Microbes Environ. Vol. 23, No. 4, 293–298, 2008 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME08521

Enumeration of Sulfur-Oxidizing Microorganisms on Deteriorating Stone of the Monuments, †

XIANSHU LI1, HIDEO ARAI2, ICHITA SHIMODA3, HIROSHI KURAISHI4, and YOKO KATAYAMA1* 1Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3–5–8 Saiwai-cho, Fuchu-shi, Tokyo 183–8509, Japan; 2Emeritus Researcher, National Research Institute for Cultural Properties, Tokyo, 13–43 Ueno Park, Taito-ku, Tokyo 110–8713, Japan; 3Department of Chemistry and Engineering, Waseda University, 3–4–1 Okubo, Shinjuku-ku, Tokyo 169–8555, Japan; and 4Ex-Tokyo University of Agriculture and Technology, 3–5–8 Saiwai-cho, Fuchu-shi, Tokyo 183–8509, Japan (Received June 5, 2008—Accepted July 31, 2008—Published online October 1, 2008)

Annual change in the density of sulfur-oxidizing microorganisms on sandstone was enumerated to know the effects on the deterioration of stone materials of the Angkor monuments in Cambodia. Samples were obtained from total 12 stations at the , , and Phnom Krom temples between 1998 and 2007. Sulfur-oxidizing microorgan- isms enumerated in a mineral salts medium supplemented with elemental sulfur as the sole energy source had a density of 101–105 MPN (g sample)−1. The sulfur-oxidizing microorganisms of the samples collected at Angkor Wat have tended to decrease in density since 2002; on the other hand, relatively constant values have been recorded in the sam- ples of Bayon and Phnom Krom. These results suggest that the sulfur-oxidizing microorganisms on the stone play an important role in the decay of the building blocks by excreting sulfuric acid. Key words: Angkor monuments, sulfur-oxidizing microorganisms, chemoorganotrophs, biodeterioration, stone weathering

The Angkor monuments in Cambodia were constructed of concrete15), were isolated from stone by Gugliandolo6). between the 9th and 13th centuries. Sandstone and laterite Surveys of organisms at the Angkor site made by the were the main building materials, and the outer surface of the Japanese Government Team for Safeguarding Angkor, buildings was usually covered with grey to yellowish brown UNESCO/Japan Trust Fund (JSA) since 1994, showed that sandstone that has higher porosity than red sandstone and cyanobacteria, algae, and lichens seem to cause the biodeteri- greenish greywacke20). The Angkor monuments were cata- oration of sandstone1,2). However, quantitative research on loged in the UNESCO World Heritage List in 1992, and inter- the biological deterioration has not yet been reported. The national cooperation has taken place because of severe dete- authors joined the JSA team in 1998 to specify the biodeteri- rioration. The Angkor monuments have been in the care of oration caused by bacteria. In this study, we focused on enu- the Authority for the Protection and Management of Angkor merating the cell density of sulfur-oxidizing microorganisms and the Region of (APSARA) since 1997. on the surface of stone materials because sulfuric acid, the Weathering of stone is a combination of physical, chemi- metabolic product of these microbes, is a strong corrosive cal, and biological processes. While biological agents such as agent. lichens, bryophytes, and higher plants are easily identified, the proliferation of microorganisms including bacteria, fungi, Material and Method and algae is difficult to estimate. Biodeterioration of stone Site description involves the production of inorganic and organic acids, the 11,12,21,22,24) Cambodia has a typical monsoon climate where the direction of formation of biofilms, and pigmentation . wind changes seasonally. Average rain fall is 200 mm per month in The microorganisms on stone have been analyzed using the wet season from May to October, and less than 50 mm per various procedures including microscopic observation, month in the dry season from November to April. Annual rainfall at enrichment and cultivation, and measurements of community the Angkor site is 1300–1500 mm per year9) and the average tem- 10) 7) perature is 25°C . The average pH of the rainfall observed in 1995 metabolism . Chemolithoautotrophic organisms of sulfur- 10) oxidizing and nitrifying bacteria have been detected on stone was 6.29 , not in the range of the definition for acid precipitation. Large animals that can be seen at the Angkor site are bats. In the surfaces of monuments. These bacteria excrete corrosive evening, about 1000 bats per day in Angkor Wat, and about 500 acids such as sulfuric acid and nitric acid which solubilize bats per day in Bayon were counted flying away in Oct. 200025). the minerals in stone. Sulfur-oxidizing bacteria of Thioba- Although the monuments are managed well at present and the cillus, important causative microorganism in the deterioration number of bats seems to be decreasing, bat droppings were still observed on the floor of the monuments in 2007. * Corresponding author. E-mail address: [email protected]; Sampling Tel: +81–42–367–5732; Fax: +81–42–367–5732. Samples were taken at 12 stations on the surface of walls, the † Part of the results reported in this paper has been presented in the base of pillars near the ground, and the entrance of monuments (Fig. Annual Reports on the Technical Survey of Angkor Monument, 1). Stone materials of Angkor Wat (samples AW1, AW2, AW3, Japanese Government Team for Safeguarding Angkor, from 1999 AW5, and AW7) and Bayon (samples BY2-1, and BY2-2) were to 2004. obtained mainly in the wet season from 1998 to 2004, 2006, and 294 LI et al.

