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Item Bioremediation of an industrial soil contaminated by hydrocarbons in microcosm system, involving bioprocesses utilizing co-products and agro-industrial wastes(2023-09-29) Conde Molina, Debora; Liporace, Franco; Quevedo, CarlaThe present study describes practical implication of bioaugmentation and biostimulation processes for bioremediation of an industrial soil chronically contaminated by hydrocarbons. For this purpose, biomass production of six autochthonous hydrocarbon-degrading bacteria were evaluated as inoculum of bioaugmentation strategy, by testing carbon and nitrogen sources included co-products and agro-industrial waste as sustainable and low-cost components of the growth medium. Otherwise, biostimulation was approached by the addition of optimized concentration of nitrogen and phosphorus. Microcosm assays showed that total hydrocarbons (TH) were significantly removed from chronically contaminated soil undergoing bioremediation treatment. Systems Mix (bioaugmentation); N,P (biostimulation) and Mix + N,P (bioaugmentation and biostimulation) reached higher TH removal, being 89.85%, 91.00%, 93.04%, respectively, comparing to 77.83% of system C (natural attenuation) at 90 days. The increased heterotrophic aerobic bacteria and hydrocarbon degrading bacteria counts were according to TH biodegrading process during the experiments. Our results showed that biostimulation with nutrients represent a valuable alternative tool to treat a chronically hydrocarbon-contaminated industrial soil, while bioaugmentation with a consortium of hydrocarbon degrading bacteria would be justified when the soil has a low amount of endogenous degrading microorganisms. Furthermore, the production of inoculum for application in bioaugmentation using low-cost substrates, such as industrial waste, would lead to the development of an environmentally friendly and attractive process in terms of cost–benefit.Item Site-specific bioremediation by bioaugmentation and biostimulation approaches on a chronically hydrocarbon-contaminated industrial soil.(2023-09) Conde Molina, Debora; Liporace, Franco; Quevedo, CarlaThe present study describes practical implication of bioaugmentation and biostimulation processes for bioremediation of an industrial soil chronically contaminated by hydrocarbons. For this purpose, biomass production of six autochthonous hydrocarbon-degrading bacteria were evaluated as inoculum of bioaugmentation strategy, by testing carbon and nitrogen sources included co-products and agro-industrial waste as sustainable and low-cost components of the growth medium. Otherwise, biostimulation was approached by the addition of optimized concentration of nitrogen and phosphorus. Microcosm assays showed that total hydrocarbons (TH) were signifcantly removed from chronically contaminated soil undergoing bioremediation treatment. Systems Mix (bioaugmentation); N,P (biostimulation) and Mix+N,P (bioaugmentation and biostimulation) reached higher TH removal, being 89.85%, 91.00%, 93.04%, respectively, comparing to 77.83% of system C (natural attenuation) at 90 days. The increased heterotrophic aerobic bacteria and hydrocarbon degrading bacteria counts were according to TH biodegrading process during the experiments. Our results showed that biostimulation with nutrients represent a valuable alternative tool to treat a chronically hydrocarbon-contaminated industrial soil, while bioaugmentation with a consortium of hydrocarbon degrading bacteria would be justifed when the soil has a low amount of endogenous degrading microorganisms. Furthermore, the production of inoculum for application in bioaugmentation using low-cost substrates, such as industrial waste, would lead to the development of an environmentally friendly and attractive process in terms of cost–beneft.Item Optimization of biomass production by autochthonous Pseudomonas sp. MT1A3 as a strategy to apply bioremediation in situ in a chronically hydrocarbon-contaminated soil.