Degradation of trimethylamine by immobilized cells of Pseudomonas putida A (ATCC 12633)

Pseudomonas putida A (ATCC 12633) capable of degrading trimethylamine (TMA) was immobilized in calcium alginate. The TMA-degrading capacity of immobilized cells was compared to free cells in batch culture by changing temperature, pH, nutrient supplementation, and initial TMA concentration. Although immobilized cells showed good removal efficiencies in wider ranges of temperature (15 °C–35 °C) and pH (6.5–8.5) than free cells, the optimal conditions for TMA removal for both free and immobilized cells were 30 °C, in buffered medium (pH 7.5) without addition of nutrients. Immobilized cells degraded up 885 mg l−1 of TMA completely whereas the free cells degraded only 295 mg l−1. For all the TMA concentrations evaluated with immobilized cells (295–885 mg l−1), after 24 h, the degradation rate started to decline. This effect was attributed to the accumulation of intracellular TMA into the cells (8.3–15.3 mg l−1) which is sufficient to inhibit the first enzyme of the aerobic degradation of TMA by P. putida, the TMA dehydrogenase. The fact that the immobilized cells showed a high stability as regards their viability and degradability of high TMA concentrations revealed a good potential of this system for treating of TMA-contaminated site.Fil: Liffourrena, Andres Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Lucchesi, Gloria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentin

Release of outer membrane vesicles in pseudomonas putida as a response to stress caused by cationic surfactants

Pseudomonas putida A (ATCC 12633), a degrader of cationic surfactants, releases outer membrane vesicles (OMVs) when grown with tetradecyltrimethylammonium bromide (TTAB) as the sole carbon, nitrogen and energy source. The OMVs exhibit a bilayer structure and were found to be composed of lipopolysaccharides, proteins and phospholipids (PLs) such as cardiolipin, phosphatidylcholine, phosphatidic acid and phosphatidylglycerol (PG). The OMVs showed a marked increase in the PG content, approximately 43 % higher than the amount registered in the parent cells from which the vesicles were derived. After growth of P. putida with TTAB, the amount of lipoprotein covalently cross-linked to the peptidoglycan showed a twofold decrease when compared with values found after growth without the surfactant [16±2 and 28±3 mg (mg cell envelope protein)-1, respectively]. This decrease in the amount of lipoprotein can be related to areas of loss of contact between the outer membrane and the peptidoglycan and, therefore, to OMV production. In addition, due to its amphiphilic nature, TTAB can contribute to OMV biogenesis, through a physical mechanism, by induction of the curvature of the membrane. Taking into account that OVMs were produced when the cells were grown under external stress, caused by the surfactant, and that TTAB was detected in the vesicles [48 nmol TTAB (nmol PL)-1], we concluded that this system of TTAB elimination is a mechanism that P. putida A (ATCC 12633) would utilize for alleviating stress caused by cationic surfactants.Fil: Heredia, Romina Marisa. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Boeris, Paola Sabrina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Liffourrena, Andres Sebastian. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Bergero, Maria Fernanda. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: López, Gastón Alberto. Universidad Nacional de Río Cuarto; ArgentinaFil: Lucchesi, Gloria Ines. Universidad Nacional de Río Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Alginate-perlite encapsulated Pseudomonas putida A (ATCC 12633) cells: Preparation, characterization and potential use as plant inoculants

Microbial immobilization can be used to prepare encapsulated inoculants. Here, we characterize and describe the preparation of Ca-alginate-perlite microbeads loaded with cells of plant growth-promoting Pseudomonas putida A (ATCC 12633), for their future application as agricultural inoculants. The microbeads were prepared by dropwise addition of a CaCl2-paraffin emulsion mixture to an emulsion containing alginate 2% (w/v), perlite 0.1–0.4% (w/v) and bacterial suspension in 0.9% NaCl (1010 CFU/mL). For all perlite concentrations used, microbead size was 90–120 μm, the trapped population was 108 CFU/g microbeads and the increase in mechanical stability was proportional to perlite concentration. Microbeads containing 0.4% (w/v) perlite were able to release bacteria into the medium after 30 days of incubation. When we evaluated how P. putida A (ATCC 12633) entrapped in Ca-alginate-perlite (0.4% (w/v)) microbeads colonized the Arabidopsis thaliana rhizosphere, an increase in colonization over time was detected (from an initial 2.1 × 104 to 9.2 × 105 CFU/g soil after 21 days). With this treatment, growth promotion of A. thaliana occurred with an increase in the amount of proteins, and in root and leaf biomass. It was concluded that the microbeads could be applied as possible inoculants, since they provide protection and a controlled release of microorganisms into the rhizosphere.Fil: Liffourrena, Andres Sebastian. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Lucchesi, Gloria Ines. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Degradation of trimethylamine by immobilized cells of Pseudomonas putida A (ATCC 12633)

