Biodegradation of Water-Soluble Polymers by Wastewater Microorganisms: Challenging Laboratory Testing Protocols

Abstract

For water-soluble polymers (WSPs) that enter environmental systems at their end-of-life, biodegradability is a key functionality. For the development and regulation of biodegradable WSPs, testing methods that are both scientifically validated and economically practicable are needed. Here, we used respirometric laboratory tests to study the biodegradation of poly(amino acids), poly(ethylene glycol), and poly(vinyl alcohol), together with appropriate low-molecular-weight reference substrates. We varied key protocol steps of commonly used testing methods, which were originally established for small molecules and tested for effects on WSP biodegradation. We found that avoiding aeration of the wastewater inoculate prior to WSP addition, incubating WSP with filter-sterilized wastewater prior to biodegradation testing, and lowering the WSP concentration can increase biodegradation rates of WSPs. Combining the above-mentioned protocol variations substantially affected the results of the biodegradation testing for the two poly(amino acids) tested herein (i.e., poly(lysine) and poly(aspartic acid)). Our findings were consistent between microbial inocula derived from two municipal wastewater treatment plants. Our study presents promising biodegradation dynamics for poly(amino acids) and highlights the importance, strengths, and limitations of respirometric laboratory methods for WSP biodegradation testing.

Keywords: Water-soluble polymers; biodegradation testing; biological wastewater treatment; environmentally benign-by-design.

Figure 1

Biodegradation of WSPs by microorganisms derived from two wastewater treatment plants (WWTPs). (A) Biodegradation curves calculated based on theoretical O₂ demand (ThOD) and measured O₂ consumption during WSP incubation using the OxiTop system. Arrows indicate extended experiments (complete data in Figure S3). Asterisks (*) indicate stopped incubations due to instrument malfunctioning. (B) and (C) Times to reach 10% and 50% biodegradation, respectively. (D) Biodegradation extents after 28 days of incubation. Error bars represent, where not stated with an asterisk (*), standard deviations of triplicates and ranges of duplicates for WWTP2 and WWTP1, respectively. Gluc: glucose, PEG: poly(ethylene glycol), PVA: poly(vinyl alcohol), Lys: lysine, PLL: ε-poly(l-lysine), Asp: aspartic acid, PAsA: poly(aspartic acid).

Figure 2

(A) Effect of inoculum washing and aeration (6 days) on WSP biodegradation. Times to reach 10% biodegradation were calculated based on theoretical CO₂ production (ThCO₂) using the BSBdigi-CO₂ system for wastewater treatment plant (WWTP) 1 inocula and based on theoretical O₂ demand (ThOD) using the OxiTop system for WWTP2 inocula. Error bars represent standard deviations of triplicate (WWTP2) and ranges of duplicates (WWTP1). Daggers (†) indicate incubations were lag phases were derived from linear interpolation due to data gaps caused by software malfunction (Figure S13). For WWTP1/PEG/6d aerated, a range is given that was visually determined (Figure S14). (B) Effect of Preincubation with filter-sterilized influent wastewater (iWW) on WSP biodegradation. Times to reach 10% biodegradation were calculated based on ThOD and measured O₂ consumption using the OxiTop system for WWTP1 and 2. Error bars represent standard deviations of triplicates where not indicated differently (*, n = 2). Gluc: glucose, PEG: poly(ethylene glycol), PVA: poly(vinyl alcohol), Lys: lysine, PLL: ε-poly(l-lysine), Asp: aspartic acid, PAsA: poly(aspartic acid).

Figure 3

WSP biodegradation at different concentrations and after pre-exposure. (A) Times to reach 10% biodegradation were calculated based on theoretical O₂ demand (ThOD) and measured O₂ consumption using the OxiTop system. Error bars represent standard deviations of triplicates or ranges of duplicates, where indicated with an asterisk (*). (B) Biodegradation curves of blank-corrected biological oxygen demand (BOD) for wastewater treatment plants (WWTP) 1 and 2. Red vertical lines mark the time points, at which test substrate was added a second time (i.e., respike). Gluc: glucose, PEG: poly(ethylene glycol), PVA: poly(vinyl alcohol), PLL: ε-poly(l-lysine), PAsA: poly(aspartic acid).

Figure 4

Combined effect of inoculum aeration, preincubation with filter-sterilized wastewater, and WSP concentration on WSP biodegradation. (A) Biodegradation curves calculated based on theoretical O₂ demand (ThOD) and measured O₂ consumption using the OxiTop system for wastewater treatment plant (WWTP) 1 and 2 inocula. (B) Times to reach 10% biodegradation. Error bars represent standard deviations of triplicates, where not indicated differently (n = #, indicated above each bar). Gluc: glucose; Lys: lysine; PLL: ε-poly(l-lysine); Asp: aspartic acid; PAsA: poly(aspartic acid).