(2022-04) Conde Molina, Debora; Liporace, Franco; Quevedo, CarlaThese days, petroleum hydrocarbon pollution has become a global problem, because of this, bioremediation is presented as a strategy for cleaning up sites contaminated with organic pollutants, and it has an increasing role in relation to the potential it presents as a non-invasive and cost-effective technology. The aim of this study is to optimize the biomass production of Pseudomonas sp. MT1A3 strain as a soil bioremediation approach for petroleum hydrocarbon polluted environments. Factorial experimental designs were employed to study the effect of several factors of composition medium and incubation conditions on biomass production. Agro-industrial wastes such as peanut oil as carbon source, NaNO3 as nitrogen source and incubation temperature were found to be significant independent variables. These factors were further optimized using Box–Behnken design. Combination of peanut oil 18.69 g/L, NaNO3 2.39 g/L and 26.06 °C incubation temperature was optimum for maximum biomass production of MT1A3 and the model validated in a bioreactor allowed to obtain 9.67g/L. Based on these results, this autochthonous strain was applied in bioaugmentation as a bioremediation strategy through microcosm designs, reaching 93.52% of total hydrocarbon removal at 60 days. This constitutes a promising alternative for hydrocarbon-contaminated soil.Item Estudio de cepas autóctonas de áreas crónicamente contaminadas con hidrocarburos para su aplicación en biorremediación.(Congreso Argentino de Microbiología Agrícola y Ambiental, 2015-11) Conde Molina, Débora; Liporace, Franco; Vázquez, Susana; Quevedo, CarlaLos hidrocarburos representan uno de los grupos contaminantes más importantes tanto por su volumen como por las consecuencias que producen a corto y largo plazo sobre el medio ambiente. En este sentido, el desarrollo de nuevas metodologías compatibles con el medio ambiente para la remediación de ambientes contaminados es un objetivo ecológico prioritario. En este trabajo se estudiaron 5 cepas aisladas de suelos crónicamente contaminados con hidrocarburos en la Refinería R.H.A.S.A., en la zona del polo petroquímico Zárate-Campana, Pcia. de Buenos Aires, con el objetivo de plantear estrategias de biorremediación sitio-específico. Las cepas TK1A2, MT1A, CO1A1, CO1A2, LG1A fueron identificadas de acuerdo con su perfil metabólico (test de API®, Biomerieux) y la secuencia parcial del gen ARNr 16S comparando contra bases de datos especializadas (EzTaxon y RDP). Además, se evaluó la capacidad degradadora de hidrocarburos de los aislamientos en cultivos en frascos Erlenmeyer conteniendo medio mínimo salino y una mezcla de hidrocarburos comerciales como fuente de carbono. También se evaluó el desarrollo en glicerol, co-producto agroindustrial de la región, como materia prima de bajo costo. Para cada uno de los aislamientos se valoraron el crecimiento microbiano y la disminución de la tensión superficial (como indicador de producción de compuestos tensioactivos) a lo largo del tiempo de incubación en presencia de las distintas fuentes de carbono. Los aislamientos fueron identificados como pertenecientes a los géneros Cellulosimicrobium, Ochrobactrum y Pseudomonas (grupos de P. fluorescens, P. stutzeri y P. aeruginosa, según Mulet y col. 2012).Todas las cepas fueron capaces de crecer en medio con hidrocarburos o con glicerol como única fuente de carbono, obteniéndose 2,60 g/L, dependiendo de la cepa. En cuanto a las mediciones de la tensión superficial, no se observaron diferencias con respecto al control cuando los microorganismos fueron crecidos en presencia de hidrocarburos como única fuente de carbono. Asimismo, la cepa LG1A mostró una disminución de la tensión superficial de 23,30 mN/m en relación al controlen los cultivos con glicerol. Estos resultados indican que todas las cepas aisladas son capaces de crecer en presencia de una mezcla de hidrocarburos o glicerol como únicas fuentes de carbono y que la cepa LG1A sería productora de tensioactivos en presencia de glicerol. En base a estos resultados, podemos concluir que las cepas aisladas ofrecen un alto potencial para su aplicación en bioprocesos conducidos a plantear distintas estrategias de remediación.Item Biodegradación de hidrocarburos por la acción de Pseudomonas sp. MT1A3, aislada de suelos crónicamente contaminado.(REDBIO, 2017-09) Conde Molina, Débora; Liporace, Franco; Giullieti, Ana María; Quevedo, CarlaLa importancia que ha adquirido la contaminación ambiental generada por el derrame de hidrocarburos en el manejo de prácticas industriales ha llevado al desarrollo de diversas metodologías de remediaciones. En nuestro país amplias zonas se ven afectadas por este tipo de contaminación. La eliminación de los contaminantes orgánicos se ha encarado mediante tecnologías probadamente efectivas que involucran tratamientos físicos y/o químicos. En la actualidad, a causa de su elevado costo y la perturbación que generan sobre las zonas afectadas, solo se aplican en ciertos casos y como primera acción de emergencia a fin de remover grandes masas de contaminantes. En este sentido un objetivo ecológico es el desarrollo de metodologías compatibles con el medio