merman et al. (1987), comparing the perfor- mances of Andersen two-stage microbial impactor with May three-stage glass impinger, indicate that although May ...
AEROBIOLOGIA
9(1993). I09- 115
Aerobiology: general and applied aspects in the conservation of art works CLAUDIA SORLINI
SU&IMAR Y. h7 this review, sources of microbial contamination o ~ r ~ J'uetors affecting airborne spoles survival, conditions that detemffne their composition and sanwling methods are considered. The relation between airborne microorganisms and microorganisms colonizing surfaces of art works is" also analyzed. Finally some advanced methods to detect and identify microorganisms responsible for alteration are suggested. Key words: Aerobiology, biodeteHoration, cultural heritage Claudia Sorlini, Dipartimento di Scienze e Tecnologie Agro-Alimentari, Ambientali e Microbiologiche, Universit~t degli Studi del Molise, Via Tiberio 2 I/A, 1-86100 Campobasso, Italy
INTRODUCTION The earth atmosphere contains a great number of solid particles which are, for the most part, of biological origin, but only a few are living organisms. These organisms consist mainly in spores of Mycetes, Bryophyta or Pteridophyta, bacteria, pollen grains, lichen propagules, musk gemmae, alga cells, vegetative cells, protozoa cysts and virus. Their diameter varies from 10 to 100 ~tm for pollens, from 2 ~tm to 10 ~tm for fungal spores, from 1 #m to 10 lam for bacteria and from 0.1 to 500 nm for viruses. Most vegetative cells are pigmented for a better protection against UV radiations. Cells and spores present in the atmosphere can live isolated or adhering to dust particles, water droplets or insects (Umbreit, 1962). It is of general opinion that microorganisms cannot grow in the atmosphere due to desiccation, solar radiations and low temperatures at high altitudes. This is the 9 (1993)
reason for the presence of only a few vegetative forms and many spores of mycetes (Olefir, 1985) that can remain in suspension even for some weeks (Gregory, 1973). The air is the most important vehicle for microorganisms to reach and form colonies on the surface of works of art. When, at the end of the fifties, research on biodeterioration of works of art began, this field was considered a branch of soil microbiology. It is not a surprise that Pochon, one of the most well known precursors of this type of research, was a soil microbiologist (Pochon et al., 1960; Pochon and Tardieux, 1962). In fact, many biodeteriorating microorganisms live in the soil and the first studies were conducted on works of art in the open air that were in direct contact with the soil microflora. Today, in the light of the knowledge acquired through research on works of art indoors, the idea that this topic is part of soil microbiology should be revised. The causes of air pollution 109
indoors and outdoors are numerous and they are not all related to soil.
MAIN SOURCES OF MICROBIAL AIR POLLUTION
Vegetative cells and spores present in the open air can come from: a) soil and plants, as a consequence of farming activities involving soil and biomass operations (Hellenbrand and Reade, 1992; Edwards et al., 1985), such as ploughing, harvesting, threshing, manure spreading etc.; b) marine and soft water aerosols due to waves and heavy rain on water surface that give rise to the formation of fine droplets carried into the atmosphere by the wind. The surface of seas and lakes being rich in microorganisms, the aerosols formed are particularly rich in microbial cells; c) land disposal, compost and solid waste treatment plants (Boutin et al., 1986); d) wastewater treatment plants, where aerosols are formed during sludge agitation. These aerosols are rich in microorganisms from intestinal origin; e) certain types of industrial production (Mattsby-Baltzer et al., 1989). Microorganisms present in close environment come from: a) outside air through doors and windows; b) dust brought in by employees and visitors; c) skin scales; d) breathing, cough and sneezes causing a dispersion of the microflora present in the throat, pharynx and even lungs. Spore and vegetative cell concentration doesn't decrease in indoor air, contrarily to open air, due to the difficulty of dispersion. The spores of many Mycetes are dispersed in outdoor and indoor air not only through man activities and passive mechanisms, such as vibrations and air draughts, but also through active propulsion mechanisms. In this case, the propulsion force is generated by the organism itself and can be determined by cell turgidity, hygroscopic twitching due to cell hydration or other 1 10
mechanisms not very well known yet. As for art works, these phenomena occur on frescoes, in particularly humid environments.
