Hitherto unknown active bacteria are involved in 2,4-dichlorophenol degradation in drilosphere and agricultural soil

2,4-dichlorophenol (2,4-DCP) is a metabolite of one of the most widely used herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D). It tends to persist in nature, and thus 2,4-DCP represents a potential soil and groundwater contaminant. Mineralization occurs in soil, mainly due to aerobic and anaerobic microbial processes. Earthworms affect the biodegradation of agrochemicals and stimulate both growth and activity of herbicide-degrading bacteria in soil. Thus, active 2,4-DCP degraders in drilosphere (i.e. burrow walls, gut content and cast) and agricultural soil were identified by 16S rRNA stable isotope probing and 454 pyrosequencing in soil columns. In situ relevant concentrations of [13C] 2,4-DCP (25 µg gdw-1) were consumed in soil within 19 and 41 days in the presence and absence of Aporrectodea caliginosa respectively. A total of 337,291 16S rRNA sequences were analyzed and consumers of 2,4-DCP were identified by relative Labelling Intensities (LIs). 23 family-level taxa indicative of Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes and Proteobacteria utilized [13C] 2,4-DCP. Dominant consumers of [13C] 2,4-DCP were Gammaproteobacteria (Pseudomonadaceae) in drilosphere and soil. Bacteroidetes (Flavobacteriaceae) were strongly labelled in cast and in 0-5 cm depth soil. Firmicutes (Clostridiaceae, Peptococcaceae and Bacillaceae) were also important [13C] 2,4-DCP consumers in the anoxic microsites of gut and lower layer of bulk soil. In oxic microcosms with drilosphere and bulk soil material 300-425 µg gfw-1 [13C] 2,4-DCP were consumed within 50 days. 12 family-level taxa were labelled, indicating a less diverse 2,4-DCP degrading community in microcosms than in soil columns. Alpha- and Betaproteobacteria (Sphingomonadaceae and Comamonadaceae) dominated [13C] 2,4-DCP consumers in microcosms of drilosphere and bulk soil material. Deltaproteobacteria (Geobacteraceae) were only detected in the absence of earthworms, indicating an inhibitory effect of soil macrofauna on deltaproteobacterial 2,4-DCP degraders. The collective data indicated that agricultural soils influenced by earthworms harbor highly diverse and hitherto unknown 2,4-DCP-utilizing microorganisms.