ambiente para la remediación de ambientes contaminados. Las capacidades de los microorganismos aeróbicos son particularmente relevantes para la biodegradación de dichos compuestos. En el presente trabajo se evaluó el perfil de crecimiento y de degradación de hidrocarburos de Pseudomonas sp. MT1A3, previamente identificada de acuerdo a su secuencia parcial del gen 16 ARNr, aislada de suelos crónicamente contaminados con hidrocarburos en la Refinería R.H.A.S.A., en la zona del polo petroquímico Zárate-Campana, Pcia. de Buenos Aires. El crecimiento de la misma fue llevada a cabo en frascos Erlenmeyer, conteniendo medio salino mínimo y como única fuente de carbono se emplearon distintas muestras de hidrocarburos, nafta (1,5%), kerosene (1,5%), diésel (1,5%) respectivamente y una mezcla de cada uno de ellos en partes iguales (HC) (4,5%). Los cultivos se mantuvieron a 25 ºC, 135 rpm. A los 4 y 10 días se estimó la biomasa de crecimiento mediante la determinación de peso seco (g/L), y se realizaron extracciones de los hidrocarburos presentes en los distintos cultivos, empleando diclorometano (1:1) con el objetivo de evaluar la biodegradación de los mismos en el tiempo. La cuantificación de los hidrocarburos se hizo por medio de cromatografía d equipo GC-2010 Plus (Shimadzu) equipado con un detector FID, una columna Petrocol DH 100 Sepulco de 100 m de longitud, 0.25 mm de diamétro interno y película de empaque de 0,5 µm. La temperatura del horno inicial fue de 45 ºC, y luego tres rampas de temperaturas de 8 ºC / min hasta 150 ºC, 4 ºC / min hasta 250 ºC, y 8 ºC / min hasta 300 ºC, finalmente se mantuvo a 300 ºC durante 15 min. La temperatura del inyector se mantuvo a 380 ºC y la temperatura del detector a 340 ºC. El gas arrastrante fue hidrógeno, trabajando a un flujo constante de 2 ml / min. Los resultados mostraron que la cepa MT1A3 presentó capacidad de degradación de determinados hidrocarburos. Con respecto a los cultivos que solo contenían nafta y diésel, se pudo observar la ausencia de 2 y 4 picos respectivamente pertenecientes a compuestos presentes en dichas fuentes de carbono luego de 4 días de inoculación comparados al control (sin inocular). Un resultado similar se observó cuando se utilizó la mezcla HC como fuente de carbono, en donde se detectó la ausencia degradación de los mismos 6 compuestos registrados anteriormente. Transcurridos los 10 días, la degradación de estos 6 compuestos fue mayor al 90 % en relación al control (cultivo sin inocular) en todas las muestras analizadas. Paralelamente, el crecimiento de MT1A3 fue de 0.69, 0.15, 1.49 y 1.76 g/L en nafta, kerosene, diésel y mezcla respectivamente a los 10 días de cultivo. En base a estos resultados, podemos tener mayor conocimiento del poder de degradación de hidrocarburos de la cepa MT1A3 para plantear futuros ensayos de aplicación en biorremediación sitio específica.Item Evaluation of a native strain isolated from chronically hydrocarbon-contaminated sites.(Universidad Nacional de General San Martín, 2016-07) Conde Molina, Débora; Giullieti, Ana María; Quevedo, CarlaThe industrial center Zárate – Campana represents one of the most important petrochemical areas in Argentina with several companies carrying out petrochemical activities. Due to spills produced in the area during last 50 years, it has generated hydrocarbon pollution on the site. In order to evaluate growth conditions of a native strain from chronically hydrocarbon-contaminated sites, in the present work we studied an environmental strain identified in the genus, which can degrade crude oil. Different cultures conditions were assayed in Erlenmeyer flasks, containing minimal salt medium and supplemented with a mixture of hydrocarbons (HC) of various molecular weights as only carbon source. Ochrobactrum sp strain (currently being a molecularly characterized) was cultivated at two temperatures: 20 and 25 ºC, and several substrate concentrations, 2, 4.5, 6, 7, 8 y 10 % V/V. Cultures were kept at 135 rpm and pH=7 during 14 days. Bacterial growth was estimated by cell dry weight method, for this a sample of 18 mL culture broth was collected at every 24 h, drying at 80ºC. Many bacteria in nature have been found to be capable of degrading crude oil, using it as their sole carbon source. Experiment showed that this strain can use HC as its carbon source. The maximum biomass concentration was obtained when the bacteria was cultured in media with 4.5 % of HC at 20ºC and 25ºC (1,33 g/L and 1,75 g/L) respectively at day 8. In addition, a significant biomass concentration was observed too when were used 6% and 7 % of HC in both temperatures between 7 and 13 days of cultured. According to these results, the environmental-derived strain Ochrobactrum sp isolated from hydrocarbon contaminated sites, has the ability to degrade crude oil at high level concentrations, and this could have potential use in bioremediation technologies.Item Bioremediation strategies based on a native strain isolated from sites contaminated with hydrocarbons.