Measurement of monomers and oligomers (≤20mer) as intermediates using LC–Orbitrap MS from marine biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) in laboratory

Degraded intermediates in the marine biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), a microbial polyester, were detected and quantified using LCOrbitrap MS. Intermediates were extracted from a biofilm (BF) attached to the biodegraded film using solvent extraction. Monomers and oligomers up to 11mer were identified on the surface of a marine biodegraded PHBH film and in the associated BF. The biodegradability and intermediate amounts of three films (ca. 90 mg) on day 9 were 13 % and 9.2 μg in the first test, 30 % and 13 μg in the second test, and 52 % and 19 μg in the third test, respectively. Differences in biodegradability and intermediate amounts between the biodegradation tests suggest that variation in the microbial community and quantity in seawater affect enzymatic degradation and mineralization rates. The amounts of 3-hydroxybutyric acid (3HB) monomer, 3-hydroxyhexanoate (3HH) monomer, and oligomers (3HB equivalent) were 67:5:28 (9.2 μg) in the first test, 69:5:26 (13 μg) in the second test, and 79:7:14 (19 μg) in the third test, respectively. The substantial occupancy of monomers in the intermediates suggests that the oligomers are rapidly enzymatically degraded into monomers, which are slowly mineralized and metabolized. Monomers were the only species identified in the pore water (free water within the BF), and the concentrations of intermediates in seawater were below the detection limit. The monomer composition (3HB:3HH) in the intermediate was consistent with the copolymer composition, indicating no selective accumulation of specific monomers.