Live-cell validity and lineage generality of the nonlinear cell size-to-carbon-mass scaling of small prokaryotes

Direct measurements of 12 cultivated prokaryotic species established that fixed, dehydrated cells obey a strongly nonlinear cell size-to-mass scaling, with carbon mass $m_{\mathrm{carbon}} = 197 \times V^{0.46}$ (fg, with volume $V$ in $\mu$m$^3$) and a carbon mass density that rises sharply as volume decreases below roughly $0.5\ \mu$m$^3$. Because these relationships were derived from chemically fixed, dehydrated material and from a small set of cultured taxa, it remains unresolved whether they hold for living cells in situ and across uncultivated prokaryotic lineages. Resolving this would fix the conversion factors that translate cell counts and biovolumes into carbon biomass for the smallest microorganisms, on which global biomass inventories of bacterioplankton, soils, and the deep biosphere depend.

Background

Microbial ecology routinely needs the carbon content of individual microbial cells: abundance data (cell counts or biovolumes from microscopy) must be converted to carbon biomass before they can enter estimates of standing stocks, growth and mortality rates, and carbon fluxes. Because cellular carbon cannot usually be measured on every cell, the field relies on empirical conversion factors — allometric equations that map a cell's volume to its carbon or dry mass, or a fixed fg C per cell assumed independent of size.

Classical allometric equations were largely derived from eukaryotic microalgae or from bulk chemical measurements of cultured bacteria, and they disagreed substantially with one another. Khachikyan et al. (2019) revisited this with direct single-cell measurements: they weighed cells of 12 environmentally relevant bacterial and archaeal species (volumes 0.011–0.705 $\mu$m$^3$) with a suspended microchannel resonator (SMR), a microfluidic chip in which a vibrating channel's resonance frequency shifts as an individual cell passes through, revealing the cell's buoyant and, on dried cells, effectively dry mass; they measured cell volumes by scanning electron microscopy (SEM) of chemically fixed, ethanol-dehydrated, critical-point-dried (CPD) cells — a preparation that replaces water with liquid CO$_2$ and evaporates it at its critical point to avoid surface-tension collapse — and they measured cellular elemental composition (C, N, O, P, S) by energy-dispersive X-ray spectroscopy. They found a strongly nonlinear carbon-mass-to-volume relationship, $m_{\mathrm{carbon}} = 197 \times V^{0.46}$ fg ($R^2 = 0.95$), so that carbon mass density ($fg\ C\ \mu$m$^{-3}$) increases nonlinearly as cells get smaller; a compilation with previously published data from larger cells suggested that above about $0.5\ \mu$m$^3$ the mass-to-volume ratio is roughly constant, while below it the density rises steeply. Compared with the models then in use, this yielded up to ~300% higher carbon estimates for the smallest cells and implied that deep-biosphere microbial carbon had been underestimated by tens to hundreds of percent.

Two limitations of the study were, however, apparent: the first was explicitly flagged by the authors, and the second is implied by the study's own extrapolation of its conversion factors to abundant uncultivated taxa that were not measured. First, the relationships were established on fixed and dried cells; the live-versus-fixed comparison was performed on only two species (a volume comparison by atomic force microscopy (AFM), a scanning-probe technique that measures live-cell length, width, and height; and a mass comparison by SMR), and although no significant differences were found, older literature suggests cells often shrink during fixation and dehydration — so the in-situ dry-mass and elemental densities of the smallest cells could be systematically overestimated. Second, only 12 cultivated species, mostly from laboratory culture in a narrow set of physiological states, were measured, leaving open whether the same scaling applies to the uncultivated lineages that dominate natural microbial communities (such as SAR11 and SAR86, two of the most abundant uncultivated marine bacterial groups; Actinobacteria, a bacterial phylum especially widespread in soils and freshwaters; and marine group I Thaumarchaeota, the ammonia-oxidizing archaea that dominate deep-ocean archaeal communities), or whether separate size- or lineage-dependent regimes are required.

Problem Statement

Determine whether the nonlinear size-to-mass relationships established for fixed, dehydrated cells of 12 cultivated prokaryotic species — in particular the carbon-mass scaling $m_{\mathrm{carbon}} = 197 \times V^{0.46}$ fg ($V$ in $\mu$m$^3$) and the corresponding carbon-mass-density relation — are valid for living prokaryotic cells in situ, and establish conversion factors between cell volume (or abundance) and cellular carbon and dry mass that are applicable to natural communities of small (<~0.5 $\mu$m$^3$) microorganisms. Concretely, this requires: (i) quantifying how chemical fixation, dehydration, and drying change the volume and mass of small prokaryotic cells across additional taxa, physiological states, and preparation protocols, beyond the two species compared in the source study, so that any over- or underestimation of in-situ dry-mass and elemental densities can be corrected; and (ii) testing the scaling itself on uncultivated and environmentally dominant lineages (e.g., SAR11, SAR86, Actinobacteria, marine group I Thaumarchaeota, soil and deep-subsurface taxa) and across growth conditions and stoichiometries, to decide whether one allometric relationship suffices or separate size- or lineage-dependent regimes are needed. A complete answer delivers empirically grounded, uncertainty-quantified conversion factors (or demonstrated correction factors relative to the 2019 equations) usable for biomass and turnover estimates in marine, freshwater, soil, and deep-biosphere settings.

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