A view of microbial life in soil

Image source

All soils teem with microscopic life that is invisible to the naked eye, and for that reason often overlooked. Yet, microorganisms are an essential component of all soils, and through their metabolic activity (mineral weathering, production and recycling of organic matter, and more) soil microbes actually play a major role in soil formation, development, and fertility.

The images presented here show diverse microorganisms inhabiting a pasture soil. I sampled this soil on the Uetliberg, in Zürich, Switzerland, in 2018. Samples were chemically fixed and observed with scanning electron microscopy. Post-acquisition pseudocoloring was applied to the images - in other words, colors were added to the original black and white images to facilitate visualization of the microbes and soil particles. (The pseudocolors do not represent the real colors of the samples.)

All images are courtesy of the Soil and Terrestrial Environmental Physics group of Dani Or at ETH Zurich. SEM image acquisition and coloring by Anne Greet Bittermann, ETH Zurich - ScopeM.

We do not know the precise identity of these microbes (we would need DNA sequencing for that), but given their shape and size some level of characterization is possible. Most of them are bacteria, which are the most abundant cellular organisms in all soils.

 

Self-organization of bacterial populations

Fluorescent bacteria in glass microchannels

In many environments, hundreds, sometimes thousands of different microbial species coexist as mixtures of cells of various sizes and shapes. For example, each human being teems with their very own and unique microbial mixture (trillions of cells), as the human microbiome project unveiled a few years ago. Microbial diversity in soil is equally – possibly even more – astounding, with a gram of rich soil capable to host thousands to millions of distinct bacterial and archaeal species, as well as hundreds of fungal and protistan species. A recent study in the magazine Nature highlights the results of the earth microbiome project, which aims at revealing the extent of  microbial diversity on our planet.


Yet, despite this wealth of diversity, microbial communities are not simply soups of species – there is order hiding behind this curtain of complexity. Actually, the more we look, the more we find patterns of microbial organization in the natural world. Some patterns are obvious and have been known for a long time, such as the distinct layers of microorganisms visible with the naked eye in sections of microbial mats. The vast majority of patterns, however, reveal themselves only at the scale of individual microbes, that is, at the microscopic scale. See, for example, the beautiful arrangements revealed in  the lab of Jessica Mark Welch at the University of Chicago. 

Microbial populations are thus often spatially organized at small scale, and in a very defined and refined way. But how does it work? Obviously, microbes don't organize following some kind of blueprint that is imposed on the community!... But what then? Part of the answer seems to reside in so-called self-organization processes. With such processes, patterns emerge from the individual behavior of cells that can only sense the conditions in their local environment and react accordingly. This in appearance simple process at the individual level can lead to seemingly complex patterns of organization at larger scale. Think of bird murmuration, or of how ants can form bridges with their own bodies! I also discussed spatial patterns of bacterial organization triggered by metabolic cooperation in a previous post.

In a recent paper whose lead author is my colleague Benedict Borer, we examined some of the basic processes that can lead to bacterial spatial self-organization. (In that study we were specifically interested in pore networks that mimic the spatial structure of soil aggregates, but the processes that matter here are valid in other kinds of environments as well.) The idea is as follows. In a given habitat, bacterial populations with distinct metabolic capabilities and food preferences would spontaneously arrange in space in order to optimize their use of the available resources. We thought there must be two necessary conditions for that. First, that the bacterial cells have some level of motility (which could be flagellar motility, such as swimming, or simply movement provide by growth and cell division). Second, that gradients of carbon and nutrients are present in the habitat (i.e. there must be some spatial heterogeneity for the microbes to respond to).  


We chose to work with two bacterial species that differ on their ability to respire: one is a strict aerobe (Pseudomonas putida), which only respires using oxygen as final electron acceptor, while the other (Pseudomonas veronii) is a facultative anaerobe that can respire with oxygen but also with nitrate as final electron acceptor. Both species are motile thanks to flagella that they can use to swim within liquid films. To facilitate observation, the two species were each tagged with a distinct fluorescent protein (green or red). The two bacterial populations were inoculated into micrometric pore networks with various structures,  and which allowed us to create gradients of oxygen (coming from the periphery of the network) and of carbon source (citrate, coming from the center of the network). Although initially well-mixed in the center of the network, after a week of incubation the two bacterial species segregated in the network to form two distinct and coexisting populations (see figure below). One grew preferentially where the oxygen was more abundant (the obligate aerobe), while the other could occupy the anoxic niche at the center of the network, which also contained more carbon.
 

