MICROORGANISMS VS. SYNTHETIC POLYMERS. ECOLOGY AND BIODEGRADATION

Despite synthetic polymers have been considered little or no deteriorable for many years, now we know they can undergo to chemical, physical and biological damage. Effects of biological degradation on polymers include masking of surface properties due to the presence of microorganisms inhabiting surfaces, embrittlement and loss of stability due to changes in chemical structure of polymers, presence of cracks and swellings due to penetration of microorganisms into the polymer matrix and changes in polymer colour due to excretion of microbial pigments. Microbial deterioration depends on the constitution and the properties of polymer materials as well as environmental conditions. Millions of tons of synthetic polymers used as adhesives, binders, coatings, inks, etc., are produced worldwide every year. In 1997 the worldwide paint and coatings industry represented a mature 50+ billion dollar market and synthetic polymers used in paint and coating industries account for approximately 45-55% of the worldwide decorative market. Deterioration of varnishes and binding media results in chemical changes that lead to an increase in the insolubility and polarity of the material, a reduction of the strength, and a change in colour, among others. As biodeterioration of synthetic polymers seriously compromises the adhesion and durability of the paint as well as its decorative/protective function, identification of the cause of synthetic polymer biodeterioration is of great importance. Although biodeterioration of synthetic polymers in objects during their lifetime should be avoided to preserve the object function, synthetic polymer materials should be susceptible to degradation once they are disposed of and treated as waste. The major ingredients of paints are the pigment, a material which provides colour, and the binding medium, a film-forming material in which the pigment particles are dispersed and forms the matrix that hardens and binds the pigments on the painted surface. All the synthetic polymers used as paint and coatings binders are belonging to different chemical classes, the most important being acrylics, polyvinylacetate and nitrocellulose-alkyds. Microorganisms can assimilate and/or degrade these synthetic polymers because their chemical bonds are the same as those found in the natural polymeric matter, which is generally easily degraded. Bioremediation is widely used for the clean-up of environmental pollutants using microorganisms and could be successfully applied for the removal of synthetic polymers, including those present in paint and coating formulations. The aims of this PhD project were to study both aspects of synthetic polymer biodegradation, in particular: • Characterise the microbial community associated to biodeterioration of an acrylic polymer used as protective and consolidant, in order to identify microorganisms potentially active against synthetic polymers. • Study bacterial degradation of nitrocellulose in order to develop a bioremediation process to remove nitrocellulose-based paints. Chapter 3 reports a case-study about how a microbial community changes in the presence or in the absence of an acrylic polymer used as consolidants. Synthetic polymers have been widely applied as consolidants and protective in cultural heritage field for the treatment of objects and buildings to prevent further deterioration. The long-term efficiency of the consolidative/protective treatments was believed influenced mainly by chemical and physical agents (e.g. UV light and temperature) and therefore lots of synthetic polymers have been used in cultural heritage conservation without testing them against biological deterioration. As a result, treated objects are sometimes in worse conditions than untreated objects and, moreover, biodeterioration of the added materials is an additional cause of damage. The study of microbial community changes due to synthetic polymer treatments and the identification of biodeteriogen microorganisms is a crucial step in developing a treatment strategy in cultural heritage conservation. In this work, we present the first molecular characterisation of a microbial community present on artistic ceramic treated with acrylics. The study was conducted on ceramic tiles of the Grande Albergo Ausonia & Hungaria façade. In 2007 the façade underwent conservation treatment to consolidate severely damaged tiles and to remove dark spots present on its surface. Tiles on the first horizontal register were then treated with the commercial synthetic resin Paraloid B72® (copolymer methylacrylate−ethylmethacrilate), as consolidant and protective product. Soon after the intervention both treated and untreated tiles showed coloured alterations caused by microorganisms between the pottery layer and the glaze. Samples from treated and untreated areas were initially observed under stereo, epifluorescence and the scanning electron microscope and analysed by Energy-dispersive X-ray spectroscopy (EDX) and micro Fourier transform infrared spectroscopy (FTIR). The results showed that the polymer, identified as Paraloid B72®, was present only in two of the nine treated samples and confirmed the presence of biological alterations in all samples. Paraloid B72® was not found on the surface of the most of the treated samples, probably because of photooxidative depolymerization and the wash out of resin from the tile surfaces. Deteriogen biofilm was present at the interface glaze-pottery in samples without any presence of Paraloid B72®, while in the two samples where we found Paraloid B72®, the biofilm was mainly in the pottery layer. Microscope techniques together with denaturing gradient gel electrophoresis (DGGE) and sequencing from total DNA extracted from Hungaria samples were used to identify sessile taxa causing coloured alteration. Our results showed that the colour of the deposit present in the tile samples and the greenish alteration on the balcony were most likely mainly due to the presence of cryptoendolithic cyanobacteria and eukaryotic algae respectively. Biodegrading microorganisms related to the presence of synthetic polymers and Paraloid B72® were also found. In particular, Phoma, an uncultured Bacteroidetes and Methylibium sp. were found in all the samples in which we detected Paraloid B72®. Melanised fungi, such as Phoma, are well known as the most damaging fungi able to attack and penetrate stone monument surfaces. Bacteroidetes show hydrolytic activity toward polymeric substances and Methylibium can grow on organic pollutants. In addition, when Paraloid B72® was present, the biofilm was located in a deeper position than in samples without any evidence of the resin. Using DGGE technique it was also proved that the microflora present on the tiles was generally greatly influenced by the environment of the Hungaria hotel. Several microorganisms related to the alkaline environment, the range of the tile pH, and related to the aquatic environment and the pollutants of the Venice lagoon were found. The rapid reappearing of microbial deterioration soon after the 2007 conservation treatment, which included the use of biocides, was likely favoured by the water content and organic substances, some of them added during the conservation treatment. Therefore, Paraloid B72® was not the best consolidant polymer to be used fort the long-time conservation of the ceramic tile of Hungaria hotel. Chapter 4 is focused on the capability of Desulfovibrio desulfuricans ATCC 13541 to attack nitrocellulose as binder in paint. Synthetic polymers used for the manufacture of paint and coatings are polymers belonging to different chemical classes and little information regarding the chemical characteristics and their variation in a population of paints are present in literature. On the base of few works, acrylics, polyvinylacetates and nitrocellulose-alkyd polymers seem to be most used as binders in spray paint formulation. The high presence of nitrocellulose, a uniformly substituted cellulose compound with varying degree of nitration, as component of paint binders should lead to a more environmental attention because the presence of nitro compounds materials in the wastewater effluent causes severe environmental problems, high toxicity and provokes serious health problems. Microorganisms are able to degrade nitrocellulose by two pathways: i) cleavage of β–1,4–glucoside bonds that produces nitrooligosaccharides of various length, normally carried out by fungi, and ii) nitrocellulose denitration that reduces the degree of nitro substitution, generally performed by bacteria. Since nitrooligosaccharides have mutagenic properties, the second pathway is preferred over the first for exploitation as a biodegradation pathway. Nitrocellulose undergoes degradation by sulphate–reducing bacteria under anaerobic conditions. In particular, sulphate–reducing bacteria of the genus Desulfovibrio decrease the amount of nitrocellulose powder in media containing this compound. Desulfovibrio spp. firstly reduces the nitration content of nitrocellulose in powder due to a nitroesterase activity of the bacteria and then reduces nitrate to ammonia through the dissimilatory nitrate reduction to ammonia (DNRA). DNRA is a two-step process involving nitrate reduction to nitrite and the subsequent nitrite reduction to ammonium by nitrate– and nitrite reductases. There are several sulphate–reducing bacteria able to reduce nitrate to nitrite, but it appears that this is not a shared feature across the genus Desulfovibrio. In contrast, the dissimilatory reduction of nitrite to ammonium seems to be widespread in Desulfovibrio. For this study, D. desulfuricans ATCC 13541 was selected because it was used in metal biosorption, and therefore the strain is resistant to the high concentrations of metals that can be encountered in paints. Nitrocellulose was selected in the form of the red spray paint by Motip–Dupli® Autocolor (colour 5–0200) that was confirmed by FTIR spectroscopy as composed by nitrocellulose and a modified polyester resin. At the end of degradation experiments, the capability of D. desulfuricans ATCC 