Loading ratio is key for biodegradation PHBV-based materials in home- composting conditions

Loading ratio is key for biodegradation PHBV-based materials in home- composting conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Loading ratio is key for biodegradation PHBV-based materials in home- composting conditions Héloïse BAZART, Valérie GUILLARD, Nathalie GONTARD, Lucile CHATELLARD This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7491274/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In the context of growing plastic pollution in the packaging sector, biodegradable polymer alternatives present viable solutions, necessitating comprehensive understanding of biodegradation in accessible systems such as home composting. This research aimed to clarify the little-studied impact of PHBV-based material loading ratios on biodegradation kinetics in home composting environments, examining both film and shredded materials shape. The materials were tested according to three usage scenarios (high, medium and low) corresponding to material-to-compost ratios of 1/3, 1/30 and 1/50, under home composting conditions using ISO 14855-1 respirometry protocols. For both material forms, loading ratios has a stronger influence on the maximum biodegradation rate rather than on the ultimate biodegradation percentage. For shredded materials, the biodegradation rate linearly decreases with the increase of the ratio, whereas no direct correlation was shown for the material in film form. This suggests a material form effect on the polymer biodegradation rate regarding its loading ratio in the compost. For films, material residue persisting after one year suggested priming effects and revealed that PHBV biodegradation in our home composting setup occurs over extended timeframes beyond those captured by standard respirometry measurements. The study revealed that high loading ratios (1/3) induced substrate inhibition across all tested materials—a previously unreported phenomenon in PHBV biodegradability research. This inhibition extended the PHBV biodegradation time required to achieve 90% ultimate biodegradation from a maximum of 365 days prescribed by the standard to 578–613 days. These results establish the existence of a maximum optimal material loading ratio, above which biodegradation becomes suboptimal. Thanks to such a threshold, a careful consideration of material introduction rates would significantly benefit to the current waste management strategies and regulatory frameworks. Plastic biodegradation Loading ratio PHBV Home composting Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Plastics are widely used in all sectors of industry for decades, but their physical-chemical durability has led to environmental accumulation since most conventional petrochemicals plastics cannot be significantly degraded through biological mechanisms in natural or anthropogenic ecosystems (i.e. composting)[ 1 ]. A large amount of persistent plastic waste is generated and accumulates on Earth. In 2019, up to 353 million tonnes of plastic waste was generated, onto the 460 million tons of plastic consumed annually, among which 40% for agri-food applications [ 2 ]. According to OECD data (2022) [ 3 ], 49% of these plastics end up in landfill alongside food waste from household consumption worldwide in 2019. This plastic waste becomes a reservoir of very persistent polymers, which serves as the origin of all past, current and future releases of plastic particles into our environment. To address these environmental concerns, a range of biodegradable materials, such as cellulose-based material, are used as an ecological alternative. In parallel emerging biodegradable polymers, such as polyhydroxyalkanoates or poly (butylene succinate) (PHA, PBS, respectively…) offer thermoplastic properties that cellulose does not have, which, in addition to other functionalities, enable materials to be tailored for specific usage and post-usage scenarios [ 4 ]. A polymer is considered biodegradable if microorganisms can ultimately metabolise it into water, carbon dioxide (and/ or methane), and biomass, within a specific timeframe and under specific environmental conditions such as pH and temperature [ 5 ]. Biodegradation of polymers can occur either on their surface or in bulk, with other parameters involve in the biodegradation rate within thickness (submillimeter or large-format 3D materials), shape, or molar mass of the starting polymer and environmental parameters like pH and temperature [ 6 – 9 ]. A polymer can be biodegradable in natural conditions including soil, aqueous environments, and/or in anthropogenic system such as industrial and home composting at varying timeframes [ 6 ]. While industrial composting is a biowaste recovery operation, which can only be carried out with the required authorizations, home composting empowers individuals to transform their kitchen and garden waste into valuable compost for personal use. Home composting offers the benefit to lower waste management costs and impacts due to a reduced volume of waste that enters the formal waste management system, being collected from households and treated. Such practice is encouraged by the European Waste Framework Directive (Article 22 (2b) of Directive 2008/98/EC)[ 10 ]. Home compostability of packaging materials enable to divert such waste from landfilling or incineration and to avoid contamination of the compost. These products are defined and regulated by the Packaging and Packaging Waste Regulation (PPWR). The systematic assessment of this biodegradation behaviour relies primarily on international standards (ASTM or ISO). The NF EN 13432:2000 [ 11 ] is a specification standard stating that a polymer can be defined as biodegradable if its biodegradation rate reaches at least 90% within 6 months in industrial composting, or 12 months in home composting. Additionally, the NF EN ISO 14855-1 [ 12 ] is an analytical and testing standard widely used that describes a respirometry method to evaluate the biodegradability of a polymer mimicking composting conditions. This approach measures the amount of CO 2 produced by the microbial community composing mature compost, the so-called inoculum, when metabolising the available organic matter. The biodegradation of a material is then estimated by comparing the amount of CO 2 produced in the presence and absence of the material in the environment. The respirometry method can be implemented under both home and industrial composting conditions, using different temperature ranges (25 ± 2°C and 55 ± 2°C, respectively). This standard specifies that the tested materials must be reduced to pieces smaller than 2 × 2 cm and that a given amount of these materials must be then added to the inoculum, equalling 1/6 of the dry weight (referred to as the material-to-inoculum ratio). Elevated temperatures reduce the time required for experiments and could explain why so many biodegradation studies have been conducted in industrial composting systems. Among the booming alternative polymers, polyhydroxyalkanoates (PHA), and in particular the poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBV), which escape the controversies surrounding non-biodegradability and non-recyclability [ 13 , 14 ], exhibit comparable thermoplasticity, tensile strength, and rigidity to conventional petrochemical polymers such as polypropylene (PP) [ 15 – 17 ]. They can be used as plastic substitutes for many applications [ 18 ]. PHBV is biodegradable in the natural environment [ 6 ]. Its use in composite formulations with cellulose, ligno-cellulosic fibers/residue has been found promising, decreasing overall costs while tailoring functional, such as barrier, properties and preserving the biodegradability [ 19 – 21 ]. The biodegradation of PHBV and its composites under aerobic conditions has been widely studied, particularly under industrial composting conditions as reviewed by [ 22 ]. However, only few studies focus on home composting environments [ 6 ]. Home composting represents a particularly relevant scenario to simplify their organic recycling but yet remains significantly under-researched compared to industrial composting applications. Understanding the mechanisms behind the biodegradation in home-composting conditions is crucial for optimising waste management strategies and end-of-life outcomes. Most published biodegradation studies reveal significant deviations from standard protocols, particularly regarding the material-to-inoculum ratio (loading ratio) which frequently differs from the recommended 1/6 without explicit justification. Studies have employed dramatically different ratios ranging from 1/55 to 1/15, with intermediate values of 1/30 [ 23 – 25 ]. These modifications are presumably implemented to accelerate biodegradation processes and obtain results more rapidly, as lower material quantities relative to inoculum are expected to enhance degradation kinetics. The impact of these ratio modifications on biodegradation timelines is substantial. While Muniyasami et al. (2016) [ 26 ], adhering to the standard 1/6 ratio, achieved 90% biodegradation in approximately 200 days, studies using reduced ratios demonstrated dramatically accelerated degradation: 65 days with 1/55 ratio [ 27 ], 95 days with 1/30 ratio [ 25 ], and 91 days with 1/15 ratio [ 24 ]. However, the effect of the loading ratio on the ultimate biodegradation value and biodegradation rate has never been quantified yet. Alongside ratio variations, material shape diversity also contributes to methodological inconsistencies. The ISO standard only specifies a maximum material size of 2 × 2 cm, suggesting the possibility to use wild range of sizes bellow this value and material shape. In the literature, the authors can use discs [ 28 ], or films [ 24 , 25 ]. Smaller particles accelerate biodegradation by shortening the fragmentation phase and increasing surface area for microbial contact which facilitates biofilm formation [ 29 ]. However, it remains unclear whether the biodegradation kinetic measured on a shredded material can be extrapolated to the same intact material such as film for instance. Additionally, the qualitative and quantitative interaction between particle size and material-to-inoculum ratio --two important factors– has not been yet investigated. In this context, this study aimed to investigate loading ratio effects on the biodegradation kinetics of PHBV-based material while controlling final shape of the material added in the compost. The research focuses on establishing quantitative relationships between various material-to-inoculum ratios (1/3, 1/30, 1/50), two material forms (films versus shredded), and biodegradation kinetic parameters such as ultimate biodegradation and biodegradation rate in home composting conditions for cellulose, Kraft paper, PHBV, and PHBV-cellulose composite polymer. 