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Review

Nano-Archives in Soils—What Microbial DNA Molecules Can Report About the History of Places

by
Johann Michael Köhler
Institute for Micro- und Nanotechnologies/Institute for Chemistry and Biotechnology, Technical University Ilmenau, PF 10 05 65, D-98684 Ilmenau, Germany
Appl. Nano 2025, 6(1), 2; https://doi.org/10.3390/applnano6010002
Submission received: 27 November 2024 / Revised: 15 January 2025 / Accepted: 22 January 2025 / Published: 27 January 2025
(This article belongs to the Collection Review Papers for Applied Nano Science and Technology)
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Abstract

:
DNA encoding the 16S rRNA of bacteria is a type of nanometer-sized information storage that can be used to characterize bacterial communities in soils. Reading this molecular ’nano-archive’ is not only of interest for characterizing recent local ecological conditions but can also provide valuable information about human impacts on soils in the past. This is of great interest for archaeology and for understanding the ecological consequences of past human activities on recent ecological conditions. Powerful sequencing methods such as the Illumina process allow many different DNA sequences to be determined in parallel and provide very efficient data sets that reflect the composition of soil bacterial communities in topsoil layers as well as in translocated and covered soils of archaeological sites such as settlements, burials or workplaces. Here, a brief overview of recent developments in the use of these molecular nano-archives for the study of archaeological soil samples is given using typical examples.

1. Introduction

The properties of soils are essential for the robustness of natural ecosystems, the fertility of land and the ability to use land for the production of food, renewable materials and biofuels [1,2]. The condition of soil is modulated by natural factors such as erosion, moisture, temperature, climate change and the activities of plants and animals [3]. Soils are also heavily impacted by industry, mining, construction, agriculture and other recent human activities [4,5,6]. Understanding the state of soils and the consequences of land use and human impacts on soils is crucial to maintaining soil fertility and resilience, managing the impacts of climate change on soils and developing ecologically and economically efficient land use strategies [7,8]. Therefore, we need to understand not only the recent responses of soils to natural changes and human activities but also the long-term effects and human impacts over centuries and millennia [9,10,11,12,13].
Soils are among the most complex and diverse natural systems [14]. On the one hand, they represent a hierarchically structured system of mineral components and pores with characteristic length scales—from about centimeters to the nanometer range—i.e., over about seven orders of magnitude. Typically, several different minerals are involved, forming a three-dimensional network of macro- and nanopores, which are partially or completely filled by water, or rather, aqueous phases with dissolved salts and many other inorganic and organic components. In addition to the abiotic components, soils typically contain an enormous diversity of biological components, including plant roots, hyphae of fungi, animals of meso- and microfauna and an enormous number of different microorganisms.
All these components carry information about the state of the habitat and the history of the places where they live. In recent years it has become increasingly clear that the composition of bacterial communities, in particular, is related not only to the recent state of the soil but also to the history of a site. The community of microscale organisms carries information resulting from the “previous experience of a place” and thus represents a type of “ecological memory” [10,13,15].
This information concerns primarily the response of different types of bacteria to the recent and past physiological conditions of the local soil environment. The development of a certain composition of soil bacterial community is dependent on many parameters such as humidity, temperature, pH, salinity, oxygen partial pressure and many further chemical components as potential substrates and contaminants. The different responses of bacteria to specific local conditions are realized by the availability of required proteins and the related gene expression. The differences in the bacterial DNA coding for different proteins depend on their taxonomical relations. Therefore, 16S rRNA is a suited tool not only for the identification of different bacteria but also for distinguishing different soil properties in recent times and the past.
The information-carrying molecules in the soil can therefore be regarded as a natural “nano-archive”. Exploiting this information can be very valuable for both ecology and archaeology [16]. DNA molecules are an important source of site-related biological information. It contains information about the types and metabolic characteristics of the soil microorganisms present in the soil. This microbial community involves recently physiologically active as well as non-active “dormant” cells [17,18]. In the following, a short review of the readout of genetic information on the single-molecule level is given and illustrated by some recently reported examples of data extraction from archaeological soil samples.

