Stable isotope fractionation of toluene under dynamic phase exchange was studied aiming at ascertaining the effects of gas-liquid partitioning and biodegradation of toluene stable isotope composition in liquid-air phase exchange reactors (Laper). The liquid phase consisted of a mixture of aqueous minimal media, a known amount of a mixture of deuterated (toluene-d) and non-deuterated toluene (toluene-h), and bacteria of toluene degrading strain Pseudomonas putida KT2442. During biodegradation experiments, the liquid and air-phase concentrations of both toluene isotopologues were monitored to determine the observable stable isotope fractionation in each phase. The results show a strong fractionation in both phases with apparent enrichment factors beyond −800‰. An offset was observed between enrichment factors in the liquid and the gas phase with gas-phase values showing a stronger fractionation in the gas than in the liquid phase. Numerical simulation and parameter fitting routine was used to challenge hypotheses to explain the unexpected experimental data. The numerical results showed that either a very strong, yet unlikely, fractionation of the phase exchange process or a – so far unreported – direct consumption of gas phase compounds by aqueous phase microorganisms could explain the observed fractionation effects. The observed effect can be of relevance for the analysis of volatile contaminant biodegradation using stable isotope analysis in unsaturated subsurface compartments or other environmental compartment containing a gas and a liquid phase.

The composition of root exudates is modulated by several environmental factors, and it remains unclear how that affects beneficial rhizosphere or inoculated microorganisms under heavy metal (HM) contamination. Therefore, we evaluated the transcriptional response of Pseudomonas putida E36 (a Miscanthus x giganteus isolate with plant growth promotion-related properties) to Cd, Pb and Zn in an in vitro study implementing root exudates from M. x giganteus. To collect root exudates and analyse their composition plants were grown in a pot experiment under HM and control conditions. Our results indicated higher exudation rate for plants challenged with HM. Further, out of 29 organic acids identified and quantified in the root exudates, 8 of them were significantly influenced by HM (e.g., salicylic and terephthalic acid). The transcriptional response of P. putida E36 was significantly affected by the HM addition to the growth medium, increasing the expression of several efflux pumps and stress response-related functional units. The additional supplementation of the growth medium with root exudates from HM-challenged plants resulted in a downregulation of 29% of the functional units upregulated in P. putida E36 as a result of HM addition to the growth medium. Surprisingly, root exudates + HM downregulated the expression of P. putida E36 functional units related to plant colonization (e.g., chemotaxis, motility, biofilm formation) but upregulated its antibiotic and biocide resistance compared to the control treatment without HM. Our findings suggest that HM-induced changes in root exudation pattern may attract beneficial bacteria that are in turn awarded with organic nutrients, helping them cope with HM stress. However, it might affect the ability of these bacteria to colonize plants growing in HM polluted areas. Those findings may offer an insight for future in vivo studies contributing to improvements in phytoremediation measures.

This work aimed to study the performance of paraquat removal by cell-immobilized ceramics. Two strains of paraquat degrading bacteria, Pseudomonas putida and Bacillus subtilis, were separately immobilized on the ceramic with and without wastewater sludge addition. Results showed that the ceramic surface with sludge has more functional groups and a more highly negative charge on the surface than the original ceramic. The ceramic with sludge had 2-3-fold of the immobilized cells higher than that of the control (without sludge) and less leaching of the immobilized cells. The sludge addition at 20% (w/w) to the ceramic provided the highest cell adhesion for both P. putida and B. subtilis. The paraquat removal efficiencies were higher than 98%, while the control ceramic could remove only 77 ± 1.2%. The immobilized cells on ceramic with sludge provided a significant degree of dissolved organic nitrogen reduction (82%) during the paraquat removal. Most organic nitrogen in paraquat was biologically mineralized (ammonified). Findings from this work suggest the superiority of ceramic with sludge in mineralizing organic nitrogen associated with paraquat.

