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.Characterization and classification of heterotrophic microbial communities based on rapid assessment of multiple solecarbon-source use, termed community-level physiological profiling (CLPP) by Lehman et al. (9), were developed over 10 years ago (6). Although CLPP is a valuable method for assessing relative change in microbial communities when properly applied (3, 12), limitations due to selective enrichment resulting from the high substrate concentrations (i.e., Ͼ100 mM) and long incubation periods (typically 1 to 4 days) restrict its present use for assessing actual differences in the physiological capabilities of microbial communities (2,8,14,16). Development of more functionally relevant CLPP will depend on the application of new detection methods that allow for rapid and direct (e.g., O 2 consumption or CO 2 production) rather than indirect (e.g., tetrazolium dye reduction) assessment of respiration, with decreased requisite incubation times and substrate concentrations. In addition, the use of undefined, proprietary reagents should be minimized so that incubation conditions can be defined and manipulated by the investigator.14 C respirometric approaches limit enrichment effects by allowing for detection of physiological response with addition of very low substrate concentrations (i.e., nanomolar levels), but even simplified assay systems (13) still have the expense associated with disposal of radioactive wastes. CLPP approaches based on CO 2 monitoring that limit incubation time have been developed (1) but require high substrate concentrations (i.e., 10 to 100 mM).Tracking the rate of O 2 consumption in the test medium may improve sensitivity, given the relatively low concentration and solubility of O 2 in water. A recently developed, fluorescencebased microplate platform for assessing dissolved oxygen (BD Oxygen Biosensor System; BD Biosciences, Bedford, Mass.) (17) could enable rapid testing of multiple substrates; the effectiveness of this system to detect known shifts in substrate utilization by mixed microbial communities is the subject of this work. The BD Oxygen Biosensor System is based on an O 2 -sensitive fluorophore, 4,7-diphenyl-1,10-phenathroline ruthenium (II) chloride, absorbed into a silicone matrix that is permeable to O 2 (17). The fluorescence of the ruthenium dye is quenched by the presence of O 2 , so the signal from the fluorophore-gel complex loaded on the bottom of the microplate wells increases in response to respiration in the overlying sample. In this work, samples of environmental systems suspended in sterile phosphate-buffered mineral salts (PBMS) ( Ϫ1 ) were inoculated into the ...

Evaluation of Biodegradability and Biodegradation Kinetics for Anionic, Nonionic, and Amphoteric Surfactants

“…In a set of follow-up experiments, samples collected at the termination of respirometry experiments were filtered (0.45 μm) and introduced to a fresh inoculum. Oxygen uptake was not measurable for any samples either by respirometry or by a microtiter-based oxygen sensor system (Garland et al 2003) after introduction to a fresh inoculum (data not shown), further indicating that metabolites formed during biodegradation of surfactants were not readily biodegradable. Complete removal of the studied surfactants in a biological treatment system may be limited or require a large liquid retention time.…”

Use of Soil Microorganisms to Improve Plant Growth and Ecosystem Sustainability

“…The reduction of redox dye and measuring the change in absorbance microplate wells after a short period of incubation (Garland, 1996, Di Salvo, García de Salamone 2008, ii. O 2 consumption on a sensitive fluorometer microplate cell (Garland et al, 2003) and iii. The production of carbon dioxide in the bottle headspace at the top of each cell (CS multi-induced respiration).…”

Detecting oxygen consumption in the proximity of Saccharomyces cerevisiae cells using self‐assembled fluorescent nanosensors

“…Optical oxygen sensing is based on the principles of the quenching effect of molecular oxygen on fluorescent ruthenium(II) complexes (Gao et al, 2004;Zhong et al, 2003) or phosphorescent metal-porphyrin dyes (Papkovsky et al, 1992, allowing for the non-invasive, reversible, and continuous detection of oxygen (Alderman et al, 2004;Garland et al, 2003;Guarino Richard et al, 2004;O'Donovan et al, 2005;O'Riordan et al, 2000;Papkovsky, 1995;Wodnicka et al, 2000). In this work, we fabricated oxygen-sensitive, fluorescent nanosensors for dynamically measuring the oxygen concentration in the proximity of individual living cells.…”

“…Oxygen consumption is correlated with many physiologically relevant processes, and therefore, oxygen concentration is often used as a surrogate for certain cell characteristics, such as their viability upon exposure to cytotoxic drugs and environmental stress (Alderman et al, 2004;Land et al, 1997;Tsukatani et al, 2003), protein synthesis capability (Wieser and Krumschnabel, 2001), mitochondria function and dysfunction (Trimarchi et al, 2000), cell growth and differentiation (Stitt et al, 2002;Yamada et al, 1985), substrate oxidation and utilization capacity (Chang et al, 1995;Cui et al, 2005;Higareda et al, 1997), sensitivity to radioactive reagents (Korystov Iu, 1983), and susceptibility to antibiotics (O'Riordan et al, 2000;Wodnicka et al, 2000). Moreover, the concentration of oxygen plays an important role in the development of microbial communities and multi-cellular organisms (Garland et al, 2003). Local oxygen tension, for instance, is a key parameter for monitoring and modeling normal chondrocyte activity and extracellular matrix production during chondrogenesis (Malda et al, 2004).…”