Combining respirometry and HPLC-HRMS to elucidate the biodegradation of polyethylene glycol and its derivatives

• PEG-dimethyl ether was shown to be biodegraded by wastewater microbes. • Biodegradation intermediates in supernatant support extracellular PEG breakdown. • Lag phase of PEG biodegradation ascribed to microbial adaptation. • HPLC-HRMS provides important complementary information to respirometry. Water-soluble polymers (WSPs) fulfil essential functions in many applications, such as home and personal care, and are often released into wastewater systems after use. Biodegradable WSPs are therefore needed and standardized biodegradation testing is crucial for their development and regulation. In this study, we combined respirometry-based biodegradation testing with high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) to obtain a process-level understanding of the biodegradation of polyethylene glycols (PEGs) by wastewater microbiomes. We used two derivatives with similar molecular weights: PEG and PEG-dimethyl ether (dmPEG). Both PEG and dmPEG exhibited substantial biodegradation, exceeding 80 % mineralization within 28 days. We demonstrate dmPEG biodegradation for the first time, while earlier reports claimed that dmPEG was non-biodegradable and that a terminal hydroxyl group is essential for PEG biodegradation. Our HPLC-HRMS analyses revealed significant shifts in the molecular weight (MW) distribution of PEG starting on day 3, implying that the initial lag phase primarily reflects microbial adaptation rather than PEG breakdown. Following this initial shift, the PEG MW distribution stabilized around 1.5 kDa and intermediates <1 kDa were not detected, which we ascribed to rapid microbial uptake and biodegradation of these low-MW species. The detection of carboxylated biodegradation intermediates in the cell-free supernatant provides evidence that certain steps in PEG biodegradation occur extracellularly. Together, our study highlights the potential of combining standardized biodegradation testing based on respirometry with HPLC-HRMS analysis to elucidate WSP biodegradation.