Microbiological analysis of mortars from the church of San Juan del Mercado at Benavente, Spain

ELSEVIER

The Scienceof the Total Environment 167(1995) 221-229

Microbiological analysis of mortars from the church of San Juan de1 Mercado at Benavente, Spain G. Arroyo* a, I. Arroyob, C. Vivar” aDepartamento

de Microbiologia II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, bInstituto de Conservation y Restauracion de Bienes Cultwales, Greco 4, 28040 Madrid, Spain

Spain

Abstract Two relatively recent techniques, epifluorescence microscopy and flow cytometry have been applied to the study of microorganisms in the mortars and frescoes of the church of San Juan de1 Mercado, Benavente, Spain. These results have been compared to those obtained with conventional techniques, the former showing clear advantages over the latter. Thus, with the aim of obtaining data, not only on the nature of the mortars themselves, but also on the metabolic products of the microorganisms, SEM-EDX and FT-IR analyses have also been carried out and considerable amounts of sulphates and nitrates have been detected. These results, taken together with the high microorganism count lead to the conclusion that the microorganisms possess high metabolic activities which necessarily influence the physico-chemical properties of the mortars. Keywords:

Mortars; Microbiological analysis;Epifluorescencemicroscopy;Flow cytometry; SEM-EDX, FT-IR

1. Introduction The Church of San Juan de1 Mercado de Benavente (Zamora, Spain) possesses three naves crowned by three semi-circular apses. Its construction was commenced at the end of the 12th century, being left, however, unfinished until some years later. Because of this, the lower part of the church is constructed with masonry (sandstone) while the upper part has been continued in brickwork at different heights, with the area covering the central nave raised above the other lateral

*Corresponding author.

naves. In the central apse, a series of paintings from the 15th century can be found as well as a fragment of a painting from the 16th century in the right-hand lateral nave (Heras Hernindez, 1973). In this study, we have investigated the mortars in the joints and in the mural painting

within the church both of which have been subject to deterioration. In recent years, there has been a growth in the use of the direct counting method employing fluorescent stains and epifluorescence microscopy, enabling the determination of the number of bacteria present in water, soils, etc. The technique has undergone various modifications with respect to the types of stains, filtration membranes

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(Zimmerman and Meyer-Reil, 1974; Jones and Simon, 1975) and microscopes used (Dayley and Hobbie, 1975; Hobbie et al., 1977; Hobbie, 1979). It is now believed that the traditional method of counting bacteria by the dish-dilution method does not, in fact, yield the true total number of living bacteria present. As a preliminary analysis, the aim was to study the degree of microbial contamination in the various mortars and mural paintings in the church, at the same time as introducing new techniques for bacteria counting in deteriorated stone materials which have been used successfully in other scientific areas. To this end, one of the objectives of this study has been to perfect the direct counting method of microorganisms using epifluorescence. Using this technique, the degree of contamination of the mortars, the total number of microorganisms present and the proportion that are metabolically active can be determined. As well as this, the diversity of the microbial communities has been determined by flow cytometry. For purposes of comparison, microorganism counts have also been carried out using dish-dilution techniques. The efficiency of the new method can thus be assessed. In addition to the counts, SEM-EDX analysis was carried out to find the composition of the different mortars and to establish a relation between the composition and the degree of contamination. Finally, the mortars were analysed using IT-IR in order to identify some of the products resulting from microbial alteration or metabolism which might reasonably be expected to exist in samples where the level of contamination is high. 2. Materials

and methods

2.1. Sampling

Samples were taken in aseptic conditions from 10 different points (Fig. 1) by scraping off the mortar from the joints with a scalpel and transporting in sterilized Petri dishes. The same method was used for the murals, using samples from the areas where the paint had come away so as to cause the least possible damage to the

167 (1995)

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original painting (NORMAL 3/80). The sample points and their characteristics are shown in Table 1. 2.2. Microorganism counts

