Eric May

Carbonate crystals precipitated by freshwater bacteria and their use as a limestone consolidant.

ABSTRACT Bacterial carbonate precipitation is known to be a natural phenomenon associated with a wide range of bacterial species. Recently, the ability of bacteria to produce carbonates has been studied for its value in the conservation of limestone monuments and concrete. This paper describes investigations of carbonate crystals precipitated by freshwater bacteria by means of histological (Loefflers methylene blue and alcian blue-periodic acid-Schiff stain) and fluorescence (CTC [5-cyano-2,3-ditolyl tetrazolium chloride]) stains, determination of cell viability inside carbonate crystals, and pore size reduction in limestone by image analysis. Carbonate crystals were found to be composed of bacteria embedded in a matrix of neutral and acid polysaccharides. Cell viability inside the carbonate crystals decreased with time. On stone, bacteria were found to form carbonate crystals, with only a few bacteria remaining as isolated cells or as cell aggregates. Pore size was reduced by about 50%, but no blockage was detected. Taken together, the results of this research provide some reassurance to conservators that biocalcification by bacteria could be a safe consolidation tool in a restoration strategy for building stone conservation.

Enhancement of physical weathering of building stones by microbial populations

Two limestones from Crete, Greece and a dolomite from Mansfield, UK were subjected to combined microbial and physical weathering simulation cycles, in an attempt to assess the contribution of each agent of decay. Sound stone discs were exposed to different temperature and wet/dry cycling regimes involving treatment with distilled water or solutions of sodium chloride or sodium sulphate. Before the weathering cycles, half of the discs were inoculated with mixed microbial populations (MMP), originally recovered from decayed building stone of Portchester Castle, Hampshire, UK. The presence of MMP greatly accelerated the rates of deterioration of stone of all treatments, measured by weight change and alteration of hydraulic properties of stone. A combination of physical and biological processes significantly enhanced the extent of decay when compared with the physical or biological agents acting alone. Populations of heterotrophic, sulphur-utilising, halotolerant and moderately halophilic bacterial populations remained large throughout the experiment. Biofilms formed by populations of microorganisms were visualised by staining and assessed by colorimetric measurement of total carbohydrate in the stone substrate. The relative contribution of microbial and physical weathering to the process is discussed.

Fat, oil and grease deposits in sewers: characterisation of deposits and formation mechanisms

Fat, oil and grease deposits (FOG) in sewers are a major problem and can cause sewer overflows, resulting in environmental damage and health risks. Often simplistically portrayed as cooling of fats, recent research has suggested that saponification may be involved in FOG formation. However there are still questions about the mechanisms effecting transformations in sewers and the role and source of metal cations involved in saponification. This study characterises FOG deposits from pumping stations, sewers and sewage works from different water hardness zones across the UK. The sites all had previous problems with FOG and most catchments contained catering and food preparation establishments. The FOG deposits were highly variable with moisture content ranging from 15 to 95% and oil content from 0 to 548 mg/g. Generally the pumping stations had lower moisture content and higher fat content, followed by the sewers then the sewage works. The water in contact with the FOG had high levels of oil (mean of about 800 mg/L) and this may indicate poor kitchen FOG management practices. FOG fatty acid profiles showed a transformation from unsaturated to saturated forms compared to typical cooking oils. This seems to relate to ageing in the sewer network or the mechanism of formation, as samples from pumping stations had higher proportions of C18:1 compared to C16. This may be due to microbial transformations by bacteria such as Clostridium sp. in a similar process to adipocere formation. There was an association between water hardness and increased Ca levels in FOG along with harder deposits and higher melting points. A link between FOG properties and water hardness has not been previously reported for field samples. This may also be due to microbial processes, such as biocalcification. By developing the understanding of these mechanisms it may be possible to more effectively control FOG deposits, especially when combined with promotion of behavioural change.