2. Materials and methods 2.1. Materials The study was conducted on PHBV materials (PHI003, 3% HV, Natureplast, Fr). Materials biodegradation was tested either with or without 20% (w/w) cellulose microfiber content (Arbocel grade B00, Rosenberg, Germany), and in two physical shapes: 2 x 2 cm pieces and powder form (> 1 mm). For comparison purposes, the cellulose microfibers and Kraft paper (2 x 2 cm, from biowaste collection bags, Sumus Italia, srl) served as positive biodegradable controls, while PET, both in 2 x 2 cm pieces and shredded shape (RAMAPET N1S, Polymix, France) was used as a negative non-biodegradable control. The PHBV and PHBV-cellulose composite films were produced by the technical center IPC of Alençon (France). The commercial PHBV (PHI003) was first mixed with boron nitride (0.5%) as a nucleating agent for all PHBV-based polymers, and with 20% w/w of cellulose fibers (B00) for the composite. The resulting powder-based mixture was then extruded using a COPERION twin-screw co-rotating extruder (ZSK 32 MC), with a screw diameter of 32 mm and a length of 1260 mm (L/D ratio of 40). The pellets produced were subsequently extruded using a Collin E45E single-screw extruder to obtain films with a flat die. The cellulose microfibers (B00) were 99.5% pure, with a bulk density of 150–185 g/l (as per DIN EN ISO 60), a skeletal density of 1.56 g/cm³, an average thickness of 15 µm, and an average length of 18 µm, according to the supplier. Kraft paper samples were pieces of biowaste collection bags that are distributed to Montpellier citizens (France) in the context of the biowaste management plan. These bags are made from Havane Kraft paper, water-based glue, and carbon-based ink (according to the supplier). The bags are biodegradable as certified by the Consorzio Italiano Compostatori (CIC), in compliance with the UNI EN 13432:2002 standard. The poly (ethylene terephthalate) (PET) films were produced by the IPC technical center using the same method described above for PHBV-based materials. PHBV, PHBV-cellulose, PET and Kraft paper films were cut into pieces of 2 x 2 cm for film shape biodegradation studies. For the shredded shape, pieces of 1 x 10 cm of PHBV, PHBV-cellulose and PET were shredded into powder using an IKA210 grinder (Germany) at high speed for a maximum of 10 seconds to prevent overheating. For this shape, the positive control used was already in powder form (cellulose B00). 2.2. Respirometry test : The biodegradability of the materials was assessed through respirometry tests following the methodology described by Bonnenfant et al., 2023 [ 16 ], which is based on the NF EN ISO 14855-1 standard [ 12 ]. The tests were conducted at a temperature simulating aerobic home compost conditions (28 ± 2°C). The microbial consortia (inoculum) came from mature compost obtained through the industrial composting of biowaste and green waste at the Aspiran industrial composting platform (France). Upon reception, the mature compost was screened to < 2 mm with a RITEC sifter. The pH of the compost was measured with a pH meter Cyberscan 510, using 5 g of compost dispersed in 5 times its volume of distilled water. The mixture was stirred for 30 min, and the pH was immediately measured in triplicate. The proportion of organic matter was characterized using an ash oven (Nabertherm, GmbH Bahnhofstr 20, 28865 Lilienthal/Bremen) at 550°C for 4 h. The water content of the compost was adjusted to 55% w/w using distilled water. A C/N ratio of 12 was determined for the compost by the composting platform. The respirometry tests were conducted in four replicates in 1 L hermetic glass jars (Le Parfait, France) containing three flasks of 60 mL each. Each experimental setup consisted of three distinct components designed to create optimal conditions for biodegradation monitoring. The first 60 mL glass flask contained the biodegradation medium, made of sieved mature compost adjusted to 55% water content (w/w), and one studied material. Three material-to-inoculum ratios of material to compost masses (i.e. substrate/inoculum) were studied for PHBV, PHBV with 20% cellulose composite, cellulose microfibers, and Kraft paper. Three material-to-inoculum ratios were tested: 1/3 and 1/30 simulating home composting where 100% and 10% respectively of food packaging are made of the tested material and collected with biowaste, and 1/50 simulating the use of the materials tested only to make organic waste bags with a capacity of 1.5 kg each. The amount of material added to each condition is described in Table 1 . As the mass of the film-shaped material is not uniform, it varies between material. The total mass of compost was adjusted to maintain consistent experimental conditions while varying the material-to-inoculum ratios. PET was introduced according to a material-to-inoculum ratio of 1/3 only to limit the space required in the incubator. For both material shapes (films, and shredded materials), four reactors containing only mature compost without any test material were used as blanks to measure the CO 2 produced from the biodegradation of the compost’s residual organic matter. Table 1 Theorical mass addition (g) of shredded or film material for each material-to-inoculum ratio Ratio 1:3 1:30 1:50 shredded film shredded film shredded film Biodegradable controls (g) 1 1.6 0.1 0.17 0.06 0.12 PHBV (g) 1.6 0.18 0.1 PHBV + 20% cellulose (g) 1.6 0.17 0.16 The second flask served as a CO 2 capture system and contained a solution of 0.2 M NaOH of precisely known volume and concentration to capture the gaseous CO 2 produced during the biodegradation of the organic carbon, according to Eq. 1. The NaOH concentration and volume were adjusted during the experiment to accommodate the rate of CO 2 production. CO 2 + 2NaOH → Na 2 CO 3 + H 2 O Eq. 1 The third 60 mL flask contained 10 mL of distilled water to maintain saturated relative humidity (RH) in the 1 L jar and preserve the compost water content. The reactors were incubated at 28 ± 2°C in IPP 110 + and IPP500 incubators (Memmert) and TS 606/4-i (WTW). All reactors were opened regularly to renew the oxygen in the jars, and to exchange the NaOH trap with a new one. The CO 2 captured by the NaOH trap was determined by titration. First, 5 mL of barium chloride solution (BaCl 2 , 20% w/w) was added to precipitate the Na 2 CO 3 according to Eq. 2. The excess of NaOH was then titrated with HCl solution at the same concentration, using thymolphthalein (0.1% in ethanol) as a colour indicator according to Eq. 3. Na 2 CO 3 + BaCl 2 → 2 NaCl + BaCO 3 Eq. 2 NaOH + HCl → NaCl + H 2 O Eq. 3 The quantity of CO 2 produced between two measurement intervals was calculated according to Eq. 4, where V NaOH is the volume of NaOH used (mL), C NaOH is the NaOH concentration (mol. L-1), VeqHCL is the equivalence volume of HCl, and C HCl is its concentration in mol. L-1. \(\:n\:CO2\:\left(mmol\right)=\:\frac{\left(\right({V}_{NaOH}\:*\:{C}_{NaOH})\:-({Veq}_{HCl}*{C}_{HCl}\left)\right)}{2}\) Eq. 4 The biodegradation rate %B was calculated using Eq. 5, where “CO 2 material” and “CO 2 blank” are the mass of CO 2 (mg) produced in the presence or in the absence of material, respectively, and “CO 2 theoretical” is the mass of CO 2 (mg) theoretically released for 100% of material biodegradation. \(\:\%B\:=\frac{\:(CO2\:material\:-\:CO2\:blank)}{{CO}_{2}\:theoretical}\:*\:100\) Eq. 5 All results were expressed as % biodegradation per g of compost. The experiment was monitored for about 12 months, and each condition was stopped earlier if a plateau of biodegradation was reached beforehand. For each kinetic, the exponential phase from the biodegradation curve was manually selected with an R 2 ≧ 0.98 and the slope of the corresponding linear regression line was calculated to determine the maximal biodegradation rate. For most conditions, a modelled curve could be fitted to the experimental data using the modified Gompertz model implemented with the “nlsLM” function from the R package “minpack.lm” (version 1.2-4) [ 30 ] in RStudio software (R version 4.4.2 (2024-10-31) [ 31 ], using Eq. 6, where y0 is the initial biodegradation value at the beginning of the experiment (in this case, 0), ymax is the ultimate biodegradation percentage reached, k is the maximal biodegradation rate previously determined, lag is the time corresponding to the lag phase, and x is the time in days. \(\:y=\:y0\:+\:\frac{\left(ymax\:-\:y0\right)\:*\:exp(\:-\:exp(k\:*\:(lag\:-\:x))}{\left(ymax\:-\:y0\right)\:+\:1)}\) Eq. 6 This model was selected because its parameters appropriately fit the experimental curve profile, including the lag phase, exponential growth phase, and final plateau. The associated curves are provided in supplementary material (section 2 , Figure S8-S13). For conditions that did not reach 90% biodegradation within the experimental timeframe, the model was used to predict the time (in days) required to reach 90%. 2.3. Polymers characteristics: Each material was characterized through molecular weight determination (SEC), carbon content analysis (CHNS-O), and thermogravimetric analysis (TGA). Additional properties including density, and particle size are summarized in Table 2 . Molecular weight (Mw) was determined by size exclusion chromatography (SEC) performed by the Technopolym platform (Toulouse, France). Samples were dissolved in chloroform at 3–5 mg. mL-1 and heated at 70°C for 1–5 h until complete dissolution. Solutions were filtered through 0.45 µm nylon membranes before analysis. SEC analysis was conducted using a UHPLC system (Ultimate 3000, Thermoscientific) equipped with an Agilent PL gel 5 µm Mixed C column. Injection volume was 50 µL with an elution flow rate of 1 mL.min-1. Detection was performed using a triple detector system: differential refractometer (Optilab Rex Wyatt, 35°C), three-angle static light scattering detector (TREOS Wyatt, 658 nm), and UV detector (Shimadzu SPD-M20A, 254 nm). Carbon content of each material (cellulose, PHBV-based materials, and Kraft paper) was determined to calculate the theoretical maximum CO 2 production for biodegradation rate calculations. The analysis was performed by the Laboratoire de Mesure Physique (LMP, Université de Montpellier, France), using a CHNS-O elemental analyser and a Sartorius Cubis Advanced MCA (precision 0,1 µg, max 2,1 g). Samples in tin capsules were loaded into a 120-position autosampler and transferred to a helium-purged inert chamber. Combustion occurred at 1150°C in a catalytic furnace, followed by reduction over hot copper at 850°C. Gases were separated using an Elementar TPD