Application of carbon compound-specific isotope analysis (carbon-CSIA) to investigate microbial transformation of glyphosate

Glyphosate (N-(phosphonomethyl)glycine) is the most applied herbicide in the world with an estimated annual application of 740 to 920 kt by 2025 extrapolating the current weed management strategy. Overuse of glyphosate in agriculture has led to frequent detection in terrestrial and aquatic environments. Concerns about glyphosate load and occurrence in the environment increase as its concentrations in water bodies tend to approach or exceed the EU drinking water threshold of 0.1 μg/L. Knowledge on the role of transformation processes on glyphosate fate in soil and water is critical to assess its impacts on the ecosystem. So far, the prevalent methods for the evaluation of glyphosate transformation include monitoring of concentration changes and detection of transformation products. However, in many cases concentration data cannot unequivocally distinguish actual elimination by transformation from other processes also reducing aqueous concentrations, such as sorption and dilution. The detection of transformation products is also often problematic due to either lack of suitable analytical methods or their fast metabolization and assimilation into the microbial biomass.
A promising complementary approach to concentration analysis is compound-specific stable isotope analysis (CSIA, e.g., ¹³C/¹²C for carbon-CSIA), which can be used to study both the cause as well as the extent of transformation of organic contaminants such as glyphosate. A proof of a shift in the stable carbon isotope ratio (¹³C/¹²C) during transformation as well as its magnitude depend on the underlying reaction mechanism and thus can be indicative for a specific transformation pathway.
Microbially driven transformation is considered as the main process driving glyphosate elimination in the environment. Two main pathways have been reported for biotransformation of glyphosate depending on the specificity of the enzyme system involved. Catalysis of C—P bond cleavage occurs by a multienzyme complex known as C—P lyase resulting in the formation of sarcosine and phosphate as primary metabolites. The second pathway involves the cleavage of the C—N bond by the enzyme glyphosate oxidoreductase (GOX) yielding  aminomethylphosphonic acid (AMPA) and glyoxylate. Even though biotransformation of glyphosate has been frequently described and a carbon-CSIA method for it was established, isotope effects associated with the different microbial transformation pathways have scarcely been reported. Evidence of isotope fractionation related to its microbial transformation could elucidate the underlying transformation pathways that govern its removal from the environment. To this end, we applied isotope analysis during glyphosate transformation by different bacterial strains. The strains hold similar or different enzymes and are aerobically cultivated under P-limiting conditions. Preliminary results so far have showed no significant carbon-isotope fractionation (<1 ‰) during glyphosate transformation by two strains following the C—P pathway. An enrichment method for glyphosate compatible with subsequent CSIA analysis is under development to accomplish precise analysis also low concentrations due to extensive transformation (C_min=20 mg/L).