The counts were carried out using two different methods, epifluorescence microscopy and directdish counting. For this, 2 g of each sample were weighed and placed in 18 ml of Tryptone Soy Broth, decimal dilutions being made subsequently. The first dilutions of each sample were incubated for 24 h at 30°C. The epifluorescence microscopy technique was applied using the AODC method (Hobbie, 1979) as follows: the samples were stained with a 0.2% concentration of fluorescent orange acridine, using 1 ml for each 10 ml of sample, for 5 min, after which 2 ml of each sample were filtered through Nucleopore filters with a pore size of 0.20 pm and a diameter of 25 mm. These were placed in a holder and a drop of immersion oil placed upon them after which they were examined by epifluorescence microscopy. In order to enhance the contrast and avoid autofluorescence, the filters were stained for several hours with a 2% solution of black stain (lrgalan Black, Merck) with 0.1% of acetic acid, and were washed twice with sterilized distilled water. The Nucleopore membranes are made of polycarbonate and have the advantage of a more uniform pore size and a smoother surface compared with those of cellulose where some bacteria remain in the interior of the filter and are unable to be detected. The bacteria were counted from 25-50 areas selected at random. The arithmetic mean (X) is calculated and multiplied by a factor F which is expressed in g/sample and is dependent on the surface of the filter and the quantity filtered. For filters with a diameter of 25 mm: F = No. of areas in the filter 25 511.19 No. of ml filtered = 2 No. microorganisms/ml = X; X x F = No. microorganisms/g The existence of different microbial communities was determined through flow cytometry. The samples filtered through nylon membranes were analysed in two different ways: applying thermal

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Fig. 1. Location of the different samples in the schematic representation of the church of San Juan de1 Mercado.

shock treatment ing any organic sample unheated. of fluorochromic propidium iodide

at 90°C for 15 min thus destroymatter present, and, leaving the To every 2 ml of sample, 0.2 ml stain were added, in this case, which intercalates with double-

chained nucleic acids, being excited at 488 nm and fluorescing at 620 nm. Five thousand cells from each sample (heated as well as the unheated ones) were passed through a flow cytometer (FACSAN FLOW cytometer;

Table 1 Samples and characteristics of stones and mortars Sample

Nature

Components

Sl s2 s3 s4 S5 S6 S7 S8 s9 SlO

Mortar Mortar Mortar Stone Mortar Mortar Mortar Mortar Mortar Mortar

Sand, lime and Sand and lime Sand and lime Sandstone Sand and lime Sand, lime and Sand, lime and Sand, lime and Sand, lime and Sand, lime and

sandstone

wallpainting wallpainting wallpainting wallpainting pigment layer

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Becton-Dickinson, San Jose) to determine the diversity and size of the microbial communities. The counts were carried out at 30°C for 24-72 h. The determination of the number of microorganisms was carried out both before and after 24 h of incubation in Tryptone Soy Broth. For Fourier-transformed infrared (ET-IR) analysis, the samples were broken up in potassium bromide ensuring a homogeneous mixture which was then submitted to a pressure of 8 tons/cm’ to form a KBr disc of 13 mm in diameter. The samples were then studied by ET-IR scanning from 4000 cm-’ to 600 cm-‘. For SEM analysis, the samples were submitted to the critical point by dehydration in acetone, submerging them in concentrations of 15, 50, 70 and 100% for 45 min. After this, they were brought to the critical point and dried by displacement of the acetone with liquid CO, which was subsequently eliminated by evaporation at 30-40°C. At the end of the process (about 45 min), the samples having attained total dryness were then coated with gold. Although EDX analysis does not require either the critical point or metal-coating, it is necessary that the samples be completely dry and fixed to the cylindrical aluminium holders with graphite.

