The seasonality of heterotrophic bacteria on sandstones of ancient monuments

brternational Bio~h'terioration 28 {1991) 49-64

The Seasonality of Heterotrophic Bacteria on Sandstones of Ancient Monuments

S. Tayler & E. May School of Biological Sciences. Portsmouth Polytechnic. UK

A BSTRA C T

The heterotrophic bacteria population occurring on decaying sandstone from ancient monuments has been examined Quantitative and qualitative observations art, pre~ented.for seasonal variation over a period of I year. The diversity of heterotrophs occurring on sand~'tone was assessed on a monthly hasis using a range of isolation media. An increase in numbers of bacteria was seen during the winter months." with both Gram-negative and Gram-positive O'pes present. Gram-positive bacteria, t:v~ecially spore-formers olethe genus Bacill us. werefound exclusively in the dry summer months. Decay activi~'. reJTectingthe potential of natural mixed populations present on the stone, also showed seasonal variation and this was related to the pattern qf change for the bacterial specie~ ohserved.

INTRODUCTION Heterotrophic bacteria have been found in large numbers on decaying building stone and it has been suggested that these bacteria are active in the decay process as a result of mineralising activities, similar to those observed with soil bacteria (Eckhardt. 1985). Paine et al. (1933) carried out an extensive survey on the distribution of heterotrophic bacteria on both sound and decayed stone. On examination of a number of different stones they found that bacterial types common to soil. water or air could be isolated in large numbers from decaying stone with Pseudomonas and Bacillus spp. being particularly prevalent. A similar study was carried out 49 International Biodeterioration 0265-3036/91/$03.50© 1991 Elsevier Science Publishers Ltd. England. Printed in Great Britain.

50

S Tayler. E. May

by Webley et al. (1963) confirming the presence of these organisms on rocks and weathered stones. The activity, of these organisms in decay of building stone has largely been ignored as stone was thought to contain few organic nutrients for their growth. However, Rosvall (1986) points out that all stonework probably possesses sufficient organic matter from soil, dust and dirt to support heterotrophic activity. Furthermore, stone may even contain endogenous diagenetic material (Eckhardt, 1985). Recently, heterotrophic bacteria have been isolated from the stonework of ancient monuments (Lewis et al.. 1985: Eckhardt, 1988: May & Lewis, 1988) and shown to be capable of causing decay under laboratory conditions (Lewis et al., 1987). To date, the seasonality of these bacteria in situ has not been studied. Previous studies have concentrated on organisms isolated on relatively few sampling occasions giving no indication how the population may change seasonally in a temperate climate. The aim of this study was to investigate whether seasonal variation occurred and if this affected the potential of heterotrophic bacteria to cause damage to stonework at different times in the year.

MATERIALS AND METHODS Portchester Castle in southern England was chosen as a suitable site for study. Samples (1 g) of Green Sandstone from single separate blocks of sound and decayed stone, in close proximity,, were taken at monthly intervals over a period of I year. Sound stone samples were collected with a chisel previously sterilised with 70% alcohol. Decayed stone was removed with sterilised tweezers. Samples were collected in sterile Petri dishes and transported to the laboratory immediately for analysis. Bacteria were recovered from the stone according to the method of Lewis et al., (1985). Direct total counts of bacteria were performed using a Helber Counting Chamber with Thoma ruling (Weber Scientific International Ltd), Cells were fixed with 0.3% formaldehyde and the suspension allowed to settle for 15 min to remove stone particles from suspension. Counts were performed in duplicate using a Wild M20 phase contrast microscope. Viable counts for a quantitative and qualitative examination of heterotrophic bacteria from stonework were carried out using the spread plate method. Suitable dilutions of the stone suspension were made in phosphate-buffered saline, pH 7.2 (Cruickshank et al., 1975) and 0.1 ml spread over the surface of solid medium. Initial studies showed that an isolation medium based on acetate as carbon source (Pringsheim &

