Uncovering the Structure and Function of Microbial Communities Formed During Periodic Tilling of TNT and DNT Co-Contaminated Soils

Environmental contamination by 2,4,6-trinitrotoluene (TNT), historically the most widely used secondary explosive, is a long-standing problem in former military conflict areas and at manufacturing and decommissioning plants. In field test plots at a former explosives manufacturing site, removal of TNT and dinitrotoluenes (DNTs) was observed following periods of tillage. Since tilling of soils has previously been shown to alter the microbial community, this study was aimed at understanding how the microbial community is altered in soils with historical contamination of nitro explosives from the former Barksdale TNT plant. Samples of untilled pristine soils, untilled TNT-contaminated soils and tilled TNT-contaminated soils were subjected to targeted amplicon sequencing of 16S ribosomal RNA genes in order to compare the structure of their bacterial communities. In addition, metagenomic data generated from the TNT tilled soil was used to understand the potential functions of the bacterial community relevant to nitroaromatic degradation. While the biodiversity dropped and the Burkholderiales order became dominant in both tilled and untilled soil regardless of tillage, the bacterial community composition at finer taxonomic levels revealed a greater difference between the two treatments. Functional analysis of metagenome assembled genome (MAG) bins through systematic review of commonly proposed DNT and TNT biotransformation pathways suggested that both aerobic and anaerobic degradation pathways were present. A proposed pathway that considers both aerobic and anaerobic steps in the degradation of TNT in the scenario of the tilled contaminated soils is presented. Importance In this study, TNT and DNT removal has been observed in field-scale experiments following periodic tilling of historically contaminated soils. The microbial community structures of uncontaminated pristine soils, untilled contaminated soils, and tilled contaminated soils were investigated using high-throughput sequencing platforms. In addition, shotgun metagenome libraries of samples from tilled contaminated soils were generated. The results indicated that a significant shift of the bacterial community at the family level between tilled and untilled contaminated soils, with tilled soils being dominated by Alcaligenaceae and untilled soils by Burkholderiacea. In-depth metagenomic analysis of samples from tilled contaminated soils, indicate the presence of genes that encode for enzymes that potentially could lead to mineralization of TNT and DNT under mixed aerobic and anaerobic periods. ### Competing Interest Statement The authors have declared no competing interest.