Community-level physiological profiling performed with an oxygen-sensitive fluorophore in a microtiter plate.
ABSTRACT Community-level physiological profiling based upon fluorometric detection of oxygen consumption was performed on hydroponic rhizosphere and salt marsh litter samples by using substrate levels as low as 50 ppm with incubation times between 5 and 24 h. The rate and extent of response were increased in samples acclimated to specific substrates and were reduced by limiting nitrogen availability in the wells.
Full-textDOI: · Available from: Jay L Garland, Mar 17, 2014
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ABSTRACT: Community level physiological profiling is a simple, high-throughput technique for assessing microbial community physiology. Initial methods relying on redox-dye based detection of respiration were subject to strong enrichment bias, but subsequent development of a microtiter assay using an oxygen-quenched dye reduced this bias and improved the versatility of the approach. Commercial production of the oxygen microplates recently stopped, which led to the present effort to develop and validate a system using a luminophore dye (platinum tetrakis pentafluorophenyl) immobilized at the bottom of wells within a 96 well microtiter plate. The technique was used to analyze three well-characterized Florida soils: oak saw palmetto scrub, coastal mixed hardwood, and soil from an agricultural field used to grow corn sillage. Substrate induced respiration was monitored by measuring respiration rates in soils under basal conditions and comparing to soils supplemented with nitrogen and various carbon sources (mannose, casein, asparagine, coumaric acid). All data was compared to a previously available commercial assay. There were no significant differences in the maximum peak intensity or the time to peak response for all soils tested (p<0.001, α=0.05). The experimental assay plates can be reused on soils up to four times (based on a deviation of less than 5%), where the commercial assay should not be reused. The results indicate that the new oxygen-based bioassay is a cost effective, open source tool for functional profiling of microbial communities.Journal of microbiological methods 11/2013; 96. DOI:10.1016/j.mimet.2013.10.016 · 2.10 Impact Factor
Polish Journal of Environmental Studies 04/2006; 15(5):669-676. · 0.60 Impact Factor
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ABSTRACT: Documented approaches for measuring soil microbial activities and their controlling factors under field conditions are needed to advance understanding of soil microbial processes for numerous applications. We manipulated field plots with carbon (C) and nitrogen (N) additions to test the capability of a respiratory assay to: (1) measure respiration of endogenous soil C in comparison to field-measured CO2 fluxes; (2) determine substrate-induced respiratory (SIR) activities that are consistent with substrate availability in the field; and, (3) report N availability in the field based on assay responses with and without added N. The respiratory assay utilizes a microplate containing an oxygen-sensitive fluorescent ruthenium dye. Respiratory activities measured with this approach have previously been shown to occur within short (6–8 h) incubation periods using low substrate concentrations that minimize enrichment during the assay. Field treatments were conducted in a randomized full-factorial design with C substrate (casamino acids, glucose, or none) and inorganic N (±) as the treatment factors. With one exception, we found that respiration of endogenous soil C in the assay responded to the field treatments in a similar manner to CO2 fluxes measured in the field. Patterns of SIR with low concentrations of added amino acid or carbohydrate substrate (200 μg C g−1 soil) were consistent with field treatments. The ratio (Nratio) of carbohydrate respiration with added N (25 μg N g−1 soil) to the same without N in the assay was significantly (P < 0.05) decreased by field N amendment. The carbohydrate Nratio exhibited a logarithmic relationship (r = 0.64, P < 0.05) with extractable inorganic soil nitrate and ammonium concentrations. These data significantly extend and support the capability of this oxygen-based respiratory assay to evaluate in situ soil activities and examine factors that limit these activities.Applied Soil Ecology 02/2013; 64:127–134. DOI:10.1016/j.apsoil.2012.11.006 · 2.21 Impact Factor