Fig. 1. Sampling location of surface materials in Bayon, Angkor Wat and Phnom Krom.

days after the sampling. Enumeration of sulfur-oxidizing microorganisms An aliquot of sample was pulverized with a sterile spatula, and then diluted (1:10) in a sterilized 0.85% NaCl solution. The suspen- sion was mixed thoroughly and then diluted ten-fold to 10−5 for inoculation. The pH of the suspension was measured in the samples obtained in 2006 and 2007. In the Angkor Wat and Bayon samples, the pH was in the range of 6.8–7.9 and in the Phnom Krom samples, 6.4–6.8. Sulfur-oxidizing microorganisms were enumerated based on the MPN method. The liquid medium23) used here was for chemolithoautotrophic sulfur bacteria with minor modifica- tions: KH2PO4, 3.0 g; (NH4)2SO4, 0.2 g; MgSO4·7H2O, 0.5 g; FeSO4·7H2O, 0.01 g; CaCl2·2H2O, 0.25 g; elemental sulfur, 10 g in 1000 mL of deionized water, pH 5.0 (WS5 medium). Elemental sulfur was sterilized separately by intermittent steaming for one hour every 24 hours three times. The diluted sample (0.5 mL) was inoculated to a 15 mL-volume test tube containing 4.5 mL of WS5 medium. The cultivation was conducted with reciprocal shaking at Fig. 2. Photograph of north side pillars at Angkor Wat showing seri- 30°C. Growth of sulfur-oxidizing microbes was confirmed by ous deterioration on the lower inner side. checking the decrease in the pH of the medium using a meter (Twin pH, Type B-212, Horiba Ltd., Kyoto, Japan) every week for two months. When the pH decreased from 5.0 to less than 4.4, the cul- 2007. All samples except AW3, which was collected from a deterio- ture was considered positive. rated wall, were collected from pillars near the ground (Fig. 2). Some samples of Phnom Krom (samples PK2 and PK3) and Bayon Enumeration of chemoorganotrophs (samples BYb, BYc, and BYd) were obtained from 2002 to 2004, Chemoorganotrophs were enumerated by the CFU method using 2006, and 2007. Samples for the dry season were obtained at AW1, one-tenth diluted Tryptosoy agar medium (E-MC83, Eiken Chemi- AW3, and AW5 in Jan. and at AW5 in Apr. 2002. cal, Tokyo, Japan) containing trypton, 1.5 g; soy peptone, 0.5 g; A small amount of surface material (approximately 0.4 g) was NaCl, 0.5 g; BactoTM agar (Bekton Dickinson, Sparks, MD, USA), obtained from a severely deteriorated area without significant cul- 15 g in 1000 mL of deionized water, pH 7.3. The diluted sample tural or artistic value. Samples were collected aseptically using a (0.1 mL) was spread onto the agar plate and cultivated at 30°C. Col- sterilized spatula onto medical paper which was sterilized in onies formed on the plate were counted during 2 to 4 weeks and the advance under UV, and then placed in a sterilized test tube. Incuba- CFU values were obtained from the mean for three plates. tion for the enumeration of microorganisms was started within 3 Sulfur-Oxidizing Microbes on Sandstone 295

