Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates / Produção significativa de vanilina e amplificação in vitro do gene ech em isolados bacterianos locais

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.

Semi-scale production of PHAs from waste frying oil by Pseudomonas fluorescens S48

The present study aimed at developing a strategy to improve the volumetric production of PHAs by Pseudomonas fluorescens S48 using waste frying oil (WFO) as the sole carbon source. For this purpose, several cultivations were set up to steadily improve nutrients supply to attain high cell density and high biopolymer productivity. The production of PHAs was examined in a 14 L bioreactor as one-stage batch, two-stage batch, and high-cell-density fed-batch cultures. The highest value of polymer content in one-stage bioreactor was obtained after 60 h (33.7%). Whereas, the two-stage batch culture increased the polymer content to 50.1% after 54 h. High-cell-density (0.64 g/L) at continuous feeding rate 0.55 mL/l/h of WFO recorded the highest polymer content after 54 h (55.34%). Semi-scale application (10 L working volume) increased the polymer content in one-stage batch, two-stage batch and high cell density fed-batch cultures by about 12.3%, 5.8% and 11.3%, respectively, as compared with that obtained in 2 L fermentation culture. Six different methods for biopolymer extraction were done to investigate their efficiency for optimum polymer recovery. The maximum efficiency of solvent recovery of PHA was attained by chloroform-hypochlorite dispersion extraction. Gas chromatography (GC) analysis of biopolymer produced by Pseudomonas fluorescens S48 indicated that it solely composed of 3-hydrobutyric acid (98.7%). A bioplastic film was prepared from the obtained PHB. The isolate studied shares the same identical sequence, which is nearly the complete 16S rRNA gene. The identity of this sequence to the closest pseudomonads strains is about 98-99%. It was probably closely related to support another meaningful parsiomony analysis and construction of a phylogenetic tree. The isolate is so close to Egyptian strain named EG 639838.

Bioremediation of chromium contaminated soil by Pseudomonas fluorescens and indigenous microorganisms.

Chromium is one of the toxic and hazardous pollutants in industrial wastewaters leading to soil contamination. In this study, the feasibility of remediating chromium contaminated soil using indigenous microorganisms and Pseudomonas fluorescens was evaluated. The contaminated soil sample was collected from Vellore and the pH, moisture content and chromium content were found to be 8.4, 22.5% and 5.1 mg/kg respectively. The effect of chromium on engineering properties showed decrease in permeability by 45.15%. For Pseudomonas fluorescens, the optimum pH, moisture content, biomass concentration and carbon source were found as 6.5, 20%, 10 mL and 10 mL/100 g respectively and for isolated mixed culture, the optimum parameters were found as 8.4, 25%, 15 mL and 15mL / 100 g respectively. Under optimum conditions, the reactor study showed 71.7% chromium reduction after 20 days. From the study, the bioremediation of chromium-contaminated soil by indigenous microorganisms was found to be a promising solution and after bioremediation, the engineering properties of the soil were found to be improved.

Antagonistic potential of fluorescent pseudomonads and control of charcoal rot of chickpea caused by Macrophomina phaseolina.

The effectiveness of plant growth promoting rhizobacteria especially Pseudomonas fluorescens isolates were tested against charcoal rot of chickpea both in green house as well as in field conditions. Most of the isolates reduced charcoal rot disease and promoted plant growth in green house. A marked increase in shoot and root length was observed in P. fluorescens treated plants. Among all the P. fluorescens isolates Pf4-99, was found most effective in the improvement of chickpea crop in green house as well as in field. Pf4-99 effectively promoted plant growth and produced indole acetic acid in culture medium. This isolate also inhibited the mycelial growth of the M. phaseolina under in vitro conditions and reduced the disease severity Potential isolate (Pf4-99) also significantly increased the biomass of the chickpea plants, shoot length, root length and protein content of the chickpea seeds. A part from these, the total number of seeds per plant and their weight were also enhanced. The colonization of Pf4-99 reduced the incidence of seed mycoflora by which indirectly enhanced the seed germination and vigour index of seedlings. The observations revealed that isolate Pf4-99 is quite effective to reduce the charcoal rot disease both in field and greenhouse, and also increases seed yields significantly Therefore, this isolate appears to be an efficient biocontrol agent against charcoal rot disease as well as yield increasing rhizobacterium.

Fe(II) speciation and its uptake by free and immobilized cells of Pseudomonas fluorescens from industrial waste water.

Studies are carried out to remove Fe(II) from wastewater using free and immobilized cells of Pseudomonas fluorescens. Experiments are carried out with free cells between 6 and 8 pH and the uptake of Fe(II) is observed to be maximum at pH 7. Further experiments are done at pH 7. Studies with free and immobilized cells revealed that immobilized cells are more efficient for the removal of Fe(II) than free cells. Fe(II) uptake with Pseudomonas fluorescens is also investigated after the addition of NaCl and MgCl2 to the cells. It is found that the uptake has increased when Sodium chloride (NaCl) and Magnesium Chloride (MgCl2) mixed cells are used. Effect of deficiency of nutrients is also studied. It is found that glucose deficient conditions inhibit Fe(II) uptake more than yeast extract deficient ones. pH also plays an important role in the transport of Fe(II) across the membrane of the cells.

