pH-Stat Titration: A Rapid Assay for Enzymatic Degradability of Bio-Based Polymers

Abstract

Bio-based polymers have been suggested as one possible opportunity to counteract the progressive accumulation of microplastics in the environments. The gradual substitution of conventional plastics by bio-based polymers bears a variety of novel materials. The application of bioplastics is determined by their stability and bio-degradability, respectively. With the increasing implementation of bio-based plastics, there is also a demand for rapid and non-elaborate methods to determine their bio-degradability. Here, we propose an improved pH Stat titration assay optimized for bio-based polymers under environmental conditions and controlled temperature. Exemplarily, suspensions of poly(lactic acid) (PLA) and poly(butylene succinate) (PBS) microparticles were incubated with proteolytic and lipolytic enzymes. The rate of hydrolysis, as determined by counter-titration with a diluted base (NaOH), was recorded for two hours. PLA was hydrolyzed by proteolytic enzymes but not by lipase. PBS, in contrast, showed higher hydrolysis rates with lipase than with proteases. The thermal profile of PLA hydrolysis by protease showed an exponential increase from 4 to 30 °C with a temperature quotient Q10 of 5.6. The activation energy was 110 kJ·mol−1. pH-Stat titration proved to be a rapid, sensitive, and reliable procedure supplementing established methods of determining the bio-degradability of polymers under environmental conditions.

Reaction pathways for the enzymatic degradation of poly(ethylene terephthalate): What characterizes an efficient PET-hydrolase?

“…Here, the pump module consisted of a syringe pump module responsible for delivering 50 mM NaOH to adjust the pH to 8.00. As described by Miksch et al 47 a typical course of NaOH consumption over time was divided into different stages. Initially, the pH of the suspension was adjusted to pH 8.00.…”

Bioplastics in the Sea: Rapid In-Vitro Evaluation of Degradability and Persistence at Natural Temperatures

“…This would counteract the formation of degradation products with carboxyl end groups, where the acidifying effect is used as a measure of degradability. Alkaline leachates would increase the pH during the blank measurement of the material (Miksch et al, 2021). This effect was observed for BPE-C-PLA and BPE-RP-PLA but not for the other bio-based polymers (data not shown).…”

“…The unit was connected to a magnetic stirrer (TM 235), a pH-electrode model A 162 2M DIN ID, and a circulation thermostat (Lauda, Lauda-Königshofen, Germany). The reaction vial was a 20-mL glass vial placed in a custom-made thermostat jacket to maintain a constant temperature (Miksch et al, 2021). pH-Stat titration was performed after Miksch et al ( 2021) whilst the rate of acidification due to the hydrolysis of the bioplastic was determined by counter-titration of a base.…”

“…These materials are based on PLA, which is the major component, accounting for at least 50% of each blend. Pure PLA is efficiently degraded by protease from Bacillus licheniformis as measured by pH-stat titration (Miksch et al, 2021). However, of the three materials containing PLA as base polymer, only BPE-C-PLA and BPE-RP-PLA showed a noticeable degradability with protease.…”

“…Such enzymes, comprising various peptidases and esterases, are present in the environment, including seawater and marine sediments (Arnosti et al, 2014;Liu and Liu, 2018;Patel et al, 2019). Hydrolases, namely proteinase K, protease, esterase, and lipase, have been used in in vitro assays on the enzymatic degradability of bioplastics (Miksch et al, 2021;Richert and Dabrowska, 2021). The pH-Stat titration is a rapid assay, which is based on maintaining the pH during a pH-affecting reaction.…”