(Argentine Society for Biochemistry and Molecular Biology, 2016-11) Conde Molina, Débora; Liporace, Franco; Vázquez, Susana; Merini, Luciano; Quevedo, CarlaThe bacterial strain studied in this work is a member of a bacterial consortium isolated from chronically hydrocarbon-contaminated site in Campana (Bs.As.). This native strain was identified as Pseudomonas sp. according to its 16S rRNA gene partial sequence. The ability of this strain to produce biosurfactants was evaluated in Erlenmeyer flasks containing a minimal saline medium (MSM) supplemented with different carbon sources: a mixture of hydrocarbons (HC; 4.5%v/v), glycerol (Gly; 2%v/v), sunflower oil (SO; 2%v/v) and peanut oil (PO; 2%v/v). Cultures were performed at 135 rpm and 25 ºC for 4 days. Bacterial growth was measured by cell dry-weight method, and biosurfactant production was estimated by direct measurement of the surface tension (ST). Results showed that bacteria was able to grow on all the carbon sources tested, reaching concentrations of 1.24g/l in HC, 7.69g/l in Gly, 3.98g/l in SO and 9.29g/l in PO. Culture supernatants showed a decrease in ST values when the strain grew on SO and PO (22.5% and 25.5%). No decrease in ST values was observed when HC and Gly were used as carbon source. As results shown, this bacterial strain can produce biosurfactants under certain culture conditions. This potential advantage could be applied in bioremediation strategy of hydrocarbon contaminated sites.Item Optimization of a site-specific biostimulation strategy using response-surface methodology to remediate a chronically hydrocarbon-contaminated industrial soil.(Argentine Society for Biochemistry and Molecular Biology, 2021-11) Conde Molina, Débora; Liporace, Franco; Quevedo, CarlaIn present times, contamination with petroleum compounds is one of the most important environmental problems, mainly in industrial areas. The Zárate-Campana petrochemical pole, Buenos Aires, Argentina, has a history of hydrocarbon contamination of more than 100 years, so it is relevant to recover these contaminated areas through the development of site-specific bioremediation technologies that are compatible with the environment. In this work, biostimulation strategy to remediate chronically hydrocarbon-contaminated soil was approached by the addition of nutrients such as nitrogen (N) and phosphorus (P) in order to evaluate the influence of these on the growth of the autochthonous microflora. Therefore, the response-surface methodology as a statistical tool was used to predict the optimum values of N and P concentration with the aim of obtaining the maximum total hydrocarbon removal in the soil. Microcosm systems were carried out into flasks containing contaminated samples belonging to RHASA refinery areas. Different N (NaNO3) and P (Na2HPO4) concentrations were added to each system according to central composite design, and incubated at 20-25°C for 90 days. Hydrocarbon concentration content was measured by gas chromatography as the response of the model. Results showed that the addition of 0.589 g N/kg and 0.304 g P/kg leads to the highest hydrocarbon removal efficiency, decreasing from 6881 ppm to 728 ppm. In addition biostimulation strategy was compared with natural attenuation and 89.71% of total hydrocarbons were removed when the biostimulation was applied, while in natural attenuation was 72.06%. In both treatments, total aerobic heterotrophic bacteria increased during 90 days, whereas the count of hydrocarbon degrading bacteria remained stable. The biostimulation strategy approached in this work showed to be a promising alternative to remediate the soils of the study site.Item Aplicación de cepas autóctonas en estrategia de bioaumento para remediar suelos crónicamente contaminados con hidrocarburos.