THE CONTROL OF DELETERIOUS BIOFILM ON ABIOTIC SURFACES: FROM A MORE SUSTAINABLE USE OF BIOCIDES TO A NEW ENVIRONMENTALLY-FRIENDLY APPROACH
Creators: name: POLO, ANDREA
Publisher: name: Universit� degli Studi di Milano
Year: 2010

description: It is well established that bacteria are the first organisms to adhere and colonize both abiotic and biotic surfaces. Their subsequent multiplication and production of exopolymeric substances (EPS) bring to biofilm formation which is believed to influence the settlement of following colonisers starting biofouling process on surfaces. Biofilm and fouling often have a deleterious effect on colonized surfaces and materials. They explicate a complex and various range of processes on abitic surfaces that cause physical damages, chemical alterations, loss of functionality and discolouration of surfaces, generating tremendous environmental and economical harm for human society. The control of the deleterious biofilm and the following fouling is today a great challenge. Currently, we have two choices: to remove biofilm by traditional methods or to research new effective antifouling approaches. 
Traditional techniques consist mainly in the application of biocides, however this practice has several disadvantages. In fact, traditional biocides are not generally specifically target against detrimental microorganisms and they are often potentially toxic both for humans and the environment. In addition, biological matter released by their use can offer a favourable substratum for subsequent colonisations. Others problems concern the development of resistance in target pest populations and the low biodegradability of these chemicals. Moreover, biocides are not always effective at low concentrations because life inside the biofilm leads to increased resistance to antimicrobial products up to 1000-fold compared to planktonic cells. Finally, current legislation in UE and USA regulates the use of biocides and lately several products have been withdrawn from the market. New antifouling strategies need to be effective, economic, safe for the public and to pose negligible risk to human health and the environment. In the last years new strategies able to control biofilm growth have been proposed as alternative to traditional active substances. However, several years will be required to set up and test satisfactory methods, so, these novel strategies can be only a long term solution. In the meantime, as short term solution, it is vital to study methods for a more sustainable use of traditional antimicrobial agents. Therefore, the aims of the PhD project here presented were:
1) to use biocides for the removal of deleterious biofilm from relevant abiotic surfaces in the most sustainable way;
2) to exploit the ability of a new promising inorganic compound, the photocatalytic titanium dioxide, as an innovative non-toxic antifouling system to control biofilm formation on abiotic surfaces. 

The study reported in the chapter 3 addresses the first aim. Generally, broad-range biocides are used to remove alterative biofilms from historical and artistic surfaces. In order to obtain a use as sustainable as possible of antimicrobial agents, the strategy has been to identify alterative microorganisms so as to use a suitable biocidal product which targets specifically the biodeteriogen agents. 
In this work, for the first time, an integrated biotechnological system that enables the cleaning of cultural heritage stone affected by both biological and chemical alteration process was used.
The study was conducted on alterations found on two stone sculptures decorating the courtyard of the Buonconsiglio Castle in Trento (Italy). Stone, especially if exposed to the weather, is subject to chemical, physical and aesthetical deterioration. In this regard, pollution and environmental parameters play an important role since they are most responsible of deterioration process. Samples from altered and unaltered areas were characterized using stereomicroscope, cross-section observations and Fourier transform infrared (FTIR) analyses. Results reveled that stone was an oolitic limestone and changes were both discolorations ascribed to biological agents and chemical alteration represented by black crusts composed mainly by gypsum with a small amount of calcite, nitrate and silicates.
Cultural and biomolecular methods were adopted to study microbial biofilm from powders samples. The cultural analyses proved that heterotrophic bacteria, fungi and prokaryotic and eukaryotic algae were present on surfaces and that in some samples the counts were quite high (up to 7 logCFU/g for bacteria and up to 6 logCFU/g for fungi). Denaturing gradient gel electrophoresis (DGGE) and sequencing from total DNA extracted allowed to identify taxa of microorganisms causing discolorations: they were Cyanobacteria, Chlorophyta green algae (Myrmecia and Friedmannia), Streptophyta green algae (Klebsormidium), microcolonial black fungi (Alternaria and Cladosporium) and other species of fungi able to deposit melanin in the cell wall (Verticillium). Fluorescent in situ hybridisation (FISH) highlighted that the Cyanobacteria generally were dominant (more than 60%) among the other prokaryotics belonging to the Bacteria domain. In this case, in order to remove the discolourations, despite the initial purpose to use a product that targeted only the small population of harmful microorganisms, we were forced to choose the biocide BIOTIN N (constituted by a mixture of tributyltin naphthenate (20% w/v) and didecyl dimethyl ammonium chloride (35% w/v)) with a broad spectrum of activity due to the taxonomical variety of the biodeteriogens.
The same traditional and biomolecular methods were carried out on the samples collected after the cleaning to verify the removal of alterative microorganisms. Treatment with the biocide resulted in a decrease of the bacterial load (up to five orders of magnitude), and neither culturable fungi nor culturable prokaryotic and eukaryotic algae grew. Indeed the DGGE profiles showed far fewer bands than before treatment and proved that Cyanobacteria and most of the green algae and dematiaceous fungi had been efficiently removed. 
Chemical alterations were removed by a biocleaning treatment, an innovative, efficient and highly selective bioremediation technology, alternative to the use of chemicals, that uses viable cells of sulphate-reducing bacteria able to remove sulphates from stone ornamental surfaces. In this work for the first time this approach was applied on limestone. Sulphate-reducing bacteria have been Desulfovibrio vulgaris subsp. vulgaris (ATCC 29579). The treatment consisted in three 12-h applications for a total duration of 36 h. Comparison of the results of the chemical analyses performed before and after the biocleaning proved that gypsum, nitrates and silicates were almost completely removed. In addition, both optical evidence and FTIR analysis showed that the limestone substratum was preserved.