Pseudomonas putida A (ATCC 12633) capable of degrading trimethylamine (TMA) was immobilized in calcium alginate. The TMA-degrading capacity of immobilized cells was compared to free cells in batch culture by changing temperature, pH, nutrient supplementation, and initial TMA concentration. Although immobilized cells showed good removal efficiencies in wider ranges of temperature (15 °C–35 °C) and pH (6.5–8.5) than free cells, the optimal conditions for TMA removal for both free and immobilized cells were 30 °C, in buffered medium (pH 7.5) without addition of nutrients. Immobilized cells degraded up 885 mg l−1 of TMA completely whereas the free cells degraded only 295 mg l−1. For all the TMA concentrations evaluated with immobilized cells (295–885 mg l−1), after 24 h, the degradation rate started to decline. This effect was attributed to the accumulation of intracellular TMA into the cells (8.3–15.3 mg l−1) which is sufficient to inhibit the first enzyme of the aerobic degradation of TMA by P. putida, the TMA dehydrogenase. The fact that the immobilized cells showed a high stability as regards their viability and degradability of high TMA concentrations revealed a good potential of this system for treating of TMA-contaminated site.Fil: Liffourrena, Andres Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Lucchesi, Gloria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentin

Identification, Cloning and Biochemical Characterization of Pseudomonas putida A (ATCC 12633) Monooxygenase Enzyme necessary for the Metabolism of Tetradecyltrimethylammonium Bromide

This study presents the first report of the purification and characterization of a monooxygenase enzyme from Pseudomonas putida A (ATCC 12633) that is responsible for the oxidation of physiologically relevant quaternary ammonium compounds, the tetradecyltrimethylammonium bromide. The degradation of tetradecyltrimethylammonium bromide by P. putida A (ATCC 12633) is initiated by N-dealkylation and catalysed by tetradecyltrimethylammonium monooxygenase (TTABMO), resulting in the formation of tetradecylalkanal and trimethylamine. Based on sequence analysis, the gene for TTABMO (ttbmo) corresponded to an ORF named PP2033 in the genome of P. putida KT2440. Mutation in ttabmo blocked the utilization of tetradecyltrimethylammonium bromide by Pseudomonas putida A (ATCC 12633) as carbon and nitrogen sources. The enzyme can be highly overexpressed in P. putida Δttabmo-T7 in active form and purified as a hexahistidine fusion protein. Like the native enzyme, the his-TTABMO was found to be a monomer with molecular mass of 40 kDa, the isoelectric point 7.3, that catalyses the breakdown of tetradecyltrimethylammonium bromide and utilized NADPH and FAD as cofactor. The biochemical properties and the analysis of the respective protein sequence revealed that TTABMO represents a typical flavoprotein monooxygenase, which is member of a flavoprotein family that is distinct from Baeyer–Villiger monooxygenases.Fil: Liffourrena, Andres Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Lucchesi, Gloria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentin

Pseudomonas putida A ATCC 12633 oxidizes trimethylamine aerobically via two different pathways

