II.  Phenomenon of microbial uncultivability: our fault or sophisticated microbial strategy?

   in collaboration with Dr. Kim Lewis

Paradoxically, the majority of microorganisms from the environment resist cultivation in the laboratory. These “uncultivables” represent 99-99.99% of all microorganisms in nature. From hundreds of thousands (or millions!) of existing microbial species only a few thousand have been isolated in pure culture and properly described.  Several groups at the division level have been identified with no known cultivable representatives. Not surprisingly, the American Society for Microbiology recognized the riddle of “uncultivable” microorganisms as one of the main challenges for research in microbiology.


Recently, we have designed and successfully tested a new cultivation methodology that provides access to many formerly “uncultivable” microorganisms (Kaeberlein et al. 2002).  Based on this advance, we are determined to resolve the nature of this puzzling biological phenomenon.


Access to uncultivated microbial majority

The concept of our novel cultivation methodology is based on a simple observation: the microorganisms appear “uncultivable” only in the laboratory, whereas in nature they seem to be growing (at least periodically).  It follows that the natural environment provides the microorganisms it harbors with the essential growth factors in appropriate combination.  We further reasoned that the uncultivable microorganisms should grow if provided with access to this natural chemical soup.  This could be done by placing “uncultivated” microorganisms into diffusion chambers and incubating these chambers in these microorganisms’ natural environment.  The chemical diffusion through the semipermeable walls of the chamber (30 nm pore size) would provide the microorganisms inside with all the naturally-occurring components. Our expectation was that these microorganisms should grow and form colonies, perhaps not even “knowing” they had ever been removed from nature.  After the chambers are retreived and open, the colonies inside can be accessed, individually subscultured into new chambers, and their pure cultures thus achieved.  In this way, the problems of media optimization/design/nutrient combination/concentration are all bypassed, and uncultivables should grow.  Our results matched our expectations: the diffusion chambers recovered 300 times more microorganisms than did the traditional Petri dish-based approach (Kaeberlein et al. 2002).  This means that we have in our hands a fundamentally new methodology to uncover, access, isolate into pure culture, and study many novel microbial forms.


<Fig. 8 The Diffusion Growth Chamber >

<Fig. 9 Diffusion Chambers vs. Petri Dish>

<Fig. 10 "Fil">

High-Throughput Microbial Cultivation

On the basis of the original diffusion chamber (Fig....) we designed and built a new device for high throughput cultivation of "uncultivable" microbial species. We call this device Isolation Chip, or for short. The ichip is an assembly of flat plates containing multiple registered through-holes. When the central plate is dipped into a cell suspension, each through-hole captures a volume of suspension containing a certain number of cells. If the suspension is appropriately diluted, this number is one cell per through-hole. Sandwiching the plate between semi-permeable barriers, such as 0.03-µm pore size filters used in the original diffusion chamber, effectively transforms the loaded plate into an array of many diffusion minichambers. In situ incubation of the loaded ichip in the cell’s original environmental habitat provides the immobilized cells with their naturally occurring growth components. Cells grow and form colonies as they do in “standard” diffusion chambers, but in ichip they form pure cultures from the start. In this way, cultivation and isolation of “uncultivables” can be achieved in large numbers and in a single step.


<Isolation Chip, or ichip, for high throughput microbial cultivation in situ. Left panel shows dipping a plate with multiple through-holes into a suspension of cells such that each hole captures (on average) a single cell, as illustrated in the central panel. Right panel shows how ichip is assembled: membranes cover arrays of through-holes from each side; upper and bottom plates with matching holes press the membranes against the central (loaded) plate. Screws provide sufficient pressure to seal the content of individual through-holes, which become miniature diffusion chambers containing single cells>

Resolving the nature of microbial uncultivability

The new cultivation methodology provided us with isolates of formerly “uncultivable” microorganisms and gave us model systems to study the nature of the phenomenon.

Working with chamber-grown colonies of “uncultivables”, we noticed that they could also be grown in Petri dishes if paired with other selected microorganisms, which we term “helpers”.  In other words, at least some “uncultivables” refuse to grow on traditional media only in isolation but readily do so as part of specific microbial consortia. 

<Fig. 11 "Fil & Archie">

<Fig. 12 "MSC 107 & MSC 16">

 

The growth pattern of microbial consortia cannot be readily explained by cross-feeding between the partners and/or removal of growth inhibitors by one of them. It seems possible that “uncultivable” microorganisms require specific signals originating from their “helping” neighbors that indicate the presence of a familiar environment. Implicit in this signaling hypothesis is that microorganisms will not grow in an unfamiliar environment even in the presence of appropriate nutrients, and this may explain why so many microorganisms cannot be isolated in pure culture on artificial media in vitro.  Presently, we are trying to identify the chemical nature of such signaling molecules.  This work is done in collaboration with Dr. J. Orjala, University of Illinois/Chicago) and Dr. P. Vouros (Northeastern University, Boston).

In parallel, we obtained variants of “uncultivable” microorganisms capable of growth in Petri dish in pure culture.  Clearly, these isolates lack the growth restriction characteristic of the parent strains.  We also learned how to transform these cultivable variants back into their original “uncultivable” state.  This gives us an excellent tool to tackle the general nature of the phenomenon by studying the growth restrictions of the original isolate, their losses in certain daughter strains, and re-acquisition of these restrictions under specific cultivation conditions.

 

Biotechnological potential of uncultivated microorganisms


Uncultivated microorganisms represent the bulk of global microbial diversity, and gaining access to these microorganisms may open an untapped source of novel biological and chemical properties. We are presently exploring several aspects of this promise. First, supported by DOE and in collaboration with Dr. Palumbo from the Oak Ridge National Laboratory in Tennessee, we are isolating “uncultivable” microorganisms important in soil bioremidiation. We are especially interested in the habitats where a number of microorganisms have been detected via rRNA approach but none has been isolated using traditional culturing techniques. Second, and also with support from DOE, we are exploring uncultivated microorganisms from soil communities on their ability to produce alternative fuel: bioethanol. We are looking for new species, as well as synergistic consortia of microbial species, that are capable of breaking down cellulose, hemicellulose, and lignin into sugars, and fermenting the sugars into ethanol. Third, we are funded by NIH to use the diffusion chamber-based approach to microbial cultivation to grow new species from the human microbiome. Specifically, in collaboration with Dr. Bruce Paster from Forsyth Institute, we aim to cultivate previously “uncultivable” species from the human oral cavity. While this effort is new, we have already isolated in pure culture three novel strains of Streptococcus and Lactobacillus. These strains are under consideration by the Broad Institute as candidates for the genome sequencing.



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