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United States Department of Agriculture

Agricultural Research Service

Research Project: ECOLOGICALLY-BASED SOIL AND CROP MANAGEMENT SYSTEMS FOR SUSTAINABLE AGRICULTURE

Location: North Central Agricultural Research Laboratory

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

Authors
item Zabaloy, Maria - UNIV NACIONAL DEL SUR
item Lehman, R
item Frey, Serita - UNIV OF NEW HAMPSHIRE
item Garland, Jay - DYNAMAC CORPORATION

Submitted to: Soil Biology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 28, 2008
Publication Date: November 12, 2008
Citation: Zabaloy, M.C., Lehman, R.M., Frey, S.D., Garland, J.L. 2008. Optimization of an oxygen-based approach for community-level physiological profiling of soils. Soil Biology and Biochemistry. 40(12):2960-2969.

Interpretive Summary: We have presented a set of research studies that optimize the use of a functional assay for soil microbial activities. This microtiter plate-based assay measures respiration by use of an oxygen-sensitive, fluorescent ruthenium dye embedded in an inert gel within the individual microplate wells. In this paper we show: (i) improved sensitivity is provided by using larger volumes of soil slurry and covering the microplate wells; (ii) respiration of endogenous carbon is detectable; (iii) disruption of soil structure during the assay procedure does not significantly affect the results; (iv) sample storage prior to assay does not markedly influence the results; (v) the assay can detect nitrogen limitation; and (vi) the assay includes the activities of soil microfungi. The attributes we have demonstrated for this assay represent substantial advances over existing functional assays of soil microbial activities.We have presented a set of research studies that optimize the use of a functional assay for soil microbial activities. This microtiter plate-based assay measures respiration by use of an oxygen-sensitive, fluorescent ruthenium dye embedded in an inert gel within the individual microplate wells. In this paper we show: (i) improved sensitivity is provided by using larger volumes of soil slurry and covering the microplate wells; (ii) respiration of endogenous carbon is detectable; (iii) disruption of soil structure during the assay procedure does not significantly affect the results; (iv) sample storage prior to assay does not markedly influence the results; (v) the assay can detect nitrogen limitation; and (vi) the assay includes the activities of soil microfungi. The attributes we have demonstrated for this assay represent substantial advances over existing functional assays of soil microbial activities.

Technical Abstract: Current approaches for rapid assessment of carbon source utilization by whole soil communities (i.e., community-level physiological profiling or CLPP) provides 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 oxygen consumption in a microtiter platform which offers flexible manipulation of experimental factors. We found that filling the wells with 240 vs. 150 µl improved the sensitivity of the system to discern both the response to a substrate amendment as low as 10 mg l-1 and unamended, endogenous respiration. The preparation of a soil slurry is needed for inoculation into the microplate. We showed that disruption of soil samples did not significantly affect the endogenous respiration in comparison to intact soil microbags in a 24-wells microplate. Storage time (up to 33 days) reduced the level of activity in intact soil microbags but not in disrupted samples. Fluorescence response to amended C-only sources (50 mg l-1) was increased by the addition of N. Moreover, we showed that the use of indigenous C and amended C substrates is N-limited shortly after soil 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 ratios of Fungal/Bacteria biomass ratio as estimated by PLFA analysis, indicating the direct estimation of the fungal biomass from cycloheximide addition is not feasible. A recommended, standardized protocol for soil analysis is provided, and further validation studies to define the underlying capabilities/biases are discussed.

Last Modified: 4/16/2014
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