SPORES AND MICROORGANISMS DIFFUSION
Spores stay in the air until their fallspeed, which, if proportional to the square of their radius, is lower than the velocity of the air current that hold them in suspension. Various physical factors contribute to determine the movement of the particles in the air. In the countryside, most airborne spores are found either at ground level or on plants, at a height where the laminar layers of air are too quiet to cause their dispersion in large quantities. Under these conditions, the quantity of spores that can be found is of about 10,000 per cubic metre of air. Their deposition onto the ground, for spores of a certain dimension, is almost immediate. In fact, 90% are deposited within 100 metres from their source. The result is that the vertical concentration profile shows a decrease with altitude. They are rarely found above 2000 metres. Particles smaller than 1 gm do not sediment. The surviving capacity increases with humidity, adequate temperature (Lyon et al., 1984; Chakraverty and Sinha, 1985; Singh and Shrivastava, 1985; Moncada et al., 1986; Martinez et al., 1986) and favourable environmental conditions. The reduction of mycete spores during the winter is generally attributed to lower temperatures (Moncada, 1986; Martinez et al., 1986; Morey, 1990). Nevertheless some cases of increase in number with decrease in temperature have also been registered (Tilak and Saibaba, 1986; Machen et al., 1991). It seems that also the light of the sun affects the number of airborne spores; for example, the spores of Phytophtora infestans reach their maximum in AEROBIOLOGIA
late morning, those of CladospoHum, Ustilago and Alternaria in the early afternoon (Bansal and Mehrotra, 1988). Indoors, glass windows by filtering short wave length radiations, that are bactericide, prevent the reduction of spores. The survival of airborne spores is increased by their adherence to protective organic matter such as skins scales or saliva drops. Lighthart and Mohr (1987) have developed a dispersion model in dynamic atmospheric conditions. Lighthart and Kim (1987) have also proposed an interesting model describing the dispersion of airborne microbial droplets up to 30 metres from their sources taking into account every environmental parameter and using Pseudomonas syringae as viable cells. The main difficulty encountered by the authors was that of the dynamics of the processes affecting microbial survival over time, which depend on the characteristics of each.microorganism. Microbial death rates are a function of ambient temperature, relative humidity, atomisation degree and vary within species. Data available is rather scarce, except for solar radiation, and is limited to a few bacterial species (Poon, 1966; Poon, 1968; Lighthart and Kim, 1989). The study conducted by Dinter and Mueller (1988) on the survival of Salmonella using an aerosol chamber is of great interest in this context.
SAMPLING AND ANALYSIS Sampling methods have been reported extensively in literature (Tilak and Saibaba, 1985; Zimmerman et a/., 1987; Laflamme, 1992; Kang and Frank, 1989; Hellenbrand and Reade, 1992). We only intend, here, to give a brief outline. The selection of a proper sampling method depends upon the survey to be done, whether, for example, a count of microbial cells instead of inert particles is needed and also implies information on the level of contamination in the air to be sampled. 9 (1993)
