Implementación de técnicas moleculares para la detección y cuantificación del agente de biocontrol Pantoea agglomerans CPA-2

Pantoea agglomerans CPA-2 es un agente de biocontrol (ACB) eficaz en el control de enfermedades de postcosecha en fruta de pepita y cítricos. No obstante, para implementar y registrar su uso como estrategia práctica de control en Europa es importante evaluar la capacidad del ACB para colonizar, persistir y propagarse en condiciones habituales de aplicación con un método de detección que permita diferenciar al antagonista del resto de la microbiota. La presente tesis doctoral tuvo como objetivo fundamental el desarrollo de técnicas moleculares para la detección y cuantificación específica de CPA-2 en su proceso de formulación y en su entorno de aplicación (pre- y postcosecha). Para cumplir este objetivo, en primer lugar se desarrolló y validó la técnica de qPCR, pero se observó la limitación de esta técnica al no discriminar entre el ADN de las células viables y las no viables de la superficie de manzanas conservadas a medio y largo plazo. Esta limitación se superó con un pre-tratamiento de las muestras con propidio de monoazida (PMA) en combinación con la qPCR (PMA-qPCR). Posteriormente, la técnica desarrollada del PMA-qPCR se aplicó para determinar la supervivencia del ACB después de su formulación por liofilización, lecho fluido y atomización pero también para evaluar su dinámica poblacional sobre la superficie de naranjas tratadas en pre- y postcosecha, siendo el primer estudio donde se aplica a un ACB. Finalmente, se estudió la persistencia de CPA-2 en tratamientos de pre- y postcosecha, confirmando su presencia mediante PCR convencional. Los resultados de esta tesis demuestran la versatilidad de las técnicas moleculares basadas en la PCR (qPCR, PMA-qPCR y PCR convencional) para detectar y cuantificar la población de CPA-2 durante su formulación y en las condiciones ambientales de aplicación. Así como la limitada dispersión y baja persistencia de CPA-2 en su entorno de aplicación.Pantoea agglomerans CPA-2 és un agent de biocontrol (ACB) eficaç en el control de malalties de postcollita en fruita de llavor i cítrics. No obstant, per implementar  i registrar el seu ús com estratègia pràctica de control a Europa és important avaluar la capacitat de l’ACB per colonitzar, persistir i propagar-se en condicions habituals d’aplicació amb un mètode de detecció que permeti diferenciar a l’antagonista de la resta de la microbiota. La present tesis doctoral va tenir com objectiu fonamental el desenvolupament de tècniques moleculars per a la detecció i quantificació específica de CPA-2 en el seu procés de formulació i en el seu entorn d’aplicació (pre- i postcollita). Per complir aquest objectiu, en primer lloc es va desenvolupar i validar la tècnica de qPCR, però es va observar la limitació d’aquesta tècnica al no discriminar entre el ADN de les cèl·lules viables i les no viables de la superfície de pomes conservades a mig i llarg termini. Aquesta limitació es va superar amb un pretractament de les mostres amb propidi de monoàcida (PMA) en combinació amb la qPCR (PMA-qPCR). Posteriorment, la tècnica desenvolupada del PMA-qPCR es va aplicar per determinar la supervivència de l’ACB després de la seva formulació per liofilització, llit fluït i atomització però també per avaluar la seva dinàmica poblacional sobre la superfície de taronges tractades en pre- i postcollita, sent el primer estudi on s’aplica a un ACB. Finalment, es va estudiar la persistència de CPA-2 en tractaments de pre i postcollita, confirmant la seva presència mitjançant PCR convencional. Els resultats d’aquesta tesis demostren la versatilitat de las tècniques moleculars basades en la PCR (qPCR, PMA-qPCR i PCR convencional) per detectar i quantificar la població de CPA-2 durant la seva formulació i en les condicions ambientals d’aplicació. Així com la limitada dispersió i baixa persistència de CPA-2 en el seu entorn d’aplicació.Pantoea agglomerans CPA‑2 is a biocontrol agent (ACB) effective in the control of postharvest diseases in pome fruit and citrus. However, in order to implement and register the use of this ACB as a practical control strategy in Europe, it is first necessary to evaluate the ability of the antagonist to colonize, persist and spread in its normal operating conditions with a screening method to identify and quantify the ACB of the rest of the microbiota. The main goal of the present thesis was the development of molecular techniques for the identification and quantification of CPA‑2 strain in its formulation and application in the environment (pre- and postharvest). To achieve this objective, the development and validation of qPCR technique was due at first, however this technique was not able to discriminate between the DNA of viable and non‑viable cells of apple surface stored at middle and long time. This limitation was overcome by treating the samples with the DNA intercalator, propidium of monoazide (PMA), combined with qPCR (PMA‑qPCR). Furthermore, PMA‑qPCR technique was applied to determine the survival of CPA‑2 after its formulation by freeze drying, spray drying and fluidized bed drying; and to assess the population dynamics of the antagonist on the surface of pre‑treated and postharvest oranges, it was the first study where this technique was applied in ACB. Finally, CPA‑2 persistence in pre‑ and postharvest treatment was studied and its presence was confirmed by conventional PCR. The results presented in this thesis demonstrate the versatility of molecular techniques based on PCR (qPCR, PMA‑qPCR and conventional PCR) to detect and quantify P. agglomerans CPA‑2 in normal operating conditions

