Where the Critters At?

Searching for Bacteria As Artifacts / My Kingdom for a Microscope!

Bacteria are funny things – diminutive in size, and rarely considered unless we consider them as the cause of disease or as casual hitchhikers in our local biome. And yet, bacteria have spent millions of years co-evolving with humans. They are a part of us, as well as organisms in their own right. And if we switch our perception, and consider them as archaeological artifacts, then it is possible that their presence will add to the human story. So, I have selected three examples from the literature to determine how we can use bacteria.

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Margesin, R., Siles, J. A., Cajthaml, T., Öhlinger, B., & Kistler, E. (2017). Microbiology meets archaeology: soil microbial communities reveal different human activities at archaic Monte Iato (sixth century BC). Microbial Ecology, 73, 925-938.

The authors examined a native (indigenous) building that was discovered in Western Sicily; the focus of the study was to examine and identify differences is the soil microbial communities within the building. Their working hypothesis was that soil microbial communities would vary in type, and that variations would reflect human activities within the building. The microbial communities were characterized by viable cell count, enzymatic activity, diversity, and community structure. The authors were able to identify different microbial communities, and differentiate human activities from their findings.

Key points included:

1. Human consumption habits left traces on the microbial environments that were unique; for example, microbial community structures could be differentiated from other deposit layers
2. Soil studies of archaeological sites generally assess pH variation, microelements, or micromorphology; failure to assess bacterial diversity impacts the biological and archaeological records
3. “Microbiological residues as part of the so-called ecofacts are, like artifacts, key indicators of consumer behavior in the past” (ecofacts is a relatively new term – do we need it, or should we stick with “artifact”, or “biological artifact”

From a critical perspective, the authors have provided a complete archaeological site review to provide a context for their biological assessment. The microbial assessment is well described, and they employed a methodology with excellent controls and replication of the results. Furthermore, each test (count, enzymatic activity, microbial community structure) was cross-verified with another test to isolate any anomalous results. Additional information on long-term soil conditions would have been useful to determine its impact on bacterial types, especially when considering bacterial types that might be considered “fragile”. Furthermore, the authors presented their data in isolation – they tested one house at one single site. Are there any sites, tested by them or other researches, that might have highlighted the accuracy of their methodology?

And, I must be honest … I miss seeing microscopic evidence of the bacteria. What would light or electron microscopy have shown if that was viewed? Sometimes, there is a reason why the “old technique” was used, and I suspect that looking at the bacteria present may have solidified claims of what is present.

Apart from my grouchiness about microscopy, the study presented provided an excellent use of the newer tools (determination of microbial biomass by cellular phospholipid fatty acids, PLFA; rapid enzymatic assays, determination of bacterial and fungal structure by Illumina amplicon sequencing) and how to use them to cross-validate results. It will definitely serve as an example of the role of bacteria as archaeological artifacts.

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Peters, S., Borisov, A. V., Reinhold, S., Korobov, D. S., & Thiemeyer, H. (2014). Microbial characteristics of soils depending on the human impact on archaeological sites in the Northern Caucasus. Quaternary International, 324, 162-171.

It’s great to read a paper that confirms a personal belief about the subject – when I started out, I held a belief that variations in microbial communities may possibly be used as a non-destructive (and rapid) means of prospecting archaeological sites. This paper confirms that suspicion. In this particular case, microbial communities were assessed across a Late Bronze Age settlement in the North Caucasus (Russia); it should be noted that the temperature of this site (cold!) was likely providential in preserving bacterial specimens. It was found that soil bacteria from areas of human habitation could be triggered to grow when glucose (sugar) was added to the growth medium; in contrast, 97% of microbes from non-human areas failed to reactivate and grow when glucose was present. The researchers were able to confirm that an area was used as a “dump area” for organic matter on the basis of enzymatic properties of the microorganisms.