2. Strategy of Sample Treatment and Extraction of Information

The information of interest is stored in the genetic material of soil microorganisms. Most recent studies have focused on the DNA encoding the so-called 16S component of ribosomal RNA, 16S rRNA. These molecules are particularly useful for assigning bacterial cells to their position in the taxonomic system [19]. The qualitative and quantitative composition of all soil bacterial 16S rRNA, therefore, reflects the specificity of a soil sample from the point of view of its bacterial community [20]. This community is strongly related to the recent ecological situation of the soil and to ecological situations in the past [21]. Several different sources of bacteria, related to natural processes as well as to past human activities, have contributed to the composition of soil bacterial communities (Figure 1).
The storage capacity of biomolecular information in soil is truly enormous. In order to estimate it, it is interesting to first look at the volume scales (Figure 2). The volume of the largest pores in a typical topsoil is in the order of 1 µL (1 mm3), while smaller pores are in the nanoliter range (1 nL = (0.1 mm)3). A soil sample with a volume of 1 mL (1 cm3) can contain up to about 108 to 109 individual bacterial cells. The volume of these cells is typically in the lower femtoliter range (1 fL = 10−15 L). The total volume of 108 bacterial cells is about 10−6 L = 1 µL, which means that it would only fill about 0.1% of the milliliter.
The size of bacterial genomes is on the order of 106 to 107 base pairs (bp), which means that the molecular information storage capacity of the DNA of a single bacterial cell is on the order of one Mbyte [22,23]. Assuming a possible content of 108 to 109 bacteria in 1 g of humus soil [24], the total information storage capacity is about 1014 to 1015 bytes or 100–1000 Tbytes. This storage capacity corresponds to the text of about 100 million to one billion books in a very large library (Figure 3). Thus, the bacterial DNA in a gram of soil can, in principle, tell us a vast amount of detail about the recent physiological and ecological situation in the soil sample, as well as the ecological history and evolution of the community of organisms at the site. The 16S rRNA is only a small part of this molecular nano-archive, but its readout is very attractive for obtaining an overview of the basic composition of the soil bacterial community.
To make use of this nano-archive, the DNA molecules associated with 16S rRNA have to be separated from all other materials. First, the total DNA from the cells is isolated and extracted from the soil sample. The 16S rRNA is selected by selective amplification using a polymerase chain reaction (PCR). After purification, quality control and molecular tagging, these DNA molecules are identified by sophisticated surface-coupled next-generation sequencing (NGS) using the Illumina method [25]. In this process, individual DNA molecules are bound to a solid surface and multiplied by a local molecular amplification process on the surface. The result is a molecular lawn composed of different spots of the same DNA molecules, with the monomolecular layer having a thickness of about 100 nm and each spot covering an area of a few square micrometers. In this way, a spot density of approximately 106/cm2 can be achieved, and typically 107 molecules can be sequenced in a few minutes.
A typical procedure of sample preparation and data processing is described in [26]. The obtained data (fastqfiles for forward and reverse aligned 16S rRNA) are converted into fasta format, and additional quality data are calculated mothur, version 1.39.5) in order to characterize the quality of sequencing and sequence alignment using the open source platform Galaxy (https://usegalaxy.org/).
The sequence data obtained were evaluated for quality and then compared with sequence data from databases containing sequences of already described or at least molecularly identified bacteria. This allows each DNA molecule to be assigned to its specific position in the taxonomic system, mostly at the genus level. If an assignment to a specific genus is not possible, an assignment to a higher taxonomic level—family, order, class or phylum—can be made in almost all cases. The recognized taxonomic types are called operational taxonomical units (OTUs). The number of reads for each OTU represents the abundance of related bacteria in the sample. The entire process is shown schematically in Figure 4.
