Effects of Enzyme Hydrolysis in Biofilm Formation and Biotic Degradation on Weathered Bioplastics

Abstract

As efforts to address plastic pollution increase, new avenues are opened for the use of biologically renewable and biodegradable plastics. With the influx of these new polymer systems, it is crucial to understand the degradation processes of these polymers, particularly through disposal systems designed to manage their waste (i.e., compost). This work seeks to characterize a multistep biodegradation system by studying how enzymatic hydrolysis impacts the formation of biofilms upon weathered biodegradable aliphatic polyesters to better understand processes that should occur in composting. Poly l-lactic acid (PLLA), after varying amounts of photochemical weathering, was exposed to the esterase proteinase K followed by exposure to suspended facultative anaerobe, Shewanella oneidensis, whose biofilms were quantified with crystal violet staining. Enzymatic hydrolysis was observed to promote the formation of a biofilm regardless of enzymatic concentration, enzyme exposure time, and state of weathering on the polymer. This trend also held true for a less commercially viable polymer like poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), which was demonstrated to be resistant to enzymatic hydrolysis. Further, we observed that the state of photochemical weathering caused variable impacts to the biodegradation of PLLA. Polymer characterization suggests that while there are changes in crystallinity and surface accessible ester linkages, increased surface area caused by photodegradation and/or enzyme hydrolysis drives the observed trends. Overall, this work demonstrates a multistep biodegradation process is more effective at breaking down biodegradable polymers than a single biotic agent, though polymer weathering influences breakdown to some extent, offering insight into the importance of managing these waste streams to ensure optimal designed biodegradability.

Figure 1

Bar graph showing biofilm growth on irradiated PLLA samples as quantified using crystal violet staining. The bars represent the average (n = 3), and the error bars represent the standard deviation. ANOVA analysis performed on no enzyme (p = 0.508) and enzyme treated (p = 0.036) samples as it relates to irradiation time. t-test performed comparing means of same UV-light doses between no enzyme and enzyme treated group with *p < 0.05.

Figure 2

Ratio of the protein to carbohydrate concentration of nonenzymatic and enzymatic treated PLLA samples at varying degrees of photodegradation. Triplicate concentrations were averaged, and error was propagated through the standard deviations of the protein and carbohydrate concentrations.

Figure 3

Optical density at 600 nm of remaining M4 nutrient broth after 3 days of biofilm growth. Bars represent the average (n = 3) of the samples with error bars representing standard deviation. ANOVA analysis performed on no enzyme (p = 0.213) and enzyme treated (p = 0.088) samples as it relates to irradiation time. t-test performed comparing means of same UV-light doses between no enzyme and enzyme treated group with *p < 0.05.

Figure 4

Leached lactic acid from PLLA samples in M4 broth as quantified with LC versus UV light degradation time and normalized to the values of the 0 h (pristine) sample. Markers represent the average (n = 3) with the error bars representing the standard deviation. “Broth Soak” are plastic samples that are similar to those in the nonenzymatically treated polymer sample whereas “Enzyme and Broth” are the samples in the enzymatically pretreated sets.

Figure 5

(A) Ester (1150–1250 cm^–1), (B) carbonyl (1710–1810 cm^–1), (C) vinyl (1600–1700 cm^–1), and (D) hydroxyl (3000–3600 cm^–1) indices of irradiated PLLA samples at varying points in the degradation process. Samples were measured in triplicate and averaged with error bars representing the standard deviation. UV: irradiation, enzyme: irradiation and 12 h in 8.2 μM proK, biofilm w/enzyme: irradiation, 12 h in 8.2 μM ProK and 3 days inoculated with S. oneidensis, biofilm w/o enzyme: irradiation and 3 days inoculated with S. oneidensis.

Figure 6

Percent crystallinity (%) versus UV light irradiation time of PLLA with differing treatments using DSC. Markers represent the average of samples (n = 3) with error bars representing standard deviation.

Figure 7

SEM images of PLLA with no or 4 h UV light irradiation and exposed to varying biological degradation treatments. Conditions are as follows, UV: irradiation with 300 nm light, enzyme: irradiation and 12 h in 8.2 μM proK, biofilm w/enzyme: irradiation, 12 h in 8.2 μM ProK and 3 days inoculated with S. oneidensis, biofilm w/o enzyme: irradiation and 3 days inoculated with S. oneidensis.

Figure 8

Bar graph showing biofilm growth on PHBV samples as quantified using crystal violet staining. Bars represent the average of samples (n = 3) with error representing the standard deviation. t-test between means reveals a significant different in the values (p = 0.0018).