International Journal of Biological Macromolecules 176 (2021) 226–232

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Enzymatic degradation and biofilm formation during biodegradation of

polylactide and polycaprolactone polymers in various environments
Agnieszka Richert ⁎, Grażyna B. Dąbrowska
Nicolaus Copernicus University in Toruń, Department of Genetics, Faculty of Biology and Veterinary Science, Lwowska 1, 87-100 Toruń, Poland

a r t i c l e i n f o a b s t r a c t

Article history: The present article presents the results of research on the susceptibility of polylactide, poly(ɛ-caprolactone) and
Received 14 October 2020 mixtures to biodegradation in conditions imitating natural extracts of compost, activated sludge, sea and river
Received in revised form 28 January 2021 water, determined by the biochemical oxygen consumption by microorganisms and susceptibility to enzymatic
Accepted 29 January 2021
degradation with the use of enzyme solutions of fungal microbial origin. Analyzes of both types of degradation
Available online 3 February 2021
were carried out over a period of seven days and in four environments: compost, activated sludge, river and
Keywords:
sea water, and four enzymatic solutions containing proteinase K, protease, esterase, and lipase. The amount of ox-
Blend PLA/PCL ygen consumed by microorganisms in the presence of the tested films was determined, as well as the weight loss
Biodegradation determined after the samples were incubated in enzymatic solutions. Images of the surface of individual samples,
Biofilm taken by fluorescence microscopy and scanning electron microscopy, confirm the formation of bacterial biofilm
Enzymes and the results of biochemical oxygen consumption by microorganisms, or weight loss. It was shown that the
Polycaprolactone compost and activated sludge extract as well as the enzymes proteinase K from Engyodontium album (synonym
Polylactide Tritirachium album) and protease from Bacillus licheniformis had the greatest impact on the biodegradation of the
tested materials.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction
The constantly increasing amount of polymer materials in the form
of waste is a problem of modern civilization. Fortunately, the growing
social awareness, changes in legal regulations, and the interest of re-
search centers in the field of environmental protection mean that biode-
gradable polymer materials are gaining more and more recognition and
interest. Biodegradable materials are used on an increasing scale in
medicine, pharmacy, biotechnology, agriculture, horticulture, and in
the packaging industry due to their valuable functional properties
such as biocompatibility, biodegradability or barrier properties [1].
Polylactide (PLA) [2,3], poly(ε-caprolactone) (PCL) have become
particularly important polymers for the economy [4–9], as well as
PLA/PCL mixtures, because according to current forecasts they can re-
place to a large extent range (mainly in packaging) those traditional
polymers that are not biodegradable. Many scientific papers present
the results of research on enzymatic degradation of polymeric materials
with the use of various enzymes [10–17].
The degradation course and rate of a given material depends on its
physicochemical properties including chemical structure, average mo-
lecular weight, chain structure, crystallinity and the activity and stability
of the enzyme used for the study as well as additional components
⁎ Corresponding author.
E-mail address: a.richert@umk.pl (A. Richert).
introduced into the polymer matrix to change the selected properties.
Biodegradability and the characteristics of the environment in which
the biodegradation process takes place are also significant.
It should be kept in mind that at least two categories of enzymes,
namely external and intracellular depolymerases, are involved in the bi-
ological degradation of polymeric materials. The degree and degree of
degradation of aliphatic polyesters depends on the type and nature of
extracellular enzymes produced by microorganisms. The decomposi-
tion of the outer layer of a given polymer occurs under the influence
of enzyme macromolecules, which have limited penetration into the in-
terior of a given material. Proteases, esterases, lipases and cutinases
which cause enzymatic degradation of polyesters by hydrolysis. In the
breakdown of PLA, they most often participate in the breakdown of pro-
tease and esterase [18–20]. At the beginning of degradation, the first se-
cretion of extracellular PLA depolymerase by the microorganisms.
However, its rapid production requires stimulation by inductors. In
this case, the inducers are amino acids, elastin or gelatin. Most inducers
have L-alanine units that are similar to PLA's L-lactic acid units in the
stereochemical position of the chiral carbon. Subsequently, the
depolymerase influences the intracellular ester bonds of PLA, which
produces oligomers, dimers and, consequently, monomers. Then these
low molecular weight compounds are absorbed by microorganisms,
and as a result of the activity of intercellular enzymes they are converted
into carbon dioxide, water and methane [21–24]. Of key importance is
the knowledge of the biochemical degradation processes of PLA, PCL

