Safety attributes of Pseudomonas sp. P26, an environmental microorganism with potential application in contaminated environments.

Currently, the selection of non-pathogenic microorganisms that lack clinically relevant antimicrobial resistance is crucial to bioaugmentation strategies. Pseudomonas sp. P26 (P26) is an environmental bacterium of interest due to its ability to remove aromatic compounds from petroleum, but its safety characteristics are still unknown. The study aimed to: a) determine P26 sensitivity to antimicrobials, b) investigate the presence of quinolone and ß-lactam resistance genes, c) determine the presence of virulence factors, and d) evaluate the effect of P26 on the viability of Galleria mellonella (an invertebrate animal model). P26 antimicrobial sensitivity was determined in vitro using the Kirby-Bauer agar diffusion method and the VITEK 2 automated system (BioMerieux®). Polymerase Chain Reaction was employed for the investigation of genes associated with quinolone resistance, extended-spectrum ß-lactamases, and carbapenemases. Hemolysin and protease production was determined in human blood agar and skimmed-milk agar, respectively. In the in vivo assay, different doses of P26 were injected into Galleria mellonella larvae and their survival was monitored daily. Control larvae injected with Pseudomonas putida KT2440 (a strain considered as safe) and Pseudomonas aeruginosa PA14 (a pathogenic strain) were included. Pseudomonas sp. P26 was susceptible to most evaluated antimicrobials, except for trimethoprim-sulfamethoxazole. No epidemiologically relevant genes associated with quinolone and ß-lactam resistance were identified. Hemolysin and protease production was only evidenced in the virulent strain (PA14). Furthermore, the results obtained in the in vivo experiment demonstrated that inocula less than 108 CFU/mL of P26 and P. putida KT2440 did not significantly affect larval survival, whereas larvae injected with the lowest dose of the pathogenic strain P. aeruginosa PA14 experienced instant mortality. The results suggest that Pseudomonas sp. P26 is a safe strain for its application in environmental bioremediation processes. Additional studies will be conducted to ensure the safety of this bacterium against other organisms.

Dibenzothiophene removal by environmental bacteria with differential accumulation of intracellular inorganic polyphosphate.

Dibenzothiophene (DBT), which belongs to the group of polycyclic aromatic heterocycles of sulfur, is a model substance to study the removal of sulfur compounds from oil due to its recalcitrance to traditional and specific removal processes. The aim of this work was to evaluate DBT bioremoval by environmental bacteria and its relationship with polyphosphate (polyP) accumulation, cell surface characteristics and bioemulsifying activity. Pseudomonas sp. P26 achieved the highest DBT removal percentage (48%) after 7 days of incubation. Moreover, positive correlations were estimated between DBT removal and bioemulsifying activity and biofilm formation. A strain-dependent relationship between the content of intracellular polyP and the presence of DBT in the culture medium was also demonstrated. The study of these bacterial characteristics, which could promote DBT transformation, is a first approach to select DBT-removing bacteria, in order to develop bioformulations that are able to contribute to desulfurization processes of petroleum-derived pollutants in the future.

Bioremediation on a chip: A portable microfluidic device for efficient screening of bacterial biofilm with polycyclic aromatic hydrocarbon removal capacity.

Polycyclic aromatic hydrocarbons (PAHs) are pollutants of critical environmental and public health concern and their elimination from contaminated sites is significant for the environment. Biodegradation studies have demonstrated the ability of bacteria in biofilm conformation to enhance the biodegradation of pollutants. In this study, we used our newly developed microfluidic platform to explore biofilm development, properties, and applications of fluid flow, as a new technique for screening PAHs-degrading biofilms. The optimization and evaluation of the flow condition in the microchannels were performed through computational fluid dynamics (CFD). The formation of biofilms by PAHs-degrading bacteria Pseudomonas sp. P26 and Gordonia sp. H19, as pure cultures and co-culture, was obtained in the developed microchips. The removal efficiencies of acenaphthene, fluoranthene and pyrene were determined by HPLC. All the biofilms formed in the microchips removed all tested PAHs, with the higher removal percentages observed with the Pseudomonas sp. P26 biofilm (57.4% of acenaphthene, 40.9% of fluoranthene, and 28.9% of pyrene). Pseudomonas sp. P26 biofilm removed these compounds more efficiently than planktonic cultures. This work proved that the conformation of biofilms enhances the removal rate. It also provided a new tool to rapid and low-cost screen for effective pollutant-degrading biofilms.

Development of low-cost formulations of plant growth-promoting bacteria to be used as inoculants in beneficial agricultural technologies.

Phosphorus is one of the main macronutrients for plant development. Despite its large deposits in soils, it is scarcely available for plants. Phosphate-solubilizing bacteria, belonging to the group of plant growth-promoting rhizobacteria (PGPR), are capable of mobilizing deposits of insoluble phosphates in the soil. The use of PGPR as inoculants provides an environmentally sustainable approach to increase crop production. The effectiveness of inoculants depends on their proper production, formulation and storage in order to ensure the application of the required number of viable microbial cells. In order to develop inexpensive technology, low-cost compounds for biomass production and protection should be used. After the biomass production process, the product should be formulated in a liquid or a solid form, taking into account required storage time, use of protectors/carriers, storage conditions (temperature, humidity, etc.), ease of application and maintenance of beneficial effects on crops. Careful determination of these optimal conditions would ensure a low-cost efficient inoculant that would promote the growth and yield of various crops.