Recent advances in membrane-based biogas and biohydrogen upgrading

Producción CientíficaBiogas and biohydrogen, due to their renewable nature and zero carbon footprint, are considered two of the gaseous biofuels that will replace conventional fossil fuels. Biogas from anaerobic digestion must be purified and converted into high-quality biomethane prior to use as a vehicle fuel or injection into natural gas networks. Likewise, the enrichment of biohydrogen from dark fermentation requires the removal of CO2, which is the main pollutant of this new gaseous biofuel. Currently, the removal of CO2 from both biogas and biohydrogen is carried out by means of physical/chemical technologies, which exhibit high operating costs and corrosion problems. Biological technologies for CO2 removal from biogas, such as photosynthetic enrichment and hydrogenotrophic enrichment, are still in an experimental development phase. In this context, membrane separation has emerged as the only physical/chemical technology with the potential to improve the performance of CO2 separation from both biogas and biohydrogen, and to reduce investment and operating costs, as a result of the recent advances in the field of nanotechnology and materials science. This review will focus on the fundamentals, potential and limitations of CO2 and H2 membrane separation technologies. The latest advances on membrane materials for biogas and biohydrogen purification will be systematically reviewed.Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/10.13039/501100011033 - (projects PID2019- 109403RB-C21)Junta de Castilla y León y Fondo Europeo de Desarrollo Regional (FEDER) - (grant CLU2017-09, UIC082, CL-EI-2021-07 and UIC 315

Postharvest Application of Potato Starch Edible Coatings with Sodium Benzoate to Reduce Sour Rot and Preserve Mandarin Fruit Quality

Starch is a biodegradable polymeric carbohydrate that can easily form films and coatings and can readily be obtained from some food industry by-products and wastes, which may contribute to the circular bioeconomy. In this work, we studied the potential of two edible coating emulsions based on pregelatinized potato starch (PPS) and glyceryl monostearate (GMS) alone (F6 and F10) or formulated with the food additive sodium benzoate (SB, 2%) (F6/SB and F10/SB) to control sour rot, an important citrus postharvest disease caused by the fungus Geotrichum citri-aurantii, and maintain postharvest quality of cold-stored ‘Orri’ mandarins. The PPS-GMS coating application was compared to dipping in water (uncoated controls) and dipping in a 2% SB (w/v) aqueous solution. The results showed that the coating F10/SB was the most promising treatment to control sour rot on mandarins, with reductions in disease incidence with respect to the uncoated control samples of 94, 69, and 55% after 2, 4, and 6 weeks of storage at 5°C, respectively. Coatings formulated without SB were ineffective. Regarding fruit quality, the coating F10 was the most effective to reduce weight loss, maintain firmness, and provide gloss on mandarins stored at 5°C for up to 6 weeks followed by a shelf-life period of 1 week at 20°C. The addition of SB to the PPS-GMS coatings adversely affected these coating properties, but the coating F10/SB still reduced weight loss compared to uncoated controls without negatively affecting the fruit physicochemical (juice titratable acidity, soluble solids content, and volatiles content) and sensory quality (overall flavor, off-flavors, external aspect). Overall, the coating F10/SB showed the greatest potential for commercial use as an efficient and environmentally friendly alternative to conventional fungicides and waxes for sour rot control and quality preservation of cold-stored mandarins