In addition to the extraction of molecular information on bacterial taxonomy, the micro-technology can be used for an empirical evaluation of the growth behavior and stress sensitivity of unknown bacteria. Therefore, a nanomolecular readout of biological data is complemented by microfluidic cultivation and characterization strategies. Typically, soil microorganisms are brought into microdroplets with volumes between about 100 pL and 1 µL. These volumes correspond to droplet diameters between about 40 µm and 1 millimeter [27,28]. These droplet-based microfluidic cultivation techniques are particularly suitable for obtaining highly resolved dose–response curves with minimal consumption of chemicals and biological material [29]. Droplet-internal concentrations of single nutrient components or chemical stressors can be applied with high accuracy and in very fine concentration steps. Droplet series or microfluidic segment sequences with hundreds or thousands of separate fluid compartments can be rapidly generated from less than 1 milliliter of nutrient solution [30]. Non-contact reading and evaluation of growth behavior or toxic effects are usually achieved by using micro-photometers and micro-fluorimeters [31]. In addition to measuring the autofluorescence of cells, analytical information from physiological processes inside the droplets can also be read out by signal transduction using microparticles with analyte-dependent fluorescence behavior, for example, to determine changes in the pH or oxygen concentration [32]. In addition, polymer microparticles doped with silver nanoparticles can be used for miniaturized Raman measurements using the SERS effect (surface-enhanced Raman spectroscopy) [33]. Microimpedance measurements are also interesting for measuring changes in the ion concentration and pH.
Microbial archaeology attracts increasing interest for archaeology concerning human pathogens, but also other microorganisms [34]. Soil samples were taken, for example, from settlement places, burial sites, the interior of urns or prehistoric or antique houses, wells, waste pits, and from handcraft, mining and metallurgy areas. Typically, a few grams or, at least one gram of soil material is taken by use of sterile sample tubes. Thus, it is possible to take samples from different archaeological layers and several points in profiles. About 0.5 to 1 gram of the sample is applied for DNA isolation. The purified DNA is then introduced into the PCR to obtain enough material for sequencing.
Cell cultivation and molecular biological analysis involve different levels of information density and corresponding length scales. On the one hand, the cultivation of cells from archaeological soil samples addresses the micrometer scale, as the typical size of soil bacteria is in the order of one or a few micrometers. Each gram of humus soil can contain up to a billion individual cells [24]. By selecting suitable cultivation conditions and stress factors, it is possible to cultivate microorganism strains of interest. The small amount of soil material required for analysis allows the composition of soil microbial communities to be distinguished with high spatial resolution. In principle, tens of thousands of soil samples could be taken from a prehistoric building floor of a few square meters. This strategy would make it possible to obtain high-resolution profiles of the lateral and vertical shifts in the composition of soil microbial communities, a method that has the potential to improve archaeology and provide much more detailed facts about the specific and everyday use of different parts of prehistoric houses, courtyards and other settlement components, as well as fortifications, craft areas and cult sites.
On the other hand, DNA extraction and molecular amplification by PCR are on the nanometer scale. Each letter in the molecular code takes up no more than about one cubic nanometer. In most cases, the approximately 1500 letters of 16S rRNA are sufficient to determine the species to which a bacterium belongs. During the PCR process, the single molecule is duplicated about 30 times, producing a sufficient number of identical copies that can be reliably sequenced. Thus, in a soil sample, low abundant and dormant bacteria can be detected in parallel with recently active and highly abundant bacteria.
Below are some examples of the use of DNA from soil bacterial communities from archaeological samples to demonstrate how these molecular nano-archives can be used to compare sites and reconstruct archaeologically interesting ancient human activities and their impact on soil bacterial communities.