The researchers took an interesting approach – they were not really concerned with WHAT species of bacteria was present; instead, they focused on how the activity of the microbes related to human activity at the site. Their methodology avoided the use of costly tests, as their growth media were relatively inexpensive.

The 3 key points I identified:

1. Measuring growth and enzymatic activity in a low-cost minimal experimental protocol can still provide useful results
2. Environmental conditions, especially soil pH and ambient temperature, may dictate the success of methodologies that determine microbial activity
3. The research supported a role for microorganisms that is fast, effective, and easy (who wants to dig holes!) when prospecting for archaeological sites.

From a critical perspective? First of all, if you’re going to go with traditional culture growth to assess microbial activity … show me the bacteria! Break out a light microscope, and do some basic taxonomy. If the supposition is that this was a farming site, then there should be evidence of Bacilli in the soil. Are there other bacteria that are uniquely associated with human or animal intervention? This information would have added to the study. And the evidence would have been strengthened if a double-blind study had been implemented – one team to collect bacteria from the real site and 2 or 3 non-sites, and a second team to conduct the bacterial analysis, and identify the actual archaeological site. The final step? If there is a role for bacteria in prospecting and identifying possible human sites … then do it. Actually prospect an area’s bacterial environment, and locate a site for study.

Again, it is difficult to be picky; after all, these data support my original contention that bacteria could play a role in non-destructive testing. Peters et al. have provided some great insight on how to make testing rapid and inexpensive. If not a foundational paper, it will support the use of bacteria and expand the methodologies that provide information.

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Boivin, S., Bourceret, A., Maurice, K., Laurent‐Webb, L., Figura, T., Bourillon, J., … & Ducousso, M. (2024). Revealing human impact on natural ecosystems through soil bacterial DNA sampled from an archaeological site. Environmental Microbiology, 26(1), e16546.

How long after human habitation can microorganisms be used to identify their activity, or trace their movements? The authors examined soil (chemical and bacterial composition) of the Roman fort of Hegra (Saudi Arabia) which had been abandoned for 1,500 years, and compared their results to a “control area” located near the fort where no human occupancy had been detected. The 4 centuries of human habitation significantly modified the soil’s chemical and bacterial composition. The area outside of the fort demonstrated relatively high bacterial diversity; however, the area inside the fort was dominated by two bacterial taxa, and both of these tax were related to humans and animals (pathogens, gut bacteria, parasites). The authors did conduct testing to rule out any impact due to chemical composition changes in the soil that might have related to human occupation.

The 3 key points I identified:

1. Although the environment is considered to be hostile for bacteria, the authors were not only able to revive bacteria, they were able to conduct sophisticated tests to count and taxonomically identify bacteria (at least to a low level; few samples were identified to the species level)
2. Samples were able to identify human activity, particularly in cases of human-animal interactions
3. The testing methodologies (polymerase chain reaction, PCR) were expensive; however, more expensive (and limited) testing may have provided more discrete identification of the bacteria

My critiques? Grrrrrr…. again, researchers appear to be relying on automated tools and scanners and such. No one is presenting microbial evidence. No matter how discrete and refined and colorimetric DNA-based assays are, they can’t beat a visual inspection that confirms if a bacteria is there … or is not there. I am impressed with their ability to detect microorganisms, and I appreciate how focused their methodology must be. I was wondering if there is a way to cost the various approaches, and determine the “sweet spot” for cost of human detection.

The authors make the claim that their methodology can be used for prospecting, and finding sites used by humans and animals with a high degree of resolution. Their methodology was certainly sufficient to work on old microbial genomes isolated from a harsh environment; I’d just like to see if there is a way to generalize it and perhaps reduce costs to be more effective.

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All of these studies directly support my perspective that bacteria, on their own, can constitute a useful archaeological artifact. My thoughts are now changing to how we can use this information. Thus far, the authors of all 3 papers have started with known sites. If the data can really be used for prospecting, then lets use it to find a novel, unknown site … that is the next, and expected step.