Amount of total organic carbon (TOC) and water content samples AW5 and AW7, 101–103 MPN (g sample)−1 was The TOC of surface materials of stone ground into powder with a measured from 1998 to 2001 and in 2007, and have not been mortar was measured with a non-dispersive infrared gas analyzer detected in other years (Fig. 3b). using 1.0 g of potassium peroxodisulfate and a modified oxidation 16) In the dry season, the density of chemoorganotrophs was vessel . Water contents of some samples collected in Aug. 2007 103–105 CFU (g sample)−1 for samples AW1, AW3, and were determined in our lab as follows: sample AW1, 0.73%; sample 2 −1 AW2, 1.14%; sample BYd, 1.39% and sample PK3, 1.65%. Water AW5 collected in Jan. 2002, and 10 CFU (g sample) for contents of other samples were not determined because of the mini- sample AW5 collected in Apr. 2002. Samples AW1 and mal amount of sample taken, however almost all samples obtained AW3 collected in Jan. 2002 showed sulfur-oxidizing micro- seemed similar to the samples for which water content was deter- organisms at a density of 102 MPN (g sample)−1. mined. Therefore, all densities were recognized as dry. The TOC of the samples collected at Angkor Wat in 2007 was as follows: 2.2 mg C g−1 for AW1 and 2.1 mg C g−1 for Results AW2. Angkor Wat Bayon Most of the lower part of pillars, up to about 40 cm from The presence of biofilms of microorganisms was observed the base, have seriously deteriorated showing thinning espe- by naked eyes at all locations except BY2-1 and BY2-2. cially the pillars on the inner side of the gallery (Fig. 2). No Similar to Angkor Wat, bat droppings were seen at the sam- obvious biofilm was observed on the surface at any of the pling stations. sampling stations. BY2-1 and BY2-2 were on the lower part of the pillars at Colonies of bacteria and fungi were observed on the agar the entrance of the tower. A density of 104–106 CFU (g sam- medium for samples collected since 1998. The density of the ple)−1 was enumerated in all samples, higher than the values chemoorganotrophs varied from 103 to 106 CFU (g sample)−1 for Angkor Wat (Fig. 4a). The density of sulfur-oxidizing in the wet season (Fig. 3a). On the other hand, the cell den- microorganisms in Bayon was in the range of 101–105 MPN sity of sulfur-oxidizing microorganisms varied with each (g sample)−1 (Fig. 4b). The TOC of BY2-1 and BY2-2 was year and/or each station. In the case of sample AW1, sulfur- 2.2 mg C g−1, and 0.5 mg C g−1, respectively. oxidizing microorganisms have been detected each year at a Samples BYb, BYc and BYd were collected from the wall density of 101–105 MPN (g sample)−1. However in the case of of the inner gallery where both biofilms with various pig-

Fig. 3. Cell density of chemoorganotrophic (a) and sulfur-oxidizing (b) microorganisms on the surface of monuments of Angkor Wat in the wet season from Sep. 1998 to Aug. 2007. Sulfur-oxidizing microorganisms were not detected in 2000. No analysis was done in 2005. Positive code of MPN for sulfur oxidizer was based on the decrease of medium pH to 4.4 from initial pH 5.0. The detection limit for MPN and CFU was 70 cells (g sample)−1 and 330 cells (g sample)−1 respectively. Error bars in Fig. 3a represent the standard deviation of data obtained in triplicate. Error bars in Fig. 3b represent 95% confidence limits. 296 LI et al.