The seasonali~' of heterotrophic bacteria on sand~'tone

51

Robinson. 1950) was superior for the recovery, of bacteria from stone. Variations in the strength of this medium were used throughout the seasonal survey to establish the effect of nutrient strength on cell recovery. The r e c o m m e n d e d strength, one-tenth strength and an increased strength medium were used. The one-tenth strength medium contained 0-001% sodium acetate and 0-05% yeast extract and bacteriological peptone. The increased strength contained 1% sodium acetate rather than the lower level of 0.01% recommended. Plates were incubated for 7 days at 25°C. After incubation the colony characteristics were assessed using a binocular microscope. Each colony type was described and ,enumerated. Representatives of each type were then removed. streaked on to fresh medium to obtain pure cultures and stored on maintenance slopes of acetate medium. Morphological and biochemical characteristics were used for presumptive identification. These included G r a m stain, potassium hydroxide test, motility by the hanging drop method, catalase test, oxidase test, oxidative/fermentation test and spore stain. Morphology was examined in liquid culture and any special arrangements of cells noted (Cowan & Steel. 1977). Presumptive identification was made using the scheme of Shewan et al. (1960) in conjunction with Bengey's Manual of Determinative Bacteriology (Bucha nan & Gibbons. 1974). Seasonal variation in bacterial types was also examined in relation to their potential to cause damage to stone under laboratory conditions. This was assessed according to the method of Lewis et al. (1988). Mixed populations of bacteria were removed from sound and decayed stone as described previously and inoculated into flasks of a glucose/peptone medium containing l cm diameter thin sandstone discs. Discs were incubated for 14 days at 25°C. They were subsequently checked for weight loss and the medium assayed for changes in pH using a Pye Unicam pH meter and levels of calcium ions using a Kent ElL calcium electrode. Monthly temperature and rainfall figures for the year of the seasonal survey are given in Table 1. Data from these studies were analysed using a B M D P statistical package implemented on a Vax mainframe computer.

RESULTS Direct total counts revealed between l0 s and 109 cells per g of stone (Fig. 1). No apparent seasonal variation could be detected. However. counts from sound and decayed stone were found to differ significantly (Kruskal-Wallis, H = 3.85. p = 0-05) with sound stone supporting the higher population.

52

S. Tavh,r. E. .~luv TABLE I Temperature and Rainfall Figures tbr the Portchester Area from November 1988 to October 1989 Month

Daily m('un t,,mperttture (°C)

Total monthly raitlfall (mm)

7-7 8.4 7-5 7.4 8-9 8. I 14.6 16.2 19.7 18.6 15.5 15.4

19 22 31 67 52 67 3 28 15 16 36 93

No,,'. Dec. Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct.

Source: Bracknell Meterorological Centre. Berkshire. UK.

The counts obtained for numbers o f viable bacteria for each month on three different strengths o f medium are given in Figs 2 and 3. Analysis o f variance tbr seasonal variation o f viable bacteria were carried out. The variation between some months was large. In July. August. September and October little or no growth occurred on some medium strengths. These were therefore omitted from the analysis o f variance. It was 10,

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Fig. 2. Viable counts of bacteria from sound stone from November 1988 to October 1989. Key: (a) reduced strength medium: (b) recommended strength medium: (c) increased strength medium. Each value is the mean of three counts for each month. necessary to take l o g a r i t h m s o f the cell n u m b e r s in o r d e r to stabilise the variance. T h e data were a n a l y s e d as a factorial e x p e r i m e n t replicated w i t h i n blocks o f time. Total viable c o u n t s o f bacteria differed significantly from season to season (F3, 39 = 11.59,p < 0.001) with a decline in n u m b e r s d u r i n g the w a r m , dry s u m m e r m o n t h s . N u m b e r s o f G r a m - p o s i t i v e (F3, 39 = 8.49, p = 0.002) a n d G r a m - n e g a t i v e bacteria (F3, 39 -- 10-07. p = 0.001) also differed significantly between seasons. A qualitative shift in the