Pursuing the Perfect Performer of Fermented Beverages: GMMs vs. Microbial Consortium

Fermented beverages are widely diverse around the world and their quality is largely based on the organoleptic characteristics developed by the metabolism of the microorganisms present during fermentation. In order to achieve controllable processes in fermented beverages along with organoleptic complexity, two divergent approaches have been followed in terms of inoculum development: (1) the inoculation of multiple microorganisms, intending to promote synergism and favor organoleptic complexity derived from the metabolic diversity, and (2) the genetic modification of a single strain with the intention that it performs multiple functions. In this chapter, we discuss these divergent approaches, their achievements and perspectives

Growth Kinetics for the Selection of Yeast Strains for Fermented Beverages

Criteria to select autochthonous yeast strains for their use in fermented beverages include their ability to dominate the media and to enhance desired sensorial characteristics and their inability to produce undesired compounds such as biogenic amines or off-odors. One of the key features in yeast selection is its Implantation, surpassing different stresses, and its fermentation performance, which requires setting up the process and monitoring it, involving important amount of resources. Methods to evaluate the tolerance of yeast strains are usually based in the qualitative measure of the growth of the microorganism in a medium containing the limiting compound after a specific time of incubation. However, studying strain growth through optical density measurements permits to estimate quantitative and comparable parameters providing an insight into the fitness of the cell to certain environment, lag phase duration, growth rate, and maximum population, among others. In the last decades, culture-independent methods have been used to evaluate the dynamic of microbial populations during fermentative process. In this chapter, a review of recent advances in the selection of fermentative yeasts as well as the utilization of kinetic evaluation and molecular strategies in conditions associated with fermented beverage for selecting yeast strains is presented

Starch-based antifungal edible coatings to control sour rot caused by Geotrichum citri-aurantii and maintain postharvest quality of ‘Fino’ lemon

BACKGROUND: Two edible coating (EC) emulsions based on potato starch (F6 and F10) alone or formulated with sodium benzoate (SB, 2% w/w) (F6/SB and F10/SB) were evaluated to maintain postharvest quality of cold-stored ‘Fino’ lemons and control sour rot on lemons artificially inoculated with Geotrichum citri-aurantii. Previous research showed the potential of these ECs to improve the storability of ‘Orri’ mandarins and reduce citrus green and blue molds caused by Penicillum digitatum and Penicillium italicum, respectively. RESULTS: The coatings F6/SB and F10/SB significantly reduced sour rot incidence and severity compared to uncoated control samples on lemons incubated at 28°C for 4 and 7 days. The F6/SB coating reduced weight loss and gas exchange compared to uncoated fruit after 2 and 4 weeks of storage at 12°C plus a shelf life of 1 week at 20 °C, without adversely affecting the lemon physicochemical quality. CONCLUSION: Overall, the F6/SB coating formulation, composed of pregelatinized potato starch, glyceryl monostearate, glycerol, emulsifiers and SB, with a total solid content of 5.5%, showed the best results in reducing citrus sour rot and maintaining the postharvest quality of cold-stored ‘Fino’ lemons. Therefore, it showed potential as a new cost-effective postharvest treatment suitable to be included in integrated disease management programs for citrus international markets with zero tolerance to chemical residues