column, and carbon as detected by thermal conductivity detector (TCD). Thermogravimetric analysis (TGA) was performed using a Mettler TGA2 apparatus (Schwerzebbbach, Switzerland) with an XP5U balance (0.0001 mg precision) to determine the maximum degradation temperature (Tdeg). Samples (5–10 mg) were analysed in triplicate under nitrogen flow (50 mL.min − 1) with heating from 25°C to 600°C à 10°C.min-1. Tdeg was determined as the temperature corresponding to the maximum of the peaks obtained from the first derivative of their TGA curve (i.e. weight loss). The particle size distribution of the resulting materials was evaluated with a laser granulometer (LS 13320 XR, Beckman Coulter) Table 2 Physico-chemical properties of the polymers used in the study. Material Cellulose Kraft paper PHBV PHBV + 20% cellulose PET shredded film shredded film shredded film Carbon content (%) 41.45 ± 0.02 40.558 ± 0.004 55.65 ± 0.06 53.24 ± 0.02 62.17 ± 0.03 Molecular weight (kDa) 453.40 X 311.3 226.55 76.25 Thickness (µm) X 100 ± 6 X 220 ± 21 X 365 ± 34 X 217 ± 14 Granulometry (µm) 58.2 ± 0.4 X 579 ± 5 X 654 ± 31 X 725 ± 38 X Tdeg (°C) 345.7 ± 7.02 X 290 ± 6.2 285.2 ± 2.2 306.9 ± 39.2 X 2.4. Statistical analysis: Statistical analyses were performed using RStudio software. Comparison between material-to-inoculum ratios and material shapes were performed through analysis of variance (ANOVA) after verifying normal distribution (Shapiro-Wilk test) and homogeneity of variances (Bartlett test, or Levene test when Bartlett test assumptions were not met, “car” R package, version 3.1-3) [ 32 ]. Tukey’s post hoc tests were used to compare means when ANOVA showed significant differences. For data with non-normal distribution, Kruskal-Wallis tests were applied, followed by log transformation to achieve normality when necessary. Statistical significance was set at p < 0.05. Graphics were generated using “ggplot2” R package [ 33 ]. 3. Results Biodegradation experiments were conducted in respirometry tests for cellulose microfibers, Kraft paper, PHBV and PHBV-cellulose composite polymer in both film and shredded shape under home composting conditions (28 ± 2°C). All experiments continued until biodegradation plateaus were reached, except for the highest ratio (1/3), for which biodegradation did not reach a plateau within the experimental timeframe for any material tested, including the cellulose control. Figure 1 shows typical biodegradation curves for shredded PHBV across different material-to-inoculum ratios, demonstrating the typical sigmoidal kinetics observed throughout the study. Figures 2 and 3 present comparative results for shredded and film material, respectively. Ultimate biodegradation percentages and time (in days) required to reach this maximum are shown in panels (a), while maximum biodegradation rates calculated from the exponential growth phase are display in panels (b). Complete kinetic profiles for all materials in both shredded and film shape are provided in supplementary material (Figure S1-S6). Except for cellulose microfibers at the 1/3 ratio, all materials (cellulose microfibers, PHBV, and PHBV-cellulose) achieved more than 90% biodegradation within 12 months, confirming their complete biodegradability according to the NF EN ISO 14855-1 standard (Fig. 2 a). The negative control (PET) showed no measurable biodegradation as anticipated (Figure S7). Cellulose microfibers at the highest material-to-inoculum ratio (1/3) reached only 83 ± 6% of biodegradation after the twelve-month experimental period, while they reached ultimate biodegradation more rapidly at 1/30 and 1/50 ratios, about 114 ± 5% and 111 ± 17% for both ratio after 168 ± 4 days and 150 ± 21 days, respectively. PHBV and PHBV-based composite with 20% cellulose biodegraded as rapidly as for the positive control at the 1/3 ratio (369 days) with an ultimate biodegradation percentage of 90 ± 3%. The 1/50 ratio yielded similar maximum degradation time for both materials: 158 ± 8 days for PHBV and 163 ± 0 days for PHBV cellulose composite. However, PHBV reached its maximum of biodegradation more rapidly for 1/30 ratio (166 ± 5 days) compared to the PHBV cellulose composite that required 221 ± 21 days. This suggests that cellulose addition may slow biodegradation kinetics when blended with PHBV at this specific ratio. For 1/30 and 1/50 ratios, maximum biodegradation percentages consistently exceeded 100%. Cellulose microfibers showed 114 ± 5% at the 1/30 ratio, and 111 ± 17% at the 1/50 ratio. PHBV reached 131 ± 2% at the 1/30 ratio and 100 ± 22% at the 1/50 ratio. The PHBV-cellulose composite achieved 124 ± 25% at the 1/30 ratio and 109 ± 20% at the 1/50 ratio. Biodegradation rates increased with decreasing material-to-inoculum ratios, as clearly demonstrated by cellulose results (Fig. 2 b): rates of 0.41 ± 0.01%/d at the 1/3 ratio, 3.46 ± 0.01%/d at the 1/30 ratio and 5.20 ± 0.01%/d at the 1/50 ratio. This trend was confirmed for PHBV and PHBV-cellulose composite, which showed similar increases between the 1/3 and 1/30 ratios. However, biodegradation rates were the same between the 1/30 and 1/50 ratios, as confirmed by statistical analysis. For PHBV, degradation rates reached 1.39 ± 0.01%/d and 1.58 ± 0.01%/d for the 1/30 and 1/50 ratios, respectively. For the PHBV-cellulose composite, biodegradation rates were 1.46 ± 0.01%/d for the 1/30 ratio and 1.77 ± 0.01%/d for the 1/50 ratio. All values are summarised in Table 3 . Overall, the highest biodegradation rates were achieved by the positive control (cellulose), regardless of the ratio tested, with rates approximately 3–4 times higher than those of PHBV-based materials, even at the highest material-to-inoculum ratio (1/3). Analysis of ultimate biodegradation, time to reach ultimate biodegradation, and maximum biodegradation rates revealed that material-to-inoculum ratios primarily impact reaction kinetics rather than final extent or time to completion in case of shredded material, as confirmed by statistical analysis. The only exception was PHBV at the 1/30 ratio, which showed a higher ultimate biodegradation percentage despite a similar rate compared to the 1/50 ratio. The PHBV-cellulose composite exhibited statistically similar ultimate biodegradation percentages across all three ratios, suggesting its biodegradation was less affected by the material/inoculum ratios compared to the other materials studied. The results also highlight the absence of correlation between maximum biodegradation rate and ultimate biodegradation extent. Consistent with the approach used for shredded materials, the experimental period for films lasted up to 370 days, with tests terminated when biodegradation rates stabilized. During the 370-day incubation period, only the PHBV-cellulose composite at ratios of 1/30 (93 ± 8%) and 1/50 (108 ± 15%), and PHBV at the 1/30 ratio (118 ± 16%), achieved the 90% biodegradation threshold required by NF EN 13432:2000 standard [ 11 ], thus meeting the compostability requirements within one year. At the highest material-to-inoculum ratio (1/3), Kraft paper, PHBV, and the PHBV-cellulose composite reached biodegradation levels of 66 ± 2%, 78 ± 12%, and 76 ± 7%, respectively, after 370 days of incubation. At the 1/50 ratio, PHBV and Kraft paper achieved biodegradation levels of 85 ± 12% and 73 ± 9%, respectively, after 266 ± 13 days and 258 ± 14 days. At the 1/30 ratio, Kraft paper achieved 89 ± 7% in 242 ± 0 days. Notably, visible fragments of PHBV and the PHBV-cellulose composite that were initially introduced at the 1/30 ratio remained in the reactors at the end of the experiment, despite reaching 100% biodegradation according to CO₂ measurements, as illustrated in Fig. 4 . Comparatively to shredded material, for films conditions, the maximum biodegradation rate tends to increase as the material/inoculum ratio decreases (i.e. when less material is present in the inoculum). Regarding maximum biodegradation rates, two distinct patterns emerged depending on material type (Fig. 3 b). For the PHBV-cellulose composite, a negative linear relationship was observed between the maximum biodegradation rate and the material-to-inoculum ratio: as the material/inoculum ratio decreased, the maximum biodegradation rate increased. Specifically, rates were measured at 1.01 ± 0.01%/d, 0.45 ± 0.01%/d and 0.35 ± 0.01%/d for ratios of 1/50, 1/30, and 1/3, respectively. In contrast, both PHBV and Kraft paper exhibited non-linear responses with the lower ratios (1/30 and 1/50) showing higher biodegradation rates than the higher ratio of 1/3. For PHBV, maximum rates were 0.55 ± 0.01%/d, 0.71 ± 0.01%/d, and 0.297 ± 0.001%/d for the ratios of 1/50, 1/30, and 1/3, respectively. For Kraft paper, maximum rates were 2.13 ± 0.01%/d, for 1/50 and 1/30 ratios but decreased dramatically at 0.32 ± 0.01%/d at the highest material concentration (Table 3 ). This represents the most substantial difference observed, with Kraft paper exhibiting significantly higher biodegradation rates than both PHBV materials at 1/30 and 1/50 ratios. Notably, the 1/3 ratios consistently reduced biodegradation rates across all materials, meaning that independently of the material shape, the 1/3 ratios slow the biodegradation rate similarly. For film materials, maximum biodegradation rates correlated with ultimate biodegradation percentages: faster biodegradation rates were associated with higher maximum biodegradation percentages. To better capture the influence of material shape on biodegradation rates, Table 3 compares all the tested materials and ratios using a 'shape effect', calculating as the relative increase in biodegradation rate between the shredded and film shapes of the same material. As expected, shredded materials exhibited significantly faster biodegradation rates under most conditions, with rates ranging from 1.6 to 3.2 times higher at material-to-inoculum ratios of 1/50 and 1/30. However, at the highest material concentration (1/3 ratio), this advantage was reduced: maximum biodegradation rates were either comparable for PHBV and PHBV-cellulose composite, or 1.3 times faster for Kraft paper. Table 3 Comparison of maximum biodegradation rate for each shape materials for the three tested material-to-inoculum ratio. The average and standard deviations were calculated based on 3 replicates. The shape effect indicates the proportional increase of the biodegradation rate between shredded and film shape (Rate shredded / rate film ). Standard deviations were deliberately rounded up to a hundredth to ensure robust conclusions and are indicated by an asterisk. Material Shape 1/50 ratio 1/30 ratio 1/3 ratio Rate (%/d) Shape effect Rate (%/d) Shape effect Rate (%/d) Shape effect Biodegradable control Cellulose powder shredded 5.20 ± 0.01* 2.4 ± 0.0 3.46 ± 0.01* 1.6 ± 0.0 0.41 ± 0.01* 1.3 ± 0.1 Kraft paper film 2.13 ± 0.01* 2.13 ± 0.01* 0.32 ± 0.01* Tested material PHBV shredded 1.58 ± 0.01* 2.9 ± 0.1 1.39 ± 0.01* 2.0 ± 0.0 0.27 ± 0.01* 0.9 ± 0.0 film 0.55 ± 0.01* 0.71 ± 0.01* 0.297 ± 0.001* PHBV + 20% cellulose shredded 1.77 ± 0.01* 1.8 ± 0.0 1.46 ± 0.01* 3.2 + 0.1 0.31 ± 0.01* 0.9 ± 0.0 film 1.01 ± 0.01* 0.45 ± 0.01* 0.35 ± 0.01* *: standard deviation rounded up to a hundredth 4. DISCUSSION This study contributes to our understanding of the relationships between the material composition and shape, their loading rate in home composting environment, and their biodegradation performance (ultimate biodegradation percentage and maximum biodegradation rate). 