3. Hints on Specific Formerly Human Activities from Observing Special Bacterial Types

Humidity and temperature are two important factors for bacterial growth and competition between different genera, species and strains. Besides them, the composition of soil bacterial communities is strongly dependent on chemical conditions. These include the availability of general or species-specific nutrients and water on the one hand and the absence of toxic substances above species- or strain-specific thresholds on the other. Important factors in the latter group are, for example, high salt concentrations, a high or low pH, toxic organic compounds and toxic metal ions, if they are bioavailable. Only a few specialized bacteria tolerate extreme conditions such as the thermophilic, acidophilic, alkaliphilic or halophilic types.
The presence of such extremophilic bacteria in the soil may indicate special conditions in the environment of the soil sample [35]. Special chemical conditions in the soil can also be indicated by bacteria that can tolerate certain heavy metal ions [36,37], halogen-substituted or aromatic organic compounds [38], or that can metabolize special substrates such as mineral oil [39,40,41]. Ammonia-oxidizing bacteria may indicate ammonia-rich soils, while sulfur-oxidizing types may indicate high levels of reduced sulfur species.
In addition to the general occurrence of certain substrate-specific or stress-tolerant bacterial types, the abundance of types is important for characterizing soil properties and interpreting the history of soil. It is, therefore, interesting to consider the rank-order functions of bacterial abundances. Typically, the rank-order functions show a strong decrease from a few high-abundance types to a few medium-abundance types to a large number of low-abundance types. The high-abundance types are obviously the most active, fast-growing bacteria under recent conditions. The lower-abundance bacteria are slow-growing cells or are in a dormant state where they have very low metabolic activity or are completely inactive, for example, in the form of spores. It is likely that a significant proportion of the less active or dormant cells represent types that were active in the past under different environmental conditions. Thus, among the less abundant types are bacteria that carry information about past environmental conditions [42].
Residues of animals and plants, but also traces of handcraft activities in buried soil, contribute to variations in soil bacteria communities. Strong effects on microbial communities can result, for example, from a concentration of cattle in stables or cattle pens. There, high concentrations of manure can cause high concentrations of nitrate and ammonia in the soil and the development of bacteria with related metabolisms. Deposition of organic residues from plants and animals and burying of related soil layers causes, typically, low local oxygen availability which induces the development of anaerobic and methanogenic bacteria. In contact zones with higher oxygen availability, methanotrophic bacteria find suited growth conditions, for example (see also below). In general, agriculture is a very important factor in the composition of soil bacterial communities. Vice versa, the microbial compositions in soils are very important for their fertility and decontamination [43,44,45,46].
Bacteria in soils that reflect past human impacts on local environmental conditions can be roughly divided into two groups: The first group includes highly abundant bacteria that are adapted to recent soil conditions. An example of this is the soil bacterial communities that live in soils that have been contaminated by high salt concentrations and in which the salt concentration has increased to the present day [47]. Highly salt-tolerant bacteria can be active in massively saline soils [48]. The second group includes bacteria that are less abundant, less active or not active at all but that have grown in the past due to specific anthropogenic soil conditions that were only temporary.
The chance of finding less abundant bacteria, which carry information about past soil conditions, is much higher in buried soils than near the surface. While the bacterial community in surface soils is constantly affected by changes in the weather, vegetation and other dynamic factors in the environment, soil layers covered by a certain thickness of material are subject to much less change and represent an ecological system with lower dynamics. Therefore, there is a high probability of finding intermediary and less abundant bacteria associated with past human-induced environmental changes.
Despite its high power, there are limitations in the use of NGS approaches to soil bacterial communities in archaeology. On the one hand, bacterial components of very low concentrations (about 1:100,000 reads) are not detected, and low-concentrated types (below about 1:10,000 reads) can hardly be used for quantitative comparisons due to stochastic effects, which have to be expected. It is not possible to read out the age of bacteria or its DNA directly from the sequence data. Even in covered soil layers, physiological processes go on, and the DNA of bacterial communities has always been discussed as a superposition of components originating from the history of soil layers and from recent microbial activities.

