## 4 Biochemical Functional Approaches  
Both growth on surface as biofilms and specific metabolic activity (i.e., acidification) of the active microorganisms are important information for the understanding and prediction of damage to the materials of cultural heritage. Earlier investigations suffered from the isolation of limited numbers of cultured bacteria in pure cultures from the general community of microorganisms due to the technical means available. There were several early attempts to quantify the activity of the microbiome based on chemical reactions [30,88]. Detection and quantifying ATP can be used as an estimation of biological activity on surfaces like paper, paintings, or other materials in swab samples using the commercially-available products of luminometers [89,90]. However, the active microbial groups and members on cultural heritage may not be the directly destructive ones responsible for the damage caused to the cultural heritage and the general purpose-driven biochemical analysis of the samples from cultural heritage only provides limited insights because a close relationship between the destruction and specific microbial group cannot be established. The \"tangible\" or physical cultural heritage, no matter whether moveable artifacts or immovable historic sites, is in a constant state of chemical transformation and equilibrium with the surrounding environmental conditions [91] and selective biochemical processes accelerate the chemical transformation of the underlying materials given the colonization by microorganisms. Selective microbial groups carry out the important biochemical transformation reactions with high specificity regulated by relevant genes and the corresponding enzymes/proteins. It is known that autotrophic microorganisms are pioneering organisms that colonized the bare surfaces of sandstone when the cultural heritage was initially built, but the roles of these microorganisms are not straight-forwardly detrimental because their protective role for the sandstone, for example from lichen, was significant in the beginning [40]. After sequestration of atmospheric CO2 onto the surface as biomass, the physical surface properties of the heritage are significantly altered, allowing for diversification of the microbial community with different physiological functions and also for accumulation of pollutants to promote the degradative microorganisms [63]. Information on these has been reviewed recently [40,92]. More recently, NO3- production and accumulation of the nitrogen cycle have been widely observed and confirmed on Angkor sandstone monuments under the tropical climate in Cambodia [32,85]. The concentration of NO3- is positively correlated with the acidity and pH value, among the different environmental variables of these sandstone monuments. The constant acidification on the cultural heritage initiates and accelerates the dissolution of mineral constituents in the sandstone and produces the phenomenon of salt attack by crystallization/efflorescence on/in the substratum material which leads to structural weakening and crack formation of the cultural heritage [16,29,30,35,70,93]. After successive sampling over a 3-year period and analyses of the ammonia-oxidizing archaea (AOA) and bacteria (AOB) by both genomic DNA and RNA from samples for real-time PCR, AOA was apparently higher than AOB on these sandstone monuments and detection of NO3- was successful [32,85]. Furthermore, a stable isotope tracer experiment by addition of the \({}^{15}\)N in the chemical forms of NH4+ or NO3- was initiated and incubated with samples collected from several Angkor monuments to quantify the nitrogen transformation rate from nitrate removal as nitrogen gas and determine the contribution of the microbial community to the removal of available nitrate. The results of this study revealed that the heterotrophic denitrification (NO3-  
via chemolithoautotrophic metabolism in laboratory experiments [61; 62].