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(c) Fig. 3. Viable counts of bacteria from decayed stone from November 1988 to October 1989. Key: (a) reduced strength medium: (b) recommended strength medium: (c) increased strength medium. Each value is the mean of three counts for each month. population was observed as can be seen in Figs 4 and 5. During N o v e m b e r , both Gram-positive and Gram-negative organisms were isolated. In July onlv Gram-positive forms, especially o f the sporeforming genus Bacillus, were recovered. This c h a n g e in the population was a gradual shift from month to month. There was also a significant difference in numbers o f bacteria isolated on the different media strengths (F2. 39 -- 10-06, p = 0.003). In almost every, case the reduced strength m e d i u m gave superior recovery. However, there was no

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(b) Fig. 4. Bacterial types isolated from (a) sound stone and (b) decayed stone, in November 1988. Key: • Gram-positive bacteria: 17 Gram-negative bacteria: [] non-subculturable. Each value is the mean of three counts for each month. A, Acinetobacter- like organisms: B. Actinohacillus: C. Actinomycetes." D, Aerococcus: E. Aeronomas/Vibrio: F. Anthrobacter: G. Bacillus: H, Chromohacterium: I, Co~.'nebac~.'erium: J. Flavobacterium/Cytophaga: K, Micrococt'us: L. Moraxella- like organisms: M. P~eudomonas Group II: N. Pseudomonas Group Ill: O. Non categorisable: P. Non subculturable.

significant difference in total n u m b e r s o f bacteria isolated on the different m e d i a strengths (F2, 39 = 10.06, p = 0.003). In almost every case. the r e d u c e d strength m e d i u m gave superior recovery. However. there was no significant difference in total n u m b e r s o f bacteria isolated from s o u n d or d e c a v e d stone ( F 1 . 3 9 = 0. 13. p = 0-717).

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Investigations into the decay potential of mixed populations of bacteria, u n d e r laboratory, conditions, showed that bacteria present during the winter a n d early spring could cause greater weight losses than those present at other times during the year (Fig. 6). Mixed populations from decayed stone caused higher weight losses than those from sound. For stone incubated with both populations of bacteria there was a tendency for levels of soluble calcium to increase with weight loss (sound: r = 0-37, n = ! 14,p < 0-001, decayed: r -- 0.61, n = ! 13,p < 0.001).

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However, as shown in Fig. 7, with the p o p u l a t i o n from decayed stone the relationship was non-linear but tended to reach an upper limit of 0-60 mg ml -~ at a weight loss in the region of 9 mg. The relationship between weight loss and pH was also non-linear but weight losses tended to be higher when pH was lower. This tendency was observed with populations from sound (r = -0.364, n = 114. p < 0-001) a n d decayed (r = -0-45, n = 113, p < 0.001) stone. The relevant scatter plots can be seen in Fig. 7. W h e n the weight loss data for both populations from s o u n d a n d decayed stone were c o m b i n e d , 43-3% of the variation in weight loss could be explained in terms of soluble calcium. However, the r e m a i n i n g 56.7% of the variation could not be a c c o u n t e d for by the parameters measured in the experimental procedure,

DISCUSSION Any investigation into the possible role of heterotrophic bacteria in the decay of stone requires an insight into the nature of mixed p o p u l a t i o n s a n d how they may change in a temperate climate. T h e survival of bacteria is d e p e n d e n t u p o n a n u m b e r of factors a n d temperature a n d levels of moisture in particular can be critical. These factors will be subject to change on a seasonal basis a n d the nature of the bacterial flora can be expected to vary. Total bacterial counts, including viable a n d non-viable cells, were not

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(a) found to significantly differ on a seasonal basis. However. viable cell counts showed significant variation indicating varying numbers of nonviable cells were counted in the direct count procedure. At the particular site studied, both the direct and viable count procedures showed sound stone consistently supported higher numbers of bacteria than decayed. Earlier work by Lewis et al. (1985) found the reverse to be true. However. more recent work by the authors of the current study have found that sound stone supports higher numbers of bacteria than does decayed at a range of sites. Viable counts of bacteria showed a significant seasonal variation in terms of numbers and types. During the cooler, wetter winter and spring months higher numbers of bacteria were recovered. These were found to be both Gram-negative (e.g. Flavobacteriurn/Cytophaga. Acinetobacter) and Gram-positive (e.g. Corvnehacterium. Micrococcus) bacteria. In the

