Size Limits of Very Small Microorganisms


Andrew Knoll, Steering Group Co-chair

How small can a free-living organism be? On the surface, this question is straightforward—in principle, the smallest cells can be identified and measured. But understanding what factors determine this lower limit, and addressing the host of other questions that follow on from this knowledge, require a fundamental understanding of the chemistry and ecology of cellular life. The recent report of evidence for life in a martian meteorite and the prospect of searching for biological signatures in intelligently chosen samples from Mars and elsewhere bring a new immediacy to such questions. How do we recognize the morphological or chemical remnants of life in rocks deposited 4 billion years ago on another planet? Are the empirical limits on cell size identified by observation on Earth applicable to life wherever it may occur, or is minimum size a function of the particular chemistry of an individual planetary surface?

These questions formed the focus of a workshop on the size limits of very small organisms, organized by the Steering Group for the Workshop on Size Limits of Very Small Microorganisms and held on October 22 and 23, 1998. Eighteen invited panelists, representing fields ranging from cell biology and molecular genetics to paleontology and mineralogy, joined with an almost equal number of other participants in a wide-ranging exploration of minimum cell size and the challenge of interpreting micro- and nano-scale features of sedimentary rocks found on Earth or elsewhere in the solar system. This document contains the proceedings of that workshop. It includes position papers presented by the individual panelists, arranged by panel, along with a summary of extensive roundtable discussions that involved the panelists for each session as well as other workshop participants.


The discussions forming the basis of this document sought to address three distinct but related issues: (1) What are the theoretical, observable, and empirically testable limits on the minimum size of organisms living on Earth today? (2) What, in theory, are the size limits on organisms not constrained by the biochemistry of extant cells? and (3) How can we recognize traces of ancient and potentially unfamiliar life in samples from other bodies in the solar system? As is evident from the summaries, there was strong consensus on the first issue, but the others remain open. The six geneticists and cell biologists in Panel 1 reached consensus on the smallest size likely to be attained by organisms of modern biochemical complexity. Free-living organisms require a minimum of 250 to 450 proteins along with the genes and ribosomes necessary for their synthesis. A sphere capable of holding this minimal molecular complement would be 250 to 300 nm in diameter,1 including its bounding membrane. Given the uncertainties inherent in this estimate, the panel agreed that 250 ± 50 nm constitutes a reasonable lower size limit for life as we know it. At this minute size, membranes have sufficient biophysical integrity to contain interior structures without the need for a cell wall, but only if the organism is spherical and has an osmotic pressure not much above that of its environment.

Panel 2 consisted of microbial ecologists asked to elucidate the smallest sizes actually observed in free-living organisms. Once again, consensus emerged from the panel's discussion. Consistent with the theoretical limits articulated by Panel 1, members of Panel 2 reported that bacteria with a diameter of 300 to 500 nm are common in oligotrophic environments, but that smaller cells are not. Nanobacteria.2 reported from human and cow blood fall near the lower size limit suggested by cell biologists; however, the much smaller (ca. 50 nm) bodies found in association with these cells may not, themselves, be viable organisms. Observations on Archaea indicate that, in general, they have size limits similar to those for Bacteria.

Two problems constrain discussions of minimal cell size in natural environments. Commonly used methods of measuring cell size have inherent uncertainties or possibilities of error. Perhaps more important, most cells found in nature cannot be cultivated. Thus, ignorance about biological diversity at small sizes remains large. These problems notwithstanding, it appears that very small size in modern organisms is an adaptation for specific environmental circumstances, including stress and scarcity of resources. Primordial organisms may or may not have been tiny, but the smallest organisms known today reside on relatively late branches of the RNA phylogeny.

Whereas Panels 1 and 2 indicated that a cell operating by known molecular rules—with DNA or maybe RNA, ribosomes, protein catalysts, and other conventional cell machinery—would have a lower size limit of 200 to 300 nm in diameter, Panel 4 suggested that primitive microorganisms based on a single-polymer system could be as small as a sphere 50 nm in diameter. There is no assurance that primordial cells would have been this small or, if they were, that such minute cells would have been more than transitory features of early evolution. Nonetheless, unless one is willing to posit that everywhere it has arisen, life has evolved a biochemical machinery comparable to that seen on Earth, the rules that govern minimum cell size may not be universal.

In fact, as explored by Panel 3, there are a number of ways that living cells or fossils might fall below the minimum size deemed likely by cell biologists and ecologists. On Mars or Europa, fossils might preserve a record of biological systems different from those we understand—perhaps early products of evolution that made do with a small complement of functional molecules. Organisms of modern biochemistry might become small by being pathogens or living in consortia—that is, by using the products of another organism's genes. Or, fossils might preserve remains that shrank after death, or parts of organisms rather than complete cells—both are common in the terrestrial record.

Of course, fossil morphologies are but one of several types of biological signature preserved in rocks. Experience with ancient terrestrial rocks shows that extractable organic molecules, minerals, fractionation in isotopic or elemental abundances, and distinctively laminated sedimentary structures can all provide indications of past life. Many of these features, however, can be mimicked by physical processes. Panel 3 concluded that a much better understanding of biological pattern formation is needed before intelligently chosen martian samples are returned to Earth. The panel also emphasized that this must go hand in hand with improved knowledge of the limits of morphological and chemical pattern formation by non-biological processes. Indigenous features of extraterrestrial samples can be accepted as biogenic only if they are incompatible with formation by physical processes.


In 2008, a small (<1 kg) sample of martian rock and soil is scheduled to be delivered to Earth by a robotic spacecraft that will be launched to Mars in 2005. Among the important questions that will be asked of these samples is, Has Mars ever been a biological planet? Our ability to address this question is directly related to our understanding of the range of morphological features that can be produced by life and by physical processes, as well as the ranges of organic chemicals, mineral forms, and sedimentary rock features that can be generated by biological and by nonbiological processes. As the results of the workshop made clear, welcome consensus has emerged among the participants regarding the size and chemical limits on modern life on Earth. But, given reasonable uncertainty about whether such features are particular products of terrestrial evolution or universal features of life, the meter stick by which the biogenicity of martian or other planetary samples is measured will likely be knowledge of the limits on physical processes—knowledge that needs to be developed before samples from Mars arrive in the laboratory.

1Contributors to the workshop have usually described relevant scale sizes or dimensions in units of nanometers (nm) or micrometers (µm), depending on the context and the features being described. For an indication of the range of relevant scale sizes, see Figure 1 in the paper by Jack Farmer, Panel 3.

2While biologists have yet to agree on a precise meaning for this term, it is generally used to refer to any single-celled microorganism proposed to have a maximum diameter in the range of tens to a few hundreds of nanometers.

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