-methylimidazolium demonstrates comparable antimicrobial and antibiofilm behavior to a cationic surfactant

<div><p>Biofilms are problematic in health and industry because they are resistant to various antimicrobial treatments. Ionic liquids are a novel class of low temperature liquid salts consisting of discrete anions and cations, and have attracted considerable interest as safer alternatives to organic solvents. Ionic liquids have interesting antimicrobial properties and some could find use in the development of novel antiseptics, biocides and antifouling agents. The antimicrobial and antibiofilm activity of 1-dodecyl-3-methylimiazolium iodide ([C₁₂MIM]I) was studied using the clinically important bacterial pathogens, <i>Staphylococcus aureus</i> SAV329 and <i>Pseudomonas aeruginosa</i> PAO1. The ionic liquid increased cell membrane permeability in both <i>S. aureus</i> and <i>P. aeruginosa</i> cells and impaired their growth, attachment and biofilm development. The ionic liquid exhibited superior antimicrobial and antibiofilm activity against the Gram-positive <i>S. aureus</i> compared to the Gram-negative <i>P. aeruginosa</i> cells. <i>Bac</i>Light™ staining and confocal microscope imaging confirmed that the ionic liquid treatment increased the cell membrane permeability of both the Gram-positive and Gram-negative bacteria. In addition, the antimicrobial and antibiofilm properties of [C₁₂MIM]I were similar or superior to those of cetyltrimethylammonium bromide (CTAB), a well-known cationic surfactant. It is concluded that the ionic liquid induced damage to bacterial cells by disrupting cell membrane, leading to inhibition of growth and biofilm formation. Overall, the results indicate that the ionic liquid 1-dodecyl-3-methylimiazolium iodide was effective in preventing <i>S. aureus</i> and <i>P. aeruginosa</i> biofilms and could have applications in the control of bacterial biofilms.</p> </div

Dual Labeling of Pseudomonas putida with Fluorescent Proteins for In Situ Monitoring of Conjugal Transfer of the TOL Plasmid

We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor P. putida cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies

Immobilization of Cr(VI) and Its Reduction to Cr(III) Phosphate by Granular Biofilms Comprising a Mixture of Microbes▿

We assessed the potential of mixed microbial consortia, in the form of granular biofilms, to reduce chromate and remove it from synthetic minimal medium. In batch experiments, acetate-fed granular biofilms incubated aerobically reduced 0.2 mM Cr(VI) from a minimal medium at 0.15 mM day−1 g−1, with reduction of 0.17 mM day−1 g−1 under anaerobic conditions. There was negligible removal of Cr(VI) (i) without granular biofilms, (ii) with lyophilized granular biofilms, and (iii) with granules in the absence of an electron donor. Analyses by X-ray absorption near edge spectroscopy (XANES) of the granular biofilms revealed the conversion of soluble Cr(VI) to Cr(III). Extended X-ray absorption fine-structure (EXAFS) analysis of the Cr-laden granular biofilms demonstrated similarity to Cr(III) phosphate, indicating that Cr(III) was immobilized with phosphate on the biomass subsequent to microbial reduction. The sustained reduction of Cr(VI) by granular biofilms was confirmed in fed-batch experiments. Our study demonstrates the promise of granular-biofilm-based systems in treating Cr(VI)-containing effluents and wastewater