High-pH and anoxic conditions during soil organic matter extraction increases its electron-exchange capacity and ability to stimulate microbial Fe(III) reduction by electron shuttling
Authors: Bai Yuge , Subdiaga Edisson , Haderlein Stefan B. , Knicker Heike , Kappler Andreas 
Published: February 2020

Abstract. Soil organic matter (SOM) is redox-active, can be microbially
reduced, and transfers electrons in an abiotic reaction to Fe(III) minerals,
thus serving as an electron shuttle. The standard procedure to isolate
organic matter (OM) from soil involves the use of alkaline and acidic
solutions and the separation of humic acids (HAs) and fulvic acids (FAs). This
process potentially leads to unwanted changes in SOM chemical and redox
properties. To determine the effects of extraction conditions on the redox
and electron-shuttling properties of SOM extracts, we prepared HA, FA, and
water-extractable organic matter (OM) extracts, applying either a
combination of 0.1 M NaOH and 6 M HCl or ultrapure water (pH 7), from soil
samples collected from the subsoil (0–15 cm, A horizon, pH 6.5–6.8) in
Schönbuch forest, Baden-Württemberg, Germany. Both chemical extractions
(NaOH∕HCl) and water extractions were done in separate experiments under
either oxic or anoxic conditions. Furthermore, we applied the NaOH∕HCl
treatment to a subsample of the water-extractable OM to separate HA and FA
from the water-extractable OM. When comparing the amount of carbon extracted
from soil by different extraction methods, we found that FA and HA
chemically extracted from the soil can make up to 34 %–40 % of the soil
organic carbon pool while the water-extractable OM only represents
0.41 %–2.74 % of the total soil organic carbon. The higher extraction
efficiency of the chemical extraction is probably due to the deprotonation
of carboxyl and phenol functional groups under high pH. Anoxic extraction
conditions also led to more extracted carbon. For water-extractable OM, 7
times more C was extracted under anoxic conditions compared to oxic
conditions. This difference was probably due to the occurrence of microbial
reduction and dissolution of Fe(III) minerals in the soil during the anoxic
water extraction and thus the concomitant release of Fe(III) mineral-bound
organic matter. To compare the redox activity of different SOM extracts, the
electron-exchange capacity (EEC) of all extracted HA, FA, and
water-extractable OM was analyzed and our results showed that, under anoxic
extraction conditions, the HA chemically isolated from the water-extractable
OM had 2 times higher EEC values compare to the water-extractable OM itself,
suggesting the potential formation of redox-active aromatic functional
groups during the extraction with NaOH under anoxic conditions by
condensation reactions between amino acids, aldehydes, and hydroxyl- and
catechol-containing molecules. We also performed a microbial Fe(III)
reduction experiment with all extracts and found that higher EEC of extracts
in turn resulted in a higher stimulation of microbial Fe(III) mineral
reduction by electron shuttling, i.e., faster initial Fe(III) reduction
rates, and in most cases also in higher reduction extents. Our findings
suggest that OM extracted with water at neutral pH should be used to better
reflect environmental SOM redox processes in lab experiments and that
potential artefacts of the chemical extraction method and anoxic extraction
condition need to be considered when evaluating and comparing abiotic and
microbial SOM redox processes.