Co-selection of antibiotic resistance genes (ARGs) by heavy metals might facilitate the spread of ARGs in the environments. Cadmium contamination is ubiquitous, while, it remains unknown the extent to which cadmium (Cd²⁺) impact plasmid-mediated transfer of ARGs in aquatic bacterial communities. In the present study, we found that Cd²⁺ amendment at sub-inhibitory concentration significantly increased conjugation frequency of RP4 plasmid from Pseudomonas putida KT2442 to a fresh water microbial community by liquid mating method. Cd²⁺ treatment (1–100 mg/L) significantly increased the cell membrane permeability and antioxidant activities of conjugation mixtures. Amendments of 10 and 100 mg/L Cd²⁺ significantly enhanced the mRNA expression levels of mating pair formation gene (trbBp) and the DNA transfer and replication gene (trfAp) due to the repression of regulatory genes (korA, korB and trbA). Phylogenetic analysis of transconjugants indicated that Proteobacteria was the dominant recipients and high concentration of Cd²⁺ treatment resulted in expanded recipient taxa. This study suggested that sub-inhibitory Cd²⁺ contamination would facilitate plasmid conjugation and contributed to the maintenance and spread of plasmid associated ARGs, and highlighted the urgent need for effective remediation of Cd²⁺ in aquatic environments.

The synergistic cooperation of microbial cells and their extracellular polymeric substances (EPS) in biofilms is critical for the biofilm’s resistance to heavy metals and the migration and transformation of heavy metals. However, the effects of different components of biofilms have not been fully understood. In this study, the spatial distribution and speciation of copper in the colloidal EPS, capsular EPS, cell walls and membranes, and intracellular fraction of unsaturated Pseudomonas putida (P. putida) CZ1 biofilms were fully determined at the subcellular level. It was found that 60–67% of copper was located in the extracellular fraction of biofilms, with 44.7–42.3% in the capsular EPS. In addition, there was 15.5–20.1% and 17.2–21.2% of copper found in the cell walls and membranes or the intracellular fraction, respectively. Moreover, an X-ray absorption fine structure spectra analysis revealed that copper was primarily bound by carboxyl-, phosphate-, and hydrosulfide-like ligands within the extracellular polymeric matrix, cell walls and membranes, and intracellular fraction, respectively. In addition, macromolecule quantification, fourier-transform infrared spectroscopy spectra and sulfur K-edge x-ray absorption near edge structure analysis further showed the carboxyl-rich acidic polysaccharides in EPS, phospholipids in cell walls and cell membranes, and thiol-rich intracellular proteins were involved in binding of copper in the different components of biofilm. The full understanding of the distribution and chemical species of heavy metals in biofilms not only promotes a deep understanding of the interaction mechanisms between biofilms and heavy metals, but also contributes to the development of effective biofilm-based heavy metal pollution remediation technologies.

Bacteria–phyllosilicate complexes are commonly found in natural environments and are capable of immobilizing trace metals. However, the molecular binding mechanisms of heavy metals to these complex aggregates still remain poorly understood. This study investigated Cd adsorption on Gram-positive Bacillus subtilis, Gram-negative Pseudomonas putida and their binary mixtures with montmorillonite using surface complexation model, Cd K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and isothermal titration calorimetry (ITC). We have shown that larger amounts of Cd are adsorbed by B. subtilis than by P. putida at pH<∼6, and Cd sorption that binding to phosphate groups plays a more important role in P. putida than in B. subtilis. This remind us that we should consider the microbe species when predict the biochemical behavior of trace metals in microbe-bearing environments. The observed Cd adsorption on the binary bacteria–clay composites was more than that predicted based on the component additivity approach. When taking bacteria–clay (1:1 mass ratio) as a representative example, an approximately 68%:32% metal distribution between the bacterial and mineral fraction was found. Both the EXAFS and ITC fits showed that the binding stoichiometry for Cd-carboxyl/phosphate was smaller in the binary mixtures than that in pure bacteria. We proposed that the significant deviations were possibly due to the physical-chemical interaction between the composite fractions that might reduce the agglomeration of the clay grains, increase the negative surface charges, and provide additional bridging of metals ions between bacterial cells and clays.