https://doi.org/10.1016/j.ijbiomac.2021.01.202
0141-8130/© 2021 Elsevier B.V. All rights reserved.
A. Richert and G.B. Dąbrowska
and the PLA/PCL blend for understanding biogeochemical cycles as well
as environmental protection and biotechnology development [25,26].
Enzymatic degradation takes advantage of the catalytic potential of
enzymes to remove toxic, hard-to-degradable compounds from the en-
vironment. As enzymes are simpler systems than whole microbial cells,
enzymatic degradation appears to be an attractive tool to support cur-
rent technologies, especially for removing degradation-resistant poly-
mers from the environment. Studies conducted in recent years
indicate the possibility of modifying bio-purification techniques by in-
troducing selected enzyme preparations to the contaminated sites that
support the transformation of difficult-to-degradable chemical com-
pounds into less toxic or completely harmless [27].
The aim of the analyzes was to check and compare the parameters of
biodegradability and enzymatic degradation of newly produced PLA,
PLA and PLA/PCL materials. The innovativeness of the work consists in
the parallel application of two research aspects of degradation (bio-
and enzymatic-) carried out on samples in a parallel manner. Namely,
on the one hand, compost, activated sludge, sea and river water extracts
simulating natural environmental conditions were used for the analysis.
On the other hand, attempts have been made to test the impact of com-
mercially available selected enzymes. The aim of this study was a com-
parative analysis of two types of degradation, a review of the mass loss,
biological consumption of oxygene by microorganism, biofilm forma-
tion and geometric structure change of the polymeric materials, that
occur under the influence of various environments and enzymes.
2. Materials and methods
2.1. Materials
Polylactide (symbol PLA), type 2003 D (NatureWorks®, USA); its
melt flow rate (MFI) was 3.4 g/10 min (2.16 kg, 190 °C), number-
average molecular weight (Mn), 156 kDa, and density (d), 1.24 g/cm3.
Poly(ɛ-caprolactone) (symbol PCL), type CAPA® 6800 (Solvay
Caprolactones, UK), MFI = 7.3 g/10 min (2.16 kg, 160 °C), Mn =
69 kDa, d = 1.1 g/cm3.
Poly(ethylene glycol) (PEG) with Mw = 1500 g mol−1 (Sigma-Al-
drich Ltd., Poznań, Poland) was used as a plasticizer (Piekary Śląskie,
Poland) was used as a solvent.
For enzymatic degradation were used the following enzymes of mi-
crobiological origin and reagents: proteinase K (≥30 units/mg) from
Engyodontium album (synonym Tritirachium album) (Blirt, Poland), pro-
tease from Bacillus licheniformis (7–15 units/mg) (Sigma-Aldrich,
Denmark), esterase from Bacillus subtilis (≥10 units/mg) (Sigma-Al-
drich, Germany), and lipase from Candida rugosa (15–25 units/mg)
(Sigma-Aldrich, Switzerland); sodium azide (NaN3) (Sigma-Aldrich,
Germany) and buffer 0.1 M Tris HCl (Blirt, Poland).
For biological degradation were used microorganisms of various en-
vironment such us compost, activated sludge, river water and seawater.
Compost (Toruń Waterworks “Centralna” Sewage Treatment Plant,
Poland), from which an extract containing a mixture of microorganisms
was made. For this purpose, up to 900 cm3 of Ringer's solution (compo-
sition [g/dm3]: NaCl - 2.250, KCl - 0.105, CaCl2 - 0.120, NaHCO3 - 0.050,
distilled water - 1 dm3) 100 g of mature compost were added. The
whole was then homogenized using a mechanical homogenizer
(Zelmer). The resulting suspension was filtered through a cloth with a
mesh size of 50 μm. The obtained filtrate was used for testing as a sus-
pension of microorganisms present in the compost.
Activated sludge (Toruń Waterworks “Centralna” Sewage Treatment
Plant, Poland). The precipitate was centrifuged at 10.000 rpm for 5 min
at 20 °C. The supernatant was removed and the pellet was resuspended
in sterile physiological saline. Then the suspension was centrifuged
again and suspended in physiological saline, the operation was repeated
twice more. The obtained suspension of washed activated sludge was
added to Ringer's solution in a ratio of 10:190.
227
International Journal of Biological Macromolecules 176 (2021) 226–232
In biodegradability tests used river water from the Vistula (Toruń,
Poland) and seawater from the Gulf of Puck (Baltic Sea, Poland).
2.2. Samples preparation
The film of PLA, PCL, PLA/PCL with poly(ethylene glycol) and chloro-
form mixtures was prepared according to the procedure described in
the patent application [28].