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drier summer months only Gram-positive forms were .found. predominantly Bacillus. This genus is particularly suited to unfavourable conditions due to its ability to produce resistant endospores. Previous work by Lewis et al. (1987) conducted during spring at Tintern Abbey. a monument in South Wales. found predominantly Gram-negative forms, particularly Flavobacterium and Pseudomonas. Strength of medium was found to have a significant effect upon recovery. The lowest strength of medium proved superior. Increasing the levels of carbon in the form of sodium acetate reduced recovery. However. the percentage of non-subcuiturable organisms was higher with the reduced strength medium, indicating there are a large n u m b e r o f bacteria on in-situ stone as yet unstudied due to their inability to grow on

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(a) conventional artificial media. It is therefore probable that high nutrient levels have an inhibitory, effect upon the cells. The effect of season on the nature of the bacterial population was found to vary its potential to cause damage to stone under laboratory conditions. Weight losses were higher during the winter and spring months when Gram-negative organisms were present. Weight losses were reduced when Gram-positive organisms became dominant during the summer and autumn months. These observations suggest that the qualitative nature of the bacterial population and not just the total number present is important in determining the potential of the population to cause decay. Populations of bacteria from decayed stone consistently caused higher weight losses than those from sound. The reason for this is as yet unclear. It is possible that the bacteria from decayed stone are more

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suited to metabolic activity under richer nutrient conditions than those from sound due to the higher levels of organic matter on decayed stone. Their survival and metabolism, and thus their ability to cause decay, may therefore be superior under the relatively rich nutrient conditions of the laboratory medium. This highlights the need for more realistic experimental conditions, especially with regard to nutrient levels. Alternatively. decayed stone may support the growth of organisms with greater potential for decay of the stone structure by products of their metabolism, whereas sound stone does not. Further work is required to determine whether the differences in the populations on sound and decayed stone are a cause or a result of damage to stone by other agencies. However. this study has shown that the quantitative and

62

S. Tavler. E. May

qualitative nature of the heterotrophic bacterial population on sandstone does vary, seasonally and is thus likely to be linked closely to changes in temperature and moisture levels associated with a temperate climate. The degree of damage to stone caused bv bacteria may thus be indirectly dependent upon season. This must be considered in any programme designed to prevent the deterioration of stone caused by heterotrophic bacteria.

REFERENCES Buchanan. R. E, & Gibbons. N. E. (Eds) (1974). Bergey~ Manual of Determinative Bacteriologo', Waverly Press Inc., The Williams & Wilkins Company. Baltimore. Cowa n. S. T. & Steel. K. J. (1977). Manual for the ldentO~cation of Medical Bacteria. Cambridge University. Press. Cambridge. Cruickshank. R.. Duguid. J. P., Marmion, B. P. & Swain, R. H. A. 11975). Medical Microbiology, Vol. II. Longman Group Ltd.. Churchill Livingstone. Edinburgh. London & New York. Eckhardt, F, E. W. (1985). Solubilisation, transport and deposition of mineral cations by microorganisms- efficient rock weathering agents. In The Chemist~" of Weathering. ed. J. I. Drever. D. Reidel. Dordrecht. pp. 161-73. Eckhardt, F. E. W. (1988). Influence of culture media employed in studying microbial weathering of building stones and monuments by heterotrophic bacteria and fungi. In 6th International Congress of Deterioration and Conservation of&one. Supplement Vol. Nicholas Copernicus University. Torun. pp. 71-81. Lewis. F. J., May, E. & Bravery. A. F. (1985). Isolation and enumeration of autotrophic and heterotrophic bacteria from decayed stone. In Proceedings of the 5th International Symposium on the Deterioration and Conservation of Stone. Vol. 2. Presses Polytechniques Romandes. Lausanne. Switzerland. pp. 633-42. Lewis, F. J., May, E., Daley, B, & Bravery, A. F. (1987). The role of heterotrophic bacteria in the decay of sandstone from ancient monuments. In The Biodeterioration of Constructional Materials, ed. L. G. H. Morton. The Biodeterioration Society, occasional publication No. 3, Lancashire Polytechnic, UK, pp. 45-53. Lewis. F. J., May, E. & Greenwood, R. G. (1988). A laboratory method for assessing the potential of bacteria to cause decay of building stone. In Proceedings of the 6th International Symposium on Deterioration and Conservation of&one. Supplement Vol. Nicholas Copernicus University, Torun. pp, 48-58. May, E. & Lewis, F. J. (1988). Strategies and techniques for the study of bacterial populations on decaying stonework. In Proceedings of the 6th International Symposium on Deterioration and Conservation of Stone, Supplement Vol. Nicholas Copernicus University, Torun. pp. 59-70. Paine, S. G., Linggood, F. V.. Schimmer, F. & Thrupp, T. C. (1933). The