The effects of humic acid (HA) on interactions between ZnO nanoparticles (ZnO NPs) and Pseudomonas putida KT2440 biofilms at different maturity stages were investigated. Three stages of biofilm development were identified according to bacterial adenosine triphosphate (ATP) activity associated with biofilm development process. In the initial biofilm stage 1, the ATP content of bacteria was reduced by more than 90% when biofilms were exposed to ZnO NPs. However, in the mature biofilm stages 2 and 3, the ATP content was only slightly decreased. Biofilms at stage 3 exhibited less susceptibility to ZnO NPs than biofilms at stage 2. These results suggest that more mature biofilms have a significantly higher tolerance to ZnO NPs compared to young biofilms. In addition, biofilms with intact extracellular polymeric substances (EPS) showed higher tolerance to ZnO NPs than those without EPS, indicating that EPS play a key role in alleviating the toxic effects of ZnO NPs. In both pure ZnO NPs and ZnO-HA mixtures, dissolved Zn²⁺ originating from the NPs significantly contributed to the overall toxicity. The presence of HA dramatically decreased the toxicity of ZnO NPs due to the binding of Zn²⁺ on HA. The combined results from this work suggest that the biofilm maturity stages and environmental constituents (such as humic acid) are important factors to consider when evaluating potential risks of NPs to ecological systems.

Bacteria based ecotoxicology assessment of manufactured nanoparticles is largely restricted to Escherichia coli bioreporters in laboratory media. Here, toxicity effects of model OECD nanoparticles (Ag NM-300K, ZnO NM-110 and TiO2 NM-104) were assessed using the switch-off luminescent Pseudomonas putida BS566::luxCDABE bioreporter in Luria Bertani (LB) medium and artificial wastewater (AW). IC50 values ∼4 mg L−1, 100 mg L−1 and >200 mg L−1 at 1 h were observed in LB for Ag NM-300K, ZnO NM-110 and TiO2 NM-104, respectively. Similar results were obtained in AW for Ag NM-300K (IC50 ∼5 mg L−1) and TiO2 NM-104 (IC50 >200 mg L−1) whereas ZnO NM-110 was significantly higher (IC50 >200 mg L−1). Lower ZnO NM-110 toxicity in AW compared to LB was associated with differences in agglomeration status and dissolution rate. This work demonstrates the importance of nanoecotoxicological studies in environmentally relevant matrices.

The feasibility of using fresh plant residues and their extracts to stimulate the bio-dissipation of polycyclic aromatic hydrocarbons (PAHs) were highlighted. Wood chip, bamboo leave, orange peel and their water-extractable organic matter (WEOM) were chosen as amendment materials. Effect of WEOM on bio-dissipation (bioaccumulation and biodegradation) of phenanthrene and pyrene from water by two bacteria were investigated. Orange peel extract demonstrated the highest efficiency for stimulating PAHs removal by bacterium B1 (Pseudomonas putida), while bamboo leave extract was the best one to enhance PAHs bio-dissipation by bacterium B2 (unidentified bacterium isolated from PAHs-contaminated soil). Amended the actual contaminated soil with 1% plant residues, PAHs dissipation were increased by 15–20%, 20–39%, 14–24%, 12–23% and 17–26%, respectively, for 2-, 3-, 4-, 5- and 6-ring PAHs via stimulating indigenous microbial degradation activity. Bamboo leave exhibited the most effective one to stimulate dissipation of PAHs in contaminated soil.

We investigated the effect of increasing CO₂ concentrations on the growth and viability of ecophysiologically different microorganisms to obtain information for a leakage scenario of CO₂ into shallow aquifers related to the capture and storage of CO₂ in deep geological sections. CO₂ concentrations in the gas phase varied between atmospheric conditions and 80% CO₂ for the aerobic strains Pseudomonas putida F1 and Bacillus subtilis 168 and up to 100% CO₂ for the anaerobic strains Thauera aromatica K172 and Desulfovibrio vulgaris Hildenborough. Increased CO₂ concentrations caused prolonged lag-phases, and reduced growth rates and cell yields; the extent of this effect was proportional to the CO₂ concentration. Additional experiments with increasing CO₂ concentrations and increasing pressure (1–5000 kPa) simulated situations occurring in deep CO₂ storage sites. Living cell numbers decreased significantly within 24 h at pressures ≥1000 kPa, demonstrating a severe lethal effect for the combination of high pressure and CO₂.