The least precise method is certainly that of gravity collection or plate sedimentation where only the cells depositing on plate containing agar medium are counted. Particles with a diameter smaller than 1 p.m, not capable of depositing, are therefore excluded. The number of colonies growing are strongly correlated to particle size, time of exposure to air and to quiescence or disturbance of ambient air. Solid impingement is a very useful method for classification of particles by size, especially using a six stage Andersen sampler. Liquid impingement offers a wide counting range and is also very precise since it allows for cell separation from aggregates but is time consuming due to the dilutions needed prior to plating. It must also be said that, the addition of betaine, a low molecular-weight organic compound, to both collection liquid and the enumeration medium has an additive effect on the colony forming ability of airborne bacteria from 2.1% to 61.3% (Marthi and Lighthart, 1990). In other cases, in order to prevent fast growing species to inhibit the slow growing ones, a non-ionic surfactant (triton N-10) was used. This compound was incorporated in the agar medium of plates used in Andersen sampler and it gave better results than rose bengal (Madelm, 1987). Membrane filtration method also offers a wide counting range, but desiccation of the biomass in the airflow, bringing about the death of the most sensitive cells, is an inconvenient and therefore reduces considerably the sensitivity of the method. Palmgren et al. (1986) report that air sampling was done on Nucleopore filters and the total number of airborne microorganisms was determined by acridine orange staining and epifluorescence microscopy. They found a high correlation between viable counts, determined in media, and total count determined by microscopy, when the airborne flora was dominated by fungal spores, while a low correlation was found for airborne bacteria. 111
Laflamme (1992) reports the performance of a Reuter centrifugal sampler and establishes its validity in sampling both small subglobose spores of Penicillium viridicatum and large irregular spores of Altelvzaria alternata. Zimmerman et al. (1987), comparing the performances of Andersen two-stage microbial impactor with May three-stage glass impinger, indicate that although May sampler reports 82% of the Andersen sampler value, the correlation between the two samplers was good. Kong and Frank (1989) comparing Andersen six-stage sieve sampler and Reuter centrifugal air sampler (RCS) showed that RCS was able to recover most bacterial types since they were distributed over various particle sizes. When airborne fungi were collected, RCS recovered significantly lower amounts than the Andersen impactor. Some attempts to evaluate airborne microbial count avoiding traditional time-consuming culture tests have been done recently. Seal and Clark (1990) have used a computerised electronic equipment for the enumeration of airborne particles divided in eight size range (from 0 to 20 tam). They found that, in ultraclean conditions, the number of particles of 5-7 gm size range correlated with the number of bacteria carrying particles, while, when the room was turbulently ventilated, the particles between 3 and 15 gm correlated with bacteria carrying particles. Thus, for these authors the electronic particles count in the 0 20 gm size range may be used for the assessment of the microbiological quality of air. The count and composition of contaminating microflora can vary considerably according to sampling points, climatic conditions, etc.. Data reported in literature are extremely disparate for both fungal spores and bacteria (Olefir, 1985; Tilak and Saibaba, 1985; Singh and Shrivastava, 1985; Moncada et aL, 1986; Martinez et aL 1986; Abdel-Hafez, 1986; Reiss, 1986; Tilak and Saibaba, 1986; de Almeida, 1988; Hunter et al., 1988; Bansal and Mherotra, 1988; Mattsby-Baltzer, 1989). 112
A particular aspect of microbiological air pollution is that a large number of vegetative cells and spores belongs to infectious or toxigenic species as Sahnonella (Dinter and Mueller, 1988), Staphylococcus aureus, Pseudomonas aeruginosa (Cosentino et al., 1990). Many Mycetes such as Aspergillus flavus (Abdel-Hafez et al., 1986) and Aspergilhts parasiticus (Chakraverty and Sinha, 1985) produce aftatoxins and other species are also very toxic (AbdeI-Hafez et aL, 1986).
