Bioremediation of Atrazine and its Metabolite Using Multiple Enzymes Delivered by a Bacillus thuringiensis Spore Display System

Atrazine (ATR), a widely used herbicide in the United States, has contaminated ground and surface water. The persistence in the environment, the carcinogenic characteristic, and the endocrine disruption activity of ATR raises public health concerns. To provide a clean source of drinking water, it is imperative to remediate ATR and its metabolites without toxic by-products. A green technology, Bacillus thuringiensis spore-based display system, has been developed to express a high density of enzymes on the spore surface. The B. thuringiensis spore can serve as a bioparticle to deliver enzymes to different environments and enhance the stability and shelf-life of enzymes expressed on their surface. A cluster of enzymes, AtzA, AtzB, AtzC, AtzD, AtzE, and AtzF, found in Pseudomonas sp. strain ADP, remediates ATR to CO2 and NH3 in the degradation pathway. AtzA-bearing spores were generated, and they successfully demonstrated the capability of converting ATR to its metabolite, hydroxyatrazine (HA) [Hsieh et al. 2020]. In this study, AtzB, a hydroxyatrazine N-ethylaminohydrolase that hydrolyzes HA to N-isopropylammelide (NiPA), was expressed on the Bacillus spores by fusing atzB to the gene encoding the attachment domain of the BclA spore surface nap layer protein. The results showed that 1 mg AtzB-bearing spores degraded 50% of HA to NiPA in water within 12 hours. The Km and Vmax of AtzB-bearing spores with HA as a substrate were 25.28 {micro}M and 1.26 nmole NiPA h-1 mg-1 spores, respectively. The optimal ratio of 1:2 for AtzA-bearing to AtzB-bearing spores was applied and successfully converted 90% ATR to NiPA in the water after 96 hours of incubation. The surface water with the addition of 34.5 nM (7.5 {micro}g L-1) of ATR was treated with a combination of AtzA-bearing and AtzB-bearing spores in a 96-hour time course study. Over 80% of applied ATR in surface water was degraded and ATR concentration was below the United States Environmental Protection Agency maximum contaminant level, 13.8 nM (3 {micro}g L-1) within 24 hours. At the end of 96 hours. ATR in surface water was completely converted to NiPA. We have successfully demonstrated the application of multienzyme bioremediation of ATR using the Bacillus spore-based display system in the surface water in the laboratory.