Methods Based on 14CO2 Uptake

These methods are free from limitations inherent in CO2 evolution, because the addition of labeled CO2 to gas phase over soil does not affect the soil trophic status, and sticky soil CO2 does not interfere with the detection of added 14CO2. An additional significant advantage is that exposure to 14CO2 does not require preliminary melting of frozen soil. A minor disadvantage inherent to the majority of techniques based on radioactive indicators is that analysis is destructive: we have to sacrifice the incubated sample, and we can't set up a continuous monitoring of 14CO2 uptake with the same sample or laboratory microcosm.

At least three groups of soil organisms are responsible for 14CO2 uptake: (i) pho-toautotrophic, (ii) chemolithotrophic (chemosynthetic), and (iii) chemoorgano-trophic (heterotrophic) organisms.

The contribution of the first group is quantified by using artificial illumination and calculating the difference between 14C-uptake under light and the dark control. Historically, photosynthetic CO2 uptake by Antarctic lichens was probably the very first reliable measurement of below-zero microbial activity in situ (Lange and Metzner 1965; Lange and Kappen 1972).

Dark CO2 fixation (DF) refers tothe activity of the second (chemosynthetic) and third (heterotrophic organisms, the most abundant in soils) microbial groups. They could be differentiated by using specific autotrophic inhibitors (acetylene, allylth-iourea, etc.) or by observing DF stimulation by the addition of oxidizeable inorganic substrates, such as H2, NH4+, S2-, S°, Fe2+, etc.

Heterotrophic microorganisms always use CO2 in biosynthetic reactions (so-called heterotrophic fixation, HF), although this process is hidden by simultaneously occurring respiratory release of CO2. Therefore, we have to add an isotope indicator to measure HF. Specifically, CO2 can be fixed in several fermentation pathways and in the anaplerotic reactions of the tricarboxylic acid (TCA) cycle via carboxylations of pyruvate or phosphoenol pyruvate (PEP) at the expense of ATP or by running a reverse TCA cycle.

Computational modeling of metabolic flux indicates that the net contribution of HF to total cellular synthesis is about 40% (Marx et al. 1996), but the empirically found stoichiometric ratio varies from 1% to 10%, with an average close to 6% (Johnson and Romanenko 1984; Santruchkova et al. 2005).

In the rest of this review, this technique will be referred to as DF because a differentiation between chemosynthetic and heterotrophic organisms has not been done. The brief protocol for testing DF is as follows: 2.0 g of frozen soil crumbled in 3-10 replicated 30 ml vials with rubber septum are incubated with 14CO2 in the headspace (at least 1,000 DPM ml-1); after a certain period (usually after 1 week of incubation at -5 to -25°C) one of the replicated vials is sacrificed. The headspace is sampled for the total CO2 (LiCor 800) and 14CO2 (1 N NaOH trap following counts with Beckman 5800 L scintillation counter) to determine isotope dilution. Then the soil is flushed with N2 and dried for 3 h at 95°C to remove non-reacted 14CO2. Finally, the fixed 14C is released and counted as 14CO2 after soil ignition at 900°C (Solid Sample Module, TOC-VE, Shimadzu) (Panikov and Sizova 2007). Linear dynamics in 14C uptake at least during the first month of incubation at -11°C has been demonstrated; the sterile (autoclaved and refrozen) control demonstrated zero retention of 14CO.

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