Stable-isotope probing (SIP) is a technique in

microbial ecology Microbial ecology (or environmental microbiology) is the ecology of microorganisms: their relationship with one another and with their environment. It concerns the three major domains of life— Eukaryota, Archaea, and Bacteria—as well as vi ...

for tracing uptake of

nutrient A nutrient is a substance used by an organism to survive, grow, and reproduce. The requirement for dietary nutrient intake applies to animals, plants, fungi, and protists. Nutrients can be incorporated into cells for metabolic purposes or excr ...

s in

biogeochemical cycling A biogeochemical cycle (or more generally a cycle of matter) is the pathway by which a chemical substance cycles (is turned over or moves through) the biotic and the abiotic compartments of Earth. The biotic compartment is the biosphere and ...

by microorganisms. A substrate is enriched with a heavier

stable isotope The term stable isotope has a meaning similar to stable nuclide, but is preferably used when speaking of nuclides of a specific element. Hence, the plural form stable isotopes usually refers to isotopes of the same element. The relative abundanc ...

that is consumed by the organisms to be studied.

Biomarkers In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, ...

with the heavier isotopes incorporated into them can be separated from biomarkers containing the more naturally abundant lighter isotope by

isopycnic centrifugation An isopycnic surface is a surface of constant density inside a fluid. Isopycnic surfaces contrast with isobaric or isothermal surfaces, which describe surfaces of constant pressure and constant temperature respectively. Isopycnic surfaces are ...

. For example, ¹³ CO₂ can be used to find out which organisms are actively

photosynthesizing Photosynthesis is a process used by plants and other organisms to convert light energy into chemical energy that, through cellular respiration, can later be released to fuel the organism's activities. Some of this chemical energy is stored in c ...

or consuming new photosynthate. As the biomarker, DNA with ¹³C is then separated from DNA with ¹²C by centrifugation.

Sequencing In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succi ...

the DNA identifies which organisms were consuming existing

carbohydrates In organic chemistry, a carbohydrate () is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula (where ''m'' may or may ...

and which were using carbohydrates more recently produced from photosynthesis. SIP with ¹⁸O-labeled water can be used to find out which organisms are actively growing, because oxygen from water is incorporated into DNA (and RNA) during synthesis. When DNA is the biomarker, SIP can be performed using isotopically labeled C, H, O, or N, though ¹³C is used most often. The density shift is proportional to the change in density in the DNA, which depends on the difference in mass between the rare and common isotopes for a given element, and on the abundance of elements in the DNA. For example, the difference in mass between ¹⁸O and ¹⁶O (two atomic mass units) is twice that between ¹³C and ¹²C (one atomic mass unit), so incorporation of ¹⁸O into DNA will cause a larger per atom density shift than will incorporation of ¹³C. Conversely, DNA contains nearly twice as many carbon atoms (11.25 per base, on average) as oxygen atoms (6 per base), so at equivalent labeling (e.g., 50 atom percent ¹³C or ¹⁸O), DNA labeled with ¹⁸O will be only slightly more dense than DNA fully labeled with ¹³C. Similarly, nitrogen is less abundant in DNA (3.75 atoms per base, on average), so a weaker DNA buoyant density shift is observed with ¹⁵N- versus ¹³C-labeled or ¹⁸O-labeled substrates. Larger buoyant density shifts are observed when multiple isotope tracers are used. Because density shifts as a predictable function of the change in mass caused by isotope assimilation, stable isotope probing can be modeled to estimate the amount of isotope incorporation, an approach called quantitative stable isotope probing (qSIP), which has been applied to microbial communities in soils, marine sediments, and decomposing leaves to compare rates of growth and substrate assimilation among different microbial taxa.

See also

References

Further reading