Poly(ethylene glycol) (PEG) with Mw = 1500 g mol−1(Sigma-
Aldrich Ltd., Poznań, Poland) was used as a plasticizer. Chloroform
(manufactured by Chempur, Piekary Śląskie, Poland) was used as a sol-
vent. The temperature of the experiment leading to the preparation of
the film was 23 °C.
2.3. Biological degradation
The investigation was based on the suspension containing a mixture
of microorganisms which can be found in mature compost. Previously
obtained composites of PLA, PCL and blend PLA/PCL were used as respi-
ratory substrates.
A sample of PLA, PCL and blend PLA/PCL (50:50) were placed in glass
measuring bottles. Subsequently, 200 ml of compost extract, activated
sludge, river water seawater separately, and 4 drops of nitrification in-
hibitor NT600 were poured into each bottle where a magnetic stir bar
was immersed. A rubber carrier with carbon dioxide absorber (0.4 g
NaOH) was then placed in each bottle. Finally, OxiTop measuring
heads were fixed, sealed bottles were placed on a stirring platform
and put into a thermostatic cabinet. The incubation was conducted for
7 days at 20 °C during which the OxiTop Controller collected the mea-
sured values every 24 h.
2.4. Biofilm formation
In order to evaluate the viability of biofilm forming bacteria, micro-
organisms retained on the filter surface were subjected to viability
staining, using a diagnostic LIVE/DEAD set (Invitrogen). Fragments of
the film (1 cm2 from each film) covered with the aqueous solutions of
dyes of LIVE/DEAD BacLight(™) Bacterial Viability Kit were incubated
for 15 min in the dark at room temperature. After the excess dye was re-
moved, the samples were placed on microscope slides and viewed
under oil immersion at ×1000 magnification using ECLIPSE 50i fluores-
cence microscope (Nikon, Japan) [17,20].
2.5. Enzymatic degradation
The samples PLA, PCL and PLA/PCL were placed into the test tubes
with lids containing reaction solution of 2 mg of the respective enzyme
(proteinase K, protease, esterase, lipase separately), 10 ml of 1.0 M Tris-
HCl buffer and 2 mg of sodium azide. Then the test tubes were incubated
at constant temperature of 37 °C [29,30].
Mass loss measurements were performed every day for a period
seven days of incubation of the samples.
After removing from the solutions, the individual samples were
thoroughly washed twice with distilled water. Then they were dried
in a laminar chamber (Bio Activa VE 120 Plus) by an exposure to a sterile
air flow; and weighed with an analytical balance of a weighing error less
than 0.1 mg. The weight loss (Δm) was calculated according to formula
(1) and expressed in percentage:
Δm ¼ fðms −mf Þ=ms g  100 ð%Þ ð1Þ
where:
ms – the original mass (mg) (determined at the beginning of the
tests) of three samples of the given film,
mf – the mass (mg) of the samples after the given time of the enzy-
matic degradation.
A. Richert and G.B. Dąbrowska
Fig. 1. Biological oxygen demand in various environments in the presence of polymers
after 7 days of biodegradation; values are expressed as mean ± SD (n = 3).
2.6. The morphology testing (SEM)
Changes in the geometrical structure of the surface of the samples
were recorded by using a scanning electron microscope (SEM) Hitachi
SU8010 (Hitachi, Japan) [31].
3. Results and discussion
3.1. Influence of environments on susceptibility on the biodegradation of
films PLA, PCL, blend PLA/PCL
The data analysis (Fig. 1) shows that the tested PLA, PCL and PLA/PCL
polymers were degraded at different rates, which depended on the type
of polymer and the environment in which the biodegradation process
took place.
The highest oxygen consumption was recorded in the presence of
PCL and the PCL/PCL mixture in the compost, it was respectively (140
mgO2/l and 132 mgO2/l after 7 days of incubation). The lowest oxygen
consumption by microorganisms was recorded in the environment of
river and sea water, for any of the materials it did not exceed 6 mgO2/
l. The biochemical wear of the background was confirmed by SEM im-
ages. Initially, pictures of the surface of PLA, PCL and PLA/PCL films
were taken before incubation (Fig. 2) and no differences between
them were noticed. After seven days of biodegradation of the film in fer-
tile environments, i.e. in compost and activated sludge, the structural
surface of the film from PLA, PCL and PLA/PCL changed. On the surface
of the PCL and PLA/PCL films, changes were seen due to the adhesion
MATERIALS:
PLA
Fig. 2. Scanning electron microscopy images of pure PLA (a), PCL (b) and PLA/PCL (c) samples (before degradation).