different techniques are shown in Table 2. The metabolically active bacteria are seen in red and the live but inactive bacteria have a green colour. The different colours are due to the proportion of RNA/DNA; if this is high, it indicates an active metabolism whereas if it is low, an inactive metabolism is suggested. An orange colouration is observed for the dead bacteria and detritus. Orange acridine intercalates with nucleic acids producing red fluorescence with RNA and green fluorescence with DNA. It can be seen that the counts obtained through dish-dilution were considerably lower than those found with the epifluorescence technique. With the former method, only viable microorganisms were counted and only from those dishes which contained 30-300 colonies. Another factor could have been that some components of the contaminating microflora might have been sublethally damaged, causing difficulties with recuperation. This, in turn, would have led to underdevelopment yielding unreliable results. The epifluorescence technique possessed the advantage of being able to detect both active and inactive microorganisms, resulting in much clearer data on the bacterial communities subjected to stress. The differences between the two methods were perceptibly lower after incubation in Tryptone Soy Broth which enabled the revitalization of many of the damaged microorganisms and produced considerably higher counts for the active microorgan-

3. Results and discussion The results obtained

from the counts using

Table 2 Count of microorganisms Sample

Sl S2 s3 s4 ss S6 s7 S8 s9 SlO

Dish count cfu/g

Microscope count no./g Direct

Incubated

Total

Active

Total

3.02 x lo6 1.2 x 106 4.7 x 106 8.03 x 106 6.5 x lo5 5.3 x 105 7.3 x 105 8.2 x lo5 9.3 x 10s

5.33 x 7.27 x 2.36 x 1.67 x 3.80 x 2.07 x 2.04 x 1.53 x 1.71 x

4.2 x 1.7 x 5.8 x 9.3 x 1.2 x 8.9 x 1.6 x 1.4 x 1.8 x

lo6 106 106 106 106 lo6 lo6 106 106

Direct

Incubated

Active 108 lo* lo9 lo9 lo9 lo9 109 109 109

3.11 x 2.87 x 1.40 x 1.67 x 3.80 x 2.07 x 2.04 x 1.53 x 1.71 x

No./g, number of cells per gram; CFU/g, Colony Forming Units per gram.

108 lo* 109 lo9 109 lo9 109 109 109

280 880 120 160 30 70 50 50 300

1.3 x 9.2 x 8.8 x 7.3 x 2.3 x 3.1 x 9.7 x 6.8 x 6.5 x

107 lo5 106 106 lo3 10s 10s lo6 lo6

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isms using the epifluorescence technique. It can therefore be seen, that under the conditions used here, this technique has clear advantages over the conventional method. It is, in addition a more rapid process, allowing the necessary data on the degree of microbial contamination to be collected in a shorter time. Epifluorescence is also able to differentiate between distinct communities and detect shapes of bacilli or cocci, etc. as well as the

SSC\Side

Scatter

--->

SSC\Side

Scatter

--->

Fig. 2. Representation cytometry.

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groupings of these microorganisms. The presence of yeasts and moulds can also be detected with relative ease. The existence of different bacterial communities with the same morphological forms was determined through flow cytometry (Figs. 2,3). The different profiles of each graph represent the various bacterial communities present. In Fig. 2, it can be seen that for samples S5 and S6, the difference between the diversity and the size of

of cellular size @SC) with respect to the granular&y (SSC) of the different samples analysed by flow

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ii! Scatter --->

SSC*ide

2 la1 ssc\SidQ

162 Scatter

l& --->

....+ 1

ssc\si& Scatter -->

‘J3:ocIR2SNWl3

U3:-15

782

Lc

7G%L

%

S4 IP M

11 ssc\Side

56 IP

182 Scatter

‘J3:G%?SNUGl6

l?P --->

1 SSC\Side

Scatter

->

ii!&] ?Qo SShSide

U31 GRR25NWi7

M

ia2

l&i Scatter

tP-

I

---:

US:GhR2StW18 78%

SSCXSidc

Scattar

L

-->

Fig. 3. Representation of the granularity with respect to the fluorescence due to the IP (FL 2) of the different samples analysed by flow cytometry (heated samples).

