ABSTRACT

Subsurface engineering environments, such as mines, tunnels and water gal-leries, may have cultural significance, in addition to their inherent geological characteristics. The 16th century Paranhos spring water tunnel located in Porto city (NW Portugal) was excavated throughout the granitic bedrock to supply water for the public fountains for more than five centuries. Nowadays, due to groundwater quality degradation and high deteriora-tion level of the granite tunnel, the water is no longer used for public purposes. This subsur-face granite heritage represents a favourable habitat for a wide variety of microorganisms, which participate in the formation processes of secondary minerals and dissolution of rock components. Due to the large number of anthropogenic contamination sources and urban surface activities along the course of the Paranhos spring water tunnel, such as garages, pet-rol stations and in situ sanitation and sewer network, this subterranean environment shows high vulnerability to contamination. In fact, previous hydrogeochemical studies showed a nitrate and sulfate-enriched composition for the groundwater probably resulting from urban drainage, sewer leakage and agricultural activities. In this study, we identify and characterize the bacterial communities in biogenic ferromanganese deposits coating the weathered subsurface granite in order to understand the relationship between their presence and the potential contamination sources. Molecular biology techniques of enrichment and isolation cultures, field emission scanning electron microscopy, transmission electron microscopy and micro-Raman spectroscopy were conducted. Thirty-five different bacterial strains were isolated, most of them belonging to Bacillus, Pseudomonas and Ensifer genera. The presence of Pseudomonas spp. is usually associated with contaminated environments, which is consistent with the contaminated sources described before. Moreover, micro-Raman analysis pointed out the presence of copper phthalocyanine, a blue pigment commonly used in the manufacture of enamels, printing inks, and automotive finishes. Its presence is consistent with the existence of garages and petrol stations on the surface of the Paranhos spring water tunnel. Electron microscopy observations of isolates and enrichment cultures for Mn- and Fe-oxidizing bacteria revealed the presence of Mn- and Fe-rich precipitates upon bacterial cells, which suggests that these bacteria induce the precipitation of Fe and Mn oxides. We were thus able to isolate bacteria probably involved in the formation of the black ferromanganese oxides coating the Paranhos granite surfaces. This clearly demonstrates that the interest in the geomicrobiology of man-made subterranean environments lies not only in the 96fact that some of them can provide useful information for general and applied microbiology but also for representing geomining sites of particular geological and cultural relevance that needs conservation and maintenance interventions.