International Journal of Biological Macromolecules 176 (2021) 226–232
of microorganisms and damage to the film, resulting from their metab-
olism (Fig. 3).
BOD is a good and reliable indicator of the intensity of organic matter
transformation in the environment. Many researchers use these mea-
surements to estimate the rate of the biodegradation process.
Interesting conclusions were reached by Massardier-Nageotte et al.
[32], who studied the biodegradation of four different types of poly-
mers, polylactic acid, polycaprolactone, a mixture of starch with
polycaprolactone and starch with poly (butadiene adipate co-
terephthalate) with activated sludge. After 28 days of incubation, PCL
was very degraded to PLA. The same authors also noted that polymers
degrade better aerobically than anaerobically. Similar studies were con-
ducted by Nakayama et al. [33], with the difference, however, that they
analyzed the biodegradation of polymers in seawater. According to the
authors, PCL, P3HB and PHBHH degraded rapidly in contrast to PLLA,
PBS and PBAT.
The degradation process of polymers is absolutely dependent on the
type of material and microorganism. The intensity of the biodegradation
process can be analyzed using SEM. This is indicated by a change in the
surface structure of the film, the formation of a biofilm involved in deg-
radation. The results of scanning electron microscopy of biofilm forma-
tion on the surface of PLA are known from descriptions [20,34–37].
In addition to biochemical oxygen consumption by microorganisms
and SEM analysis, the bacterial biofilm formed on the surface of poly-
mers can be analyzed using the fluorescence microscopy technique
[20]. The results of the obtained analyzes are shown in Fig. 4.
The greatest number of microorganisms, including live can be ob-
served in the photos presenting films that were incubated in the extract
of the compost and the sludge, much less the material was subjected to
biodegradation in seawater and river. On the other hand, the material
on which the greatest changes were observed was the foil made of
PCL and PLA/PCL.
3.2. Influence of various enzymes on degradation PLA, PCL and blend PLA/
PCL samples
The results of testing the weight loss of PLA, PCL and PHB samples
treated with proteinase K and proteases after 7 days are presented in
Table 1. They show that these losses are most intense during the first
days of enzymatic degradation. The weight loss of PLA and PLA/PCL
films under the influence of proteinase K is the highest among all ana-
lyzed samples and used enzyme solutions.
Similar observations as to the intensity and speed of enzymatic deg-
radation on biodegradable plastics were demonstrated by Żenkiewicz
et al. [38]. The study of the morphological structure of the surface of
PCL PLA/PCL
228
A. Richert and G.B. Dąbrowska
MATERIALS:
PLA PCL
d
Fig. 3. The surface of PLA, PCL and PLA/PCL films after 7 days of biodegradation in compost (a), activated sludge (b), river water (c), sea water (d).
PLA, PCL and PLA/PCL was started with making SEM images of the sur-
face of these polymers in their original state, before the start of biodeg-
radation and enzymatic degradation (Fig. 4). The changes in the surface
structure of individual polymer materials after seven days of degrada-
tion in solution of proteinase K, protease, esterase and lipase are
shown in Fig. 5.
When analyzing the obtained images of the film surface after degra-
dation in the proteinase K solution, it can be noticed that the largest
changes occur in the PLA film (Fig. 5a), the porous structure covering
the entire surface, pores of various depths and sizes up to about 3 μm,
229
International Journal of Biological Macromolecules 176 (2021) 226–232
PLA/PCL
smaller in the PCL film PLA/PCL (Fig. 5b, c), pores predominantly from
about 0.2 to 3 μm, mostly oval, cover only part of the surface, with
smooth areas between them).
The results of research on the analysis of the surface structure of bio-
degradable polymer materials were presented in literature [39,40,42].
Enzymatic degradation is a frequently undertaken research in order
to understand the mechanisms of the degradation processes. However,
there are many problems associated with it, namely: obtaining pure en-
zymes from microorganisms, selecting microorganisms to obtain a pure
enzyme, selecting an enzyme for the polymer material, maintaining
A. Richert and G.B. Dąbrowska International Journal of Biological Macromolecules 176 (2021) 226–232

