The overall goal of this procedure is to quantify the maximum potential rates at which extracellular enzymes in soils degrade their target substrates. This is accomplished by first combining field moist soil and buffer in a blender, and then pipetting the mixed samples into a deep well sample plate and deep well standard plates. The deep well plates are incubated and centrifuged before 250 microliters is transferred from each well of the deep well plates to corresponding wells of black flat bottomed.
96 well plates. The three plates are then red on the fluorimeter and the resulting data are analyzed. Ultimately, potential soil extracellular enzyme activities are determined by the rate at which fluorescent dye is released from the substrate by an enzyme catalyzed reaction where higher fluorescence indicates more substrate degradation.
The main advantage of this technique over existing methods is the ability to measure enzyme activities in a large number of samples by taking advantage of the deep 96 well microplate format. We first had the idea for this method when we saw the opportunity to increase sample volume compared to standard microplate by adapting other protocols to the deep well plate format. The implications of this technique allow us to better link microbial physiology to ecosystem scale processes.
Visual demonstration is critical for this technique because there are many nuances and sample handling that are not easy to follow in a written procedure. This method can help answer key questions in the fields of microbial ecology and soil bio geochemistry, such as how do soil microbes respond to changes in their environment and how do their physiological responses affect critical ecosystem processes, such as decomposition and nutrient cycling. Though this method can provide insight into decomposition of soil organic matter and and nutrient cycling and terrestrial ecosystems, it can also be applied to marine and freshwater systems.
Generally, individuals new to this method will struggle with pipetting skills and time sensitivity issues related to this protocol. Furthermore, the large amount of data generated in this procedure is sometimes overwhelming for first time users. To begin assay setup, label three deep well plates as sample MUB standard for four methyl and beone and MUC standard for seven amino four methyl coumarin.
Pour each standard and substrate into separate clean pre-labeled reservoirs oriented in rows. Then pipette the appropriate MUB standard into corresponding wells of the MUB standard plate as listed in the text protocol. Repeat this process for the MUC standards into corresponding wells of the MUC standard plate to prep soil slurries.
For each soil sample, weigh 2.75 grams of field moist soil before adding to a blender. Add 91 milliliters, a 50 millimolar buffer, blend on high for one minute. Pour the blender contents into a clean glass bowl at least as wide as an eight channel pipette with a stir bar.
Soil slurry may be sead through an about one millimeter strainer before pouring into the bowl. Place the bowl on a stair plate and mix the soil slurry. Then pipette 800 microliters of soil slurry into the first column of the sample plate.
Do the same for the first column of the MUB standard and MUC standard plates. Rinse the blender, stir plate, and stir bar with deionized water or buffer between soil samples. Repeat this process for each soil sample.
Moving to the next column with each subsequent sample. Next, pipette the appropriate substrate into the corresponding wells of the sample plate. As listed in the text protocol, prepare a sample plate for each incubation temperature to be tested.
Seal the deep well plates with plate mats. Invert each sealed plate by hand until the solution is thoroughly mixed. Place the plates in the appropriate incubator for the required incubation time period as listed in the text protocol record the initial time as time zero, as well as the appropriate incubation times at the end of each incubation period.
Centrifuge the sealed plates for three minutes at approximately to 2, 900 G.After centrifugation transfer, 250 microliters from each well of the incubated deep well plates into the corresponding wells of black flat. Bottom 96 well plates. One black plate will be used for each incubated deep well plate.
It is important to transfer samples from the incubated deep well plates into the corresponding wells in the black flat. Bottom 96 well plates following manufacturer's instructions for the flora metric plate reader used. Set the excitation wavelength to 365 nanometers and the emission wavelength to 450 nanometers.
Load one of the standard plates, set the fluorimeter to automatic gain and read the plate. Then set the gain to manual and decrease the value from optimal to the next lowest number rounded to the nearest five. For each standard plate, repeat the measurement twice for each plate.
Next, load the sample plate. Set the gain to automatic and run the sample plate. Rerun the sample plate manually to match the highest gain for each of the standard plates.
Begin data analysis by entering standard curve fluorescence data into a spreadsheet for the MUB and MUC standard. Convert the micromolar concentrations to micromoles for each sample. Standard curve fluorescence data, calculate the slope y intercept and R squared values for MUB and MUC standard concentrations.
Acceptable r squared values should exceed 0.98 for each sample. Standard curves can be visualized in a scatter plot with fluorescence reads plotted as the dependent variable and standard concentration plotted as the independent variable. Enter the sample plus substrate raw fluorescence data into a new spreadsheet.