Fig. 4. Cell density of chemoorganotrophic (a) and sulfur-oxidizing (b) microorganisms on the surface of monuments of Bayon in the wet season. Samples BY2-1 and BY2-2 were enumerated from Sep. 1998 to Aug. 2007 and BYb, BYc, and BYd, from Aug. 2003. Sulfur-oxidizing microorgan- isms were not detected in 2000. No analysis was done in 2005. Positive code of MPN for sulfur oxidizer was based on the decrease of medium pH to 4.4 from initial pH 5.0. The detection limit for MPN and CFU was 70 cells (g sample)−1 and 330 cells (g sample)−1 respectively. Error bars in Fig. 4a represent the standard deviation of data obtained in triplicate. Error bars in Fig. 4b represent 95% confidence limits. mentations and serious deterioration were observed. The Freshly quarried sandstone obtained as a negative control TOC of BYc and BYd was 9.6 mg C g−1, and 7.8 mg C g−1, from the Angkor site in 1998 showed no decrease in pH in respectively. Sulfur-oxidizing microorganisms were in the the WS5 medium. Therefore, the decrease in pH of sandstone range of 102–104 MPN (g sample)−1 (Fig. 4a, b). in the WS5 medium was considered to be caused by the dete- riorated sample. Uchida and Suda18) reported 46.8 mg g−1 of Phnom Krom sulfate in deteriorated sandstone at the Angkor site, about The stone materials of Phnom Krom showed exfoliation, 400 times higher the level in freshly quarried sandstone (0.119 and most of the relief was detached. A large number of bats mg g−1). Arroyo et al.3) showed that a considerable amount of have been observed flying away from one of the buildings in sulfate found on the decayed stone was produced by sulfur- the evening, and many bat droppings were found inside, indi- oxidizing bacteria. Therefore, the high sulfate concentration cating the building is a roost. Sample PK2 was collected in deteriorated sandstone at the Angkor site was considered from lower part of a pillar at the entrance of a small temple to be produced by sulfur-oxidizing microorganisms. located close to the east gate. Sample PK3 was collected The source of sulfur may be bat droppings at the Angkor from the surface of the inner wall of the temple. site. Hosono et al.8) used an isotope to study the sandstone of The density of chemoorganotrophs was in the range of the Angkor monuments, and found that the S (sulfur) and P 104–106 CFU (g sample)−1 and numbers were relatively sta- (phosphorus) components of sandstone are mainly from bat ble. The density of sulfur-oxidizing microorganisms was in droppings. As mentioned above, the average pH of rain water the range of 102–104 MPN (g sample)−1 (Fig. 5a, b). Both of at the Angkor site was 6.29, not in the range of acid precipi- them were detected every sampling year, and no significant tation. Therefore, the high concentration of sulfate found in difference was seen between the two samples. The TOC of deteriorated stone was considered to originate from biologi- PK2 and PK3 was 0.9 mg C g−1, and 3.0 mg C g−1, respec- cal change and not from a reaction of sulfide contained in the tively. atmosphere. These results indicated that the bat droppings on the monuments are one of the sources of salts containing Discussion sulfur. Our results indicated relatively constant cell densities of In this study, we investigated the annual change in the cell sulfur-oxidizing microorganisms in Bayon and Phnom density of sulfur-oxidizing microorganisms inhabiting the Krom. However, sulfur-oxidizing microorganisms in sample surface of deteriorated sandstone and producing sulfate. AW5 at Angkor Wat had tended to decrease in density since Sulfur-Oxidizing Microbes on Sandstone 297

Krom was found. There was little or no significant change in the density of chemoorganotrophs at the Angkor site, with values in the range of 103–106 CFU (g sample)−1. Tayler and May17) reported a similar density of chemoorganotrophs, 104–106 cells g−1, at Portchester Castle in southern England, although the medium used was different. In the dry season, the density of chemoorganotrophs was lower than that in the wet season although no obvious differ- ence was found in the density of sulfur-oxidizing microor- ganisms. Uchida et al.19) showed the water content of sand- stone to be up to 14% in the wet season and 3% in the dry season. Water is an important environmental factor both for the growth of microorganisms and for the diffusion of salts by capillary action. Therefore, the low water content of sand- stone in the dry season was correlated to the low density of microorganisms. Tayler and May17) studied the seasonal vari- ation of microorganisms on decayed stone and reported higher numbers of bacteria in the wetter winter than dry sum- mer. However, the lower water content in the dry season was also considered to cause the concentration of sulfuric acid in stone and lead to much more serious deterioration. A novel species of sulfur-oxidizing bacteria using thio-