4.1. Material-to-inoculum ratio influences the biodegradation kinetics In this study, we systematically evaluated three distinct material loading ratios: 1/3, 1/30, and 1/50, each representing different realistic loading scenarios. The 1/3 ratio simulates an extreme case where 100% of single-use plastic packaging is replaced by biodegradable alternatives and fully collected through biowaste systems in France (see supplementary material, section 4 ). The 1/30 ratio represents one-tenth of this scenario, to evaluate linear relationship between ratios, while the 1/50 ratio models more realistic conditions, such as 1.5 kg of apples packaged in biodegradable plastic bags typically available in French retail markets. Our results demonstrate that material-to-inoculum ratios fundamentally control biodegradation kinetics rather than ultimate biodegradability, and this last one seems more impacted by the material shape. Each material exhibited optimal loading ratios, particularly pronounced in film configurations: ratios of 1/30 or below for PHBV and Kraft paper, and 1/50 or below for PHBV-cellulose composites. Most significantly, the highest ratio (1/3) induced substrate inhibition phenomenon well-established in microbiology but never demonstrated before in material biodegradability studies. Under these high-loading conditions, extrapolated biodegradation curves predicted extended degradation periods of 578 ± 238 days for PHBV films and 613 ± 169 days for PHBV-cellulose composite films to achieve > 90% biodegradation as the ISO 13432-2 [ 11 ] standard requires (see supplementary material, Figure S14). It let suppose that at optimal ratios, compost microbial community are able to process all available substrates including material while excessive material quantities (1/3 ratio) either saturate inoculum capacity or create inhibitory conditions. It lets suppose that at optimal ratios, compost microbial community are able to process all available substrates including material while excessive material quantities (1/3 ratio) either saturate inoculum capacity or create inhibitory conditions. This loading-dependent performance was consistently observed across both material shapes, though the magnitude of shape effects varied with ratio conditions. Under such high loading ratios, material fragments may remain in the mature compost, but they will likely continue to biodegrade once the compost is used and applied to the field, when the ratios will consequently decrease. Given that the ISO standard recommends a 1/6 ratio and our results show limitations at 1/3 ratios, adhering to recommended guidelines is crucial for proper biodegradation of these materials. The inhibitory effects observed at elevated ratios emphasize the importance of maintaining optimal conditions in domestic composting systems. However, scaling these laboratory findings to home composting presents challenges, as domestic composters have limited control over the quantity of biodegradable plastics added to their bins. This necessitates awareness and moderation when disposing of biodegradable materials in home compost. Fortunately, exceeding optimal ratios would primarily extend decomposition times rather than prevent biodegradation entirely. Future research should validate these findings in real domestic composting conditions to provide practical guidelines for home composters. 4.2. Material shape effects within the context of loading ratio optimization While material-to-inoculum ratio serves as the primary determinant of biodegradation kinetics, material shape provides secondary but significant acceleration effects, particularly at intermediate loading ratios. Material particle size and biodegradation rates are interconnected through underlying mechanisms that become most apparent under optimal loading conditions. Previous studies demonstrate that reducing polymer granulometry enhances enzymatic hydrolysis and increases biodegradation rates, as observed in aqueous aerobic systems [ 34 , 35 ] and in soil [ 8 ], though this finding was not confirmed by Yang et al. (2005) [ 36 ] in industrial composting, highlighting behavioural diversity across different studies. The fragmentation of the material (film to shredded film shape) increased the surface area exposure, enabling enhanced microbial accessibility and enzymatic contact [ 29 ]. This enhancement stems from bypassing initial biodegradation stages devoted to material fragmentation under physicochemical phenomena, especially mechanical stress [ 37 ]. However, the magnitude of these shape effects diminished under high loading conditions, particularly at the highest ratio, where substrate inhibition overshadowed surface area advantages. When biodegradable materials are reduced to sufficiently fine particles, and introduced at appropriate ratios, microorganisms can efficiently consume them within optimal composting timeframes. While these results suggest potential benefits of pre-treatment processes for post-use biodegradable materials, such processes require specific infrastructure and are difficult to implement and expensive [ 34 ]. For PHBV-cellulose composites, the interaction between loading ratio and shape became particularly complex. Mechanical shredding may release cellulose fractions separately from PHBV, and given cellulose's higher degradation rate, this separation potentially accelerates overall composite biodegradation, but only under appropriate loading conditions where microbial communities can effectively process the increased substrate availability. The "shape effect" (Table 3 ) quantifies material shape impacts on biodegradation rates for identical materials (PHBV or PHBV + 20 cellulose) under specific loading ratios in home composting. This provides realistic approaches for evaluating plastic waste fate and establishing PHBV material guidelines for composting applications. When biodegradable materials are reduced to sufficiently fine particles, and introduced at appropriate ratios, microorganisms can efficiently consume them within optimal composting timeframes. Finally, this significant influence of material shape on biodegradation rates highlights the importance of using appropriate control shapes when applying NF EN ISO 14855-1 standard [ 12 ]. Control materials—paper sheets, cellulose papers, or starch films—should match tested polymer physical shapes and be evaluated under comparable loading ratios to ensure valid respirometry comparisons. 4.3. Limitations of respirometry analysis in presence of priming effect Assessment accuracy appeared to be influenced by specific material-to-inoculum ratio conditions. Nearly all shredded material conditions and two film conditions exhibited biodegradation percentages exceeding 100%, indicating CO₂ production greater than theoretically expected based on tested material carbon content in respirometry assays. This phenomenon was most evident at intermediate ratios, where optimal loading conditions appeared to maximize the "priming effect". The priming effect, first identified by Löhnis (1926) [ 38 ], refers to CO₂ overproduction from adding easily biodegradable organic matter to soil [ 39 ] or mature compost [ 40 ]. This addition stimulates microbial activity and enhances recalcitrant organic matter degradation, producing additional CO₂. Notably, for PHBV and PHBV-cellulose composite films at the 1/30 ratio, film fragments remained in compost despite an ultimate biodegradation percentage greater than 100% (Fig. 4 ), strongly supporting the priming effect theory and demonstrating the disconnect between CO₂ production and actual material disappearance. The magnitude of CO₂ overproduction varied systematically with material-to-inoculum ratios. Under high loading conditions, substrate inhibition appeared to suppress both material degradation and priming effects, resulting in more conservative CO₂ measurements. Conversely, optimal ratios maximized both material biodegradation and compost organic matter stimulation, leading to the highest CO₂ overproduction. Under shredded conditions across all ratios, visual identification of remaining materials became nearly impossible due to particle size, making material decomposition status assessment challenging regardless of loading conditions. In the literature, similar CO₂ overproduction occurred during biodegradation of diverse materials, including biodegradable (PHA, PBS) and hydrolytically degradable polymer (poly (lactic acid)) [ 41 ] and cellulose/starch [ 42 ]. This CO₂ overproduction may stem from additional metabolic processes within mature compost, particularly nitrogen cycle processes [ 41 ]. Since tested polymers lack nitrogen, microorganisms must biodegrade native compost organic matter to acquire nitrogen, simultaneously inducing CO₂ production through surrounding organic matter degradation [ 43 ]. Additionally, considering that compost remained unstirred for one-year, anaerobic pockets could form near film materials due to their barrier properties, creating favourable conditions for anaerobic community proliferation and CO₂ production through denitrification. These possible metabolic process accumulations suggest needs for more precise CO 2 quantification during our respirometry tests. However, in this study, distinguishing CO₂ from material versus compost biodegradation was impossible. Moreover, CO 2 monitoring provides indirect biodegradation quantification that may introduce measurement errors. The standard addresses this limitation through 90% thresholds accounting for potential errors. To address indirect quantification issues and improve respirometry accuracy, the literature suggests several differentiation approaches: ¹⁴C isotope labelling for CO 2 distribution quantification [ 44 ], biomass growth quantification through insoluble protein content measurement [ 45 ]. The NF EN ISO 14855-1 standard specifies that vermiculite addition could also limit material CO 2 overproduction, but, unfortunately, it will not prove that all CO 2 derives solely from material. Respirometry approaches such as those presented in the current study and by NF EN ISO 14855-1 focus on the use of mature compost with consistent characteristics (C/N ratio, pH, organic matter content). However, extrapolating results to broader applications like home composting presents challenges when materials must be processed alongside easily biodegradable sources such as biowaste. A key question remains whether microorganisms preferentially degrade biowaste or biodegradable materials. Conclusion and perspective This research investigated the critical role of material-to-inoculum ratio in biodegradation kinetics under home composting conditions using ISO 14855-1 respirometry protocols. The material-to-inoculum ratio emerged as the primary determinant of biodegradation performance, with each material exhibiting distinct optimal loading ranges: 1/50 or below for PHBV-cellulose composites, and 1/30 to 1/50 for PHBV and Kraft paper films. Most significantly, this study revealed that high loading ratios (1/3) induce substrate inhibition—a novel phenomenon in polymer biodegradability research—dramatically extending degradation times to 578–613 days for PHBV-based film materials. This loading-dependent behaviour was consistently observed across both material shapes, though shape effects provided secondary acceleration under optimal ratio conditions. Respirometry data showed CO2 levels exceeding theoretical values through priming effects within the composting environment, highlighting the importance of further research into these ratios and priming mechanisms to refine biodegradation quantification approaches. These findings emphasize the critical need for controlled material loading in composting applications, improved assessment methodologies for reliable polymer biodegradation evaluation, and careful consideration of material introduction rates in waste management policies. Reassuringly, excess ratios would predominantly delay decomposition timelines without blocking the biodegradation pathway entirely. During the preparation of this work the authors used Claude AI (Anthropic, 2024) to improve the readability and language of the manuscript. After using this tool/service, the authors reviewed and edited the content as needed and takes full responsibility for the content of the published article. 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. The authors thank the PLANET facility ( https://doi.org/10.15454/1.5572338990609338E12 ) run by the IATE joint research unit for providing process experiment supports: granulometer Beckman Coulter, Nabertherm ash oven and RITEC sifter. Declarations Funding This work was funded by the French National Research Agency (ANR) under the ANR-21-CE43 project BioCyPlast. Author Contribution H.B: Conceptualization, formal analysis, investigation, methodology, writing - original draftV.G: Conceptualization, Supervision, writing - review & editingN.G: Conceptualization, Project administration, Supervision, writing - review & editingL.C: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Validation, Writing - review & editing Data Availability Data are provided at this link: https://entrepot.recherche.data.gouv.fr/privateurl.xhtml?token=ff8b2c30-670c-4f29-9902-faf4babc9d1b References Ritchie, Hannah, Samborska, Veronika, Roser, Max. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7491274","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512404054,"identity":"1b29be6b-42be-4aea-986a-7a01ba15f42c","order_by":0,"name":"Héloïse BAZART","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYBACAwbmBoYEMAMMbBLAVEIBPi2MyFoS0hIY2EC0AQEtDAgthyFaGPBoMWdvbHzw4I8NkHH84efKH+fz+OW7Ez88MGCQ5xc7gFWLZc/BZoPEtjQgIyFZ8kzC7WLJNt7NEkCHGc6cnYDdYTcS2yQSGw4zGBxIOCDZkHA7ccMx3g0gLQkGt3Fouf+wTSLhz38Gg/MPm382JJwDadn8A6+WG4xALWwHgIxkNqAtB0BatuG35UwiyC/JPJYznrFZNqQlJ85sy91mkWAggdsvxw8ffPjjj52cOX/645sNNnaJ/cxnN9/8UWEjzy+NXQsM8KALSOBVPgpGwSgYBaMAPwAABtZjyVLUHtkAAAAASUVORK5CYII=","orcid":"","institution":"UMR IATE, Université de Montpellier","correspondingAuthor":true,"prefix":"","firstName":"Héloïse","middleName":"","lastName":"BAZART","suffix":""},{"id":512404057,"identity":"98dec8cf-24c5-4361-b798-e810a2eea511","order_by":1,"name":"Valérie GUILLARD","email":"","orcid":"","institution":"UMR IATE, Université de Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Valérie","middleName":"","lastName":"GUILLARD","suffix":""},{"id":512404058,"identity":"bcc959cb-b307-4d31-9da5-94826e7f8547","order_by":2,"name":"Nathalie GONTARD","email":"","orcid":"","institution":"UMR IATE, INRAE","correspondingAuthor":false,"prefix":"","firstName":"Nathalie","middleName":"","lastName":"GONTARD","suffix":""},{"id":512404059,"identity":"f1036f9c-6931-4d0a-9111-f47b304552b9","order_by":3,"name":"Lucile CHATELLARD","email":"","orcid":"","institution":"UMR IATE, Université de Montpellier","correspondingAuthor":false,"prefix":"","firstName":"Lucile","middleName":"","lastName":"CHATELLARD","suffix":""}],"badges":[],"createdAt":"2025-08-29 19:53:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7491274/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7491274/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91063769,"identity":"afcd928a-d0ee-42e2-bd81-d1709fed889d","added_by":"auto","created_at":"2025-09-11 09:28:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66880,"visible":true,"origin":"","legend":"\u003cp\u003eTypical biodegradation kinetic curves obtained in the present study with the example of shredded PHBV for the three material-to-inoculum ratios studied. Each point represents the mean and standard deviation based on 3 replicates.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7491274/v1/991ea456bdb6862c91447351.jpg"},{"id":91062748,"identity":"596053a1-49cd-48f7-a32e-3a54d8abee69","added_by":"auto","created_at":"2025-09-11 09:20:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":104977,"visible":true,"origin":"","legend":"\u003cp\u003eBiodegradation parameters calculated for the three materials in shredded shape at 3 material-to-inoculum ratios with (a) ultimate biodegradation percentage achieved (%) and the time required (days) for each material at the three material-to-inoculum and (b) maximum biodegradation rates calculated from the slopes of the corresponding linear regression line. The average and standard deviations were calculated based on 3 replicates. Biodegradation test lasted 373 days in total. Statistical significance was assessed using appropriate tests, with differences considered significant at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7491274/v1/c1a74ac2f1918dea11bb24d5.jpg"},{"id":91062750,"identity":"57a40720-54f3-4a90-b3cf-0bcb2b3798e5","added_by":"auto","created_at":"2025-09-11 09:20:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":102629,"visible":true,"origin":"","legend":"\u003cp\u003eBiodegradation parameters calculated for the three materials in film shape at 3 material-to-inoculum ratios with (a) ultimate biodegradation percentage achieved (%) and the time required (days) for each material at the three material-to-inoculum and (b) maximum biodegradation rates calculated from the slopes of the corresponding linear regression line. The average and standard deviations were calculated based on 3 replicates. Biodegradation test lasted 370 days in total. Statistical significance was assessed using appropriate tests, with differences considered significant at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7491274/v1/bb4d3fdb4995e03bb0503b03.jpg"},{"id":91063770,"identity":"a068e05e-6ef4-411b-87d8-1df057e1ec49","added_by":"auto","created_at":"2025-09-11 09:28:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116827,"visible":true,"origin":"","legend":"\u003cp\u003eRemaining material at 349 days of the experimental period for PHBV 1/30 conditions.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7491274/v1/c23b4ee60d98370a64be7402.jpg"},{"id":91766481,"identity":"5e74d912-498f-432e-a96e-3b5fe7fe91a1","added_by":"auto","created_at":"2025-09-20 12:31:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1236615,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7491274/v1/fe0a7a97-b9ab-4612-a065-d9f9030d8ed2.pdf"},{"id":91062753,"identity":"59d479bb-1dab-4c48-a52e-03a95a9cdfe9","added_by":"auto","created_at":"2025-09-11 09:20:54","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2020713,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterialbazartetal2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7491274/v1/fc559e0294428fe957e0114a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Loading ratio is key for biodegradation PHBV-based materials in home- composting conditions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlastics are widely used in all sectors of industry for decades, but their physical-chemical durability has led to environmental accumulation since most conventional petrochemicals plastics cannot be significantly degraded through biological mechanisms in natural or anthropogenic ecosystems (i.e. composting)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A large amount of persistent plastic waste is generated and accumulates on Earth. In 2019, up to 353\u0026nbsp;million tonnes of plastic waste was generated, onto the 460\u0026nbsp;million tons of plastic consumed annually, among which 40% for agri-food applications [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to OECD data (2022) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], 49% of these plastics end up in landfill alongside food waste from household consumption worldwide in 2019. This plastic waste becomes a reservoir of very persistent polymers, which serves as the origin of all past, current and future releases of plastic particles into our environment. To address these environmental concerns, a range of biodegradable materials, such as cellulose-based material, are used as an ecological alternative. In parallel emerging biodegradable polymers, such as polyhydroxyalkanoates or poly (butylene succinate) (PHA, PBS, respectively\u0026hellip;) offer thermoplastic properties that cellulose does not have, which, in addition to other functionalities, enable materials to be tailored for specific usage and post-usage scenarios [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. A polymer is considered biodegradable if microorganisms can ultimately metabolise it into water, carbon dioxide (and/ or methane), and biomass, within a specific timeframe and under specific environmental conditions such as pH and temperature [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBiodegradation of polymers can occur either on their surface or in bulk, with other parameters involve in the biodegradation rate within thickness (submillimeter or large-format 3D materials), shape, or molar mass of the starting polymer and environmental parameters like pH and temperature [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A polymer can be biodegradable in natural conditions including soil, aqueous environments, and/or in anthropogenic system such as industrial and home composting at varying timeframes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile industrial composting is a biowaste recovery operation, which can only be carried out with the required authorizations, home composting empowers individuals to transform their kitchen and garden waste into valuable compost for personal use. Home composting offers the benefit to lower waste management costs and impacts due to a reduced volume of waste that enters the formal waste management system, being collected from households and treated. Such practice is encouraged by the European Waste Framework Directive (Article 22 (2b) of Directive 2008/98/EC)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Home compostability of packaging materials enable to divert such waste from landfilling or incineration and to avoid contamination of the compost. These products are defined and regulated by the Packaging and Packaging Waste Regulation (PPWR).\u003c/p\u003e\u003cp\u003eThe systematic assessment of this biodegradation behaviour relies primarily on international standards (ASTM or ISO). The NF EN 13432:2000 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] is a specification standard stating that a polymer can be defined as biodegradable if its biodegradation rate reaches at least 90% within 6 months in industrial composting, or 12 months in home composting. Additionally, the NF EN ISO 14855-1 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] is an analytical and testing standard widely used that describes a respirometry method to evaluate the biodegradability of a polymer mimicking composting conditions. This approach measures the amount of CO\u003csub\u003e2\u003c/sub\u003e produced by the microbial community composing mature compost, the so-called inoculum, when metabolising the available organic matter. The biodegradation of a material is then estimated by comparing the amount of CO\u003csub\u003e2\u003c/sub\u003e produced in the presence and absence of the material in the environment. The respirometry method can be implemented under both home and industrial composting conditions, using different temperature ranges (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 55\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, respectively). This standard specifies that the tested materials must be reduced to pieces smaller than 2 \u0026times; 2 cm and that a given amount of these materials must be then added to the inoculum, equalling 1/6 of the dry weight (referred to as the material-to-inoculum ratio). Elevated temperatures reduce the time required for experiments and could explain why so many biodegradation studies have been conducted in industrial composting systems.\u003c/p\u003e\u003cp\u003eAmong the booming alternative polymers, polyhydroxyalkanoates (PHA), and in particular the poly(3-hydroxybutyrate-\u003cem\u003eco\u003c/em\u003e-3-hydroxyvalerate) (PHBV), which escape the controversies surrounding non-biodegradability and non-recyclability [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], exhibit comparable thermoplasticity, tensile strength, and rigidity to conventional petrochemical polymers such as polypropylene (PP) [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. They can be used as plastic substitutes for many applications [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. PHBV is biodegradable in the natural environment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Its use in composite formulations with cellulose, ligno-cellulosic fibers/residue has been found promising, decreasing overall costs while tailoring functional, such as barrier, properties and preserving the biodegradability [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe biodegradation of PHBV and its composites under aerobic conditions has been widely studied, particularly under industrial composting conditions as reviewed by [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, only few studies focus on home composting environments [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Home composting represents a particularly relevant scenario to simplify their organic recycling but yet remains significantly under-researched compared to industrial composting applications. Understanding the mechanisms behind the biodegradation in home-composting conditions is crucial for optimising waste management strategies and end-of-life outcomes.\u003c/p\u003e\u003cp\u003eMost published biodegradation studies reveal significant deviations from standard protocols, particularly regarding the material-to-inoculum ratio (loading ratio) which frequently differs from the recommended 1/6 without explicit justification. Studies have employed dramatically different ratios ranging from 1/55 to 1/15, with intermediate values of 1/30 [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These modifications are presumably implemented to accelerate biodegradation processes and obtain results more rapidly, as lower material quantities relative to inoculum are expected to enhance degradation kinetics.\u003c/p\u003e\u003cp\u003eThe impact of these ratio modifications on biodegradation timelines is substantial. While Muniyasami et al. (2016) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], adhering to the standard 1/6 ratio, achieved 90% biodegradation in approximately 200 days, studies using reduced ratios demonstrated dramatically accelerated degradation: 65 days with 1/55 ratio [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], 95 days with 1/30 ratio [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and 91 days with 1/15 ratio [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the effect of the loading ratio on the ultimate biodegradation value and biodegradation rate has never been quantified yet.\u003c/p\u003e\u003cp\u003eAlongside ratio variations, material shape diversity also contributes to methodological inconsistencies. The ISO standard only specifies a maximum material size of 2 \u0026times; 2 cm, suggesting the possibility to use wild range of sizes bellow this value and material shape. In the literature, the authors can use discs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], or films [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Smaller particles accelerate biodegradation by shortening the fragmentation phase and increasing surface area for microbial contact which facilitates biofilm formation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, it remains unclear whether the biodegradation kinetic measured on a shredded material can be extrapolated to the same intact material such as film for instance. Additionally, the qualitative and quantitative interaction between particle size and material-to-inoculum ratio --two important factors\u0026ndash; has not been yet investigated.\u003c/p\u003e\u003cp\u003eIn this context, this study aimed to investigate loading ratio effects on the biodegradation kinetics of PHBV-based material while controlling final shape of the material added in the compost. The research focuses on establishing quantitative relationships between various material-to-inoculum ratios (1/3, 1/30, 1/50), two material forms (films versus shredded), and biodegradation kinetic parameters such as ultimate biodegradation and biodegradation rate in home composting conditions for cellulose, Kraft paper, PHBV, and PHBV-cellulose composite polymer.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eThe study was conducted on PHBV materials (PHI003, 3% HV, Natureplast, Fr). Materials biodegradation was tested either with or without 20% (w/w) cellulose microfiber content (Arbocel grade B00, Rosenberg, Germany), and in two physical shapes: 2 x 2 cm pieces and powder form (\u0026gt;\u0026thinsp;1 mm). For comparison purposes, the cellulose microfibers and Kraft paper (2 x 2 cm, from biowaste collection bags, Sumus Italia, srl) served as positive biodegradable controls, while PET, both in 2 x 2 cm pieces and shredded shape (RAMAPET N1S, Polymix, France) was used as a negative non-biodegradable control. The PHBV and PHBV-cellulose composite films were produced by the technical center IPC of Alen\u0026ccedil;on (France). The commercial PHBV (PHI003) was first mixed with boron nitride (0.5%) as a nucleating agent for all PHBV-based polymers, and with 20% w/w of cellulose fibers (B00) for the composite. The resulting powder-based mixture was then extruded using a COPERION twin-screw co-rotating extruder (ZSK 32 MC), with a screw diameter of 32 mm and a length of 1260 mm (L/D ratio of 40). The pellets produced were subsequently extruded using a Collin E45E single-screw extruder to obtain films with a flat die.\u003c/p\u003e\u003cp\u003eThe cellulose microfibers (B00) were 99.5% pure, with a bulk density of 150\u0026ndash;185 g/l (as per DIN EN ISO 60), a skeletal density of 1.56 g/cm\u0026sup3;, an average thickness of 15 \u0026micro;m, and an average length of 18 \u0026micro;m, according to the supplier. Kraft paper samples were pieces of biowaste collection bags that are distributed to Montpellier citizens (France) in the context of the biowaste management plan. These bags are made from Havane Kraft paper, water-based glue, and carbon-based ink (according to the supplier). The bags are biodegradable as certified by the Consorzio Italiano Compostatori (CIC), in compliance with the UNI EN 13432:2002 standard. The poly (ethylene terephthalate) (PET) films were produced by the IPC technical center using the same method described above for PHBV-based materials.\u003c/p\u003e\u003cp\u003ePHBV, PHBV-cellulose, PET and Kraft paper films were cut into pieces of 2 x 2 cm for film shape biodegradation studies. For the shredded shape, pieces of 1 x 10 cm of PHBV, PHBV-cellulose and PET were shredded into powder using an IKA210 grinder (Germany) at high speed for a maximum of 10 seconds to prevent overheating. For this shape, the positive control used was already in powder form (cellulose B00).