AEROMICROBIOLOGY ART
AND
WORKS
OF
There is a strong correlation between aerobiology and biodeterioration of works of art considering that the air, as we have seen earlier, is the main vehicle for the dispersion of microorganisms. However, not all airborne microbial particles are capable of growing on the surface of works of art bringing about their deterioration. This depends on the microorganism characteristics, the chemical composition of the substrate, the gas present in the air and on climatic conditions, especially relative humidity and temperature. Another important factor is the number and variety of microbial species present in the atmosphere. It is also possible that, even in cases of high levels of pollutants, works of art are not damaged due to low values in relative humidity and temperature, although some species, such as xerophilic fungi, can grow with low water activity (0.6), and psycrophilic can grow at low temperature. From a preliminary survey conducted on seventy papers on aeromicrobiology and on about one hundred on biodeterioration of stone artworks (Caneva et al., 1991; Locci, 1972; Paleni and Curri, 1972; Sorlini et al., 1982; Strzelczyk, 1981), a preliminary list of airborne microorganisms can be done, based on the frequency of their presence in air and on the AEROBIOLOGIA
stone surface of works of art. Table I shows that some species are frequent in the air as well as on works of art. Table L Frequency of fungi in the air and on the stone
surface of works of art (data fi'omliterature). Genus Aspergitlus Penicillium A hernaria Claclosporimn
A h"
Stone
++++
+++++
++++
+++++
++++
+++++
++++
+++++
Thricoderma
++
Rhizopus Fusarium Rhodotorula Toruhl Chaetonlium Stachybotris Plloma Alu'eobasidiunl
++ + + + +
veO, high; + + + + + + tow; + rare
+++++
+ ++ + + + ++
high: + + + meclium;
Studies on the correlation between microbiological pollution and biodeterioration of works of art are, for the moment, too scarce to allow conclusions. However, it is possible to say that large particles that transport microorganisms are more harmful insofar as they can sediment on the surfaces with greater facility and rapidity. On the other hand, the microorganisms with particular nutritional requirements, some of which are pathogens, cannot easily grow on works of art due to unfavourable conditions. The development of new time-saving techniques is necessary for a better evaluation of biodeterioration processes. ATP determination could be adopted for a quick enumeration of total viable airborne particles. This molecule is, in fact, a good index of the number of microorganisms. In this case air sampling is carried out with a liquid impinger and an aliquot of sus9 (1993)
pended microbial cells undergoes ATP extraction with appropriate reagents. ATP is determined by the amount of light emitted after addition of luciferine-luciferase enzyme system (Salusbery et aL, 1989). This technique is rapid and results can be obtained within a few minutes giving a global value of microbial pollution both for Schizomycetes and Mycetes. The only disadvantage is the lack of specificity. The identification of some airborne species in relation to their potential damaging effect on specific materials could be useful. For example, in indoor environment rich in cellulose (libraries, museums with tapestries or paintings, etc.) cellulolytic microorganisms, responsible for biodeterioration of these materials, could be identified even if other types of microorganisms, capable of growing on organic matter used in wood and cellulose manufactures, can also be damaging, in environments rich in wood. In environments with high relative humidity and temperature, algae capable of damaging frescoes and stone works can be investigated (Wee and Lee, 1980). Their identification can be done with enrichment culture or using Raman spectroscopy that allows for detection of chlorophyll in samples even at low concentration. More advanced genetic techniques could be tested for the identification of airborne microorganisms that are particularly deteriogens for stone works like Thiobacil&s thioparus and T. thioxidands. Polymerase Chain Reaction, based on the amplification of defined fragments of DNA, is an interesting technique for the identification of microorganisms, even if they are present in very small number. This technique is highly sensitive and specific and assures an answer within few hours as compared with weeks using traditional techniques. The knowledge of the number and species of airborne microorganisms living outdoors or indoors is essential for the prevention of the biodeterioration of works of art. A decrease in | ]3
m i c r o b i a l c o n t a m i n a t i o n can b e o b t a i n e d using e l e c t r o s t a t i c air purifiers. M a x i m u m
levels o f
m i c r o b i a l air p o l l u t i o n s h o u l d b e e s t a b l i s h e d b y e x p e r i m e n t a l tests in o r d e r t o k e e p t h e d e t e r i o r a t i o n p r o c e s s u n d e r c o n t r o l a n d to m i n i m i z e t h e risk. I n d e e d , a z e r o risk can b e a t t a i n e d o n l y t h r o u g h sterile c o n d i t i o n s w h i c h a r e u n t h i n k a b l e o u t d o o r s a n d in i n d o o r e n v i r o n m e n t s o p e n to public. It m u s t also b e e m p h a s i z e d that plants, that a r e r e s p o n s i b l e f o r air p o l l u t i o n (such as p l a n t s for industrial and urban waste water treatment a n d so on), s h o u l d b e l o c a t e d far a w a y f r o m w o r k s of art in o r d e r to a v o i d , b e s i d e s l a n d scape degradation, biological degradation.
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