Fig. 5. Cell density of chemoorganotrophic (a) and sulfur-oxidizing sulfate as an energy source was isolated from volcanic depos- −1 (b) microorganisms on the surface of monuments of Phnom Krom in the its in Miyake-Jima (TOC content, 4 mg g ; water content, wet season from Aug. 2002 to Aug. 2007. No analysis was done in 3.6%)13). Moser and Olson14) and Badawy4) reported 103 cells 2005. Positive code of MPN for sulfur oxidizer was based on the g−1 of autotrophic sulfur-oxidizing bacteria in soils. decrease of medium pH to 4.4 from initial pH 5.0. The detection limit 5) for MPN and CFU was 70 cells g−1 sample and 330 cells g−1 sample, Chapman also reported that the density of sulfur-oxidizing respectively. Error bars in Fig. 5a represent the standard deviation of bacteria (neutrophilic Thiobacillus spp.) was in the range of data obtained in triplicate. Error bars in Fig. 5b represent 95% confi- 102–103 cells g−1, the bacteria being found in 84% of agricul- dence limits. tural soil samples. Therefore, the sulfur-oxidizing microor- ganisms can be detected easily in many kinds of soils and 2002. The density of sulfur-oxidizing microorganisms on the even in volcanic environment. However, on the sandstone stone surface was considered to have a relationship with the surface the density of sulfur-oxidizing microorganisms was cleaning of bat droppings at the Angkor site. Till 1999, there considered to differ according to location. For example, sul- were many bats living at the Angkor site and bat droppings fur-oxidizing microorganisms have been detected in Bayon were observed in the temples. With the increasing number and Phnom Krom, but varied by location in Angkor Wat. of visitors, APSARA that constructed in 1995 conducted a This may have a relationship with environmental factors thorough cleaning of Angkor Wat. The bat droppings on the such as the infiltration of rain water through crannies in the ground were swept away and new droppings were cleaned wall, changes in humidity and temperature caused by sun- quickly. This may explain the decreasing trend seen in light, and the supply of organic materials on the stone. How- Angkor Wat especially at stations AW5 from 2001. The den- ever at this time, details are still unknown. sity of sulfur-oxidizing microorganisms in the samples col- Our data on the annual change in microbial density clearly lected in Bayon increased after 2002 and remained relatively showed that microorganisms with sulfur-oxidizing activity stable at high levels [around 105 MPN (g sample)−1], differ- inhabited the stone and should be considered to contribute to ent from the density values of Angkor Wat. Bat droppings in the deterioration. Bayon were cleared by APSARA similar to Angkor Wat from 2003 to 2007. The TOC was approximately 4 times Acknowledgements higher in the Bayon (samples BYb, BYc, and BYd) than Angkor samples. The high TOC value may be due to This research was supported in part by the Japanese Government Team for Safeguarding Angkor and a Grant-in-aid for Scientific chemolithoautotrophs, chemoorganotrophs, algae and lichen Research (No. 19251001) from The Ministry of Education, Culture, on the surface of the stone, providing organic and inorganic Sport, and Technology of Japan. We thank the APSARA Authority substances and water and supporting the sulfur-oxidizing and Ministry of Environment, Kingdom of Cambodia, for sampling. microorganisms. In Phnom Krom, a large number of bats We also thank Dr. Ken-ichiro Suzuki, Director of NITE Biological have been observed flying away in the evening from one of Resource Center, Japan for valuable advice. the buildings where samples were not collected. However, no obvious bat droppings were observed on the floor of the References sampling location. In addition, algae and lichen were not 1) Arai, H. 1994. Studies on biodeterioration and its control in Angkor observed at the sampling site. No clear reason for the contin- sites (Part 1)—investigation of main organisms related to deteriora- uous detection of sulfur-oxidizing microorganisms at Phnom tion of stone materials. Insect and Fungus Damage to Cultural Proper- 298 LI et al.

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