Fungal biodeterioration of colour cinematographic films of the cultural heritage of Cuba

Until recently, cinematographic film was largely cellulose-triacetate-based. However, this material is highly susceptible to biodeterioration, thus placing historic film collections, an important part of the cultural heritage of many countries, at risk. In the present study, samples taken from several biodeteriorated color cinematographic films belonging to the collection of the Cuban Institute for Cinematographic Industry and Arts (ICAIC) were investigated. Infrared spectroscopy showed that all films were of the same composition, i.e., a gelatin emulsion coating one side of a cellulose-triacetate-based film support. The films were analyzed by environmental scanning electron microscopy and scanning electron microscopy to determine the degree of biodeterioration and the type of colonizing microorganisms. Significant fungal colonization was found on both sides of the films in all samples, with a higher concentration of fungi on the gelatin emulsion side. Epifluorescence microscopy of fluorochrome-dyed films demonstrated that some of the fungi were still active, indicating that the films under study, and probably others at the ICAIC, are at risk of further deterioration. Fungi were identified by molecular biology techniques. The fungi mainly responsible for the observed biodeterioration were those belonging to the genera Aspergillus and Cladosporium, although other genera, such as Microascus and Penicillium, were identified as well. In accordance with the findings described herein, the existing guidelines for the prevention and control of film biodeterioration are discussed.

Diseño y evaluación de un procedimiento on-line para la biorremediación de aguas radiactivas en centrales nucleares

Recientemente se ha demostrado la existencia de microorganismos en las piscinas de almacenamiento de combustible nuclear gastado en las centrales nucleares utilizando técnicas convencionales de cultivo en el laboratorio. Estudios posteriores han puesto de manifiesto que los microorganismos presentes eran capaces de colonizar las paredes de acero inoxidable de las piscinas formando biopelículas. Adicionalmente se ha observado la capacidad de estas biopelículas de retener radionúclidos, lo que hace pensar en la posibilidad de utilizarlas en la descontaminación de las aguas radiactivas de las piscinas. En la presente tesis se plantea conocer más profundamente la biodiversidad microbiana de las biopelículas utilizando técnicas de biología molecular como la clonación, además de desarrollar un sistema de descontaminación a escala piloto con el objetivo de valorar si el proceso podría resultar escalable a nivel industrial. Para ello se diseñaron y fabricaron dos biorreactores en acero inoxidable compatibles con las condiciones específicas de seguridad sísmica y protección frente a la radiación en la zona controlada de una central nuclear. Los biorreactores se instalaron en la Central Nuclear de Cofrentes (Valencia) en las proximidades de las piscinas de almacenamiento de combustible nuclear gastado y precediendo a las resinas de intercambio iónico, de forma que reciben el agua de las piscinas permitiendo el análisis in situ de la radiación eliminada del agua de las mismas. Se conectó una lámpara de luz ultravioleta a uno de los biorreactores para poder comparar el desarrollo de bipelículas y la retención de radiactividad en ambas condiciones. En estos biorreactores se introdujeron ovillos de acero inoxidable y de titanio que se extrajeron a diversos tiempos, hasta 635 días para los ovillos de acero inoxidable y hasta 309 días para los ovillos de titanio. Se analizaron las biopelículas desarrolladas sobre los ovillos por microscopía electrónica de barrido y por microscopía de epifluorescencia. Se extrajo el ADN de las biopelículas y, tras su clonación, se identificaron los microorganismos por técnicas independientes de cultivo. Asimismo se determinó por espectrometría gamma la capacidad de las biopelículas para retener radionúclidos. Los microorganismos radiorresistentes identificados pertenecen a los grupos filogenéticos Alpha-proteobacteria, Gamma-proteobacteria, Actinobacteria, Deinococcus-Thermus y Bacteroidetes. Las secuencias de estos microorganismos se han depositado en el GenBank con los números de acceso KR817260-KR817405. Se ha observado una distribución porcentual ligeramente diferente en relación con el tipo de biorreactor. Las biopelículas han retenido fundamentalmente radionúclidos de activación. La suma de Co-60 y Mn-54 ha llegado en ocasiones al 97%. Otros radionúclidos retenidos han sido Cr-51, Co-58, Fe-59, Zn-65 y Zr-95. Se sugiere un mecanismo del proceso de retención de radionúclidos relacionado con el tiempo de formación y desaparición de las biopelículas. Se ha valorado que el proceso escalable puede ser económicamente rentable. ABSTRACT The existence of microorganisms in spent nuclear fuel pools has been demonstrated recently in nuclear power plants by using conventional microbial techniques. Subsequent studies have revealed that those microorganisms were able to colonize the stainless steel pool walls forming biofilms. Additionally, it has been observed the ability of these biofilms to retain radionuclides, which suggests the possibility of using them for radioactive water decontamination purposes. This thesis presents deeper knowledge of microbial biofilms biodiversity by using molecular biology techniques such as cloning, and develops a decontamination system on a pilot scale, in order to assess whether the process could be scalable to an industrial level. Aiming to demonstrate this was feasible, two stainless steel bioreactors were designed and manufactured, both were compatible with seismic and radiation protection standards in the controlled zone of a nuclear plant. These bioreactors were installed in the Cofrentes Nuclear Power Plant (Valencia) next to the spent nuclear fuel pools and preceding (upstream) ion exchange resins. This configuration allowed the bioreactors to receive water directly from the pools allowing in situ analysis of radiation removal. One ultraviolet lamp was connected to one of the bioreactors to compare biofilms development and radioactivity retention in both conditions. Stainless steel and titanium balls were introduced into these bioreactors and were removed after different time periods, up to 635 days for stainless steel balls and up to 309 days for titanium. Biofilms developed on the balls were analyzed by scanning electron microscopy and epifluorescence microscopy. DNA was extracted from the biofilms, was cloned and then the microorganisms were identified by independent culture techniques. Biofilms ability to retain radionuclides was also determined by gamma spectrometry. The identified radioresistant organisms belong to the phylogenetic groups Alphaproteobacteria, Gamma-proteobacteria, Actinobacteria, Deinococcus-Thermus and Bacteroidetes. The sequences of these microorganisms have been deposited in GenBank (access numbers KR817260-KR817405). A different distribution of microorganisms was observed in relation to the type of bioreactor. Biofilms have essentially retained activation radionuclides. Sometimes the sum of Co-60 and Mn-54 reached 97%. Cr-51, Co-58, Fe-59, Zn-65 and Zr-95 have also been retained. A radionuclide retention process mechanism related to biofilms formation and disappearance time is suggested. It has been assessed that the scalable process can be economically profitable.