4. Application Examples

In recent years, several studies on the characterization of soil microbial communities from archaeological sites have shown that the analysis of genetic data from soil microorganisms can provide interesting additional information on past human activities. In addition to powerful analyses of human remains and human pathogens [42,49,50], looking at soil microbial communities can help to understand the nature and intensity of local human impacts on the soil and the specific features of sampling sites in different strata and closely adjacent points. Some examples of such studies are considered below (overview in Table 1).
One of the most important impacts of early human activity on local soil conditions was the concentration of animals due to pastoralism. The presence of cattle is always associated with a massive release of manure and usually also with the deposition of organic matter from feed, straw and animal litter. It forms a strong impact on the memory of soil [64]. High concentrations of urea, nitrate, ammonia, phosphate, carbohydrates, lipids and proteins are deposited in the soil. This is accompanied by a high consumption of oxygen. As a result, mostly anaerobic conditions have developed. Thus, microbial communities with ammonia-oxidizing bacteria, nitrate-reducing bacteria, sulfur-related types, methanogenic and hydrogen-oxidizing bacteria have developed. Such bacterial communities have been identified, for example, in the soils of Bronze Age settlements that were archaeologically investigated in the North Caucasus region. Analyses of bacterial DNA from these settlements provided clear evidence of animal husbandry [30,39]. Probably, the detection of DNA indicating the presence of rumen-associated bacteria can be interpreted as the former presence of domestic animals. Such bacterial types could indicate the use of prehistoric walls to protect livestock. Similar compositions of bacterial communities have also been found in a building identified as a stable and in layers of former road surfaces in the Roman city of Carnuntum (Austria)—a large settlement and important military and administrative city located near the banks of the Danube. The presence of specific bacterial types in soil samples from archaeological excavations at this site also provides evidence for the processing of fish, for example [63].
Bacterial communities indicative of former high concentrations of manure and deposition of residues from the processing of animal material were also observed in soil samples from a historical tannery area in the city (medieval suburb) of Jena. In addition to nitrogen- and sulfur-related types, a large number of specific OTUs were found at this site, including comparatively high concentrations of archaea, methanogenic and methanotrophic types, such as Dada and Zixibacteria [60]. Differences in the composition of the fillings of the archaeological vats and their surroundings indicate an increase in beta diversity due to former human activities.
In addition to the feces of livestock, former latrines are also characterized by typical components of bacterial communities. For example, the study of soils representing residues from latrines of medieval houses in the cities of Riga and Jerusalem allowed us to identify bacteria related to the intestinal flora of human intestines [55]. Analyses of the bacterial genes of latrine contents led to conclusions about the former human diet and diseases of the medieval populations of the respective cities.
An increase in bacterial diversity due to human influence has already been observed by R. Margesin et al. in the study of soils from different layers of an Iron Age settlement in Sicily. They found clearly different compositions of soil bacterial communities in the different soil layers [16]. An increase in beta diversity may also result from burial sites, where the contents of urns create different conditions for bacterial communities in the local soil. A similar result was obtained by S. Voyron et al. by studying the fungal communities in soil samples from a 6th- or 7th-century burial mound in Japan. They confirmed the translocation of soil material and the burial of former topsoil material under layers of translocated soil [58]. The translocation of soil material was also associated with the refilling of a pre-industrial exploratory shaft for coal mining, which was found and investigated archaeologically near Bennstedt (Germany). Bacterial DNA analyses of these soil samples from the shaft, its surroundings and the coal seam revealed very different compositions of bacterial communities [26].
The detection of a strong increase in cyanobacteria in the sediments of a 4000-year-old lake in northeastern Germany could be explained by a strong increase in nutrients in the lake, which favored bacterial growth. This massive change in environmental conditions could be correlated with strong deforestation in the lake area and intensive development of agriculture in the Early Bronze Age [61].
A specific archaeological question has been addressed by the study of bacterial communities on the stones of ancient buildings. G. Chimienti et al. were able to show that the bacterial communities living on the sun-exposed stone surfaces of a medieval church in Siponti (Italy) contained both thermophilic and radiation-resistant species [52].
The traces of human impact in the composition of soil bacterial communities can also reflect specific human activities in different places. Not only recent industrial mining but also pre-industrial or medieval mining caused strong changes in local soil conditions by translocating material and stressing soils through changes in nutrient spectra, increasing the salinity, decreasing the pH or contamination by toxic heavy metals. The deposition of ash from pre-industrial salt production in the saltworks of Bad Dürrenberg (Germany) caused a high content of halophilic bacteria in the associated soils [62]. The impact of ancient metalworking on the soil was demonstrated in the case of a sample from a metalworking site in the medieval castle of Altenburg (Germany). From this sample, a strain of Rhodococcus erythropolis was isolated and studied by microfluidic cultivation in nutrient media with stepwise varied metal ion concentrations. These studies revealed very high tolerances to nickel and cobalt, suggesting an adaptation of this strain to the specific local human-induced exposure situation [57].
The studies discussed here demonstrate that the analysis of soil bacterial communities can be applied to a wide variety of archaeological situations (Figure 5). They illustrate that the characterization of bacterial DNA from the molecular nano-archives in soils can be considered a general strategy to complement traditional methods in archaeological investigations.