Progressive changes in water and sediment quality in a wetland system for control of highway runoff

Innovative wetland based systems were designed and installed on the Newbury Bypass, Berkshire, England to provide flow balancing and pollution control for road runoff. The systems were monitored over 18 months to evaluate performance, pollutant removal processes and offer improved design and operation codes for this new application of wetlands. Water quality, sediment accumulation rates, and metal concentrations in size-fractionated, settling solids and deposited sediments were determined in parts of the system to provide information on spatial and temporal variability. The results presented here show that over the long term, there were progressive changes in parts of the system for BOD and COD and for metal concentrations in the sediment fractions, which occurred with linear (or semi log-linear) rates, despite variability in flow rates, retention times and in pollutant loading to the system. Future work will continue monitoring to increase the data set, examine possible processes contributing to the regression constants, and test the potential use of the regressions in system modelling. Attempts at modelling road runoff treatment using wetlands must allow for progressions, since the systems can only be effective if they retain removed metals in the sediment sink.

A feasibility study of a Salix viminalis gravel hydroponic system to renovate primary settled wastewater

A Salix viminalis/gravel system based on hydroponics was developed for wastewater renovation in order to avoid the problems of soil damage and pollution associated with long-term application of wastewater to soil. For such a system to work the mineral elements applied must match closely the requirements of the tree species. To examine this the growth and nutrient uptake of S. viminalis in wastewater was compared with that in Long Ashton nutrient solution (1/4 strength). S. viminalis grew more slowly in wastewater than in Long Ashton solution, but exhibited no obvious deficiency or toxicity symptoms. Since industrial wastewaters often contain metals, the extent to which copper might inhibit wastewater treatment in this system was also examined. S. viminalis was grown in wastewater amended with 10 and 100 ppm copper. Trees were unaffected by wastewater with 10 ppm copper when compared to trees grown in wastewater alone. Wastewater containing with 100 ppm copper was too toxic for the trees to thrive and wastewater treatment was reduced. Treatment efficiencies for unamended wastewater were 57.7% for nitrogen, 90.6% for phosphorus and 24.9% for potassium. These efficiencies are much greater than those quoted for a Salix/soil system, and thus Salix/gravel systems may have potential for wastewater treatment in environmentally sensitive areas or situations.

Influence of Environmental Temperature on Biocalcification by Non-sporing Freshwater Bacteria

The process of biocalcification, recognised as precipitation of calcium carbonate, has been described as a widespread phenomenon associated with a wide range of different bacterial species. This biocalcifying activity, and factors that affect it, have been widely studied in moderately halophilic bacteria but there is a lack of information on factors that affect biocalcification by freshwater bacteria. In this paper, we study how temperature can affect biocalcification by freshwater bacteria that potentially could be used for the process of bioconsolidation during conservation. Ten isolates were characterised by standard biochemical and API 20NE tests. Their biocalcifying activity was studied at temperatures between 10 and 40°C in B4 liquid medium. Mineralogical and quantitative analyses of the crystals were carried out by XRD, and morphological studies by SEM. Biocalcification only occurred when bacteria were present and were able to grow. Carbonate precipitation by bacteria increased with time and temperature of incubation. Temperature affected not only the amount of precipitation but also crystal quality and morphology. As bioconsolidant agents, these organisms could be applied to stone when the temperature does not exceed 40°C depending on the type of isolate.

Investigations of the localisation of bacterial activity on sandstone from ancient monuments

Methods were investigated for the determination of activity levels of bacteria on sandstone using the reduction of 2-(4-iodophenyl)-3-(4 nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan as a measure of dehydrogenase activity. A microscopy technique, based on use of acridine orange with bright-field illumination, was developed which gave a good visual image of bacterial cells, including those containing INT-formazan. Surveys at two monuments, Portchester Castle and Tintern Abbey, were carried out using this method which showed that between 20.7 and 51.9% of bacterial cells present in situ were active. Extraction of INT-formazan directly from the stone using methanol indicated that bacteria were tightly bound to stone particles and that microscopic methods would underestimate counts of bacteria. Surveys of five monuments using the extraction method showed that microbial populations on sandstone in situ were active but activity could not be related to decay state of the stone.