Also include the incubation time and soil dry weight for each sample, subtract the standard curve intercept values from the corresponding sample fluorescence values, and then divide by the slope of the corresponding standard curve. Using the standard line equation, multiply the sample micromoles by 91 milliliters, which is the buffer volume used in the soil slurry. Divide the value obtained by the sample specific incubation time and dry soil mass.
Finally multiply the value obtained by 1000 to get the desired units. Results are shown from an experimental climate study comparing soils that experienced ambient climate conditions to soils that were exposed to elevated carbon dioxide and heating treatments shown here are potential extracellular enzyme activities or EEAs at the prairie heating and elevated carbon dioxide enrichment site in control plots and treatment plots. In this example, potential carbon nitrogen and phosphorus enzyme acquisition activities assay at zero to five centimeter soil depths did not differ by experimental treatment.
However, at five to 15 centimeter soil depths, several potential EEAs did differ significantly. For example, the carbon degrading enzymes beta one four glucocide and beta D cello biolace were lower in the elevated carbon dioxide and heating treatment plots when compared to the ambient climate conditions plots the nitrogen and phosphorus mineralizing enzymes were also lower in the treatment plot when compared to ambient climate conditions. At five to 15 centimeter soil depths.
Calculating and plotting the sum of all carbon nitrogen or phosphorus cycling potential EEAs can be a useful approach to observe broader patterns regarding potential soil carbon, nitrogen, and or phosphorus cycles. In this example, the sum of beta one four glucco, beta D cello, bio hydrolase, beta xlo, and alpha one four glucco potential EEAs was calculated to represent potential carbon cycling activities. The sum of beta one four N acetyl glucose amid dase and L leucine amino peptidase was calculated to represent potential nitrogen cycling activities.
Phosphatase was used to represent potential phosphorus cycling activities. Potential EEAs for total carbon, nitrogen and phosphorus cycling trended lower in the elevated carbon dioxide and heating treatment plots compared to the ambient climate conditions plots at the five to 15 centimeter soil depths. However, this trend was only significant for total nitrogen and phosphorus cycling activities, and soil EEAs did not significantly differ among the treatment plots.
At zero to five centimeter soil depths. The results suggest contrasting trends in enzyme activity, functional group among the treatment plots in response to soil depth, the ratio of potential EEAs is one way to assess microbial nutrient demands. Here, soil enzymes, tochi geometry, carbon to nitrogen, carbon to phosphorus or nitrogen to phosphorus activities significantly differs among the treatment plots at zero to five centimeter soil depths.
However, potential enzyme carbon to phosphorus and nitrogen to phosphorus ratios were higher in the elevated carbon dioxide and heating treatment plots. Compared to ambient climate conditions at the five to 15 centimeter soil depths temperature can strongly influence soil EEAs. Yet in typical lab assays, soil enzymes are measured at a single temperature that may not correspond to in C two temperature conditions.
Aran plots are used to visualize activation energy and are plotted using the logarithm of enzyme activity as a function of the inverse temperature converted to degrees kelvin on the x axis. Activation energy is commonly defined as the minimum energy required to catalyze a chemical reaction and is used here as a proxy for the temperature sensitivity of enzyme catalyzed reactions. Higher activation energy indicates enzyme temperature sensitivity.
Likewise, activation energies directly correspond to Q 10. Temperature coefficient values the potential enzyme kinetics for carbon, nitrogen and phosphorous EEAs were assessed among both treatment plots at the two soil depths, and the findings demonstrated that the temperature sensitivity of EEAs was not significantly different among treatment plots at either soil depths. Once mastered 12 samples can be inoculated in the 96 wheel microtiter plates in 30 minutes or less.
Furthermore, after the prescribed incubation times, you can typically spin, transfer and read the plates again on the urometer within half an hour While attempting this procedure. It's important to pipette consistently. Any air in pipetting will adversely affect your results Following this procedure.
Other methods like DNA extraction and sequencing can be performed in order to answer additional questions related to the role of soil microbial community composition in soil function after its development. This technique paved the way for researchers in the field of microbial ecology to explore nutrient cycling and biogeochemical processes in terrestrial ecosystems. After watching this video, you should have a really good idea of how to quantify potential enzyme activities.
Furthermore, you should be able to interpret the data to reflect potential microbial responses to factors such as climate and different soil types within different ecosystems. Don't forget when working with fluorescent materials, precautions should always be taken to minimize light exposure due to the light sensitivity of the fluoro moiety.
