Soil Biology Lab

Nitrogen is an essential element being required by all living creatures. It is a key structural component of critical organic molecules such as amino-acids, nucleic acids and proteins. Most of the N found in soil is part of SOM being released and transformed by the action of multiple microorganisms that decompose decaying organic matter. N exists in the soil in many different forms that transform very easily from one form to another. Main transformation channels in soil are:

• N mineralization; convention of organic N to inorganic forms :
• N immobilization: uptake or assimilation of inorganic N forms by microbes and other soil organisms
• Nitrification: conversion of ammonium (NH₄⁺) to nitrite (NO₂^-) and then nitrate (NO₃^-)
• Denitrification: conversion of nitrate to nitrous oxide (N₂O) and to dinitrogen gas (N₂)

Potential Mineralizable Nitrogen in soil (anaerobic and aerobic incubation)

Relevance: Nitrogen (N) mineralization is the capacity of the soil to transform the organic N  that is in soil organic matter to inorganic N. It is often used as an index of the N available to plants in terrestrial ecosystems.

Description of the technique: Potential Mineralizable Nitrogen (PMN) measures N mineralization after an aerobic incubation period over which accumulated inorganic N is used to calculate the rate on N mineralization during that period.

Denitrification

Relevance: Denitrification is the reduction of the nitrogen oxides: nitrate (NO₃^-), to gases nitric oxide (NO), nitrous oxide (N₂O) and dinitrogen(N₂)

Description: Potential denitrification measures maximum denitrification capacity of a soil at a reference temperature. It is measured in the lab under anaerobic conditions, at constant temperature  and with non-limiting N. Actual denitrification measures soil denitrification capacity under standardized lab conditions, without the addition of external N.

Soil is the largest pool of terrestrial organic carbon (C). The abundance of organic C is a key factor controlling soil fertility and plant productivity. Soil organic C storage is controlled by the balance between the C input (via plant material and/or organic amendments) and C losses (from the soil biota catabolic activity).

Soil Organic Matter

Relevance: Soil organic matter (SOM) is the largest pool of terrestrial C, containing threefold carbon than either the atmosphere or the terrestrial vegetation. SOM is key to determine soil fertility and plant productivity.

Description: Soil samples are dried at 105°C and the organic matter content of the soil is assessed gravimetrically by dry combustion of the organic material in a furnace at 550°C.

SOM Carbon Pools

Relevance:  Different pools of soil organic matter are distinguished in the soil that differ in their activity vs. stability. In general, the active pools correlate better with biological activity and respond relatively rapidly to management changes, whereas more stable pools are more indicative of the capacity of the soil to store carbon. SOM fractionation allows separation into different SOM pools that can subsequently be analysed for composition or biological activity.

Description:

1. Chemical extraction:
2. Physical separation: Physical SOM fractionation is based on the separation of relatively fresh organic matter (Particulate Organic Matter – POM) through soil dispersion and sieving from the finer organic matter and organo-mineral complexes that represent more stable pools.

Water Extractable Carbon

Relevance: The most labile fractions of soil organic matter (SOM) are water-soluble organic substances. The solubility of the different compounds depends on the temperature of the solvent and therefore we distinguish between Cold Water Soluble Carbon (WSC) and hot water extractable carbon (HWC).

Description:

1. Cold Water Soluble Carbon (CWEC)
2. Hot Water Extraction Carbon  (HWEC): C is extracted using a hot 2 M KCl extraction. HWC is a heterogeneous pool of organic C  containing labile and more stable components than WSC that form a reserve of nutrients and energy for plants and microorganisms.

POXC : Permanganate oxidizable carbon (POXC; i.e. active C)

Relevance:  Permanganate oxidizable carbon (POXC) is a method to estimate the active C fraction of total SOC. POXC relates to most measures of soil microbial activity, including microbial biomass C, substrate induced respiration, soluble carbohydrate C, and total SOC and POC. POX-C is a good indicator to changes in management or environmental variations (Culman et al 2012).

Description:

Total Soil Organic Carbon (SOC)

Relevance:

Description: Total Soil Carbon is measured with a CN element analyser using dry combustion. In non-calcareous soils the Total Soil Carbon content corresponds to the Carbon present in the soil organic matter. In calcareous soils, however, the Total Soil Carbon consists of both organic and inorganic Carbon.

Phosphatase activity in soil

Relevance: Phosphorus (P) is an essential element for life, which is derived from the weathering of minerals in parent rock material. Organisms can assimilate only dissolved phosphate and therefore, phosphatase activity plays a fundamental role in the transformation of P from SOM into available forms. Phosphatase enzymes are produced by bacteria, fungi and plant roots and serve to transform complex and sometimes unavailable forms of organic P into assimilable phosphate.