The two bacterial populations, initially well-mixed, grow and segregate in the pore network as function of their respiration metabolism. From Borer et al., 2018

Interestingly, this spatial organization did not occur when carbon source and oxygen were provided together (i.e., there was no counter-gradients of oxygen and carbon). Another intriguing result was that the coexistence of the two species, as seen in the figure, could not been achieved in well-mixed liquid environments (vials or flasks), because one of the species would always win over and dominate the community (see below). This illustrates how the habitat spatial structure can help limit competition and maintain species coexistence.

Experimental and modeling results show competitive exclusion in homogeneous cultures (a) and coexistence in structured pore networks (b). The percentage corresponds to the connectivity in the network (100% is highest connectivity).

Admittedly, our system is artificial and our community only composed of two species... Yet,  the basic mechanisms that we observed most probably also apply to more complex environments and could participate to the final structure of the community. Bacteria have a remarkable faculty of optimizing their distribution and activity in complex habitats, which we could demonstrate with our simple mathematical and experimental models.
 

Cooperation shapes the spatial patterns of bacterial organization

Cooperative bacterial strains colonizing a surface

Bacteria colonize surfaces in all environments. That could be the surfaces of soil aggregates, of rocks in a stream bed, of plant leaves, of animal skin, or that could be the surface of your showerhead... On such surfaces microbes establish complex communities ('biofilms') that can contain many different interacting species. These various species are usually not randomly distributed in the biofilm, but rather organized depending on their environmental preferences (for example some like well-aerated areas, others not so much...) and on the type of interactions that they have with each other. This can result in complex patterns of organization that manifest at the microscopic scale and up to the millimeter scale. Such patterns are not trivial, as they can sustain microbial activity and functions that would not be possible in a well-mixed environment, which has importance for biotechnology applications as well.

In a new study published this month, we examined the role of cooperation in shaping spatial patterns of bacterial organization on wet surfaces. The paper is available online and is entitled 'Cooperation in carbon source degradation shapes spatial self-organization of microbial consortia on hydrated surfaces'. Our idea was that a feeding dependency between two partners would directly control their distribution in space, hence imposing a specific pattern. We used a simple model system made of two bacterial strains that could grow using the chemical compound toluene (a hydrocarbon), but only when they were working together as a 'team' (a bacterial consortium in the jargon).

Symplasmata: a curious case of multicellularity in bacteria

Cells in a symplasmatum and surrounded by a capsule,
seen with transmission electron microscopy.

'Curiouser and curiouser’, famously said Lewis Carrol’s Alice, as she was experiencing some very peculiar events in Wonderland. I have sometimes felt like Alice when I was studying the curious behavior of the bacterium Pantoea agglomerans [1], during my time in the Lab Leveau at UC Davis.

At first sight, Pantoea agglomerans looks quite ordinary. It grows as rods a few micrometers long, it can swim with flagella and it feeds on all sorts of sugars. It belongs to the family Enterobacteriaceae, and thus it is a distant cousin of E. coli. You can find P. agglomerans in all sorts of environments, but it is particularly good at colonizing the surface of plants, and in certain cases it competes with pathogens and thus keeps its plant host healthy (that is, it can serve as a biocontrol agent). Because it is a very good leaf colonizer, we have used it in many studies of bacterial life in the ‘phyllosphere’ (the aerial surfaces of plants), such as the one described in this previous post. 

Now here’s what special, and actually seemingly unique, about Pantoea bacteria. Under certain conditions, instead of dividing and spreading as individual cells, the bacteria stay close together and form an aggregate containing up to hundreds of tightly packed cells. Aggregation is not uncommon in bacteria but, in the case of Pantoea, cells are constrained by a fibrillar layer, and surrounded by a thick capsule made of polysaccharides, which indicates some level of cooperation and resources sharing (see image on top of the post). The resulting sausage-shaped structures are called symplasmata [2]. Interestingly, the species name 'agglomerans' (forming into a ball), which was coined by the great Dutch microbiologist and botanist Martinus Beijerinck in a paper dating from 1888, probably refers to the species' ability to form symplasmata [3]. Although symplasmata have been known for a very long time, their importance and function in the environment is still a mystery. We have observed symplasmata on bean leaf surfaces, and others have described them attached to the roots of rice plants (Achouak et al., 1994). What is their ecological role? Does it benefit the plant as well? We do not know yet. 

