Community-Level Physiological Profiling Performed with an Oxygen-Sensitive Fluorophore in a Microtiter Plate

Dynamac Corporation, Mail Code DYN-3, Kennedy Space Center, FL 32899, USA.
Applied and Environmental Microbiology (Impact Factor: 3.67). 06/2003; 69(5):2994-8. DOI: 10.1128/AEM.69.5.2994-2998.2003
Source: PubMed


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.

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Available from: Jay L Garland, Mar 17, 2014
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    • "While useful for rapid discrimination of microbial communities , the functional relevance of the assay was limited due to the strong enrichment bias associated with the high microbial growth level required for dye reduction. Subsequent improvements (Garland et al., 2003) based on the detection of respiration using an O 2 sensitive fluorophore dye reduced the problem of enrichment bias (Garland et al., 2012). Other alternative CLPP approaches have been developed based on monitoring of CO 2 production in microwells (Degens et al., 2001; Campbell et al., 2003). "
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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.
    Full-text · Article · Nov 2013 · Journal of microbiological methods
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    • "Profiling microbial respiration on various substrates provides an insight into microbial functional diversity (Chapman et al., 2007; Degens and Harris, 1997). Microbial respiration has been assessed by O 2 consumption (Garland et al., 2003), by CO 2 production (Cheng and Coleman, 1989), and by coupling the two (Sierra and Renault, 1995). Respiration is more often characterized by CO 2 measurements, which are easier and sensitive (Dilly, 2001). "
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    ABSTRACT: The MicroResp™ method allows soil respiration and microbial community physiological profiles to be determined colorimetrically in microplates. This method, however, neglects CO2 storage in the agar gel carrying the colorimetric indicator, and calcite dissolution associated with CO2-induced change in soil solution pH. Our objective was to improve the method by taking into account CO2 in the gel in the calculation of microbial respiration, describing the effect of microbial CO2 on the pH of the soil solution and calcite dissolution, and checking whether CO2 distribution among calcite, soil solution, air and gel is near equilibrium after incubation. We propose a thermodynamic equilibrium model describing (a) distribution of CO2 among calcite, soil solution, gel and air, (b) dissociations of water, carbonic acid, cresol red, and substrates in the gel and soil solution, (c) exchange of adsorbed cations with H3O+ in the gel, and (d) calcite dissolution in soil. In-gel experiments were designed to calibrate the model, quantify the rate of CO2 exchange with air, and compare conservation procedures. On-soil experiments were designed to check whether calcite dissolution is near equilibrium and whether the model predicts the effect of CO2 on the pH of the solution. In-microplate experiments were designed to assess the effects of incubation period and soil quantity on estimated microbial respiration. The model can describe the distribution and speciation of CO2 in the gel, the soil solution and the air space of each microplate well. Initial properties of the gel vary with storage: soda lime partly extracts CO2 supplied as NaHCO3, and dries out the gel, which can skew the calibration. When incubation is over, the proportion of microbial CO2 in the gel is higher at lower microbial respiration. Incubations shorter than 4 h underestimate microbial respiration due to the slow diffusion of CO2 in the gel. CO2 in the soil solution cannot be overlooked; it decreases the soil pH and may promote calcite dissolution in calcareous soil. It is important to precisely estimate initial CO2 air fraction and to control temperature, which affects both thermodynamic constants and microorganisms.
    Full-text · Article · Oct 2013 · Geoderma
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    • "Microbial inocula from litter, rhizosphere and bulk soil communities were also prepared from the two CO 2 treatments. We used the BD-oxy system (BD Oxygen Biosensor System, BD Biosciences, Bedford, MA, USA (Garland et al., 2003; V€ ais€ anen et al., 2005; Zabaloy et al., 2008) to evaluate microbial respiration. The system uses a fluorophore that fluoresces as O 2 is consumed during the 48 h incubation. "
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    ABSTRACT: Rising atmospheric carbon dioxide (CO2) could alter the carbon (C) and nitrogen (N) content of ecosystems, yet the magnitude of these effects are not well known. We examined C and N budgets of a subtropical woodland after 11 yr of exposure to elevated CO2. We used open-top chambers to manipulate CO2 during regrowth after fire, and measured C, N and tracer 15N in ecosystem components throughout the experiment. Elevated CO2 increased plant C and tended to increase plant N but did not significantly increase whole-system C or N. Elevated CO2 increased soil microbial activity and labile soil C, but more slowly cycling soil C pools tended to decline. Recovery of a long-term 15N tracer indicated that CO2 exposure increased N losses and altered N distribution, with no effect on N inputs. Increased plant C accrual was accompanied by higher soil microbial activity and increased C losses from soil, yielding no statistically detectable effect of elevated CO2 on net ecosystem C uptake. These findings challenge the treatment of terrestrial ecosystems responses to elevated CO2 in current biogeochemical models, where the effect of elevated CO2 on ecosystem C balance is described as enhanced photosynthesis and plant growth with decomposition as a first-order response.
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