RESUMEN
The current study aimed to characterize Arthrobacter sp. Sphe3 ability to reduce Cr(VI) in suspended cell cultures as well as in immobilized form using Ca-alginate beads. Adaptation studies in the presence of 5 mg L(-1) Cr(VI) showed a significant increase in specific growth rate from 0.25 to 0.3 h(-1) and bioremoval percentage from 64% to 94% (p<0.05), whereas Arthrobacter sp. Sphe3 could tolerate up to 50 mg L(-1) Cr(VI). Optimization of culture conditions resulted in complete reduction of 45 mg L(-1) Cr(VI) at 30 °C, pH 8 and 10 g L(-1) of glucose. High glucose concentrations helped at reducing (80±2.4)% of initial 100 mg L(-1) Cr(VI), whereas the bacterial strain could tolerate 850 mg L(-1) Cr(VI). Cr(III) formation was first evidenced by the appearance of a green insoluble precipitate in the medium. Cell biomass was successfully immobilized in Ca-alginate beads that were evaluated for their stability. Cell release was sharply decreased when 4% Na-alginate was used under non-shaking conditions. Biotransformation efficiency was enhanced when 25-50 mg cells mL(-1) Na-alginate from the exponential growth phase were collected and co-encapsulated with either 1% glucose and 0.5% (NH4)2SO4, or 1% LB medium. Immobilized biocatalyst could be reused up to 6 continuous cycles in the presence of 10 mg L(-1) Cr(VI), but its performance was lowered at higher metal concentrations comparing with free cells that significantly maintained their reducing ability up to 300 mg L(-1) Cr(VI).
Asunto(s)
Arthrobacter/metabolismo , Cromatos/metabolismo , Alginatos , Arthrobacter/crecimiento & desarrollo , Biomasa , Biotransformación , Células Inmovilizadas/metabolismo , Cromo/metabolismo , Medios de Cultivo , Ácido Glucurónico , Ácidos HexurónicosRESUMEN
The bioremediation of petroleum-contaminated soil was investigated at laboratory scale, using three different approaches. The first approach comprised biostimulation of indigenous microorganisms. The second approach involved combination of biostimulation of indigenous microorganisms and bioaugmentation by inoculation with free cells of petroleum degrading Pseudomonas aeruginosa strain Spet. The third was a variation of the second, in which inoculation with encapsulated cells in starch and sodium alginate of P. aeruginosa strain Spet was applied. The bioremediation of the original hydrocarbon-contaminated soil (3.5% dry weight) and that of diluted with clean natural soil at 1:1 w/w were investigated. By providing sufficient moisture, nutrients and aeration by stirring in the original contaminated soil, total concentration of n-alkanes was reduced by 94% after 191 days of treatment and total concentration of 16 polycyclic aromatic compounds by 79%, while for the 1:1 diluted soils biodegradation reached 89% and 79%, respectively. The results showed that bioaugmentation with free or encapsulated P. aeruginosa cells and/or soil dilution had no significant effect on biodegradation.
Asunto(s)
Biodegradación Ambiental , Restauración y Remediación Ambiental/métodos , Petróleo/metabolismo , Pseudomonas aeruginosa/metabolismo , Microbiología del Suelo , Contaminantes del Suelo/metabolismo , Secuencia de Bases , Cromatografía de Gases , Cartilla de ADNRESUMEN
A novel halophilic bacterium, designated strain MSS4(T), was isolated from the solar salterns of Mesolongi, Greece. The micro-organism, a motile, Gram-stain-positive, aerobic rod, proliferated at salinities of 1.0-4.0 M NaCl, with optimal growth at 2.5 M NaCl. Endospores were not observed. Strain MSS4(T) showed optimal growth at 37 degrees C and pH 8.0. The G+C content of its DNA was 47.2 mol%. The polar lipid pattern of strain MSS4(T) consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidic acid and phosphatidylethanolamine. It possessed anteiso-C(15 : 0), C(18 : 0), C(16 : 0) and anteiso-C(17 : 0) as the major fatty acids (altogether representing 84.7 % of the total). The predominant isoprenoid quinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid. 16S rRNA gene sequence analysis showed that the new isolate has 96.1 % similarity to Bacillus qingdaonensis CM1(T) and Bacillus aidingensis 17-5(T), 95.5 % to Bacillus salarius BH169(T) and lower similarity to other Bacillus species. These results justify the assignment of strain MSS4(T) to a novel species within the genus Bacillus, for which the name Bacillus halochares sp. nov. is proposed. The type strain is MSS4(T) (=LMG 24571(T) =DSM 21373(T)).
