Clomazone biodegradation in soil microcosms : evaluation of bioaugmentation and biostimulation with native bacteria from rice crop soils in Chaco, Argentina

Abstract

Clomazone (CMZ) is a selective pre and post emergence herbicide indicated for application in rice crops. Its incorrect use has been causing environmental problems, such as soil, groundwater and surface water persistent contamination. CMZ-resistant/tolerant native bacterial strains were previously isolated from rice crop soils in Las Palmas, Chaco, which were able to degrade this herbicide in batch cultures. The aim of this study was to evaluate CMZ biodegradation under controlled experiments in soil microcosms (MC) by bioaugmentation (the addition of specific microorganisms) and biostimulation (the addition of nutrients to increase microbial population activity) procedures. For this purpose, 30 MC were prepared by triplicates using 150 g of sterile and non-sterile rice crop soils under different treatments: control (non-treated MC), bioaugmentation, and a combination of bioaugmentation and biostimulation. The soil used exhibited a phosphorus deficit (8.4x10-4%), therefore biostimulated MC were designed by adding a stock solution of K2HPO4 1M to achieve the optimal C:N:P ratio of 100:10:1. For bioaugmented-MC, inocula consisted of strain G1 or a mixed culture of the isolates G1, H2, and H3, selected according to previous results. The MC were incubated at 30 °C and monitored for a period of 60 days. Bacterial growth was assessed by viable cell counts every 7 days, while CMZ concentration in soil was quantified by HPLC at regular intervals to determine CMZ removal. Results indicated that sterile soil control MC showed no detectable microbial growth until day 15, and CMZ concentration remained stable throughout the 60 days, confirming its abiotic stability under these conditions. In contrast, the other experimental conditions evidenced bacterial growth over time however no CMZ removal was registered in non sterile soil control MC. In G1 MC, G1 attained a maximum growth of 1×102 CFU/g soil at 14 days and of 1×103 CFU/g soil in P-supplemented MC, suggesting that P availability is a limiting factor. Furthermore, the G1-H3-H2-bioaugmented MC also achieved cell development of 1×103 CFU/g soil at 14 days, both with and without P-amendment indicating a greater intrinsic growth capacity of the consortium and a lesser dependence on external P supplementation. A clear predominance of isolate H2 was observed, followed by H3 and, to a lesser extent, G1, suggesting a dynamic of competition among the strains. Regarding the CMZ concentration analysis by HPLC, differentiated results were obtained: G1-MC did not show significant agrochemical degradation under any treatment condition. However, in the G1-H3-H2-MC a 14% CMZ removal was observed at 35 days without biostimulation, while in P-supplemented MC, the removal percentage increased to 26% at 35 days. These results suggest that the microbial consortium with P-amendment is effective in CMZ degradation in MC, being a factor to consider for the herbicide removal in polluted and productive soils.