13541 to adhere onto the surface of the paint was assessed by epifluorescence microscopy observations and confirmed by FTIR–ATR spectroscopy that showed the presence of proteinaceous material on the painted surface. Nitrocellulose degradation was followed indirectly by measuring nitrate, nitrite and ammonia concentration in the cultural medium and directly by stereoscope microscopy observation, FTIR spectroscopy and colourimetric measurements of the paint layer. The results proved that, even if slight abiotic degradation of the paint as a consequence of the long immersion time in the culture medium was noticeable, D. desulfuricans was active against Autocolor paint. In particular the bacteria acted with high specificity on the N–O bond of the nitro–substituted cellulose. Moreover, after incubation with D. desulfuricans, paint detachment and fading of Autocolor paint slides were clearly perceptible at first glance. The colour fading of Autocolor paint, proved by changes in CIELAB colour parameters, could be caused by the degradation of the paint and the removal of nitro groups from the nitrocellulose molecule. In this work, changes in nitrate– and nitrite reductase activity in D. desulfuricans incubated in the presence or in the absence of nitrocellulose as binder of Autocolor paint were also evaluated. It was assessed that the activity of nitrate reductase was equivalent while nitrite reductase activity was higher in D. desulfuricans grown in the presence or in the absence of Autocolor paint. In Chapter 5 the developement of a new primer pair specific for nrfA gene in Desulfovibrio genus is reported. DNRA or nitrate ammonification is an anaerobic process in which nitrate is reduced to ammonia with nitrite as intermediate. The ability to carry out DNRA is phylogenetically widespread. Many sulphate-reducing bacteria are able to perform respiratory ammonification in the presence of nitrate when sulphate is absent and/or in low concentration. The first step of DNRA, the nitrate reduction to nitrite, is usually performed by the periplasmic nitrate reductase NapAB, while the second step, the nitrite reduction to ammonium is catalysed by the pentaheme cytochrome c nitrite reductase NrfA. In Desulfovibrio spp., nitrite reductase NrfA plays an important role for those strains able to use nitrate and nitrite rather than sulphate as electron acceptor, and for those bacteria capable of reducing nitrite but unable to reduce nitrate. In fact, nitrite is a very toxic compound and the additional function of nitrite reductase allows Desulfovibrio spp. to survive in environments containing nitrite up to millimolar concentrations. As the presence of the nitrite reductase in the Desulfovibrio genus is widespread, the gene nrfA, that encods for the key enzyme of the second step of the DNRA pathway, could be used as a marker for this dissimilatory process. Multiple alignment of nrfA sequences of Desulfovibrio species, available from two different databases, showed several consensus sequences. nrfA primers were designed into these conserved sequences using Primer3 software and in silico tested by BlastN tool from NCBI and ThermoPhyl software. The results showed that the best primer pair was nrfA-F2 – nrfA-R5. The selected primer pair was then tested firstly on Desulfovibrio and Desulfomicrobium strains from culture collection and secondly on two environmental samples. The results proved that a 850bp, identified by sequencing as a nrfA gene fragment, was successfully amplified using the new primer pair. nrfA gene sequences obtained from NCBI and KEGG databases, the two environmental samples and Desulfovibrio and Desulfomicrobium culture collection strains were then clustered in OTUs. The results showed that there was a high diversity in nrfA sequences clustered in OTUs and there were OTU groups not represented by any sequence present in databases, confirming that more work should be done to study nrfA gene and DNRA pathway in Desulfovibrio genus. In conclusion, this project showed that: • Biotechnology provides valid tools to study changes in microbial community structure due to the presence of synthetic polymers and to identify synthetic polymer biodeteriogen microorganisms. • Desulfovibrio desulfuricans ATCC 13541 is able to degrade nitrocellulose as binder in paint and likely performs this degradation by the DNRA pathway. • The new nrfA primers could help for isolating and obtaining more sequences of the nrfA gene from both culture collections and environmental samples and for studying dissimilatory nitrate reduction to ammonia (DNRA) pathway. Further studies on synthetic polymer biodeterioration should be made for the selection of the best one to be applied for each specific use in order to prevent the loss of polymer function once it has been applied to a surface. On the other hand, D. desulfuricans is a promising bacterium in nitrocellulose-based paint bioremediation and nrfA gene could be used to study DNRA metabolism or nitrite detoxifying in bacteria of Desulfovibrio genus, in order to improve nitrocellulose bioremediation process.