MATERIALS:
PLA PCL PLA/PCL

Fig. 4. Biofilm formation on polymers PLA, PCL and blend PLA/PCL in (a) compost, (b) activated slugde, (c) sea water, (d) river water. Live cells are green, dead cells are rEd.)

purity in the enzyme-polymer system during the experiment, especially
long-distance experiments.
4. Conclusions
The best degradation environment turned out to be compost and ac-
tivated sludge, and the most susceptible materials to biodegradation
were PCL and the PLA/PCL mixture. The use of commercially available
enzymes degrades PLA more efficiently than the use of consortia of en-
vironmental microorganisms (as shown by the weight loss). SEM ana-
lyzes have shown that PLA is degraded most efficiently after
incubation with proteinase K or protease. Research has shown that the
consortia of microorganisms present in compost and activated sludge
are more useful in biodegradation of polymer materials than those
from sea or river water.
Declaration of competing interest

The authors declare that they have no known competing financial

Table 1
Collective comparison of selected properties of PLA, PCL and blend materials.
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Δm [mg] after 7 days enzymatic degradation PLA PCL Blend PLA/PCL

Proteinase K 21.13 1.55 11.11 Acknowledgement

Protease 4.49 1.28 2.54
Esterase 2.29 1.91 2.00
This research was supported by funds provided by Nicolaus Coperni-
Lipase 1.75 1.62 1.50
cus University in Toruń (Toruń Poland) to maintain research potential.

230
A. Richert and G.B. Dąbrowska International Journal of Biological Macromolecules 176 (2021) 226–232

MATERIALS:
PLA PCL PLA/PCL

Fig. 5. SEM images of PLA, PCL and blend PLA/PCL samples after 7 days incubation with proteinase K (a), protease (b), esterase (c), and lipase (d).

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