(Congreso Argentino de Microbiología Agrícola y Ambiental, 2021-09) Conde Molina, Débora; Liporace, Franco; Quevedo, CarlaUno de los problemas ambientales más importantes es la contaminación con hidrocarburos del petróleo, principalmente en las zonas industriales. El polo petroquímico Zárate -Campana, Buenos Aires, Argentina presenta un historial de contaminación con hidrocarburos de más de 100 años, es por eso que resulta importante la recuperación de estas áreas contaminadas a partir del desarrollo de tecnologías de biorremediación sitio específicas, compatibles con el medio ambiente. En este trabajo se estudiaron 6 cepas de bacterias degradadoras de hidrocarburos TK1A2, MT1A3, LG1A, AG1A, CO1A1 y CO1A2, aisladas de sitios crónicamente contaminados con hidrocarburos e identificadas en los géneros Pseudomonas, Cellulosimicrobium y Ochrobactrum. A fin de evaluar la producción de biomasa y biosurfactantes, las cepas aisladas fueron cultivadas en medio mínimo salino con diferentes fuentes de carbono (mezcla de hidrocarburos, glucosa, glicerol, suero de leche bovino, suero de leche ovino, aceite de girasol refinado, aceite de girasol alto oleico, aceite de maní crudo, aceite de maní frito o aceite de camelina) y de nitrógeno (NaNO3, NH4Cl urea). Si bien todas las cepas fueron capaces de crecer en todas las fuentes de carbono y nitrógeno ensayadas, en presencia de aceite de maní crudo, exhibieron mayor producción de biomasa. Con respecto a las fuentes de nitrógeno estudiadas, las cepas MT1A3, LG1A, AG1A y CO1A2 tuvieron mayor crecimiento en NaNO3, mientras que las cepas TK1A2 y CO1A1 crecieron manera similar en las fuentes de nitrógeno. MT1A3, TK1A2, CO1A1 y CO1A2 no mostraron ser productoras de biosurfactantes en las todas las condiciones ensayadas, mientras que en los medios de cultivos con LG1A y AG1A se registró disminución de la tensión superficial con respecto a los controles en varios de los medios formulados. Resultando ser AG1A en glicerol la mejor condición para producir biosurfactantes. Partiendo de estos resultados, todas las cepas fueron cultivadas en un mismo medio de cultivo con el objetivo de emplear esta mezcla en estrategias de bioaumento en un sistemas de microcosmos para la remoción de hidrocarburos de un suelo crónicamente contaminado. El tratamiento mostró que las bacterias aeróbicas heterótrofas totales aumentaron durante los 90 días que duró el ensayo, mientras que el crecimiento de bacterias degradadoras de hidrocarburos se mantuvo estable. Paralelamente, se observó una degradación de 84,17% de hidrocarburos totales, cuando se aplicó el bioaumento, mientras que en el control (atenuación natural) fue del 72,06%. En base a estos resultados, las cepas estudiadas muestran un gran potencial para ser usadas como bioaumento en sistemas de microcosmos, siendo una alternativa prometedora combinada con una estrategia de bioestimulación para remediar los suelos del sitio de estudio.Item Strategies to develop healthier industrial white slice diary bread through fat, sugar and flour substitution(2021-04) Conde Molina, Débora; Quevedo, Carla; Arqueros, ValeriaIntroduction. Industrial white slice dairy bread (WSDB) is an interesting target for developing a reduced calorie bread as it contains fat and sugar in its formulation. Three commercial substitutes were evaluated in this study in order to improve nutritional profile of bread. Materials and methods. Toler Fat Less Saladas (TFLS) as fat substitute, Granofiber Sweet (GS) as sugar substitute and Granofiber Sym 200 (GS200) as flour substitute, were assessed by analyzing the dough fermentative properties using Rheofermentometer, and the dough behaviour properties on mixing–heating–cooling using Mixolab. Additionally, loaf specific volume and texture profile were measured as baking quality parameters. Results and discussion. In case of fat substitution, TFLS caused a similar effect to fat, showing high starch gelatinization. In agreement, fat and TFLS provided WSDB with similar changes in crumb texture, which led to improve the quality of bread preservation at short shelf life of end-product (5 and 10 days). Otherwise, sugar substitution influenced mainly gas production during fermentation stage. However, dough development was similar when sugar or GS was added in WSDB, thereby increasing loaf specific volume. These results may indicate that GS applied in a complex formulation bread as WSDB, provides a suitable effect like sugar. Concerning flour replacement, several rheology changes and a significant decrease of gas production occurred on GS200 added-dough. However, GS200 showed an important contribution in gas retention capacity, influencing in good dough development. Therefore, bread loaf specific volume of WSDB+GS200(3%) and WSDB+GS200(6%) showed satisfactory results, indicating the potential capacity to use GS200 on WSDB recipe to formulate high fiber bread. Conclusions. We conclude that substitutes evaluated in this work can be used for breadmaking to improve nutritious quality of bread for health benefits.