In early summer 2022 and 2023, we will collect soil from each plot using a 10 cm soil core to a depth of 10 cm. In restored plots, we will collect the cores from the space between plants to avoid collecting nursery soil. For the restored and low-quality reference sites, we will collect 5 soil samples per plot from each site. For the high-quality reference site, we will collect 8 samples. For each site in each year, soil samples will be bulked together and sieved to 2mm before analysis.
Approximately 10 grams of soil will be reserved and used for enzyme assays to determine the amount of potential enzyme activity of ß-glucosidase, ß-N-acetylglucosaminidase, and leucine aminopeptidase between the sites. Briefly, soil samples are mixed with enzyme-specific substrates that stimulate enzyme activity, then incubated, and a microplate reader will be used to observe their fluorescence.
Another 10 grams of soil will be used to calculate the amount of inorganic nitrogen. The samples will be mixed with 40 ml of 0.5 M K2SO4, mixed on a shaker table for 4 hours, and filtered via vacuum through a 1-micron glass fiber filter, and frozen until analyzed with a continuous flow analyzer. Similarly, 10 grams of air-dried soil will be used to calculate DOC and Total dissolved nitrogen (TDN) using aqueous extraction methods. Soil samples will be added to 40 ml of distilled water, mixed on a shaker table for 4 hours, and filtered via vacuum through a 1-micron glass fiber filter. The sample will then be frozen until further processing using a Shimadzu analyzer to determine the concentration. DON will be estimated as TDN minus inorganic N.
The remaining soil will be ground for the TC and TN analysis. For each plot, 15-20 mg of soil will be placed in tin foil capsules and run through an elemental analyzer. Samples of known carbon and nitrogen content will be run as standards, and some samples will be run more than once for quality control totaling roughly 100 runs. All soil samples will be processed at the D’Antonio and Schimel labs at University of California, Santa Barbara using in-house equipment.
To determine the effect of treatment (restored, low quality reference, high quality reference) and year (2022 and 2023) on TC, TN, DOC, TOC and each enzyme’s activity, I will construct linear models with each metric as the dependent variable, treatment and year as explanatory variables, and replicate plots as a blocking effect (Ex: DOC ~ treatment + year + (1|replicate).
Results and findings will be written up into a report or manuscript at the end of the two years of sampling and will contribute to collaborative project outputs with researchers from other institutions. There will be regular conversations with researchers at UC Davis and Santa Barbara Botanical Gardens to update them on progress and to evaluate how the soils study interacts with other research components.