Optimization of an oxygen-based approach for community-level physiological profiling of soils

Abstract

Current approaches for rapid assessment of carbon source utilization by whole soil communities (i.e., community-level physiological profiling or CLPP) provide a limited, biased view of microbial communities with little connection to in situ activities. We developed an alternative CLPP approach based upon fluorometric detection of dissolved oxygen consumption in a microtiter platform which offers flexible manipulation of experimental factors. In the attempt to reduce oxygen re-dissolution, the wells were filled with liquid to very near the top and sealed. We found that filling the wells with 240 vs. 150 µl of sample improved the sensitivity of the system to discern both the response to a substrate amendment as low as 10 mg l-1 and un-amended, endogenous respiration. The preparation of a soil slurry facilitates inoculation into the microplate. Disruption of soil samples had a limited effect on the endogenous respiration in comparison to intact soil microbags in a 24-well microplate. Storage time (up to 33 days) reduced the level of activity in intact soil microbags but not in disrupted samples. A microcosm fertilization experiment was set to study the effects of N availability on the respiratory response in the plates. The use of soil organic carbon (SOC) and amended C-substrates (50 mg l-1) was increased by the addition of nitrogen (N) in the plate, and appeared N-limited shortly after microcosm fertilization. The addition of the eukaryotic inhibitor cycloheximide delayed the initial increase in fluorescence (time to minimum response) of several C sources (casein, acetate, asparagine, coumaric acid), varying among soils, which could be explained by the fungal use of these compounds. However, the extent of the inhibition caused by cycloheximide did not increase at higher fungal to bacteria ratios as estimated by PLFA analysis, indicating that the direct estimation of the fungal biomass from cycloheximide addition is not feasible. This paper provides an optimized, standardized protocol for soil analysis, and sets the basis for further validation studies that will continue to define the underlying capabilities/biases of this approach.