5. Conclusions

The examples show that the composition of local soil bacterial communities contains specific information about the history of sites. This information is related to ancient human impacts on the soil, resulting in specific local environmental conditions for bacterial growth and competition between different strains. This information content of soil can be harvested by extraction and analysis of bacterial DNA. This biomolecular source represents a type of molecular nano-archive that stores data about the past of local soils, thus preserving information about the changes in local ecological conditions due to human impacts in prehistoric and historic times. The most commonly used DNA sequences, encoding 16S rRNA, are well suited for a rough taxonomic classification of the components of bacterial communities. This provides insight into the character and diversity of soil bacterial communities influenced by humans and the associated past human activities at the sample sites. However, the stored reservoir of bacterial genes contains many more genes that can, in principle, tell us much more about the microbial strains and, in addition, about their biochemical and physiological potential. It is expected that this source of data will be developed and used much more by archaeologists in the future.

Funding

This research received no external funding.

Acknowledgments

I would like to thank my colleagues Jialan Cao, Linda Ehrhardt, P. Mike Günther, Steffen Schneider, and Frances Möller for their fruitful cooperation and discussions.

Conflicts of Interest

The author declares no conflicts of interest.

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Figure 1. External sources contributing to the composition of soil bacterial communities. Different shapes, sizes and colors symbolize the high variety in soil bacteria and its properties.
Figure 1. External sources contributing to the composition of soil bacterial communities. Different shapes, sizes and colors symbolize the high variety in soil bacteria and its properties.
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Figure 2. Different volume scales in soil have to be reconsidered: The pores in 1 mL of humus soil can offer space for about hundred million to billion bacterial cells. Different shapes, sizes and colors symbolize the high variety in soil bacteria and its properties.
Figure 2. Different volume scales in soil have to be reconsidered: The pores in 1 mL of humus soil can offer space for about hundred million to billion bacterial cells. Different shapes, sizes and colors symbolize the high variety in soil bacteria and its properties.
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Figure 3. Storage capacity of molecular-encoded information in 1 g of soil: The DNA of contained bacteria possesses a principal storage capacity corresponding to a huge library with one billion books.
Figure 3. Storage capacity of molecular-encoded information in 1 g of soil: The DNA of contained bacteria possesses a principal storage capacity corresponding to a huge library with one billion books.
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Figure 4. Process chain for extracting molecular information from bacterial DNA coding for 16S rRNA for taxonomical identification of bacteria in the form of operational taxonomical units (OUT) and analyses of quantitative compositions of soil bacterial communities by use of so-called next-generation sequencing (NGS, Illumina process); different colors symbolize different DNA molecules coding for 16S rRNA.
Figure 4. Process chain for extracting molecular information from bacterial DNA coding for 16S rRNA for taxonomical identification of bacteria in the form of operational taxonomical units (OUT) and analyses of quantitative compositions of soil bacterial communities by use of so-called next-generation sequencing (NGS, Illumina process); different colors symbolize different DNA molecules coding for 16S rRNA.