Description: Production of p-nitrophenol by hydrolysis of p-nitrophyenyl phosphate is measured photometrically.

European Earthworms quantification and identification

Relevance: Earthworms are major drivers of soil ecosystem processes strongly influencing litter decomposition and soil structure via burrowing. Besides, they also stimulate microbial decomposers activity, mix and aggregate soil, increase soil water infiltration, improve water holding capacity, provide channels for root growth, and bury and shred plant residues. Overall, they are considered ecosystem engineers and are important indicators of soil quality/ health.  

Description: Hand sorting, counting, weighing and visual identification of earthworm species.

European Nematodes extraction and identification

Relevance: Nematodes are non-segmented worms. They are among the most numerous and diverse of the multicellular organisms found on earth. They have different soil functions that cover different trophic levels since there are nematodes that feed on bacteria or fungi, nematodes that feed on nematodes, or other smaller organisms, and nematodes that feed on roots. This last group has being widely study because are responsible for plant diseases, however, most nematodes in the soil are not plant parasites; beneficial nematodes contribute to decomposition and help controlling plant diseases.

Description: Nematodes are extracted using the Oostenbrink funnel technique.

Enchytraeids

Relevance: Enchytraeids are small (1-30 mm), unpigmented worms. They are involved in the soil food web since they feed on bacteria, fungi, and protists and decompose organic matter and influence the soil structure due to their burrowing activity, faecal pellet production and the mixing of mineral and organic matter.

Description: Enchytraeids are extracted using the Tullgren extractor funnel.

Staining and quantification of arbuscular mycorrhizal fungi

Relevance:  Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with two-thirds of all land plants and are widespread in most terrestrial ecosystems. AMF are well known to transfer nutrients (specially P) and water to the host plant in exchange of photosynthetic C compounds. Additionally, AMF also provide with other essential functions such as plant defence against pathogens, improvement of soil structure, and enhance plant functional diversity.

Description: Percentage of AMF root length colonization is measured by staining the roots for microscope visualization and count using the McGoningle line intersection method.

Collembola

Relevance: Collembola (springtails) are microarthropods which, together with mites, constitute an important component of soil mesofauna in all terrestrial ecosystems. They play an important role in plant litter decomposition processes and in forming soil microstructures.

Description: Collembola are extracted using the Tullgren extractor funnel.

Microbial catabolic profile – MicroResp©

Relevance: MicroResp© is a technique to measure multi substrate induced respiration (Multi-SIR) in soils. This method provides useful information about catabolic capacity of the microbial community to respire various carbon substrates, the soil microbial biomass and its functional diversity. The resulting catabolic profiles can be used to compare different soil types or treatment effects on the same soil type.

Description: MicroResp© measures the amount of CO₂ respired by the soil microorganisms as a response to the applied C source using a colorimetric detection system.

Soil Respiration Potential

Relevance: Soil Respiration Potential is a measure of soil’s ability to transform organic carbon to inorganic carbon (CO₂) under optimal moisture and temperature conditions.

Description: Soil Respiration Potential is assayed in aerobic incubation under environmental conditions that are near optimal.

Chloroform fumigation in soil samples for biomass N

Relevance: Chloroform fumigation is used to estimate microbial biomass, C and N. Microbial biomass acts both as a nutrient reservoir and as a catalytic force in decomposition. It  is crucial for understanding nutrient fluxes within and between ecosystems.

Description: Chloroform fumigation kills most soil organisms and destroys their membranes and cell walls. During fumigation microbial cells are lysed and N is extracted and measured on the segmented flow analyser (SFA).

Relevance: Molecular microbial analysis relies on extracting and characterizing nucleic acids and other cellular components, such as phospholipid fatty acids (PLFA). Molecular approaches allow to detect and characterize the enormous diversity of soil microbes.

PCR-based assays

Relevance: PCR techniques allow the amplification of specific regions of the DNA selected by primers that can target different taxonomic resolution levels. The most common target for characterizing microbial communities are the rRNA genes. These include SSU rRNA genes (16S in bacteria and archaea or 18S in eucarya), and the interanl transcribed spacer (ITS) region  for fungi.

Description: PCR based analysis include Sanger’s sequencing, fingerprinting methods (TRFLP) and high throughput sequencing (Illumina).

Phospholipid fatty acids (PLFA)

Relevance: PLFAs are short-chain fatty acids that can be isolated only form active microorganisms. PLFAs extraction and identification enables characterization of the microbial community structure and measurement of relative abundance of total microbial community as well as fungi, bacteria (gram + and gram -) and actynomicetes.

Description: