# A critical review of the state of knowledge on factors responsible for the degradation in soils of polyhydroxyalkanoates

Ludovic Dulac, Mikaël Kedzierski, Stéphane Bruzaud

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Ludovic Dulac, Mikaël Kedzierski, Stéphane Bruzaud. A critical review of the state of knowledge on factors responsible for the degradation in soils of polyhydroxyalkanoates. Polymer Testing, 2025, 149, pp.108861. 10.1016/j.polymertesting.2025.108861. hal-05072706

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Submitted on 19 May 2025

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# A critical review of the state of knowledge on factors responsible for the degradation in soils of polyhydroxyalkanoates

Ludovic Dulac, Mikaël Kedzierski, Stéphane Bruzaud*

<figure><img src="image_2.png" /></figure>

Université Bretagne Sud, Institut de Recherche Dupuy de Lôme (IRDL), UMR CNRS 6027, 56321 Lorient, France

## ARTICLE INFO

### Keywords:

- Polyhydroxyalkanoates
- Degradation
- Degradation kinetics
- Degradation protocols

## ABSTRACT

Plastic pollution in the environment, particularly in soils, has become a major ecological concern due to its persistence and potential risks to ecosystems and human health. To mitigate this issue, biodegradable polymers have been developed as sustainable alternatives to conventional plastics. Among them, polyhydroxyalkanoates (PHA) stand out due to their promising biodegradability under diverse environmental conditions. However, their degradation is governed by a complex interplay of intrinsic factors (e.g., chemical structure, crystallinity, molar mass and polydispersity, physical state ...) and extrinsic factors (e.g., temperature, humidity, oxygen, micro-organisms ...), which remain only partially understood. Despite significant research efforts, the methodologies used to assess PHA degradation are often inconsistent or suboptimal, limiting the comparability and reliability of results. This review aims to provide a comprehensive analysis of the impact of these factors on PHA degradation in soils and suggests innovative experimental approaches to further the studies of these degradation processes.

## 1. Introduction

Plastics have become indispensable in our daily lives, whether in food packaging, agriculture, construction, textiles, or other areas, due to their lightness, low cost, and durability [1]. While these properties are beneficial for industrial applications, they pose significant environmental threats. Their resistance to natural degradation implies persistence in the environment over decades, thereby polluting seas and soils. To counteract this growing issue, sorting centers have been established to facilitate waste recycling [2,3]. However, they handle only a small fraction of the total waste stream, with the majority still directed toward incineration or landfilling. A portion of this waste may also inadvertently escape into natural environments due to poorly regulated landfills, littering, and other mismanagement practices [4]. Once exposed to environmental conditions, plastics undergo various degradation processes, including abiotic mechanisms (such as UV exposure, thermal degradation or chemical reactions like hydrolysis) as well as biotic degradation through microbial activity (Fig. 1).

Depending on the polymer's erosion pathway, it will occur at the surface in the case of biotic degradation, and throughout the bulk in the case of abiotic degradation [5]. Depending on the mechanism, the consequences on the mechanical properties will vary. In the case of surface erosion, the loss will occur linearly. In the case of bulk erosion,

there will be a significant drop once a critical molar mass value is reached [6]. (Fig. 2).

These processes generate plastics more brittle, friable and fragmentable, leading to the formation of microplastics (<5 mm). However, unlike marine contamination, where plastic pollution is widely documented, soil contamination by plastics remains less visible although just as worrying [8]. To curb the problem of plastic pollution, industries and academics have developed biodegradable polymers intended to replace synthetic ones like polyethylene (PE) or polypropylene (PP). Those biodegradable polymers can be rapidly converted into biomass, carbon dioxide (CO2), and water by microorganisms. But the term biodegradable can be misleading and should not be understood as "disposable in nature" [9]. For example, poly (lactic acid) (PLA), the most common biodegradable polymer, slowly degrades in soils [10,11], but much more rapidly in industrial compost [12]. The main reason is its high glass transition temperature (Tg ~ +55 °C) which makes PLA very stable below it. Among these biodegradable polymers, one family stands out: polyhydroxyalkanoates (PHA) (Fig. 3).

PHA are biodegradable in all environments [13], as well as biobased, since they are derived from bacterial fermentation [14]. Their discovery dates back to Maurice Lemoigne in 1926 [15], and interest in this family of molecules has grown ever since. By adjusting the bacterial culture medium, it is possible to modulate the chemical structure of PHA, resulting in two main PHA families: short-chain-length (scl-PHA) and

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: stephane.bruzaud@univ-ubs.fr (S. Bruzaud).</footnote>
**Abbreviations:**

Polyhydroxyalkanoates PHA
Poly(3-hydroxybutyrate) PHB
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PHBHV
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) P3HB4HB
Poly(3-hydroxybutyrate-co-3-hydroxykexanoate) PHBHHx

medium-chain-length (mcl-PHA). For instance, Cupriavidus necator can produce PHB (scl-PHA) when grown on glucose as a substrate [14], and this strain is also capable of synthesizing PHBV (scl-PHA) using more unconventional feedstocks, such as microalgae-derived starch and levulinic acid [16]. In the case of mcl-PHA, Pseudomonas sp. is typically used for [17]. Although sugars remain the most traditional substrates, there is a growing interest in using industrial waste streams - such as palm oil mill effluents and paper mill wastewater effluents - as alternative resources [18,19]. Scl-PHA are the most widespread PHA and have a side chain containing fewer than four carbon atoms, making them crystalline, rigid, and brittle, whereas mcl-PHA (much rarer due to their lack of commercial availability) have four or more atoms on the side chain, making them more amorphous, ductile, and flexible. PHA has the advantage over other biodegradable polymers that many micro-organisms possess the enzymes to degrade them [20]. This degradation occurs primarily through biotic processes and a surface-driven mechanism [21]. Microorganisms first colonize the material's surface, forming a biofilm that secretes PHA depolymerases. These depolymerases are made up of three sites: one to bind to the surface, one to perform catalysis and one to link the two. Due to their large size, depolymerases cannot penetrate the sample but instead break down polymer chains at the surface into soluble products (i.e., monomers or oligomers), which are then assimilated by microorganisms. Moreover, Iwata et al. [22] have shown that for PHB, depolymerases adsorb randomly to the surface, with no preference for edges or the like. In soil, different types of microorganisms are capable of degrading PHA, such as fungi and bacteria [23]. First studies of degradation of PHA in soils began in the 1990s [24,25]. The aim was to gain a better

understanding of how intrinsic (i.e. crystallinity, physical state, molar mass, shape ...) and extrinsic parameters (i.e. temperature, humidity, micro-organisms ...) influence degradation kinetics. From these pioneering experiments, a large number followed to identify the impact of each factor (Table 1). The problem is that there is currently no standardized protocol, although some experiments are based on ISO standards. The results obtained by each team may be difficult to compare, due to differences in test parameters. What's more, the study of intrinsic and extrinsic parameters is often not optimized. The various factors are not isolated from each other. Nevertheless, better understanding the role and weight of each parameter would be an asset, as it could potentially allow for the design of PHA-based objects, such as packaging, with controlled or programmed degradation. All of this would make PHA very attractive, making them even more competitive compared to conventional plastics. The aim of this review is first to explain the role of each parameter on PHA degradation kinetics. Secondly, protocols with new ideas from the literature will be proposed to optimize the study of each factor.

## 2. Methodology

The literature reviewed was sourced from various scientific databases (Scopus, ResearchGate, Google Scholar) and editorial websites, using combinations of the following terms: "soil biodegradation", "soil degradation", "polyhydroxyalkanoates", "poly (3-hydroxybutyrate)", "crystallinity", "UV", "temperature" and "humidity" among others. This review exclusively considered peer-reviewed experimental studies, with reviews consulted only for supplementary insights. The literature search produced numerous articles, of which more than 125 were selected following screening for relevance and English-language criteria. Following this, a detailed evaluation was conducted on the remaining articles to exclude those not aligned with the primary objectives of our study, retaining only the most pertinent and insightful contributions.

## 3. Influence of intrinsic parameters on the degradation

Intrinsic parameters refer to the physico-chemical properties specific to the polymer, including crystallinity, molecular weight, surface roughness, hydrophobicity and shape. A key challenge in studying their

<figure><img src="image_3.png" /><figcaption>Fig. 1. Degradation pathways of polymer.</figcaption></figure>
impact on degradation kinetics is their interdependence, modifying one
parameter can unintentionally influence others. For instance, adjusting
crystallinity may also alter the roughness of the samples. Therefore, the
proposed protocols do not guarantee isolation of the effect of one
parameter but attempt to minimize variability.

3.1. *Crystallinity*

3.1.1. *Effect of the crystallinity on degradation kinetics*

Polymers can be classified as either amorphous or semi-crystalline. In the amorphous state, the chains are arranged randomly, creating free volume that makes it easier for degrading agents to diffuse and access ester functions. This structural openness thus facilitates chemical and enzymatic hydrolysis. By contrast, semi-crystalline polymers contain both amorphous and crystalline domains. In the crystalline domains, chains are tightly packed in an ordered arrangement, impeding the penetration of water and enzymes and consequently slowing down degradation. Although many enzymatic degradation studies [38–40] have shown that amorphous regions break down more readily than crystalline zones, these findings stem from controlled laboratory conditions that may not always reflect real-world environments. Because biodegradation involves numerous interdependent factors, having a higher crystallinity does not necessarily mean a polymer will degrade more slowly. Some studies [24,41–43] have reported an influence of crystallinity on degradation, whereas others [44] have reported no significant effect. For instance, Boyandin et al. [23], who examined the biodegradation of polyhydroxybutyrate (PHB) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) in Vietnam, observed that PHB (x 61 %) degraded before PHBHV (x 50 %). They attributed this difference to local soil microorganisms being better suited to PHB. Conversely, Volova et al. [26] tested PHB (x 78 %) and PHBHV (x 60 %) and found that PHBHV degraded first. The multifactorial nature of biodegradation was also highlighted by Arcos-Hernandez, who investigated PHBHV samples with different HV contents. Interestingly, the least crystalline variant (47 % HV, x 14 %) showed the slowest degradation - 48 % mass loss after 15.9 weeks - whereas a sample with 12 % HV (x 58 %) reached 67 % mass loss. Surface morphology differences were proposed as a key factor in this discrepancy (Table 1). These examples underscore the need for caution when attempting to predict degradation kinetics based solely on crystallinity. An additional complication arises from how experiments are conducted: most studies either compare distinct copolymers [23,25,26,41] or examine a single

copolymer containing varying amounts of co-monomers [31]. Both approaches often alter multiple parameters at once (e.g., roughness, hydrophobicity, molecular weight, chemical structure), making it difficult to isolate the specific role of crystallinity. A more direct method would be to adjust crystallinity within a single PHA composition, but this strategy has yet to be explored. Other features of crystallinity, such as spherulite size and chain conformation, have been studied even less and typically only in the context of enzymatic degradation [45]. Findings regarding spherulite size remain contradictory [39,45]. For their part, Wen and Lu [31] found that early-stage degradation occurs within both the spherulite core and its outer region, areas where amorphous domains prevail. Regarding chain conformation, planar zigzag structures appear to degrade more readily than helical ones [46], likely due to reduced steric hindrance, which allows enzymes greater accessibility to the ester functions.

3.1.2. *Protocols*

To modify the PHA crystallinity without changing the rate of co-
monomers, several methods are available. The first, simple to perform,
involves annealing. Samples formed by solvent casting or injection
molding or extrusion are placed in an oven at a temperature between T<sub>g</sub>
and T<sub>m</sub>, near the T<sub>c</sub>. This thermal energy promotes chain reorganization
and the formation of new crystalline regions. In De Koning et al.'s [47]
study about PHB, crystallinity increased from 60 % to 68 % after 350
min at 115 °C. Kurusu et al. [48] observed a similar increase in crys-
tallinity from 63 % to 75.8 % for PHB after 1000 min at 125 °C. How-
ever, with the annealing method, there is a risk that surface roughness
may also be affected [49], inhibiting or accelerating degradation ki-
netics. Annealing also has the drawback of creating crystalline lamellae
of different sizes, a parameter that has an impact on kinetics [38]. The
second method to modify crystallinity involves solvent casting,
following the work of Anbukarasu et al. [50]. They used acetic acid
instead of chloroform to create PHB films. The polymer is dissolved in
acetic acid at 160 °C for about 1 h and then poured into a Petri dish
maintained at a specific temperature. By modulating the temperature of
the Petri dish, crystallinity varies. With a Petri dish maintained at 80 °C
they obtained 60.4 % crystallinity, compared to 78 % at 160 °C. The key
point of this method is roughness: between samples made at 140 °C and
160 °C, roughness is roughly equal to 5 nm, while the difference in
crystallinity is significant: 70 % at 140 °C compared to 78 % at 160 °C.
Byun and co-workers [51] have worked on PLA and like Anbukarasu
et al. have shown that by changing solvent, it is possible to modify

<figure><img src="image_4.png" /><figcaption>Fig. 2. Effects of surface and bulk erosion on polymer properties over time. Inspired on W. Meereboer et al. [7] and Burkersroda et al. [5].</figcaption></figure>
crystallinity. In their study, they made 3 films with chloroform, dichloromethane and a dichloromethane/acetonitrile mixture. They obtained crystallinity values of 24.03 %, 14.25 % and 46.15 % respectively. This work provides further avenues for modulating PHA crystallinity by solvent casting.

## 3.2. Shape, accessible surface and thickness

### 3.2.1. Effect on degradation kinetics

Depending on the geometry of the samples, the accessible surface area to microorganisms will vary. For the same volume, the accessible surface area of a film will be greater than that of a bead [52]. The larger the surface area, the more accessible sites there will be for enzymes and therefore a difference in degradation kinetics. In Boyandin et al.'s study [23], granules and films were compared. For PHB films, degradation was almost complete after 300 days, while the granules were only half-degraded (Table 1). They showed that microbeads had a significant decrease in molecular weight compared to films. Is this due to better enzyme penetration into the spheres or a different degradation mode? Additional analyses by SEM will help clarify the phenomenon. Porous samples, though less studied [28,30], also fall into this category. Increasing specific surface area increases available sites for biotic or abiotic interactions, speeding up kinetics. Another crucial yet often overlooked parameter is the thickness of the samples. Since PHA degradation primarily occurs at the surface, thicker samples will degrade more slowly [6,29]. However, it is common in the literature to find studies [31,53] where sample thickness varies significantly, sometimes differing by a factor of two or three between analyzed specimens. Therefore, controlling sample thickness is a critical step in the preparation of PHA films to ensure reliable and comparable degradation studies.

### 3.2.2. Protocols

The first protocol is about sample shape and thickness control and the second on porosity. To study the impact of shape, the comparison between microbeads and films seems the most appropriate as they are both easy to produce. The synthesis of microbeads is carried out using the emulsion-evaporation method, following the approach described by Volant et al. [54]. Initially, PHA granules or powders are completely dissolved in chloroform or dichloromethane. The polymer solution is then poured into a 2 % PVA solution under mechanical stirring at 4000 RPM. The emulsion time is about 15 min. The formed microbeads are then dried by solvent evaporation by gently stirring the solution for 24 h. A sieve is then used to collect them, and they are passed through a lyophilizer. It seems that the type of polymer used affects the final diameter of the microbeads. Microbeads size can be determined by SEM or laser diffraction analysis. For film formation, solvent casting is

frequently used [55–57] because it is easy to perform. The granules or powder of the desired PHA formulation are dissolved in chloroform and stirred until complete dissolution (Fig. 4a). The solution is then poured into a container like a Petri dish and left to evaporate until the film forms (Fig. 4b). The evaporation rate can be controlled by adding an aluminum lid pierced by a few holes. To control the thickness of the film, the following formula (Eq. (1)) can be used:

$$m = \frac{\rho \times h}{\pi \times r^2} \qquad (1)$$

with $\rho$ the density of the PHA (g/cm³), $h$ the desired thickness of the film (cm) and $r$ the radius of the Petri dish (cm).

The synthesis of porous PHA (Fig. 5) can be achieved through non-isothermal polymerization-induced phase separation [58] (NIPS). The protocol is based on Kang et al.'s work [58]. Firstly, PHA powder or pellets are dissolved in CHCl₃. After complete dissolution, THF is added, and the solution is left to stand until a gel forms. The gel is then washed with methanol and water to remove any solvent residue.

## 3.3. Molecular weight

### 3.3.1. Effect on degradation kinetics

Chain length and molecular weight of a polymer are correlated: the higher the molecular weight, the longer the chain. What Numata et al. [59] have shown is that enzymes preferentially attack chain ends because ester functions are more accessible there. Therefore, for samples with the same weight, the one with the lowest molar mass (i.e. the more chain ends) should have the fastest degradation rate. The issue is that all their tests have been carried out by enzymatic degradation. In soil, the link between molecular weight and degradation kinetics is not established [26].

### 3.3.2. Protocols

To study the influence of molecular weight, it is recommended to use PHB. By changing production methods (substrates, strain ...) PHB with different molecular weights can be obtained [60,61]. For copolymers (PHBHV, PHBHHx, P3HB4HB ...), it seems much more complicated to change the molar mass by modifying the production processes while maintaining the same co-monomer ratio across different samples to avoid influencing other parameters. Chemical hydrolysis (acidic or basic) is another way of manipulating molecular weight. For example, Tapadiya et al. [62] used a 2M NaOH solution at 30 °C on PHB films. During their experiment of hydrolysis, they noted an increase of crotonic acid, a subproduct of degradation: 14, 18.5 and 32 % after 21, 52 and 92h respectively. Due to basic hydrolysis, random chain scission will take place, giving the formation of oligomers and crotonic acid. The greater the number of scissions, the greater the amount of crotonic acid.

<figure><img src="image_5.png" /><figcaption>Fig. 3. Chemical structures of (a) PHB, (b) PHBHV, (c) PHBHHx, (d) P3HB4HB.</figcaption></figure>
<table><caption>**Table 1**
PHA degradation as a function of parameter studied.</caption><thead><tr><th>PHA</th><th>Parameter/Value</th><th>Shape</th><th>Conditions</th><th>Test time (days)</th><th>Mass loss (%)</th><th>Ref.</th></tr></thead><tbody><tr><td>Crystallinity (%)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHB</td><td>61</td><td>Films</td><td>Field soil, 26–31 °C, humidity 70–84 %, 15 cm depth</td><td>300</td><td>100</td><td>[23]</td></tr><tr><td>PHBV</td><td>51</td><td></td><td></td><td></td><td>60</td><td></td></tr><tr><td>PHB</td><td>78</td><td>Films</td><td>Field soil, 21 °C; 28 °C, 50 % moisture content, 2 cm depth</td><td>35</td><td>60; 95<br>90; 100</td><td>[26]</td></tr><tr><td>PHBHx (12 % HV)</td><td>60</td><td></td><td></td><td></td><td>92; 100</td><td></td></tr><tr><td>PHBHx (12 % HHx)</td><td>56</td><td></td><td></td><td></td><td>100; 100</td><td></td></tr><tr><td>P3HB4HB (10 % 4HB)</td><td>50</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHBHV (12 % HV)</td><td>58</td><td>Films</td><td>Soil-compost mixture, 25 °C, 80 % WHC</td><td>111</td><td>67<br>54<br>48<br>62<br>49<br>49</td><td>[27]</td></tr><tr><td>PHBHV (43 % HV)</td><td>39</td><td></td><td></td><td></td><td>54</td><td></td></tr><tr><td>PHBHV (47 % HV)</td><td>14</td><td></td><td></td><td></td><td>48</td><td></td></tr><tr><td>PHBHV (52 % HV)</td><td>29</td><td></td><td></td><td></td><td>62</td><td></td></tr><tr><td>PHBHV (64 % HV)</td><td>42</td><td></td><td></td><td></td><td>49</td><td></td></tr><tr><td>PHBHV (72 % HV)</td><td>40</td><td></td><td></td><td></td><td>49</td><td></td></tr><tr><td>Shape</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHB</td><td>–</td><td>Films</td><td>Field soil, 26–31 °C, humidity 70–84 %, 15 cm depth</td><td>300</td><td>100</td><td>[23]</td></tr><tr><td>PHBV</td><td>–</td><td>Pellets</td><td></td><td></td><td>55</td><td></td></tr><tr><td>PHB</td><td>–</td><td>Film</td><td>Garden soil, 30 °C, humidity 80 %</td><td>42</td><td>62<br>100</td><td>[28]</td></tr><tr><td>PHB nanofiber</td><td></td><td>Nanofiber</td><td></td><td></td><td></td><td></td></tr><tr><td>Thickness (mm)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHB</td><td>0,03; 0,11; 0,18</td><td>Films</td><td>Forest soil, 27 °C</td><td>56</td><td>77; 33.5; 13.5<br>64.3; 54.6; 9.5<br>54.2; 21.4; 11.4<br>100; 21.5; 15.8</td><td>[29]</td></tr><tr><td>PHBHx (8 % HHx)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHBHx (12 % HHx)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHBHx (21 % HHx)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Porosity (%)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHB</td><td>6<br>80</td><td>Films<br>Fibers</td><td>Garden soil, 22 °C, 60 % moisture content</td><td>9</td><td>0<br>65.5</td><td>[30]</td></tr><tr><td>Roughness (Sq (nm))</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P3HB4HB (5 % 4HB)</td><td>–</td><td>Films</td><td>Garden soil, room temperature, 20 % moisture content</td><td>60</td><td>54.4<br>69.7<br>79.9<br>93.4<br>82.0</td><td>[31]</td></tr><tr><td>P3HB4HB (7 % 4HB)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P3HB4HB (10 % 4HB)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P3HB4HB (15 % 4HB)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>P3HB4HB (20 % 4HB)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHB</td><td>3.66</td><td>Films</td><td>Farm soil, 30 °C, 20 % moisture content</td><td>56</td><td>33.31<br>50.34</td><td>[32]</td></tr><tr><td>PHB – Plasma treatment</td><td>39.81</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Humidity</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHBV (4 % HV)</td><td>–</td><td>Films</td><td>Garden soil, 28 °C, 15 % moisture content<br>Garden soil, 28 °C, 20 % moisture content<br>Garden soil, 28 °C, 25 % moisture content<br>Garden soil, 28 °C, 30 % moisture content</td><td>56</td><td>12<br>23.6<br>95<br>95</td><td>[33]</td></tr><tr><td>PHA</td><td>–</td><td>Films</td><td>Garden soil, 28–32 °C, 10 % moisture content<br>Garden soil, 28–32 °C, 20 % moisture content<br>Garden soil, 28–32 °C, 30 % moisture content<br>Garden soil, 28–32 °C, 40 % moisture content<br>Garden soil, 28–32 °C, 50 % moisture content</td><td>30</td><td>50.2<br>72.6<br>93<br>96.4<br>91.5</td><td>[34]</td></tr><tr><td>Temperature</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHB</td><td>–</td><td>Films</td><td>Forest soil 28 °C; 37 °C; 60 °C<br>Sandy soil 28 °C; 37 °C; 60 °C<br>Activated sludge soil 28 °C; 37 °C; 60 °C<br>Farm soil 28 °C; 37 °C; 60 °C</td><td>25</td><td>10.5; 7.1; 4.9<br>5.8; 10.0; 4.5<br>68.9; 98.9; 30.5<br>41.3; 68.8; 14.8</td><td>[35]</td></tr><tr><td>PHB</td><td>–</td><td>Films</td><td>Clay soil, 20 °C; 30 °C; 40 °C</td><td>200</td><td>19.7; 38.7; 36.6</td><td>[36]</td></tr><tr><td>PHB</td><td>–</td><td>Films</td><td>Field soil, 21 °C; 28 °C<br>50 % moisture content, 2 cm depth</td><td>35</td><td>60; 95<br>90; 100<br>92; 100<br>100; 100</td><td>[26]</td></tr><tr><td>PHBHx (12 % HV)</td><td>–</td><td>Films</td><td>Farm soil, 30 °C; 50 °C<br>WHC 40 %</td><td>30</td><td>95.5; 41.7</td><td>[37]</td></tr><tr><td>PHBHx (12 % HHx)</td><td>–</td><td>Films</td><td>Forest soil, 28 °C; 37 °C; 60 °C</td><td>25</td><td>10.5; 7.1; 4.9</td><td>[35]</td></tr><tr><td>P3HB4HB (10 % 4HB)</td><td>–</td><td>Films</td><td>Farm soil, 30 °C; 50 °C<br>WHC 40 %</td><td>30</td><td>95.5; 41.7</td><td>[37]</td></tr><tr><td>PHB Soil composition</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHB</td><td>–</td><td>Films</td><td>Clay soil, 20 °C<br>Laterite soil, 20 °C<br>Saline soil, 20 °C<br>Sandy soil, 20 °C<br>Tarine soil, 20 °C<br>Compost, 20 °C<br>Sewage sludge, 20 °C</td><td>200</td><td>19.7<br>21.7<br>13.9<br>17.6<br>8.8<br>14.8<br>20.6</td><td>[36]</td></tr><tr><td>UV</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHB</td><td>–</td><td>Films</td><td>Forest soil 28 °C<br>Sandy soil 28 °C<br>Activated sludge soil 28 °C<br>Farm soil 28 °C</td><td>25</td><td>10.5<br>5.8<br>68.9<br>41.3</td><td>[35]</td></tr><tr><td>UV</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PHB</td><td>–</td><td>Films</td><td>Garden soil, 30 °C, humidity 80 %, 30 min exposition UVA<br>Garden soil, 30 °C, humidity 80 %</td><td>42</td><td>68<br>62</td><td>[28]</td></tr></tbody></table>
<figure><img src="image_6.png" /><figcaption>Fig. 4. Solvent casting method: (a) powder or granules are mixed with a compatible solvent such as chloroform. The solution is left to stir overnight to ensure complete dissolution (b) the solution is then poured into a Petri dish where, after the solvent evaporates, the polymer film can be recovered.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 5. SEM images of PHB samples made by NIPS method with different ratio of CHCl<sub>3</sub> – THF. (a) 70/30 CHCl<sub>3</sub> – THF, x1000; (b) 70/30 CHCl<sub>3</sub> – THF, x5000; (c) 57/43 CHCl<sub>3</sub> – THF, x1000; (d) 57/43 CHCl<sub>3</sub> – THF, x5000. The porosity of these samples is around 97 %. Reproduced with permission from Kang et al. [58].</figcaption></figure>

Thus, an increase in the quantity of crotonic acid over time is linked to the presence of increasingly short chains within the material, so lower molar masses of PHA. Yu and Marchessault [63], as well as Yu et al. [64], used the same basic hydrolysis principle to reduce molecular weight. But Yu et al. [64] also demonstrated that basic hydrolysis works better than acidic hydrolysis, explaining that in a basic solution, re-esterification is not thermodynamically feasible, whereas it is in an acidic environment. The samples produced by these different methods will have different molecular weights but also possibly different crystallinities. For some, crystallinity decreases as molecular weight increases [65]; for others, it remains constant [66] or follows a bell curve [67].

## 3.4. Surface morphology

### 3.4.1. Effect on degradation kinetics

Surface irregularities can serve as anchoring points for microorganisms and facilitate biofilm development [68]. These irregularities can result from different factors: co-monomer content, evaporation rates in solvent casting, etc. Wen et al. publication [31] compared different P3HB4HB (5% 4HB, 7% 4HB, 10% 4HB, 15% 4HB and 20% 4HB) and found that the copolymer with 15% 4HB degraded quicker than the

other one, even the 20% 4HB which is less crystalline. They explain this by the fact that 20% 4HB has a very smooth surface, preventing microorganisms from interacting well. Arcos-Hernandez et al. [27]. employed AFM to better understand how surface morphology influences the degradation kinetics of various PHBHV samples they produced. Their PHBHV (12% HV) displayed a relatively smooth surface, suggesting fewer crystalline domains and thereby a faster degradation rate. In contrast, the PHBHV (43% HV) exhibited a lamellar surface morphology, which resulted in a slower degradation rate.

### 3.4.2. Protocols

To modulate surface roughness, controlling the solvent evaporation during the solvent-casting process is a way. A higher evaporation rate increases the surface roughness, while a slower rate produces a smoother surface [69]. To regulate the evaporation rate, an aluminum lid with holes can be used; by adjusting the number and/or size of these holes, the evaporation rate - and consequently, the sample's roughness - can be modified. An alternative method is to choose a different solvent. For example, Strawhecker et al. [69] reported that dichloromethane, which has a higher saturation vapor pressure than chloroform at 20 °C (0.4570 bar vs 0.2350 bar), evaporates more rapidly, resulting in a rougher surface (144 nm vs 79 nm, respectively). However, it is essential
to assess crystallinity to ensure minimal variations between samples, as modifying roughness may also impact other intrinsic parameters [69]. Annealing is another effective method for modifying surface roughness without altering other intrinsic parameters. Wang et al. [49] investigated this phenomenon in PP and demonstrated that annealing temperature influenced roughness without affecting crystallinity. The application of thermal energy facilitates stress relaxation and local molecular rearrangements, allowing for the perfection of crystalline structures. This process increases the number of chains in a rigid amorphous state, which are randomly distributed and contribute to surface alterations. Using X-ray diffraction (XRD), Wang et al. [49] observed no significant differences in crystallinity between samples annealed at 125 °C and 145 °C. However, roughness increased significantly, from 67.70 nm at 125 °C to 147.34 nm at 145 °C. Similarly, De Koning et al. [47] examined the effect of annealing time on the crystallinity of PHB. Their findings indicate that PHB samples with identical crystallinity can be obtained by adjusting both temperature and annealing duration. For instance, a sample annealed at 115 °C for 60 min and another at 128 °C for 10 min both exhibited a crystallinity rate of 65.9 %. Based on Wang et al.'s study, it can be inferred that these two PHB samples have different surface roughness levels. Thus, annealing is the most suitable approach for achieving variations in roughness while preserving other intrinsic material properties.

# 4. Influence of extrinsic parameters on the degradation

Extrinsic parameters refer to the environmental conditions affecting polymer degradation, such as temperature, humidity, soil composition, microbial activity, UV exposure, pH, and oxygen levels. As with intrinsic parameters, some of these factors will be explored in detail, and protocols will be proposed to better assess their influence on degradation kinetics.

## 4.1. Humidity

### 4.1.1. Effect on degradation kinetics

Water is crucial for microbial growth, and variations in its availability directly influence microbial activity and degradation kinetics [70]. When humidity levels are too low, microbial metabolism slows down; conversely, if the humidity is too high—approaching saturation—anaerobic conditions may develop, which also reduces microbial metabolism [71]. Another important consideration is the way humidity is reported in literature. Some studies refer to water content [72], while others use the maximum water-holding capacity (WHC) [73]. Since soils differ in composition and thus in their water retention abilities [74], WHC is generally the preferred metric to replicate experiment conditions. ISO 17556:2019 recommends a range of 40 %–60 % WHC. However, Chinaglia et al. [75] compared conditions at 25 % WHC, 50 % WHC, and 80 % WHC, observing increased degradation kinetics at 80 % WHC. Patil et al. [34] compared five different humidity levels: 10 %, 20 %, 30 %, 40 % and 50 %. Maximum degradation was observed at 40 % with 96.4 % degradation, compared to 50.2 % degradation at 10 %. In their case, they refer to water content, not WHC, so achieving higher degradation at 40 % than at 50 % could indicate that the soil was close to saturation at 50 %. Studies [33] on this extrinsic parameter seem to agree that the higher the water content, without reaching saturation, the faster the kinetics.

### 4.1.2. Protocols

To avoid introducing additional variables, it is essential to use a single PHA formulation, one type of soil, and maintain a constant temperature throughout the experiment. The experimenter can choose both the temperature and the soil type. For humidity, at least three values should be selected to provide a broad dataset. For example, using 25 % WHC, 50 % WHC, and 80 % WHC would cover a wide range, although more closely spaced values may be chosen for a more detailed study.

While numerous approaches can be used to evaluate the water retention capacity of soil, ISO 11721-1:2001 describes one of the most accessible methods [76]. To ensure that the water content remains constant during the experiment, gravimetric measurements will be performed. In this procedure, a few grams of soil are sampled and placed in an aluminum dish, which is then heated in an oven at 105 °C for at least 24 h. By recording the mass of the empty aluminum dish (g), the mass with wet soil (g), and the mass with dry soil (g), the water content (%) can be calculated using the following formula (Eq. (2)):

$$ Water\ content = \frac{(weight\ of\ wet\ soil) - (weight\ of\ dry\ soil)}{(weight\ of\ dry\ soil) - (weight\ of\ empty\ aluminium\ dish)} \quad (2) $$

Similarly, it is crucial to weigh the container at the beginning of the experiment to record its initial mass. Then, on a weekly basis, water will be added according to the mass lost, which reflects the rate of evaporation.

## 4.2. Temperature

### 4.2.1. Effect on degradation kinetics

Like humidity, temperature is a key factor in the development of microorganisms. In soils, microorganisms are mesophilic [77], meaning they grow best between 20 °C and 45 °C. When approaching or exceeding these extremes, microbial activity is greatly slowed, thereby slowing degradation kinetics [78]. Therefore, when designing experiments, it is crucial to strike a balance between promoting rapid PHA degradation and avoiding enzyme denaturation. ISO 17556:20219 advises maintaining 25 °C all along, but some studies have demonstrated enhanced degradation at higher temperatures. Manna and Paul [36] compared the degradation of PHB in eight different soils with three different temperatures: 20 °C/30 °C/40 °C. Through mass loss measurements, they observed the highest degradation at 30 °C across all soils, due to higher microbial activity at that temperature (Fig. 6). In clay soil, for example, degradation reached 19.7 % at 20 °C after 200 days compared to 38.7 % at 30 °C. A study [26] comparing PHB, PHBHV (12 % HV), PHBHx (12 % HHx), and P3HB4HB (10 % 4HB) at 21 °C and 28 °C showed greater degradation at 28 °C than at 21 °C after 35 days, regardless of the copolymer. Nishide et al. [37]. conducted experiments at 30 °C and 52 °C and observed faster degradation at 30 °C. By isolating micro-organisms, they were able to observe an abundance of fungi at 30 °C, whereas at 52 °C, their presence was much lower. Soil temperature will therefore influence the development of micro-organisms and hence degradation kinetics. Kim and co-workers [35] conducted a similar experiment by raising the temperature around 60 °C. As the limits temperature for microbial activity were exceeded, a slowdown in degradation kinetic was noticed. While enzymatic hydrolysis is the main degradation process for PHA, high temperatures can also catalyze chemical hydrolysis. Mergaert et al. [24] showed that for PHBHV (10 % HV), degradation reached 100 % after 200 days at 40 °C compared to 32 % at 28 °C. However, at 40 °C, molecular weights showed a drastic drop, indicating abiotic degradation (chemical hydrolysis), which was not observed at 28 °C. In some African, Middle Eastern, or Southeast Asian countries, these high temperatures (25 °C and above) can be found in soils [79] (between 0 and 5 cm) correlating these results with reality. However, few studies [80] have replicated conditions found in colder climates, leaving a knowledge gap in this area.

### 4.2.2. Protocols

It is essential to use the same PHA formulation, a single type of soil, and maintain a consistent WHC throughout the study. The study should be conducted at three different temperatures to obtain a wide range of data. The choice of these three temperatures will depend on the specific goals of the study. If the aim is to simulate real-world conditions, temperatures such as 15 °C/20 °C/28 °C could be chosen to simulate
<figure><img src="image_8.png" /><figcaption>Fig. 6. Comparison of the effect of temperature on PHB degradation in eight different soils. Figure inspired by the results of Manna et Paul [36].</figcaption></figure>

European soil temperatures [79] for example. Alternatively, the range could be extended up to 40 °C, choosing 25 °C/30 °C/40 °C if the objective is to show the effect of temperature on degradation kinetics. It is also possible to narrow the temperature range to determine whether there is a significant difference between two close temperatures. The choice of soil will depend on what the experimenter has access to, and the recommended WHC is 80 %.

## 4.3. UV exposure

### 4.3.1. Effect on degradation kinetics

Ultraviolet (UV) radiation is highly energetic, and, over time, damages polymers exposed to it through various photo-oxidation mechanisms, such as Norrish I and II reactions. These processes lead to chain scission and the formation of intermediate radicals that may further react with other PHA chains, perpetuating additional chain scission via radical initiation [81]. Although UV-induced degradation affects the thermal and mechanical stability of the samples, this initial degradation does not necessarily result in accelerated overall degradation kinetics. Through chain scission, the polymer chains become more mobile, which can allow them to reorganize and form new crystalline regions, a phenomenon known as chemi-crystallization. Since enzymes preferentially attack amorphous regions, a reduction in these areas due to UV exposure could influence the degradation kinetics [59]. Rudnik and Briassoulis [11] highlighted this phenomenon by conducting two parallel experiments. In one, PHA samples were buried in soil; in the other, samples were exposed to sunlight for 2–3 months and then buried in the soil. The UV-exposed samples showed an increase in crystallinity, but no faster degradation compared to samples buried directly in the soil. Similarly, D.S. Rosa et al. [82] observed no impact of UV radiation on kinetics. Their explanation lies either in crosslinking or that the fragmented chains of PHB samples were just small enough to reorganize and crystallize without accelerating the degradation process. In contrast, Sadi et al. [83] observed accelerated kinetics, although this effect was not immediate. Their study compared the properties of the UV-exposed surface layer with those of a deeper, less affected layer. The surface layer showed increased crystallinity and chain scission, while the deeper layer, though also exhibiting chain scission, experienced less chemi-crystallization. As PHA degradation is a surface phenomenon, the enzymes must first degrade the more crystalline surface layer. Once this layer is removed, the deeper layers, which have undergone a chain scission but have no chain rearrangement, are easier to degrade.

However, the study concluded at only a 6 % mass loss after 300 days, leaving open the intriguing question of how degradation kinetics would evolve once the deeper, UV-unaffected layers were reached.

Furthermore Altaee et al. [28] noted that PHB exposed to UV exhibit surface modifications such as cracks and changes in roughness, which could facilitate biofilm development [84]. For aging tests, some combine UV radiation and water spraying [81], other alternate between the two [84], and others expose the samples to UV radiation alone [28].

### 4.3.2. Protocols

There are two primary methods for the photodegradation of samples. The first involves exposing them to UV radiation outdoors, where they are also subject to environmental factors such as precipitation. While this approach reflects natural conditions, it lacks control over variables like weather fluctuations. The second method employs test chambers that facilitate accelerated aging under precisely monitored settings. This controlled approach is generally preferred, as it ensures consistency across different studies and reduces variability in the results.

The proposed protocol follows method A-1 of the ISO 4892-3 standard, using UV-A 340 nm lamps to simulate the global solar spectrum. Each testing cycle consists of 8 h of dry UV-A exposure, followed by 4 h of condensation in darkness. Samples are exposed for predetermined durations based on the experimental design. After completing each exposure period, the samples are buried in soil according to a specific protocol established by the research team and then compared to unaged control samples. It is critical that aged and virgin samples share the same initial characteristics, ensuring that only the impact of UV exposure is being measured.

## 4.4. Soil composition

### 4.4.1. Effect on degradation kinetics

Soil composition influences water retention capacity, pH, and the presence of microorganisms. For example, Deroiné et al. [85], showed that PHBHV degradation in foreshore sand took 600 days, much longer than in agricultural soils or others. The authors attributed this prolonged degradation period to the low water retention capacity of sand and a gradual decline in microbial populations. Studies by Hamarashid [86] et al. and Briasoulis [87] et al. have shown that soil texture plays an important role. Clay soils, for example, retain organic matter and nutrients more effectively than sandy soils, resulting in faster degradation of biodegradable polymers. Kim et al. [35] examined four different soils
and showed that PHB samples placed in activated sludge lost 68.9 % of their weight in 25 days, compared to 10.5 % in forest soil, all at 28 °C. This difference is due to fewer microorganisms and lower activity in forest soil than in activated sludges. These studies [29,35] suggest that some soils are more prone to rapidly degrading PHA than others, due to more intense microbial activity or greater diversity within communities. Other research [88,89] highlights that the microbial population varies with soil depth. The surface layer (0–15 cm) is the area with the highest microorganism density. Additionally, the soil's water retention capacity, which varies with soil composition, can strongly influence microorganism growth, as demonstrated by Boyandin et al. [42] Volova et al. [80] investigated the degradation of PHB in two distinct ecosystems: a tropical climate in southwestern India and a continental climate in eastern Siberia. These environments differ in temperature and humidity, but more notably in soil type and the associated microbial communities. In Siberia, the soil is classified as meadow-chernozem heavy loamy, whereas in India, it is red ferritic. The degradation process was slower in India due to lower moisture levels and reduced nutrient availability, both of which limited microbial activity. Microbial populations varied significantly between the two locations across all examined groups, including copiotrophic, prototrophic, oligotrophic bacteria, nitrogen-fixing bacteria, and mycelial fungi. In Siberia, copiotrophic bacteria reached (57.7 ± 8.6) × 10⁶ CFU/g, whereas in India, their abundance was markedly lower at (2.3 ± 0.8) × 10⁶ CFU/g. This disparity in microbial density translated into distinct degradation kinetics: the average degradation time in India was 126.4 days, nearly twice as long as in Siberia (64.8 days). This study underscores the significant influence of geographical conditions on extrinsic factors, which in turn affect microbial activity and ultimately determine the degradation kinetics of PHB.

### 4.4.2. Protocols

To investigate the influence of soil composition, at least two soil types should be compared: those with fine textures (e.g., clay loam, loam, silty clay loam) and those with coarse textures (e.g., silty loamy sand, sandy loam). Studies [86,87] have shown that degradation kinetics can vary significantly between these texture classes. For each soil type, several parameters must be characterized: soil textural class (acording to USDA or USCS standards), pH, total organic matter, total nitrogen, electrical conductivity, water-holding capacity (WHC), available ions (e.g., phosphorus, potassium, calcium, magnesium, sodium, sulfate, CaCO₃), and the abundance of bacterial and fungal colony-forming units (CFUs). In conducting experiments, a WHC of 80 % is recommended, along with a temperature of 28 °C.

## 5. Conclusion

Consumer societies are increasingly relying on plastics for a wide range of applications, including packaging, construction, and textiles. As a result, plastic production continues to rise [90]. These materials, with their diverse properties, have revolutionized numerous industries, making modern societies highly dependent on them. However, their end-of-life management remains a significant challenge. In response to plastic pollution, biodegradable polymers such as PLA or PHA have been developed as potential alternatives. Due to their bio-based nature and highly interesting biodegradability, PHA is at the heart of many efforts to expand their use. They are found in packaging or consumables, but PHA represents only a small share of biodegradable plastics, barely 5 %, with the market being dominated by PLA [91]. Many studies have been conducted to better understand the degradation of PHA in soils [92], in a somewhat disorganized manner. Besides standardized tests such as ISO, ASTM, and others, researchers have conducted experiments both in laboratories and outdoors, in environments that can differ radically from one another. As a result, there is now a vast database on PHA degradation in soils, but it is quite difficult to compare the results due to the lack of test standardization. The objective of this review was to elucidate

the role of both intrinsic and extrinsic parameters in influencing the degradation kinetics of PHA. For each parameter, optimized protocols has been proposed to enhance their study, addressing limitations observed in the current literature. For future publications on this topic, it will be essential to provide as much detailed information as possible. Many studies compare the effects of copolymers without reporting their crystallinity rate, while others fail to indicate mass loss for all samples or omit crucial parameters such as temperature and humidity. As the number of publications on PHA degradation in soils continues to grow, greater rigor in data reporting is necessary to ensure comparability between studies and to better understand the influence of each factor. A clearer understanding of intrinsic and extrinsic parameters will, in turn, facilitate the development of PHA with controlled or even programmable biodegradation. In parallel, addressing the management of PHA waste is now a pressing issue. While some PHA are certified for industrial or home composting, universal biodegradability does not exist [9]. Gaining deeper insight into the role of each factor would help waste treatment stakeholders optimize their processes to accelerate degradation, ultimately enabling the design of PHA with structures tailored to their intended end-of-life. Additionally, standardizing experimental protocols is a key area to explore. However, this should not merely replicate certification standards (ISO, ASTM, etc.), which often define conditions that do not reflect real environmental contexts. Instead, establishing common criteria - such as sample size, thickness, or water-holding capacity - would improve the comparability of studies while maintaining relevance to real-world conditions.

## CRediT authorship contribution statement

**Ludovic Dulac:** Writing – original draft, Conceptualization. **Mikaël Kedzierski:** Writing – review & editing, Writing – original draft, Project administration, Conceptualization. **Stéphane Bruzaud:** Writing – review & editing, Writing – original draft, Project administration, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

No data was used for the research described in the article.

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