COMPARATIVE STUDIES OF MICROBIAL COMMUNITIES ON STONE MONUMENTS IN TEMPERATE AND SEMI-ARID CLIMATES

Climate is recognised to play an important part in influencing the activity of microorganisms on stone in monuments and other objects of cultural value. In the UK and Greece, the numbers and distribution of heterotrophic bacteria were not strongly related to seasonal changes in temperature and rainfall. At Portchester Castle, qualitative changes in bacterial populations have been observed; actinomycetes were found only on decayed stone and dominant on stone from Tell Basta, in semi-arid Egypt. In the Minoan Palace at Petras and the fortifications of Khania, higher counts of halotolerant heterotrophic bacteria were found in sheltered areas on stone showing other biological growths and salt efflorescence. For all monuments, stones of lower mechanical strength supported higher bacterial counts and electron microscopy showed extensive sheets of biofilm. Estimations of carbohydrate in stone could also be related to whether the sites were exposed or protected. There is evidence to suggest that variations in the nature of bacterial populations may be dependent on season in temperate regions and in the Mediterranean climate related to the location within the monument. Biofilm production and perhaps halotolerance provide means by which bacteria resist adverse changes in moisture levels. Extreme fluctuations in moisture may induce major shifts in bacterial populations selecting filamentous, spore-forming types that penetrate deeper into the stone.

Constructed Wetlands for Wastewater Treatment in Colombia

A modular wastewater treatment system has been installed for the Colombian Coffee Growers Federation (FEDERACAFE) at the Fundacion Manuel Mejia residential training centre. This system was designed by the University of Portsmouth, UK, and based on previous experience with the Gravel Bed Hydroponic (GHB) constructed wetland system. The steep topography of the site posed constraints on the design and a modular system composed of a rock filter in gabions for primary treatment, GBH beds for secondary treatment and a pond for tertiary treatment was selected. This system was designed to follow the contours of the site and maximise treatment. The GBH reed bed was planted with Typha angustifolia in early 1997. The system is being monitored by CENICAFE, the research division of FEDERACAFE. Monitoring of the system has shown large BOD removals from more than 350 to less than 20 mg/l. Initial results have also suggested 4 log cycle reductions in indicator bacteria. It is hoped that the success of this system will result in the replication of the treatment process at many villages in the Colombian coffee growing region where conventional sanitation technologies are impractical.

The use of sound velocity determination for the non-destructive estimation of physical and microbial weathering of limestones and dolomites

Two limestones from Crete, Greece and Mansfield dolomite from the UK were tested experimentally for their resistance against physical and microbial attack. XRD and SEM were used for the assessment of their mineralogy, original structure and weathering patterns. Stone discs subjected to physical, microbial and combined physical/microbial weathering simulation cycles were treated with distilled water, NaCl and Na2SO4solutions, alone or in combination with mixed microbial populations (MMP). Measurements of weight change, mechanical alteration and viable bacterial counts were augmented by the non-destructive technique of sound velocity transmission. Both longitudinal (Up) and shear (Us) waves velocities were mostly stable or slightly accelerated in the case of distilled water and NaCl treatments, but with the Na2SO4weathering regime, involving expansion due to hydration and dehydration, they decreased. However, a marked decrease was found whenever MMP were present. These observations suggest a possible contribution of microorganisms to physically induced deterioration of stone. Correlation of Upand Usto weight, other mechanical properties and bacterial counts highlighted the importance of surface hardness in predicting mechanical behaviour. Furthermore, the evidence suggests that porosity size might influence the type of stone attack and a possible contribution of sulphur bacteria to deterioration. Sound transmission velocity proved to be an effective diagnostic technique to assess the mechanical state of building stones.