Fluorescent bacteria under the microscope

Pantoea agglomerans and Pseudomonas syringae bacteria

Some time ago I made experiments growing two bacterial species on a gel surface, using fluorescence to distinguish between them. Since some of these pictures looked nice to me, I decided to share them here!

Here's some information about the bacteria and how the images were taken:

Pantoea agglomerans and Pseudomonas syringae are two bacterial species that live in association with plants: the former as a harmless inhabitant of plant leaves and the latter as a pathogen that can colonize the inside part of the plants. Because it is not easy to visualize these bacteria in their natural environment (the surface of plant leaves), it is common to use fluorescently-tagged strains. I discussed this type of research in a previous post.

Bacteria in a wastewater treatment plant

Bacteria in activated sludge from a wastewater treatment plant

It is a remarkable fact that we fully depend on microbes to treat or sewage water. In every wastewater treatment plant, from the simplest to the most modern ones, the essential activity is biological and is mainly carried out by bacteria. In modern plants, sewage water is directed to large aerated tanks in which the pollution is consumed by a mixture of microbes and organic matter known as  activated sludge.

The video below shows how an aerated tank looks like. You don’t want to take a swim in there… 

For a microbiologist, a wastewater treatment plant is a delight: I’ve never seen another environment with as much diversity in the size and shapes of cells. It is a true microbial jungle containing countless bacteria and many protozoans that feed on them.

Unwanted guests

Microbial contaminants (three species) on a plate

Microbiologists use Petri dishes, filled with a variety of agar media, to grow microbes. Even a small lab can produce hundreds of plates every week… And it is quite common to store in the fridge those of these plates that are not directly inoculated for a future use. Thus, when you open a microbiologist's fridge, you may find columns of Petri plates labeled with the medium and antibiotics they contain, waiting to be covered with a suspension of microbes.

Sometimes, when you store them for long periods, you can have the disagreeable surprise to find unwanted guests on your plates, that is, contaminant microbes!

Filamentous bacteria under the microscope

Filamentous bacteria from soil, seen with phase contrast microscopy.

It's pretty easy to isolate soil bacteria: take a scoop of soil, mix it with some water, then plate the liquid on a Petri dish and incubate it overnight at 25-30 °C. Voilà. 

The isolated bacterial species will vary with the conditions (type of medium, temperature); here I found many filamentous bacteria on the plate. They look a little bit like filamentous fungi (since they also form a mycelium), but usually you can easily tell them apart (with a microscope) because of their smaller diameter. 

The mechanics of bacterial cluster formation on plant leaf surfaces as revealed by bioreporter technology

Green and red fluorescent bacteria on the surface of a leaf

Our new publication is out there as an early view in Environmental Microbiology, and I shamelessly take this opportunity to write about it here!

Here's the story. The plant foliage is colonized by a crowd of microbes (with bacteria on the front line—up to 10⁸ bacteria per gram of leaf has been reported!). Some of them can be pathogenic, hence a threat, but most of them are harmless or even favorable inhabitants of the plant ecosystem.

Bacteria form large clusters (aggregates) of cells on the surface of leaves, but the mechanism of formation of such structures is not very well understood. What we want to know is how these bacteria grow and colonize this specific environment at the microscale, that is, at their own scale.

First, bacteria have to land on the leaf. Wind, rain, insects can all contribute to bring microbial visitors onto the leaf surface (what we call the phyllosphere), usually few cells at a time. Once on the leaf, these immigrants will grow at the expense of the plant sugars available on the leaf and, if the conditions are favorable, rapidly multiply to form clusters of up to thousands of cells. Do these clusters result from the random aggregation of cells or do they result from the reproduction of a single bacterium? The two mechanisms—that we call aggregative and replicative—are fundamentally different but not necessarily mutually exclusive. 

Thanks to fluorescence microscopy it is possible to visualize glowing bacteria on the surface of a leaf. One difficulty, however, is that we cannot follow the same microarea of leaf overtime… We are limited to a snapshot view, and so it's impossible to decide whether a given cluster was formed through aggregative or replicative behavior. For this reason we developed techniques that would enable us to deduce a posteriori what was the mechanism of formation.