these communities is very slight, whereas for samples S4 and S3, the difference is quite clear. In Fig. 3 (heated samples), the effect of heating on the viability of the microbial communities is shown. There does exist, however, thermoresistant groups, probably largely made up of spores. The method of observation using flow cytometry has proved itself to be a quick and statistically acceptable technique, allowing also the simulta-

neous recording of other useful physico-chemical parameters although these are irrelevant to this work (Back and Kroll, 1991; Vesey et al., 1993). It would be of great value to continue using this analytical technique in order to establish its efficiency definitively. The action of microorganisms upon mortars uses the same mechanism as those on any other stone monument, even though these may differ in

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their chemical composition (Caneva et al., 1991). Taking into account the results shown in Table 2, we can surmise that the microbial contamination was high in all the samples taken, with slight variation from sample to sample. The data in themselves are not indicative that a biological alteration is taking place in the mortar, but it is evident that amongst the microorganisms can be found both chemotrophs and heterotrophs, as both have guaranteed access to the necessary raw materials, some supplied by the mortar and others gathered from contact with the ground, dust, air, human activities, etc. (Eckhardt, 1985). One can conclude, therefore, that high levels of contamination favour and accelerate the acceleration of the vital activities of microorganisms (Lewis et al., 1987). Different microbial groups have been detected using SEM, including bacilli and cocci shapes, etc. which back up the evidence obtained from epifluorescence microscopy (Fig. 41. The results obtained from SEM-EDX showed that the mortars were composed of lime and sand. Large amounts

227

of Cl, Na and K were detected in sample S2 (Table 3) which attested to the outward mobilization of salts in the form of epifluorescences. The existence of halotolerant microorganisms in stone materials of monuments, and their influence on degradation processes within the material has previously been investigated (Schostak and Krumbein, 1992). Considerable amounts of S and P were also detected. The former probably arose from sulphates, whose presence was later confirmed by FT-IR, and the latter could have been derived from organic residues produced by the microorganisms. For sample S5, similar results were obtained to those of sample S2, although smaller quantities of Cl and P were present. Samples S9 and SlO furnished fewer additional elements over the original components of the mortar, suggesting that in this area, although appearing more deteriorated, having even lost paint in some places, the mortar itself is less altered than at other points in the church. It can be confirmed as a result of the FT-IR analyses, that the mortars of both the joints and

Fig. 4. SEM micrograph showing different types of microorganisms in a mortar.

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Table 3 Elemental composition of the samples obtained from SEM-EDX Sample

Sl s2 s3 s4 s5 S6 s7 S8 s9 SlO

SEM-EDX Si

Ca

Cl

Mg

S

P

K

Al

Fe

Na

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++

++ + + + +

+ + + + + + + + +

+++ + +++ -

++ + + + + + + +

+++ + + +++ + + + + +

++ +++ + + +++ + + + ++ ++

+ +++ + + + + +

+ + + -

-

the murals of the apses and the right-hand lateral nave (lime and sand) are mainly composed of quartz and carbonates. In sample S2 (Table 41, sulphates have been detected and in sample S3, sulphates and nitrates in significant quantities. Samples S6, S9 and SlO possess hardly any products of alteration and contain almost solely the components of the mortar itself. This, together with the data from SEM-EDX, shows that these mortars are less altered than the joint mortars even though microorganism counts were high in the former. The presence of sulphates and nitrates in samples S2, S3, S4 and S5 could be due to the presence of microorganisms of the S and N cycles which are known to oxidize ammonia first to nitrites and then the nitrates. Furthermore, nitrates are catalysts in the reduction of SO, to

sulphides and subsequently to sulphates. The microorganisms of the S and N cycles show synergic action (Bock et al., 1988). 4. Conclusions

The epifluorescence technique has given access to a simple and rapid way of obtaining microorganism counts, showing undoubted advantages over conventional counting methods, in addition to providing information on the active microbial population of a sample. Flow cytometry has enabled the distinction between different microbial communities and can be used in the identification of species once the reference curves have been obtained. Finally, through SEM-EDX and FT-IR tech-

