The objectives of this grant were to determine the responses of the microflora to contamination of historic limestone materials with atmospheric pollutants. We also studied the effects of these interactions on the deterioration of the materials.
In the first year of this project we did a detailed comparative analysis of the effect of sulfur and hydrocarbons on the microflora of limestone tombstones in a polluted and unpolluted area of Massachusetts. We found that the populations of bacteria and fungi were significantly smaller on limestone in the polluted area. Similarly, the diversity of the microflora was much smaller on the stone in the polluted area. Conversely, the populations of bacteria capable of utilizing sulfur compounds or hydrocarbons were much larger on the stone from the polluted areas, presumably as a result of the presence of a plentiful supply of sulfur and hydrocarbons deposited from air pollution on the limestone. We found that these bacteria were capable of utilizing very small quantities of atmospheric pollutants and producing significant quantities of acid.
During the second year we investigated the corrosive processes in detail. We isolated and identified the predominant microorganisms growing on the stone in the polluted area. The predominant bacteria belong to the genera Bacillus, Vibrio and Xanthomonas. The major groups of fungi include Aureobasidium and Cladosporium. We inoculated these predominant microorganisms onto sterilized limestone, and exposed the samples to sulfur and hydrocarbons in our environmental chamber at a temperature of 30 degrees and 80% relative humidity. In these accelerated tests we found that the predominant microorganisms grow to large populations on the limestone at low concentrations of sulfur and hydrocarbons. Significant quantities of corrosive acids were produced in less than a month in these experiments.
We carried out an extensive scanning electron microscope study of the growth of these organisms on the limestone. Both sulfur and hydrocarbon degrading populations were investigated for their ability to attack limestone. We found that a complex interaction of fungi and bacteria is involved in the limestone attack by both the sulfur and hydrocarbon-utilizing microfloras. Our data indicate that the initial penetration is by fungi that grow into the pores of the stone. However, these fungi act as Trojan horses, carrying a large population of bacteria into the interior of the stone.
During the final year of the project we tested a new innovative method of quantitative analysis of the effects of pollutants on limestone. We used a new high power computer assisted tomography instrument at the Harvard Medical School. This instrument was designed to analyze solid materials non-destructively. It has proved to be an excellent analytic tool in our research.
Our data showed that stone treated with products of acid rain increased in volume. When we exposed this stone to other microbial acids, no loss in volume or voids within the stone were detected. We concluded that the minerals formed by the interaction of sulfates with the limestone provided protection from the action of microbial acids. However, this does not suggest that acid rain provides protection to the stone. Ultimately gypsum minerals exfoliate and cause limestone deterioration.
Our research yielded an important discovery in terms of the microbial ecology of these materials. We frequently detected very small striated microorganisms in our samples. These bacteria were less than one micrometer in size. They were not culturable. We did not find any previous references to their occurrence. These observations suggest that limestone exposed to atmospheric pollutants may harbor an unusual microflora either capable of living on the resultant minerals or involved in their formation.