Influencia de la concentración salina y la temperatura en la composición de ácidos grasos de membrana de Pseudomonas fluorescens GNP-OHP-3 / Influence of salinity and temperature on fatty acid composition of Pseudomonas fluorescens GNP-OHP-3 membrane

Las bacterias responden a los cambios ambientales modificando su composición, para evitar el daño que dichos cambios podrían ejercer. Una de las modificaciones más importantes es la variación de la composición de los ácidos grasos de las membranas celulares, que le permite mantener la homeoviscosidad ante situaciones de estrés. Trabajos previos han estudiado la acción de la temperatura, presión hidrostática y diferentes solventes sobre cepas de Pseudomonas putida. En este trabajo se estudió la acción conjunta de la temperatura y la salinidad sobre la composición de ácidos grasos de membranas celulares de Pseudomonas fluorescens GNP-OHP-3, una cepa bacteriana aislada de un hábitat contaminado con petróleo. Pseudomonas fluorescens GNP-OHP-3 respondió a las variaciones de temperatura modificando los ácidos grasos de sus membranas de manera similar a lo descripto en otros integrantes de su género: ante el aumento de temperatura se observó un incremento de ácidos grasos saturados y una disminución de los ácidos grasos insaturados. En el rango de concentraciones salinas ensayadas las variaciones de los ácidos grasos mayoritarios fueron en general erráticas. La respuesta de los ácidos grasos ciclo propano pudo expresarse con ecuaciones matemáticas que permitieron predecir el porcentaje de estos ácidos en relación a la concentración de cloruro de sodio.

The bacteria respond to environmental changes modifying their composition. One of the most important modifications is the variation on fatty acid composition of cellular membranes to maintain the homeoviscosity. The action of temperature, hydrostatic pressure and solvents on Pseudomonas putida has been thoroughly studied. In this paper, the combined action of the temperature and salinity on fatty acid composition of cellular membranes of Pseudomonas fluorescens GNP-OHP-3, a bacterial strain isolated from a petroleum contaminated habitat, was studied. The modifications in the fatty acid composition of Pseudomonas fluorescens GNP-OHP-3 membrane were similar to those described for other members of Pseudomonas: an increase in saturated fatty acids and a decrease in unsaturated fatty acids were observed with the increase of the temperature. Variations of main fatty acids were in general erratic in the range of assayed saline concentrations. The variation of cycle propane fatty acids could be expressed with mathematic equations that allowed to predict their percentage in relation to sodium chloride concentration.

Biodegradation of benzidine based azodyes Direct red and Direct blue by the immobilized cells of Pseudomonas fluorescens D41.

Benzidine based azodyes are proven carcinogens, mutagens and have been linked to bladder cancer of human beings and laboratory animals. The textile and dyestuff manufacturing industry are the two major sources that released azodyes in their effluents. The dye, Direct blue contains two carcinogenic compounds namely benzidine (BZ), 4-amino biphenyl (4-ABP), while the dye Direct red has benzidine (BZ). Among 40 isolates of Pseudomonas fluorescens screened, one isolate designated as D41 was found to be capable of extensively degrading the dyes Direct blue and Direct red. Immobilized cells of P. fluorescens D41 efficiently degraded Direct red (82%) and Direct blue (71%) in the presence of glucose.

Tratamiento de semillas de algodón con pseudomonas spp. fluorescentes en el biocontrol de rhizoctonia solani / Cotton seed treatment with fluorescent pseudomonas spp. in rhizoctonia solani biocontrol

Fue evaluado el efecto del tratamiento de semillas de algodón con pseudomonas spp. fluorescentes para el control de rhizoctonia solani. Primeramente se seleccionaron 67 cepas de pseudomonas para inocular semillas de algodón de la variedad precoce-1. Las semillas fueron sumergidas en suspensiones bacterianas (10/8 células/ml) preparadas en solución de MgSo40.1M y posteriormente sembradas en bandejas con suelo sin esterilizar e infectado con el patógeno (cepa RS-4), en densidad de 50 mg de sustrato (arroz) colonizado/Kg de suelo. La evaluación de la intensidad de la enfermedad se realizó a los 10 días después de la siembre, utilizándose una escala de valores de 0 a 4, donde 0= sin síntomas y 4= máximo de síntomas. Las cepas de pseudomonas p-8, CR-27, CB-38, CB-33 y P-5, fueron más eficaces en el control de las cepas de r. solani (RS-4, RS-5 y RS-6), con densidades de inóculos de 50, 100 y 150 mg/kg de suelo. La cepa CB-33 ejerció mayor biocontrol que la cepa RS-4 del fitopatógeno, así como las cepas RS-5 y RS-6 respectivamente. Las cepas bacterianas presentaron mayores niveles de control que el fungicida quintozene en todas las condiciones

Produccíon de pioverdina por Pseudomonas fluorescens en sangre humana a 4ªC: asociacíon a proteínas y efecto citotóxico del pigmento / Production of pyoverdin by Pseudomonas fluorescens in human blood at 4 degrees C: association with proteins and the cytotoxic effect of the pigment

Se purificó pioverdina de pseudomonas fluorescens desrrollada en medio sintético y se practicó un ensayo de toxicidad pra leucocitos totales humanos (LTH); se observó un alto índice de muerte y lisis celular, dependiendo de la concentración y tiempo de incubación. Dosis sublíticas redujeron la capacidad fagocítica de los LTH. Hubo síntesis del pigmento en sangre inoculada con cepas de P. fluorescens a 4-C. la precipitación salina del plasma indicó que las fracciones ricas en globulina y albúmina retuvieron pioverdina en distintos porcentajes (46% y 37% respectivamente). Mediante diálisis se observó que la fracción con albúmina mantenía mayor cantidad del pigmento unido e en estado de agregación