In general, the cases of biofilms with large taxonomical variety (as the biofilms on the sculptures from Buonconsiglio Castle in Trento) are very frequent. Therefore, due to the side-effects of biocides, the development of alternative strategies for the prevention and control of deleterious biofilm becomes imperative. They must allow to protect materials from biodeterioration, and thus preserve their usefulness for as long as possible. In addition, new approaches must be safe for human beings and the environment. The study reported in the chapter 4 refers to this second aim of the project. The ability of photoactivated titanium dioxide (TiO2), a promising alternative to biocide for the biofilm control and prevention, was investigated. The biocidal activity of TiO2 against planktonic cells thanks to its strong photocatalytic properties has been reported since 1985. TiO2 per se is non-toxic, as the tests in rats prove, and has been approved by the American Food and Drug Administration (FDA) for use in human food, drugs, cosmetics and food contact materials. Moreover, it is considered an environmentally friendly photocatalyst, it is relatively inexpensive, chemically stable and effective under weak solar irradiation in ambient atmospheric environment. 
Few attention was dedicated to study the potential of photocatalytic TiO2 against the bacterial biofilm so far. The aim was to investigate the ability of photocatalytic TiO2 as a new non-toxic antifouling nanotechnology to deter and prevent the attachment and biofilm formation of selected bacteria on TiO2-coated surfaces. 
Aeroxide P25 (Degussa) was employed as source of TiO2. The effects of both photocatalyst nanopowder suspensions (3g/l concentration) and thin TiO2-film applied on glass coverslides by sol-gel method were valued. During the experiments TiO2 was photoactivated by a lamp emitting radiation over a UV-A wavelength range with light intensity similar to outdoor solar irradiation (between 3000 and 500 ?W/cm2). An efficient protocol for the photoactivation of TiO2 was set up degrading the dye rhodamine B. Thus activity was investigated on Bacillus cereus-group sp. (Gram-positive) and P. stutzeri and P. aeruginosa (Gram-negative) planktonic cells. The results proved that photoactivated TiO2 provoked a significant decrease of CFU/ml. Biocidal activities of nanopowder suspension in demineralised water in Bacillus sp., P. stutzeri and P. aeruginosa were respectively 1-log reduction after 24 h, 2-log reduction after 30 min and 1-log reduction after 2 h compared to non-photoactivated TiO2. TiO2 thin film also produced a complete disinfection of P. aeruginosa planktonic cells in 24 h. Finally, the activity of photoactivated TiO2 was investigated on P. aeruginosa biofilm at various formation steps both at the solid-liquid and at the solid-air interface. It was proved that neither TiO2 nanopowder nor photocatalytic film showed any biocidal activity on P. aeruginosa biofilm at all the interfaces investigated. The experiments have demonstrated that the lack of cell inactivation by photocatalytic action on biofilm was not due to 1) the presence of phosphates that could block active sites on the catalyst surface, 2) scavenge oxidative radicals produced at the surface, nor to insufficient presence of O2 at the TiO2 surface, which maintains charge transfer in photocatalytic reactions, 3) biofilm growth that screens UV-A light, deactivating TiO2, 4) insufficient build-up of photocatalytically-generated reactive species necessary for cell inactivation. In addition, confocal laser scanning microscope analysis demonstrated that not even exopolysaccharides produced by biofilm cells were the cause of fail inhibition since almost absent in the very young tested biofilms. The only possible explanation for these findings was that the cells, when live in sessile form, invoke a genetic response that imparts them an increased resistance to oxidative stress generated by photoactivated TiO2. 

In conclusion, the studies reported here have demonstrated that: 
a) Biotechnologies could facilitate a more sustainable use of biocides addressing the choice toward a suitable product that targets only the biodeteriogen microorganisms.
b) The biocleaning treatment is an effective technology, alternative to use of chemicals, to remove selectively sulphates from stone. 
c) The photocatalytic TiO2 is not a good candidate to develop an effective technology that is alternative to traditional biocides for the control of deleterious biofilm. 

Further studies with other promising environmentally-friendly compounds may provide new ways to move forward in the search and to pursue the goal of an efficient surface coating methods able to prevent biofilm formation or, at least, to interfere with their inconvenient increased resistance to biocides, respecting the human health and environment.
The research for new non-toxic antifouling strategies continues.