The seasonalitv 01"heterotrophic bacteria

on

.~and.~tone

63

relationship of microorganisms to the decay of stone. Philosophical Transactions o1"the Royal Socie~'. B222, 97-127. Pringsheim, E. G. & Robinson. C. F. (1950). Observations on two very large bacteria. Ca~'ophanon latum Peshkoff and Lineola Ionga (nomen provisorum). Journal of General Microbiolo~,. 4, 198-209. Rosvall, J. (1986). Air Pollution and Conservation: Safeguarding our Architectural Heritage. Workshop. Rome. October 1986, University of Gothenburg. Shewan, J. M., Hobbs, G. & Hodgkiss. W. (1960). A determinative scheme for the identification of Gram-negative bacteria with special reference to the Pseudomonaceae. Journal of Applied Bacteriology'. 23, 379-90. Webley, D. M. (1963). The microbiology of rocks and weathered stones. Journal of'Soil Science. 14, 102-12.

DISCUSSION WITH REVIEWERS Q. Do you have any data to suggest the relevant i m p o r t a n c e ofbacterial attack on stone as c o m p a r e d to fungal, algal, or lichen attack'? U n d e r what circumstances would you expect one, or more, to predominate? A. O u r work has been mainly restricted to bacterial activity, particularly the heterotrophs, but our electron microscope studies have indicated that fungi a n d algae are both present in the microbial c o m m u n i t i e s , especially on the surface of decaying stone. We have detected fungal h y p h a e penetrating down several centimetres into the blocks. Sally Tayler's work has indicated that moisture levels are of critical importance in exerting selective pressure on the microbial population. Dry weather leads to a p r e p o n d e r a n c e of Gram-positive bacteria and some fungi. Interestingly, this p o p u l a t i o n appears less aggressive in laboratory, tests with stone discs. Q. In the conservation field, as you are probably aware, m o n e y is always limited a n d conservators need to address the most deleterious e n v i r o n m e n t a l aspects first in a conservation effort, C a n you give any evidence or i n f o r m e d o p i n i o n s as to the rela'tive i m p o r t a n c e of biodeterioration as c o m p a r e d to acid rain, wind erosion, a n d water damage? A. This is a difficult question a n d at the heart o f t h e biologist's problem to get the message across. We feel that biodeterioration m e c h a n i s m s play some part but clearly the scale is difficult to assess. Perhaps the activity of aggressive m i c r o o r g a n i s m s accentuates the effects of the chemical a n d physical agencies. Currently, we are c o n d u c t i n g field trials with blocks i m p r e g n a t e d with decay species to see if the process is accelerated. Q. What were the controls for m e a s u r i n g weight loss in stone during

64

X Yavler. E. May

winter a n d s u m m e r m o n t h s ? Are the weight losses attributable to just the microbes or was weather a factor'? A. O u r experiments are all laboratory, tests where weather is excluded as a factor. We are assessing the capacity of the p o p u l a t i o n prevalent at that time o f year to bring about a weight loss in stone discs in a culture system. T h u s the weight losses are reflecting the potential of the microbial c o n s o r t i u m to do damage. Losses are greatest in winter, at a time w h e n w e t / d ~ a n d freeze/thaw regimes are also dominant. D a m a g e agencies at this time seem to be in phase.