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Figure 5. Biomolecular soil nanoarchives can supply specific information from archaeological places of very different character.
Figure 5. Biomolecular soil nanoarchives can supply specific information from archaeological places of very different character.
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Table 1. Examples of analyses of microbial communities in soil samples from archaeological places.
Table 1. Examples of analyses of microbial communities in soil samples from archaeological places.
Sampling Area/Sampling SiteArchaeological SituationBacterial CommunitiesInterpretationRef.
Northern CaucasusBronze Age settlementsmetabolic active bacterialivestock herdingS. Peters et al., 2013 [51]
Siponti (Italy)medieval churchextremophiles, UV-adaptedadaptation to stone surfaceG. Chimienti et al., 2016 [52]
Monte lato, Sicily (Italy)Iron Age settlementdifferent metabolic featuresdifferentiation of archaeol. stratalR. Margesin et al., 2017 [16]
SchöpsIron Age burial fieldenhanced diversity inside urnsBeta-diversity enhancementJ.M. Köhler et al., 2018 [53]
Monte lato. SicilvIron Age settlementhighly diverse Proteo- and Actino- bacteria-dominated communitiesdifferentiation of archaeol. stratalJ.A. Siles et al., 2018 [54]
Riga, Jerusalem (Latvia, Israel) medieval cities, latrineshuman gut-related communities intestinal flora of pre-indust. populationS. Sabin et al., 2020 [55]
Places in Thuringia (Germany)prehistoric rampartsrumen-associated typeslivestock in rampartsJ.M. Köhler et al., 2020 [56]
Altenburgmedieval metal workerhighly metal-tolerant strainselection pressure by Ni and CoJ. Cao et al., 2021 [57]
Tobiotsuka Kufun (Japan)Burial moud (6th–7th century)high fungal diversitytranslocated and buried top soilsS. Voyron et al., 2022 [58]
Bennstedtpreindustrial coal
prospection shaft		three different communities in coal seam, shaft and top soilcommunity reflecting
material translocation		L. Ehrhardt et al., 2022 [26]
North Caucasus (Russia) ancient agriculture areasthermophile and manure-relatedanimal dungN. Borisov et al., 2022 [59]
Jena (Inselplatz)historical tannery areahigh diverse communities with abundant sulfur- and nitrogen-related typesresidues of tannery and dying activities, manureJ.M. Köhler et al., 2023 [60]
Tiefer See (Germany)early Bronze Age land usecyanobacteria in lake sedimentsdeforestation due to human impact E.C. Nwosu et al., 2023 [61]
Bad Dürrenbergash deposit of historical
Saline		high content of salt-tolerant typescommunities adapted to highly salted depositsJ.M. Köhler et al., 2024 [62]
Petronell-CarnuntumRoman citydiverse local communitiessite-specific bacteriaJ.M. Köhler et al., 2024 [63]
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Köhler, J.M. Nano-Archives in Soils—What Microbial DNA Molecules Can Report About the History of Places. Appl. Nano 2025, 6, 2. https://doi.org/10.3390/applnano6010002

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Köhler JM. Nano-Archives in Soils—What Microbial DNA Molecules Can Report About the History of Places. Applied Nano. 2025; 6(1):2. https://doi.org/10.3390/applnano6010002

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Köhler, Johann Michael. 2025. "Nano-Archives in Soils—What Microbial DNA Molecules Can Report About the History of Places" Applied Nano 6, no. 1: 2. https://doi.org/10.3390/applnano6010002

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Köhler, J. M. (2025). Nano-Archives in Soils—What Microbial DNA Molecules Can Report About the History of Places. Applied Nano, 6(1), 2. https://doi.org/10.3390/applnano6010002

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