The present study examined the aerobic metabolism of trimethylamine in Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine. In both conditions, the trimethylamine was used as a nitrogen source and also accumulated in the cell, slowing the bacterial growth. Decreased bacterial growth was counteracted by the addition of AlCl3. Cell-free extracts prepared from cells grown aerobically on tetradecyltrimethylammonium bromide exhibited trimethylamine monooxygenase activity that produced trimethylamine N-oxide and trimethylamine N-oxide demethylase activity that produced dimethylamine. Cell-free extracts from cells grown on trimethylamine exhibited trimethylamine dehydrogenase activity that produced dimethylamine, which was oxidized to methanal and methylamine by dimethylamine dehydrogenase. These results show that this bacterial strain uses two enzymes to initiate the oxidation of trimethylamine in aerobic conditions. The apparent Km for trimethylamine was 0.7 mM for trimethylamine monooxygenase and 4.0 mM for trimethylamine dehydrogenase, but both enzymes maintain similar catalytic efficiency (0.5 and 0.4, respectively). Trimethylamine dehydrogenase was inhibited by trimethylamine from 1 mM. Therefore, the accumulation of trimethylamine inside Pseudomonas putida A ATCC 12633 grown on tetradecyltri- methylammonium bromide or trimethylamine may be due to the low catalytic efficiency of trimethylamine monooxygenase and trimethylamine dehydrogenase.Fil: Liffourrena, Andres Sebastian. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Salvano, Mario Armando. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Lucchesi, Gloria Ines. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Immobilization of a microbial consortium on Ca-alginate enhances degradation of cationic surfactants in flasks and bioreactor

Free cells and Ca-alginate encapsulated cells of Aeromonas hydrophila MFB03 removed in shake flasks up to 65% of tetradecyltrimethylammonium bromide (TTAB) 30 mg l−1. While planktonic cells were unable to utilize 50 mg l−1 benzalkonium chloride (BAC) after 48 h of incubation, the immobilized cells of A. hydrophila MFB03 removed up to 90% of BAC (25–210 mg l−1) in the same period. A microbial immobilized consortium formed by A. hydrophila MFB03 and Pseudomonas putida A (ATCC 12633) was able to degrade, after 24–30 h, 65% of TTAB and 100% of BAC, both added at 50 mg l−1. BAC was completely removed by the consortium encapsulated in a stirred tank bioreactor at 30 °C, 100 rpm and 0.024 g beads ml−1 of medium, after 12 h of incubation; reaching a degradation rate five times greater than the one obtained in shake flasks (0.415 vs. 0.089 mg−1 h−1, respectively). The system completely removed, with the same efficiency, 50 mg l−1 of BAC for 3 consecutive cycles. These results indicate that the use of Ca-alginate beads containing cells of the consortium can be considered a proper method to achieve degradation of cationic surfactants.Fil: Bergero, Maria Fernanda. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Liffourrena, Andres Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Opizzo Balza, Bianca. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Fochesatto, A.S.. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; ArgentinaFil: Lucchesi, Gloria Ines. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Practical class to evaluate the adsorption of Al(III) ions from aqueous solutions using non-viable bacterial biomass trapped in an agar-agar matrix

The contamination of wastewater with metals and the use of biotechnologies to remove them is commonly discussed in theoretical biology, microbiology and biotechnology classes aimed at undergraduate students. This work introduces an easy, economical and safe practical laboratory class related to this topic. The practice consists in performing batch assays to remove Al(III) from aqueous solutions by using non-viable bacterial biomass trapped in agar-agar beads. Students prepare these beads and analyse Al(III) adsorption on the basis of: pH, time of contact, and Al(III) concentration. Free Al(III) is quantified spectrophotometrically through a simple colorimetric method. The data obtained are used to calculate and graphically represent the amount of metal adsorbed to the beads. Finally, students analyse and discuss the results to determine the optimal adsorption conditions. Moreover, since Al(III) becomes much more attached to beads with trapped biomass than to beads without microorganisms, the class serves to demonstrate one of the advantages of using immobilised microbial cells in adsorption processes. This class was satisfactorily implemented in an upper-level course for undergraduate students of biological sciences. They were able to properly execute all the experiments and calculations proposed and to become acquainted with useful experimental design to remove a metal.Fil: Boeris, Paola Sabrina. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Bergero, Maria Fernanda. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Heredia, Romina Marisa. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Liffourrena, Andres Sebastian. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Lucchesi, Gloria Ines. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentin