Soil Restoration and Monitoring Guidelines

Soil microbes break down the organic matter through catabolic processes. Additionally, they play a pivotal role in nutrient cycling, soil structure formation and overall soil fertility. Microorganisms are highly responsive to environmental changes, making them valuable indicators for assessing forest restoration. Various methods are available for determining soil microbial diversity, including culturing, microscopy, DNA-methods and image analysis (Vogt et al., 2015). In SUPERB, we report the potential catabolic activity/diversity and metabolic activity. 

Assessing catabolic microbial activity and diversity is essential for understanding soil health. By evaluating potential catabolic activity, we can quantify the efficiency of microbial communities in mineralizing organic substrates, thereby releasing essential nutrients and contributing to the soil’s nutrient pool (Vogt et al., 2015). Within SUPERB, we selected the Biolog EcoPlate™ method to measure this parameter (Gaublomme et al., 2006). Microorganisms inoculated onto different carbon sources leave a response pattern over a time (Biolog, 2023). To ensure accurate assessment, samples were collected in a sterile way by using disposable gloves, with all equipment sterilized using ethanol 90% and a gas burner. Samples were stored under cool conditions (7°C) and analyzed withing 14 days (FunDivEUROPE, 2013). In the lab, the fresh samples were being diluted and pipetted onto the Biolog EcoPlates™, containing 31 different carbon sources and 1 control in triplicates. The inoculated plates were placed in an incubator at 25°C for a period of 48 hours. The absorbance was measured after filling the ecoplates (day 0) using a VERSAmax microplate reader (OD590 nm) and on day 3 and 5. With the obtained data the average well color development and the Shannon diversity index can be expressed.

The metabolic activity is a sensitive indicator of many belowground processes and ecological interactions. Soil respiration is an indirect index for soil biological activity and can be used to assess microbial biomass (Vogt et al., 2015). Soil microorganisms respire carbon dioxide as a byproduct of metabolism while degrading organic matter and cycling nutrients (Rieke, Cappellazzi, et al., 2022). Within SUPERB, the microbial biomass was measured by incubating rewetted dried and sieved soil (10-15 gram) in a jar during 24 hours at 24°C (Comeau et al., 2023; Moebius-Clune, 2016; Soil health institute, 2022; Vogt et al., 2015). After 24 hours, CO2 levels were measured using the LICOR 7810. The measurement was done by collecting the gas with a syringe and injecting the sample into the closed loop of the LICOR system (Comeau et al., 2023). The CO2 levels were calculated by using the volume of the closed loop and the CO2 concentration before and after the injection. A more cost-effective and simpler alternative for measuring carbon levels (compared to the LICOR) is the Checkpoint Dansensor.

Additionally, the biomass of the fine roots can also be determined. Fine roots are a key indicator of belowground productivity and nutrient uptake, as fine roots are primarily responsible for water and nutrient absorption. Measuring fine root biomass helps assess ecosystem functioning, soil-plant interactions, and the impact of environmental changes or restoration on root dynamics (Likulunga et al., 2022; Magalhães & Mamugy, 2020; Vogt et al., 2015). Within SUPERB, fine root sampling was conducted using the same methodology as for soil carbon (FunDivEUROPE, 2011). Because laboratory analysis of fine roots can be labor-intensive and time-consuming, we selected a less intensive sampling method to balance feasibility with data quality. For each sample, fine root picking was carried out for 5 minutes on the dried but unsieved soil, and fine root biomass was calculated by dividing the mass of the collected roots by the total volume of the dried sample (Likulunga et al., 2022; Magalhães & Mamugy, 2020).