Table 4 Composition of the mortars obtained using FT-IR spectroscopy Sample

Sl s2 s3 s4 SS S6 s7 S8 s9 SlO

FI-IR Silicates

Nitrates

Sulphates

++ + +++ +++ +++ +++ +++ +++ +++ +++ +++

++ ++ ++ +++ -

+++ +++ +++ +++ -

-

-

G. Ar~oyo et al. /Science of the Total Environment 167 (1995) 221-229

niques, it has been possible to ascertain the elemental composition of mortars as well as products from both the metabolic activity of microorganisms and the alteration induced by the action of microorganisms upon the support. Acknowledgements

We are grateful to the CICYT (project PAT 91-0722) under whose sponsorship this work has been carried out. We would also like to thank Angela Artega and Dolores Gayo for their invaluable technical help with the FT-IR, Felipe Montero with the SEM-EDX and Albert0 Alvarez with the flow cytometry. References Back, J.P. and R.G. Kroll, 1991. The differential fluorescence of bacteria stained with a&dine orange and the effects of heat. J. Appl. Bacterial., 71: 51-58. Bock, E., W. Sand, M. Meincke, B. Wolters, B. Ahlers, C. Meyer and F. Sameluck, 1988. Biologically induced corrosion of natural stones - strong contamination of monuments with nitrifying organisms. In: D.R. Houghton, R.N. Smith and H.O.W. Eggins (Eds.), Biodeterioration 7. Elsevier Applied Science, Barking, UK, pp. 436-440. Caneva, G., M.P. Nugari and 0. Salvadori, 1991. Biology in the Conservation of Works of Art. ICCROM, Rome, 182 PP. Daley, R.J. and J.E. Hobbie, 1975. Direct counts of aquatic bacteria by a modified epifluorescence technique. Limnol. Oceanogr., 20: 875-882. Eckhardt, F.E.W., 1985. Mechanism of the microbial degrada-

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tion of minerals in sandstone monuments, medieval frescoes and plaster. Proceedings of Vth International Congress on Deterioration and Conservation of Stone, Laussanne, Vol. 2, pp. 643-652. Heras Hernandez, D., 1973. Cat&logo Artistic0 Monumental y Arqueoldgico de la Di6cesis de Zamora. Zamora. Hobbie, J.E., 1979. Activity and bacterial biomass. Arch. Hydrobiol. Beih. Ergebn. Limnol., 12: 59-63. Hobbie, J.E., R.J. Daley and S. Jasper, 1977. Use of nucleopore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbial., 33: 1225-1228. Jones, J.G. and B.M. Simon, 1975. An investigation of errors in direct counts of aquatic bacteria by epifluorescence microscopy, with reference to a new method for dyeing membrane filters. J. Appl. Bacterial., 39: 1-13. Lewis, F.J., E. May, E. Daley and A.F. Bravery, 1987. The role of the heterotrophic bacteria in the decay of sandstone from ancient monuments. Biodeterioration of constructional materials. In: L.H.G. Morton (Ed.), Proceedings of the Summer Meeting of the Biodeterioration Society, pp. 45-54. NORMAL 3/80 (Normativa Manuffatti Lapidei CNR-ICR, Italia). Materiali Lapidei: Campionamento, 6 pp. Schostak, V. and W.E. Krumbein, 1992. Occurrence of extremely halotolerant and moderate halophilic bacteria on salt impaired wallpaintings. In: J. Delgado Rodrigues, F. Henriques and F. Telmo Jeremias (Eds), Proceedings of the 7th International Congress on Deterioration and Conservation of Stone. Laboratorio National de Engenharia Civil, Lisbon, Portugal, Vol. 1, pp. 551-560. Vesey, G., J.S. Slade, M. Byrne, K. Shepherd, P.J. Dennis and C.R. Fricker, 1993. Routine monitoring of Cvptosporidium oocystes in water using flow cytometry. J. Appl. Bacterial., 75: 87-90. Zimmerman, R. and L. Meyer-Reil, 1974. A new method for fluorescence staining of bacterial populations on membrane filters. Kiel. Meeresforsch., 30: 24-27.