This contribution provides the verification of a simple procedure on quantitative separate estimation of 1) root respiration, 2) rhizomicrobial respiration, and 3) microbial respiration from soil organic matter (SOM) decomposition in non-sterile soils. The method is based on growing C₄ plants on C₃ soil or vice versa. Hence, the d¹³C values of SOM, maize roots, microbial biomass, and total CO₂ efflux from the soil are used to determine the three fractions of CO₂. These contributions of root respiration, rhizomicrobial respiration and respiration derived from SOM decomposition to total soil CO₂ efflux can be calculated according to isotopic mass balance of microbial biomass and CO₂.

Maize was grown on a C₃ soil in the laboratory for 40 days and trapping of total CO₂ was started nine days after germination. Samples of bulk soil, maize roots, microbial biomass (chloroform-fumigation-extraction method), and total CO₂ (trapped in NaOH) were taken five times during the growth period and analyzed for d¹³C values at an isotope ratio mass spectrometry unit.

At the end of the experiment, d¹³C values of maize roots and total CO₂ efflux were significantly different, but very close at –15.8 and –16.9‰, respectively. The d¹³C value of SOM was –26.8‰ and the one of microbial biomass was –23.7‰. Using these d¹³C values according to the suggested approach, root respiration contributed to 91%, rhizomicrobial respiration to 4%, and SOM decomposition to 5% of the CO₂ efflux. Compared to recent studies, which used different approaches, rhizomicrobial respiration and SOM decomposition were underestimated by our approach. This could be due to 1) a ¹³C fractionation between microbial biomass and its CO₂ or 2) a small portion of active microbial biomass in the rhizosphere contributing to the CO₂ efflux. Checking the first reason for underestimation by incubation of C₃ soil and trapping CO₂, we found a fractionation of 3‰ between microbial biomass and respired CO₂, leading to an accumulation of the heavier isotope in the CO₂. Correcting the calculation by this fractionation, rhizomicrobial respiration increased by 6%. Checking the second reason for underestimation, we calculated that only a hypothetical portion of 60% active microbial biomass led to a rhizomicrobial respiration of 43%, which is comparable to reported results of other studies. However, this portion of active microbial biomass is non-realistic, since other studies showed that less than 10% of total microbial biomass is active and the remainder is dormant.

The suggested method based on ¹³C natural abundance for partitioning of total CO₂ efflux from the soil into three sources fails due to the small part of active microbial biomass in the bulk soil and rhizosphere. This led to a discrepancy between d¹³C of microbial biomass and of microbially respired CO₂.