The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of β-galactosidase activity constitutively.Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of β-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to β-galactosidase activity .The method was found to be sensitive enough to determine 1.7 x 10(6) CFU mL-1 in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.

Production and partial characterization of serine and metallo peptidases secreted by Aspergillus fumigatus Fresenius in submerged and solid state fermentation

Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 ºC with an inoculum of 1 x 10(6) spores and yielded 1500 active units (UµmL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 ºC and yielded 40 UµmL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 ºC, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase.

Efecto biofertilizante del preparado: residuos vegetales - bacteria nativa diazótrofa, sobre las variables biométricas en plántulas de Rhapanus sativus / Biofertilizer effect of the prepared from vegetales wastes - diazotroph native bacterium on biometrics variables of Rhapanus sativus seedlings

El uso de bioinoculantes a base de microorganismos con potencial biofertilizante representa una alternativa económicamente viable y de producción limpia para el sector agrícola. El objetivo del presente trabajo fue evaluar el efecto biofertilizante de un preparado elaborado con residuos sólidos vegetales (RSV) procedentes del mercado y la bacteria nativa diazótrofa Azotobacter A15M2G. Se elaboraron biopreparados utilizando diferentes concentraciones de bacteria (106, 107 y 108 UFC) en un medio de cultivo obtenido a partir del 25% p/v de cada uno de los siguientes RSV: Brassica oleracea (repollo), Lactuca sativa (lechuga) y Allium fistulosum (cebollín). Los biopreparados fueron evaluados en plantas de rábano (Rhapanus sativus) en invernadero, utilizando un diseño estadístico completamente al azar de 5 tratamientos con 3 repeticiones: T1, control; T2, semillas pregerminadas tratadas con RSV al 25% p/v; T3, semillas pregerminadas con bioinoculante de 106 UFC; T4, semillas pregerminadas con bioinoculante de 107 UFC; T5, semillas pregerminadas con bioinoculante de 108 UFC. Se evaluó: número de hojas, área foliar, longitud de la planta, longitud de la raíz y peso seco de toda la planta (ensayos por triplicado). Se observó un incremento altamente significativo en peso seco para T5 (0,88 g) y T4 (1,10 g); y diferencias significativas en el área foliar, para los mismos tratamientos, con un valor superior a 2000 cm2. El biopreparado con bacterias nativas y RSV mejoró el crecimiento y desarrollo de las plantas de rábano, pudiéndose dar un valor agregado a estos residuos y de esta manera obtener un biofertilizante potencialmente utilizable en otros cultivos.

The use of bioinoculantes from microorganisms with biofertilizer potential, represents an economically viable alternative and of clean production for the agricultural sector. The aim of this study was to evaluate the effect of biofertilizer preparation obtained from vegetable solid waste (RSV) of the market and the native bacteria Azotobacter A15M2G diazotroph.Biological cultures were prepared using different inoculum concentrations, 106, 107 y 108 UFC in a culture medium obtained from 25% w / v of each of the following substrates: Brassica oleracea (cabbage), Lactuca sativa (lettuce) and Allium fistulosum (chives). The microbial inoculants were evaluated in radish plants (Rhapanus sativus) in greenhouse using a completely randomized design of 5 treatments with 3 replicates: T1, pre-germinated seeds without any treatment; T2, pre-germinated seeds treated with the dye waste vegetables 25% w / v; T3, pre-germinated seeds treated with bacterial concentration bioinoculants to 106 UFC; T4, pre-germinated seeds treated with bacterial concentration bioinoculants to 107 UFC, and T5, pre-germinated seeds treated with bacterial concentration bioinoculants to 108 UFC. Assessed variables were: number of leaves, leaf area, plant length, root length and dry weight of the entire plant (all assays in triplicate). The results showed a highly significant increase in dry weight, for T5 (0.88 g) and T4(1.10 g); and significant differences in leaf area for the same treatments, with a value greater than 2000 cm2, compared to others. The biopreparado from native bacteria and RSV improved the growth and development of the radish plants, being able to give a added value to these residues and to obtain a potentially usable biofertilizer in other cultures.

Effect of carbon source and precursors on the production of plant growth regulators by azotobacter chroococcum [R19] and bacillus megaterium var. phosphaticum [R44]

An experiment was carried out to examine the effect of carbon source, tryptophan and adenine concentrations on the production of growth regulators by A. chroococcum [R19] and B. megaterium var. phosphaticum [R44]. Mannitol and glucose were the best carbon sources for the production of plant growth regulators [PGRs] by A. chroococcum and B. megaterium var. phosphaticum, respectively. A. chroococcum produced higher amounts of zeatin and kinetin compared to those produced by B. megaterium var. phosphaticum while B. megaterium var. phosphaticum produced higher amounts of [9R] benzyl adenine and [9G] bcnzyl adenine compared with those produced by A. chroococcum. Production of auxins, gibberellic acid [GA[3]] and cytokinins was increased with increasing tryptophan concentration. The highest amounts of PGRs produced by the two strains were obtained with tryptophan at 1000 micro M. Highest amounts of PGRs produced by A. chroococcum and B. megaterium var. phosphaticum at 10 and 100 micro M of adenine, respectively. Also, the produced amounts of gibberellic acid and cytokinin than that produced by B. megaterium var. phosphaticum. Generally, obtained data showed that the application of the optimal conditions together gave highest amounts of PGRs as compared with the other individual factors. This result is logic and was anticipated

Isolation and characterization of azotobacter strains from brazilian soils

Thirty-eight Azotobacter spp strains isolated from Brasilian soils were characterized for the foollowing properties : utilization of different carbon sources, diffusible pigment production, melanin production, sodium-dependent growth, siderophore production, streptomycin resistance and antibiotic-like substances production. Four strains were identified : one as A. armeniacus and three as A. paspali.

Ocorrência de plasmídios em linhagens bacterianas fixadoras assimbióticas de nitrogênio / Detection of plasmids in non-symbiotic nitrogen fixers bacterial strains

Foi feito um estudo dos genes envolvidos com o processo de fixaçäo assimbiotica de N2, em populaçöes bacterianas selvagens do solo, com o intuito de se verificar a ocorrência de genes nif plasmidiais, que pudessem ser transferidos para outros sistemas näo fixadores. Foi observada a presença de plasmídios nos isolados naturais, 2-2II, 5-3 e 40-1 com pesos moleculares de 64, 40 e 92 Md, respectivamente, revelados por eletroforese em gel de agarose. Em nenhuma destas linhagens, se detectou a presença de genes plasmidiais envolvidos com o processo de fixaçäo de N2, uma vez que após a eliminaçäo dos plasmídios com agentes de "cura", estas continuaram seu desenvolvimento normal em meio isento de nitrogênio incorporado. A maior porcentagem de eliminaçäo de plasmídios foi conseguida atraves do tratamento com temperatura de 44§cC(2 a 10%), seguida do tratamento com brometo de etídio (1 a 5%), sendo menores as obtidas com acriflavina (0,5 a 1%) e acridina laranja (0,2 a 0,8%)