A Philosophy of Sampling
Think .... (like a microbe)
In my previous post, I set out a list of what I thought are Big Questions that research in fundamental microbial ecology should be attempting to resolve. That immediately brings to mind the experimental approaches that are most likely to do so. There are, of course, many layers to consider. Here and in the next two posts I give my thoughts on three elements that in my mind are linked: sampling strategies, experimental approaches and means to experimentally constrain a microbial ecosystem for testing.
A Philosophy of Sampling
Sampling is something all experimental microbial ecologists do, but it is worthwhile to reflect upon whether the samples we collect are ‘representative’ (and understand what they are representative of) as well as appropriate for the particular study. One’s view of the world shifts with the scale at which we view it – remember that here I am asking you to “think like a microbe.”
Microbial ecologists are asking questions – what do we want to know; can it be studied at the scale where decisive factors are operating; and does the investigation lead to generalities that may apply to other systems.
Perhaps the most important consideration for sampling is how homogeneous or heterogeneous the system is at the scale of the microenvironment that microbes are directly experiencing. Here I discuss three major habitats that differ in this regard – planktonic habitats of lakes and oceans, unsaturated soils, and the gastrointestinal tracts of animals.
Planktonic habitats:
The surface zones of oceans and lakes may represent a best-case scenario for spatial homogeneity -- this layer is well mixed by wind-driven turbulence and convective forces from heating and cooling. In the ocean, this mixed zone may range from 30-100 m in depth depending upon the latitude and season. In lakes of course it is shallower, depending upon the fetch (maximum length of open water the wind can travel). For Lake Mendota, with a fetch of ~8 km, the epilimnion is 10 m deep in the summer. Although homogeneous in theory, the hydrodynamics may be more complicated depending upon morphometry, currents as well as small-scale processes. A variety of messenger-activated sampling devices are available to collect water samples from discrete depths.
Below this zone, temperature decreases at up to 1° C / m and thereby generates a density gradient that can be quite stable. Strong gradients of chemicals and specific microbes can arise at a scale of tens of cm here in the metalimnion. The situation resembles a less compressed analog of some microbial mats. Discrete sampling of these layers can be accomplished via tubing lowered to depth and water retrieved with a peristaltic pump on the boat.
Unsaturated soils
If the surface mixed zone of a lake is a best-case for spatial homogeneity, soils are the worst-case. In unsaturated soils, the distribution of water is not continuous -- thereby limiting diffusion of nutrient resources and other chemicals between microenvironments. Soil properties such as texture and porosity will influence which sites are ‘habitable’ and in which there are adequate water and nutrient resources for microbial activity. In a thoughtful perspective, Smercina et al. (2021) described soils as a mosaic of hot spots and hot moments. At the bulk level, soil is a desert that contains thinly distributed oases of water and nutrients where a microbial community can flourish. They highlight two important microenvironments – the rhizosphere at a sub-mm scale and intact soil micro- or macro-aggregates in which mineral or organic surfaces combine with pore structure to provide microhabitats. It feels likely that the bulk scale comprises a metacommunity sensuLeibold et al. (2014): sets of local communities linked by dispersal. There are statistical models that can infer the relative importance of different ecological processes such as dispersal across a habitat (Stegen et al., 2013)
Clearly it is technically challenging to sample at these scales. I am not advocating abolition of bulk soil sampling methods – but it is important to keep in mind that what is sampled is not “the” microbial community but rather an integration of very many potentially-different microbial communities. Any conclusions derived from these samples should keep that in mind.
Gastrointestinal tract
Modern microbial ecology had its roots in the digestive system – Hungate’s studies of the rumen. Over the past 20 years, there has been heightened interest in the human gastrointestinal tract as the role of the microbiome in human health has been recognized. It is important to note that the sampling regimes used by rumen microbiologists and those studying the human GI tract are quite different. To a first approximation, the rumen is a well-mixed reactor and experimentalists can access its contents via a fistula surgically installed into the rumen. In contrast, the colon of the human GI tract more closely approximates a plug flow reactor. For reasons of convenience (for both the investigator and the subject), samples of fecal matter are analyzed – effectively the output of the reactor.
What struck me in sorting through the literature is I could find no assessment of how representative fecal samples were of the entire colon. The transit time through the colon is ~35 hours. Surely there are changes in resource quality during this time that could produce succession in the microbial community. While unethical in humans, analysis of colon sections along its length in an omnivore such as the pig could provide insights. I found one report that analyzed proximal and distal contents of the pig colon (Crespo-Piazuelo et al. 2018). Although the differences in community composition were smaller between these samples than with upper tract samples, there remained clear separation between the proximal and distal colon communities.
A core principle in analysis of plant/animal-microbe interactions is that close physical associations may have disproportionate impact. The rhizosphere in plants is a case in point. Restricting colon analysis to lumen samples misses the distinct environment present at the intestinal wall – not only does it contain unique glycoprotein resources (mucin) but is a radial source of O2 that can be scavenged by bacteria near the wall. Ethical issues abound in obtaining authentic samples, so animal systems would be a preferred model for experimentation.
Ending remarks
I have been critical of some practices here but my intent is to be constructive – to think of what the dimensions of an authentic habitat is from the microbes’ perspective. If you choose to sample on a larger scale, consider the impact that has on conclusions you derive from analyses of homogenized communities. For example, a grab sample of unsaturated soil will contain a large number of physically isolated communities. Hence, it makes no sense to discuss the ‘interactions’ between them. In a planktonic sample, individual cells are distantly separated but still do interact via their competition for dissolved nutrients in the milieu.
If one is interested in questions in which the microscale is where the action is, it behooves one to look at technologies that permit sampling and analysis of more finely grained samples. These have greatly improved over the past 20 years.
I have neglected discussion of an equally important aspect of sampling – appropriate replication. At the experimental design phase, what level of differences do you imagine there will be among your treatments and how many replicates are necessary to have confidence that any differences found are not just due to chance?
Today’s Zen
Kwakwakaʼwakw dancer -- Alert Bay, British Columbia
References
Crespo-Piazuelo, D., Estellé, J., Revilla, M. et al. (2018) Characterization of bacterial microbiota compositions along the intestinal tract in pigs and their interactions and functions. Sci Rep 8, 1272. https://doi.org/10.1038/s41598-018-30932-6
Leibold, M. A., Holyoak, M., Mouquet, N., Amarasekare, P., Chase, J. M., Hoopes, M. F., et al. (2004). The metacommunity concept: a framework for multi-scale community ecology. Ecol. Lett. 7, 601–613. doi: 10.1111/j.1461-0248.2004.00608.x
Smercina DN, Bailey VL and Hofmockel, KS (2021) Micro on a macroscale: relating microbial-scale soil processes to global ecosystem function. FEMS Microbiology Ecology 97: fiab091 https://doi.org/10.1093/femsec/fiab0
Stegen, J.C. Xueju Lin, Fredrickson JK , Chen X, Kennedy DW, Murray CJ, Rockhold ML, Konopka A. (2013) Quantifying community assembly processes and identifying features that impose them, The ISME Journal 7: 2069–2079, https://doi.org/10.1038/ismej.2013.93