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.2. Respirometry test\u003c/b\u003e:\u003c/h2\u003e\u003cp\u003eThe biodegradability of the materials was assessed through respirometry tests following the methodology described by Bonnenfant et al., 2023 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], which is based on the NF EN ISO 14855-1 standard [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The tests were conducted at a temperature simulating aerobic home compost conditions (28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C). The microbial consortia (inoculum) came from mature compost obtained through the industrial composting of biowaste and green waste at the Aspiran industrial composting platform (France). Upon reception, the mature compost was screened to \u0026lt;\u0026thinsp;2 mm with a RITEC sifter. The pH of the compost was measured with a pH meter Cyberscan 510, using 5 g of compost dispersed in 5 times its volume of distilled water. The mixture was stirred for 30 min, and the pH was immediately measured in triplicate. The proportion of organic matter was characterized using an ash oven (Nabertherm, GmbH Bahnhofstr 20, 28865 Lilienthal/Bremen) at 550\u0026deg;C for 4 h. The water content of the compost was adjusted to 55% w/w using distilled water. A C/N ratio of 12 was determined for the compost by the composting platform.\u003c/p\u003e\u003cp\u003eThe respirometry tests were conducted in four replicates in 1 L hermetic glass jars (Le Parfait, France) containing three flasks of 60 mL each. Each experimental setup consisted of three distinct components designed to create optimal conditions for biodegradation monitoring. The first 60 mL glass flask contained the biodegradation medium, made of sieved mature compost adjusted to 55% water content (w/w), and one studied material. Three material-to-inoculum ratios of material to compost masses (i.e. substrate/inoculum) were studied for PHBV, PHBV with 20% cellulose composite, cellulose microfibers, and Kraft paper. Three material-to-inoculum ratios were tested: 1/3 and 1/30 simulating home composting where 100% and 10% respectively of food packaging are made of the tested material and collected with biowaste, and 1/50 simulating the use of the materials tested only to make organic waste bags with a capacity of 1.5 kg each. The amount of material added to each condition is described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. As the mass of the film-shaped material is not uniform, it varies between material. The total mass of compost was adjusted to maintain consistent experimental conditions while varying the material-to-inoculum ratios. PET was introduced according to a material-to-inoculum ratio of 1/3 only to limit the space required in the incubator.\u003c/p\u003e\u003cp\u003eFor both material shapes (films, and shredded materials), four reactors containing only mature compost without any test material were used as blanks to measure the CO\u003csub\u003e2\u003c/sub\u003e produced from the biodegradation of the compost\u0026rsquo;s residual organic matter.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTheorical mass addition (g) of shredded or film material for each material-to-inoculum ratio\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRatio\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e1:3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e1:30\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e1:50\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eshredded\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003efilm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eshredded\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003efilm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eshredded\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003efilm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBiodegradable controls (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePHBV (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePHBV\u0026thinsp;+\u0026thinsp;20% cellulose (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe second flask served as a CO\u003csub\u003e2\u003c/sub\u003e capture system and contained a solution of 0.2 M NaOH of precisely known volume and concentration to capture the gaseous CO\u003csub\u003e2\u003c/sub\u003e produced during the biodegradation of the organic carbon, according to Eq.\u0026nbsp;1. The NaOH concentration and volume were adjusted during the experiment to accommodate the rate of CO\u003csub\u003e2\u003c/sub\u003e production.\u003c/p\u003e\u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2NaOH \u0026rarr; Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO \u003cem\u003eEq.\u0026nbsp;1\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe third 60 mL flask contained 10 mL of distilled water to maintain saturated relative humidity (RH) in the 1 L jar and preserve the compost water content.\u003c/p\u003e\u003cp\u003eThe reactors were incubated at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C in IPP 110\u0026thinsp;+\u0026thinsp;and IPP500 incubators (Memmert) and TS 606/4-i (WTW). All reactors were opened regularly to renew the oxygen in the jars, and to exchange the NaOH trap with a new one.\u003c/p\u003e\u003cp\u003eThe CO\u003csub\u003e2\u003c/sub\u003e captured by the NaOH trap was determined by titration. First, 5 mL of barium chloride solution (BaCl\u003csub\u003e2\u003c/sub\u003e, 20% w/w) was added to precipitate the Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e according to Eq.\u0026nbsp;2. The excess of NaOH was then titrated with HCl solution at the same concentration, using thymolphthalein (0.1% in ethanol) as a colour indicator according to Eq.\u0026nbsp;3.\u003c/p\u003e\u003cp\u003eNa\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;BaCl\u003csub\u003e2\u003c/sub\u003e \u0026rarr; 2 NaCl\u0026thinsp;+\u0026thinsp;BaCO\u003csub\u003e3\u003c/sub\u003e \u003cem\u003eEq.\u0026nbsp;2\u003c/em\u003e\u003c/p\u003e\u003cp\u003eNaOH\u0026thinsp;+\u0026thinsp;HCl \u0026rarr; NaCl\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO \u003cem\u003eEq.\u0026nbsp;3\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe quantity of CO\u003csub\u003e2\u003c/sub\u003e produced between two measurement intervals was calculated according to Eq.\u0026nbsp;4, where V\u003csub\u003eNaOH\u003c/sub\u003e is the volume of NaOH used (mL), C\u003csub\u003eNaOH\u003c/sub\u003e is the NaOH concentration (mol. L-1), VeqHCL is the equivalence volume of HCl, and C\u003csub\u003eHCl\u003c/sub\u003e is its concentration in mol. L-1.\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:n\\:CO2\\:\\left(mmol\\right)=\\:\\frac{\\left(\\right({V}_{NaOH}\\:*\\:{C}_{NaOH})\\:-({Veq}_{HCl}*{C}_{HCl}\\left)\\right)}{2}\\)\u003c/span\u003e\u003c/span\u003e \u003cem\u003eEq.\u0026nbsp;4\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe biodegradation rate %B was calculated using Eq.\u0026nbsp;5, where \u0026ldquo;CO\u003csub\u003e2\u003c/sub\u003e material\u0026rdquo; and \u0026ldquo;CO\u003csub\u003e2\u003c/sub\u003e blank\u0026rdquo; are the mass of CO\u003csub\u003e2\u003c/sub\u003e (mg) produced in the presence or in the absence of material, respectively, and \u0026ldquo;CO\u003csub\u003e2\u003c/sub\u003e theoretical\u0026rdquo; is the mass of CO\u003csub\u003e2\u003c/sub\u003e (mg) theoretically released for 100% of material biodegradation.\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\%B\\:=\\frac{\\:(CO2\\:material\\:-\\:CO2\\:blank)}{{CO}_{2}\\:theoretical}\\:*\\:100\\)\u003c/span\u003e\u003c/span\u003e \u003cem\u003eEq.\u0026nbsp;5\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAll results were expressed as % biodegradation per g of compost. The experiment was monitored for about 12 months, and each condition was stopped earlier if a plateau of biodegradation was reached beforehand.\u003c/p\u003e\u003cp\u003eFor each kinetic, the exponential phase from the biodegradation curve was manually selected with an R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;≧\u0026thinsp;0.98 and the slope of the corresponding linear regression line was calculated to determine the maximal biodegradation rate. For most conditions, a modelled curve could be fitted to the experimental data using the modified Gompertz model implemented with the \u0026ldquo;nlsLM\u0026rdquo; function from the R package \u0026ldquo;minpack.lm\u0026rdquo; (version 1.2-4) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] in RStudio software (R version 4.4.2 (2024-10-31) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], using Eq.\u0026nbsp;6, where y0 is the initial biodegradation value at the beginning of the experiment (in this case, 0), ymax is the ultimate biodegradation percentage reached, k is the maximal biodegradation rate previously determined, lag is the time corresponding to the lag phase, and x is the time in days.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:y=\\:y0\\:+\\:\\frac{\\left(ymax\\:-\\:y0\\right)\\:*\\:exp(\\:-\\:exp(k\\:*\\:(lag\\:-\\:x))}{\\left(ymax\\:-\\:y0\\right)\\:+\\:1)}\\)\u003c/span\u003e\u003c/span\u003e \u003cem\u003eEq.\u0026nbsp;6\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThis model was selected because its parameters appropriately fit the experimental curve profile, including the lag phase, exponential growth phase, and final plateau. The associated curves are provided in supplementary material (section \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Figure S8-S13). For conditions that did not reach 90% biodegradation within the experimental timeframe, the model was used to predict the time (in days) required to reach 90%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Polymers characteristics:\u003c/h2\u003e\u003cp\u003eEach material was characterized through molecular weight determination (SEC), carbon content analysis (CHNS-O), and thermogravimetric analysis (TGA). Additional properties including density, and particle size are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eMolecular weight (Mw) was determined by size exclusion chromatography (SEC) performed by the Technopolym platform (Toulouse, France). Samples were dissolved in chloroform at 3\u0026ndash;5 mg. mL-1 and heated at 70\u0026deg;C for 1\u0026ndash;5 h until complete dissolution. Solutions were filtered through 0.45 \u0026micro;m nylon membranes before analysis. SEC analysis was conducted using a UHPLC system (Ultimate 3000, Thermoscientific) equipped with an Agilent PL gel 5 \u0026micro;m Mixed C column. Injection volume was 50 \u0026micro;L with an elution flow rate of 1 mL.min-1. Detection was performed using a triple detector system: differential refractometer (Optilab Rex Wyatt, 35\u0026deg;C), three-angle static light scattering detector (TREOS Wyatt, 658 nm), and UV detector (Shimadzu SPD-M20A, 254 nm).