Asunto(s)
Bacillus/genética , Bacillus/clasificación , Bacillus/crecimiento & desarrollo , Bacillus/aislamiento & purificación , Composición de Base , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Grecia , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Monosacáridos/metabolismo , Nitratos/metabolismo , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Agua de Mar/microbiología , Esporas Bacterianas/fisiología , Ureasa/metabolismoRESUMEN
A novel phenanthrene-degrading bacterium, designated strain Sphe3(T), was isolated from a creosote-contaminated soil in Greece. Cells were non-motile, Gram-positive, aerobic, and rod- to coccus-shaped. The strain was isolated on the basis of formation of a clear zone on agar plates sprayed with phenanthrene. Optimal growth occurred at 30 degrees C. The G+C content of the DNA was 65.7 mol%. The polar lipid pattern of strain Sphe3(T) consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant fatty acids were iso-C(15 : 0), anteiso-C(15 : 0), iso-C(16 : 0), C(16 : 0) and anteiso-C(17 : 0), representing >86 % of the total fatty acids. The predominant isoprenoid quinone of strain Sphe3(T) was menaquinone-8 (MK-8). Based on 16S rRNA gene sequence analysis, strain Sphe3(T) showed 99 and 98.9 % similarity to the type strains of Arthrobacter oxydans and Arthrobacter polychromogenes, respectively. Strain Sphe3(T) showed 91 % similarity to homologues of A. oxydans and A. polychromogenes based on recA gene sequence analysis. Based on 16S rRNA and recA gene sequence analysis and DNA-DNA hybridization analysis, as well as physiological and chemotaxonomic characteristics, it is concluded that strain Sphe3(T) represents a novel species of the genus Arthrobacter, for which the name Arthrobacter phenanthrenivorans sp. nov. is proposed. The type strain is Sphe3(T) (=DSM 18606(T) =LMG 23796(T)).
Asunto(s)
Arthrobacter/clasificación , Arthrobacter/metabolismo , Fenantrenos/metabolismo , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S , Rec A Recombinasas/genética , Microbiología del Suelo , Especificidad de la EspecieRESUMEN
A polycyclic aromatic hydrocarbon (PAH)-degrading bacterial strain Spyr1 was isolated from Greek creosote polluted soil by an enrichment method using pyrene as sole carbon and energy source. Spyr1 was identified as Mycobacterium sp. based on 16S rDNA analysis and it was capable of degrading pyrene, fluoranthene, fluorene, anthracene, and acenaphthene. The effect of entrapment in glass beads and alginate/starch mixtures on the survival and pyrene degradation ability of Spyr1 cells in liquid suspensions and soil microcosms was tested and compared with that of freely suspended cells. In general, free cells showed higher degradation of pyrene and other PAH than immobilized cells. However, immobilized cells could better tolerate PAH and they maintained their viability and PAH degradation capability for at least 1 year after storage at 4 degrees C. Entrapped cells in glass beads exhibited better pyrene biodegradation performance than alginate/starch entrapped cells in liquid suspensions and could be used effectively for at least ten repeated cycles. Alginate/starch entrapped cells exhibited better yields than glass beads entrapped cells for removing pyrene as well as mixtures of PAH in soil microcosms.
Asunto(s)
Mycobacterium/clasificación , Mycobacterium/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Microbiología del Suelo , Biodegradación Ambiental , Células Inmovilizadas , Mycobacterium/aislamiento & purificación , Especificidad de la EspecieRESUMEN
We investigated the applicability of the green fluorescent protein of Aequorea victoria as a reporter for gene expression in the strictly fermentative Gram-negative ethanologenic bacterium Zymomonas mobilis and in the moderately halophilic bacterium Halomonas elongata. We have succeeded to express a mutated gene of green fluorescent protein under the control of different promoters in Z. mobilis and H. elongata grown under various glucose or salt concentrations, respectively. Our results demonstrate that gfp can serve as an easily assayable reporter gene in both organisms. Maximum fluorescence was obtained in Z. mobilis grown aerobically and in H. elongata grown under elevated salt concentration in solid medium. For both bacteria the fluorescence obtained was higher when the gfp gene was placed under the control of a native promoter.
