Studying biology in deep soil layers is challenging and the scientific documentation and understanding of water and nutrient uptake from there is quite limited. Deep Frontier has developed unique research facilities and developed equipment and methods that allow studying deep roots in these facilities.

Isotope tracers and resource uptake
The project used minirhizotron methods for measuring roots and 15N isotope labelling for measuring deep root nitrogen activity. In addition, Deep Frontier developed methods for injecting or placing isotope tracers into the soil in order to obtain realistic data on their uptake and to develop ingrowth core methods. With such methods, we could place soil samples temporarily in soil where roots can grow into the samples, for example take up tracers added to it, and then recollected the soil samples for analysis of roots and nutrient content. Focus was on uptake of mobile and immobile nutrients as well as uptake of water.

Deep Frontier tested a range of stable isotope tracers and developed methods in semi-field conditions to study uptake for later use in field conditions. With modern mass spectrometers, one can measure a range of isotopes simultaneously with high precision.

Studying water uptake by use of isotope labelled water posed certain challenges compared to nutrient tracers. Methods to overcome these challenges were developed as well. Deep Frontier developed methods based on sampling evaporating water or plant sap water, which allowed very short term non-destructive measurements, or longer term studies by exploiting the fact that oxygen from water is built into plant carbohydrates during the photosynthesis process.

Deep Frontier analyzed the data from root observations, crop development and resource use and their interaction in order to enhance our understanding of their relationship.

Ingrowth cores and root microbiomes
In field conditions the use of ingrowth cores are essential for research in deep soil, hence methods were developed that allowed for repeated sampling. Also, protocols for extracting DNA from soil and quantifying plant roots from extracted DNA was created.

Additionally, Deep Frontier studied microbiology below 2 meters depth. This improved our understanding of deep root microbiology and colonization strategies in deep soil layers.

Carbon deposition and soil biology
Regarding the processes affecting deposition and decomposition of organic matter and carbon in deep soil layers, to quantify the effect of deep rooted crops on soil carbon stocks below 1 meter depth and to study root interactions with soil (micro)biology, the activities of Deep Frontier to this regard were:

• Development of methods for measuring deposition and turnover of root derived carbon in deep soil layers, including a simplified model for quantifying soil carbon storage 
• Quantification of carbon sequestration with deep rooted species 
• Methods for studying roots and their interactions with soil biological communities and how deep soil and rhizosphere biology are affected by deep rooting crop species.

Deep rooted species and cropping systems
Deep Frontier identified deep rooted species with a significant activity between 2 and 4 meters depth in the soil and included these into cropping systems as crops, cover crops or intercrops in mixed stands together with more conventional crop species.

Systems where deep rooted perennial crops are grown in rows and common crops, such as cereals, were grown on the area between the deep rooted rows and compared to cereal crops grown in monoculture. Also, competition and complementarity between deep rooting crops and the companion cereal crops was studied.