2007 Annual Report 1a.Objectives (from AD-416) Although microbial processes in soil are important to sustaining agricultural production systems, methods for assessing soil microbial activity are limited. The purpose of this project is to optimize and validate a simple and rapid approach for monitoring microbial respiration in soil. Rapid assessment of carbon source utilization by whole communities (i.e., community-level physiological profiling or CLPP) has proven to be an effective tool for quick discrimination among soil types or treatments, but function as estimated by the redox dye reduction in Biolog plates provides a limited, biased view of microbial communities with little direct connection to in situ activities. This proposal involves an alternative, more functionally relevant CLPP approach based upon fluorometric detection of oxygen consumption. The fluorescence of an oxygen-sensitive fluorophore-gel complex loaded on the bottom of the microplate wells increases in response to respiration in the overlying sample (i.e., aqueous soil slurries). A peak in fluorescence can be detected within hours as opposed to days in response to much lower substrate concentrations (i.e., 1 mM vs >100 mM), thereby greatly reducing selective enrichment bias compared to the Biolog technology. While initial results with the method have established functional relevance, issues remain with respect to the optimization and validation of the method for soil analysis. Can the assay be improved to detect response to lower levels of supplementation (i.e., less than ~50 ppm threshold observed to date) and to measure low levels of endogenous (i.e., unsupplemented) respiration? How much of an effect does disruption of the soil structure into a slurry for inoculation have on the measured response, and are there practical approaches to overcome this disruption? With or without such improvements in sensitivity or sample processing, the method requires validation in the field. Specifically, does substrate use as measured in the BDOxy plates reflect differences in in situ substrate availability and use? Can oxygen consumption without carbon supplementation consistently detect endogenous respiration, and how do these rates compare to alternative measures of respiration such as soil carbon dioxide flux? Can fungal and bacterial respiration be effectively distinguished through the use of selective inhibitors, and do the trends reflect independent assessments of fungal and bacterial biomass? Can nitrogen limitation in agricultural soils be effectively determined by evaluating substrate use with and without N supplementation? Our overall goals are to provide the soil microbiological community with an understanding of the underlying capabilities/biases of the approach as well as a recommended, standard protocol. 1b.Approach (from AD-416) We proposed to optimize the assay by evaluating approaches to both improve sensitivity and reduce bias. While the BDOxy system allows for testing substrate concentrations 100 fold lower than other substrate induced respiration approaches, further improvements in sensitivity would allow even lower levels of substrate addition and should improve the ability to measure endogenous levels of respiration. The most likely approach for increasing the sensitivity is eliminating oxygen diffusion into the plate. The reduction in fluorescence following depletion of the substrate clearly shows that the observed response is a balance between oxygen consumption and re-aeration of the plate; eliminating diffusion should allow for lower levels of oxygen consumption to be detected. To date, BDOxy-CLPP analysis of soil has involved making a soil slurry, which obviously disrupts the soil structure. The extent to which this disruption affects the response has not been assessed, and we propose to do so in the present study by comparing the response of intact soil bags directly immersed into the BDOxy plates vs. soil slurries created from the same soil bags. We also propose to validate various capabilities of the assay. Endogenous respiration as estimated by oxygen consumption in unsupplemented samples will be compared to independent measures of soil respiration based on carbon dioxide production. Specifically, we propose to perform lab incubations of soils in sealed flasks, and correlate carbon dioxide flux measurements with BDOxy-CLPP response over an extended period of incubation. The functional relevance of the assay will be verified by analyzing substrate-induced responses in soils amended with different substrates. The potential for distinguishing fungal versus bacterial activity in soil will be done by using fungicides in the assay, and relating the degree of inhibition to independent measures of fungal and bacterial biomass. The lack of ancillary proprietary ingredients in the BD plates allows for ready manipulation of factors such as nitrogen availability; we will use this feature to test for nitrogen limitation in long term N-amendment students in both agricultural and forest soils. 3.Progress Report This report documents research conducted under a Reimbursable Agreement between ARS and Dynamac Corporation with funding from the CSREES-NRI program. Additional details of research can be found in the report for the in-house associated project 5447-12620-002-00D, Ecologically-Based Soil and Crop Management Systems for Sustainable Agriculture. The objectives of this research project “Optimization and Validation of Community-Level Physiological Profiling Based on Oxygen Consumption for Determining Microbial Respiration” are to optimize and validate a new approach for a functionally relevant community-level physiological profiling (CLPP) of soil microbial communities using a rapid O2 consumption-based technology performed in a laboratory assay. Progress in FY-07 relates to section 2.3.2 of the proposal: “Quantifying the Effects of Sample Processing” which describes an experiment using soil microbags. The soil microbag experiment was designed to quantify the effect of soil disruption on soil microbial activities measured in the laboratory assay. Initially, one-hundred and six soil microbags were constructed of nylon spawn sac netting that was sealed with Atlas-Mike’ poly “magic thread.” The soil was collected from the Eastern South Dakota Soil and Water Research Farm on a plot where soybeans had been recently harvested. After removal of the top 10 cm of soil (Barnes sandy clay loam), about 5 kg of soil was collected from this depth, sieved (4.75 mm) at field moisture, and homogenized. Each soil microbag was filled with 2.0 ± 0.1 g of field wet, sieved, homogenized soil. The resulting microbags each had a nominal volume of 1 cm3 and were kept at 4 °C until all were completed (< 1 wk). The 106 soil microbags were buried at 10-cm depth in a 3-m long trench in the same soybean stubble field where the soil was originally collected. Fifty soil microbags were excavated after 1 month burial and used in initial assays to test the effects of soil disruption and to test the effect of carbon amendment on soil microbial activities. A second set of 50 soil microbags were excavated after eight months burial for the same tests. Test results showed that endogenous carbon utilization could easily be detected in 1:1 dilutions (in water) of these soils. Carbon amendment, 50 mg/l of either sucrose or casamino acids, did not stimulate activity. Disruption of soil microbags that were buried for 1 month stimulated greater activity, while the converse was true with microbags buried for 8 months. Additional soil microbags (210) were constructed as above and buried to support additional analyses.