Asunto(s)
Halomonas/genética , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Zymomonas/genética , Fluorescencia , Dosificación de Gen , Expresión Génica , Genes Reporteros/genética , Proteínas Fluorescentes Verdes , Halomonas/metabolismo , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Zymomonas/metabolismoRESUMEN
AIM: The aim of this work was to construct a Zymomonas mobilis mutant capable of simultaneous ethanol and ice nuclei production from agricultural by-product such as sugar beet molasses, in steady-state continuous culture. METHODS AND RESULTS: A sucrose-hypertolerant mutant of Z. mobilis strain CP4, named suc40, capable of growing on 40% (w/v) sucrose medium was isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. Plasmid pDS3154 carrying the inaZ gene of Pseudomonas syringae was conjugally transferred and expressed in suc40. The potential for simultaneous ethanol and bacterial ice nuclei production was assessed in steady-state continuous cultures over a range of dilution rates from 0.04 to 0.13 h(-1). In addition, the fatty acid and phospholipid profile of the three strains was also investigated. Ethanol production up to 43 g l(-1) was achieved at dilution rates below 0.10 h(-1) in sugar beet molasses. Ice nucleation activity gradually increased with increasing dilution rate and the greatest activity, -3.4 log (ice nuclei per cell), was observed at the highest dilution rate (0.13 h(-1)). Both mutant strains displayed a different fatty acid and phospholipid profile compared with the wild-type strain. CONCLUSIONS: The ability of the mutant and recombinant plasmid-containing strains to grow on high sugar concentrations and in high osmotic pressure environments (molasses) can be attributed to their phospholipid and fatty acid contents. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking into account that sugar beet molasses is a low cost agricultural by-product, the simultaneous ethanol and bacterial ice nucleation production achieved under the studied conditions is considered very promising for industrial applications.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Etanol/metabolismo , Melaza , Pseudomonas/genética , Zymomonas/metabolismo , Biomasa , Chenopodiaceae , Conjugación Genética , Medios de Cultivo , Ácidos Grasos/análisis , Hielo , Mutación , Presión Osmótica , Fosfolípidos/análisis , Sacarosa/metabolismo , Zymomonas/química , Zymomonas/genética , Zymomonas/crecimiento & desarrolloRESUMEN
Release of ice nuclei in the growth medium of recombinant Halomonas elongata cells expressing the inaZ gene of Pseudomonas syringae was studied in an attempt to produce cell-free active ice nuclei for biotechnological applications. Cell-free ice nuclei were not retained by cellulose acetate filters of 0.2 microm pore size. Highest activity of cell-free ice nuclei was obtained when cells were grown in low salinity (0.5-5% NaCl, w/v). Freezing temperature threshold, estimated to be below -7 degrees C indicating class C nuclei, was not affected by medium salinity. Their density, as estimated by Percoll density centrifugation, was 1.018 +/- 0.002 gml(-1) and they were found to be free of lipids. Ice nuclei are released in the growth medium of recombinant H. elongata cells probably because of inefficient anchoring of the ice-nucleation protein aggregates in the outer membrane. The ice+ recombinant H. elongata cells could be useful for future use as a source of active cell-free ice nucleation protein.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/genética , Halomonas/genética , Pseudomonas/genética , Proteínas Bacterianas/análisis , Proteínas Bacterianas/biosíntesis , Clonación Molecular , Medios de Cultivo , Congelación , Halomonas/crecimiento & desarrollo , Immunoblotting , Proteínas Recombinantes/biosíntesis , Cloruro de SodioRESUMEN
Metal complexes of thiamine pyrophosphate (TPP) of the general formula [M2(TPPH)2Cl2]x4H2O (M = Zn2+, Cd2+) were isolated from methanolic solutions and characterized by elemental analysis, FT-IR, and multinuclear NMR spectroscopies. The data provide evidence for the bonding of the metals to the N(1') atom of the pyrimidine ring and to the pyrophosphate group. The stability constant measurements of TPP and 2-(alpha-hydroxyethyl)thiamine pyrophosphate (HETPP) metal complexes in aqueous solution imply the formation of dimeric complex species similar to the isolated solid products. They indicate also that HETPP forms more stable metal complexes than does TPP. To evaluate the coenzyme action of TPP and HETPP metal complexes, enzymic studies have been done using pyruvate decarboxylase apoenzyme. TPP metal complexes do not bind to the apoenzyme, unlike the Zn(II)-HETPP complex which can act as coenzyme. Considering these results, possible functional implications for thiamine involvement in catalysis are discussed.
Asunto(s)
Cadmio/química , Piruvato Carboxilasa/metabolismo , Tiamina Pirofosfato/análogos & derivados , Tiamina Pirofosfato/química , Zinc/química , Apoenzimas/química , Apoenzimas/metabolismo , Catalasa/química , Catalasa/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Activadores de Enzimas/farmacología , Concentración de Iones de Hidrógeno , Ligandos , Espectroscopía de Resonancia Magnética , Espectrofotometría Infrarroja , Espectrometría RamanRESUMEN
Exponentially growing cells of Zymomonas mobilis normally exhibit a lag period of up to 3 h when transferred from 0.11 M (2%) to 0.55 M (10%) glucose liquid medium. A mutant of Z. mobilis (CU1Rif2), fortuitously isolated, showed more than a 20-h lag period when grown under the same conditions, whereas on 0.55 M glucose solid medium, it failed to grow. The growth of CU1Rif2 on elevated concentrations of other fermentable (0.55 M sucrose or fructose) or nonfermentable (0.11 M glucose plus 0.44 M maltose or xylose) sugars appeared to be normal. Surprisingly, CU1Rif2 cells grew without any delay on 0.55 M glucose on which wild-type cells had been incubated for 3 h and removed at the beginning of their exponential phase. This apparent preconditioning was not observed with medium obtained from wild-type cells grown on 0.11 M glucose and supplemented to 0.55 M after removal of the wild-type cells. Undelayed growth of CU1Rif2 on 0.55 M glucose previously conditioned by the wild type was impaired by heating or protease treatment. It is suggested that in Z. mobilis, a diffusible proteinaceous heat-labile factor, transitionally not present in 0.55 M glucose CU1Rif2 cultures, triggers growth on 0.55 M glucose. Biochemical analysis of glucose uptake and glycolytic enzymes implied that glucose assimilation was not directly involved in the phenomenon. By use of a wild-type Z. mobilis genomic library, a 4.5-kb DNA fragment which complemented in low copy number the glucose-defective phenotype as well as glucokinase and glucose uptake of CU1Rif2 was isolated. This fragment carries a gene cluster consisting of four putative coding regions, encoding 167, 167, 145, and 220 amino acids with typical Z. mobilis codon usage, -35 and -10 promoter elements, and individual Shine-Dalgarno consensus sites. However, strong homologies were not detected in a BLAST2 (EMBL-Heidelberg) computer search with known protein sequences.
Asunto(s)
Glucosa/metabolismo , Zymomonas/crecimiento & desarrollo , Zymomonas/genética , Transporte Biológico , Conjugación Genética , Medios de Cultivo , Farmacorresistencia Microbiana/genética , Cinética , Sistemas de Lectura Abierta , Mapeo Restrictivo , Zymomonas/metabolismoRESUMEN
Ice nucleation protein was partially purified from the membrane fraction of E. coli carrying inaZ from Pseudomonas syringae. The ice nucleation protein was totally localized in the bacterial envelope and was extracted by either salt (0.25 M NH4Cl) or the nonionic detergent Tween 20. The extracted protein was partially purified by sequential passage through DEAE-52 cellulose and Sephacryl-S400 columns. The activity of the purified protein was lost after treatment with phospholipase C, and its activity was subsequently restored by addition of the naturally occurring lipid phosphatidylethanolamine. These results suggest that ice nucleation proteins have a requirement for lipids that reconstitute a physiological hydrophobic environment similar to the one existing in vivo, to attain and maintain a structure that enables ice catalysis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/metabolismo , Pseudomonas/genética , Proteínas Bacterianas/química , Escherichia coli/genética , Genes Bacterianos , Fosfolípidos/análisisRESUMEN
Ergosterol, lanosterol and two further unidentified sterols were detected and quantified in Schizosaccharomyces pombe cell extracts. In cells grown under anaerobic conditions, the levels of these sterols were dramatically reduced with a concomitant increase of their squalene precursor as compared with cells growing under aerobic conditions. Presence of ethanol resulted in a decrease in the sterol content under aerobic conditions. On the contrary, under anaerobic conditions presence of ethanol resulted in a three-fold increase of total sterols. Lanosterol was the main constituent of this elevation. It is suggested that lanosterol in parallel with unsaturated fatty acids is responsible for maintaining membrane integrity of S. pombe cells growing in the presence of ethanol.