\u003c/p\u003e\u003cp\u003eCarbon content of each material (cellulose, PHBV-based materials, and Kraft paper) was determined to calculate the theoretical maximum CO\u003csub\u003e2\u003c/sub\u003e production for biodegradation rate calculations. The analysis was performed by the Laboratoire de Mesure Physique (LMP, Universit\u0026eacute; de Montpellier, France), using a CHNS-O elemental analyser and a Sartorius Cubis Advanced MCA (precision 0,1 \u0026micro;g, max 2,1 g). Samples in tin capsules were loaded into a 120-position autosampler and transferred to a helium-purged inert chamber. Combustion occurred at 1150\u0026deg;C in a catalytic furnace, followed by reduction over hot copper at 850\u0026deg;C. Gases were separated using an Elementar TPD column, and carbon as detected by thermal conductivity detector (TCD).\u003c/p\u003e\u003cp\u003eThermogravimetric analysis (TGA) was performed using a Mettler TGA2 apparatus (Schwerzebbbach, Switzerland) with an XP5U balance (0.0001 mg precision) to determine the maximum degradation temperature (Tdeg). Samples (5\u0026ndash;10 mg) were analysed in triplicate under nitrogen flow (50 mL.min \u0026minus;\u0026thinsp;1) with heating from 25\u0026deg;C to 600\u0026deg;C \u0026agrave; 10\u0026deg;C.min-1. Tdeg was determined as the temperature corresponding to the maximum of the peaks obtained from the first derivative of their TGA curve (i.e. weight loss).\u003c/p\u003e\u003cp\u003eThe particle size distribution of the resulting materials was evaluated with a laser granulometer (LS 13320 XR, Beckman Coulter)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePhysico-chemical properties of the polymers used in the study.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMaterial\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCellulose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eKraft paper\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003ePHBV\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003ePHBV\u0026thinsp;+\u0026thinsp;20% cellulose\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003ePET\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eshredded\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003efilm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eshredded\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003efilm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eshredded\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003efilm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarbon content (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e41.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40.558\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e55.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e53.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e62.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMolecular weight (kDa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e453.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e311.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e226.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e76.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThickness (\u0026micro;m)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e220\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e365\u0026thinsp;\u0026plusmn;\u0026thinsp;34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e217\u0026thinsp;\u0026plusmn;\u0026thinsp;14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGranulometry (\u0026micro;m)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e579\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e654\u0026thinsp;\u0026plusmn;\u0026thinsp;31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e725\u0026thinsp;\u0026plusmn;\u0026thinsp;38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTdeg (\u0026deg;C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e345.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e290\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e285.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\u003cp\u003e306.9\u0026thinsp;\u0026plusmn;\u0026thinsp;39.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Statistical analysis:\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using RStudio software. Comparison between material-to-inoculum ratios and material shapes were performed through analysis of variance (ANOVA) after verifying normal distribution (Shapiro-Wilk test) and homogeneity of variances (Bartlett test, or Levene test when Bartlett test assumptions were not met, \u0026ldquo;car\u0026rdquo; R package, version 3.1-3) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Tukey\u0026rsquo;s post hoc tests were used to compare means when ANOVA showed significant differences. For data with non-normal distribution, Kruskal-Wallis tests were applied, followed by log transformation to achieve normality when necessary. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Graphics were generated using \u0026ldquo;ggplot2\u0026rdquo; R package [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eBiodegradation experiments were conducted in respirometry tests for cellulose microfibers, Kraft paper, PHBV and PHBV-cellulose composite polymer in both film and shredded shape under home composting conditions (28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C).\u003c/p\u003e\u003cp\u003eAll experiments continued until biodegradation plateaus were reached, except for the highest ratio (1/3), for which biodegradation did not reach a plateau within the experimental timeframe for any material tested, including the cellulose control. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows typical biodegradation curves for shredded PHBV across different material-to-inoculum ratios, demonstrating the typical sigmoidal kinetics observed throughout the study.\u003c/p\u003e\u003cp\u003eFigures 2 and 3 present comparative results for shredded and film material, respectively. Ultimate biodegradation percentages and time (in days) required to reach this maximum are shown in panels (a), while maximum biodegradation rates calculated from the exponential growth phase are display in panels (b). Complete kinetic profiles for all materials in both shredded and film shape are provided in supplementary material (Figure S1-S6).\u003c/p\u003e\u003c/p\u003e\u003cp\u003eExcept for cellulose microfibers at the 1/3 ratio, all materials (cellulose microfibers, PHBV, and PHBV-cellulose) achieved more than 90% biodegradation within 12 months, confirming their complete biodegradability according to the NF EN ISO 14855-1 standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The negative control (PET) showed no measurable biodegradation as anticipated (Figure S7). Cellulose microfibers at the highest material-to-inoculum ratio (1/3) reached only 83\u0026thinsp;\u0026plusmn;\u0026thinsp;6% of biodegradation after the twelve-month experimental period, while they reached ultimate biodegradation more rapidly at 1/30 and 1/50 ratios, about 114\u0026thinsp;\u0026plusmn;\u0026thinsp;5% and 111\u0026thinsp;\u0026plusmn;\u0026thinsp;17% for both ratio after 168\u0026thinsp;\u0026plusmn;\u0026thinsp;4 days and 150\u0026thinsp;\u0026plusmn;\u0026thinsp;21 days, respectively. PHBV and PHBV-based composite with 20% cellulose biodegraded as rapidly as for the positive control at the 1/3 ratio (369 days) with an ultimate biodegradation percentage of 90\u0026thinsp;\u0026plusmn;\u0026thinsp;3%. The 1/50 ratio yielded similar maximum degradation time for both materials: 158\u0026thinsp;\u0026plusmn;\u0026thinsp;8 days for PHBV and 163\u0026thinsp;\u0026plusmn;\u0026thinsp;0 days for PHBV cellulose composite. However, PHBV reached its maximum of biodegradation more rapidly for 1/30 ratio (166\u0026thinsp;\u0026plusmn;\u0026thinsp;5 days) compared to the PHBV cellulose composite that required 221\u0026thinsp;\u0026plusmn;\u0026thinsp;21 days. This suggests that cellulose addition may slow biodegradation kinetics when blended with PHBV at this specific ratio.\u003c/p\u003e\u003cp\u003eFor 1/30 and 1/50 ratios, maximum biodegradation percentages consistently exceeded 100%. Cellulose microfibers showed 114\u0026thinsp;\u0026plusmn;\u0026thinsp;5% at the 1/30 ratio, and 111\u0026thinsp;\u0026plusmn;\u0026thinsp;17% at the 1/50 ratio. PHBV reached 131\u0026thinsp;\u0026plusmn;\u0026thinsp;2% at the 1/30 ratio and 100\u0026thinsp;\u0026plusmn;\u0026thinsp;22% at the 1/50 ratio. The PHBV-cellulose composite achieved 124\u0026thinsp;\u0026plusmn;\u0026thinsp;25% at the 1/30 ratio and 109\u0026thinsp;\u0026plusmn;\u0026thinsp;20% at the 1/50 ratio.\u003c/p\u003e\u003cp\u003eBiodegradation rates increased with decreasing material-to-inoculum ratios, as clearly demonstrated by cellulose results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb): rates of 0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d at the 1/3 ratio, 3.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d at the 1/30 ratio and 5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d at the 1/50 ratio. This trend was confirmed for PHBV and PHBV-cellulose composite, which showed similar increases between the 1/3 and 1/30 ratios. However, biodegradation rates were the same between the 1/30 and 1/50 ratios, as confirmed by statistical analysis. For PHBV, degradation rates reached 1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d and 1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d for the 1/30 and 1/50 ratios, respectively. For the PHBV-cellulose composite, biodegradation rates were 1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d for the 1/30 ratio and 1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d for the 1/50 ratio. All values are summarised in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eOverall, the highest biodegradation rates were achieved by the positive control (cellulose), regardless of the ratio tested, with rates approximately 3\u0026ndash;4 times higher than those of PHBV-based materials, even at the highest material-to-inoculum ratio (1/3).\u003c/p\u003e\u003cp\u003eAnalysis of ultimate biodegradation, time to reach ultimate biodegradation, and maximum biodegradation rates revealed that material-to-inoculum ratios primarily impact reaction kinetics rather than final extent or time to completion in case of shredded material, as confirmed by statistical analysis. The only exception was PHBV at the 1/30 ratio, which showed a higher ultimate biodegradation percentage despite a similar rate compared to the 1/50 ratio. The PHBV-cellulose composite exhibited statistically similar ultimate biodegradation percentages across all three ratios, suggesting its biodegradation was less affected by the material/inoculum ratios compared to the other materials studied. The results also highlight the absence of correlation between maximum biodegradation rate and ultimate biodegradation extent.\u003c/p\u003e\u003cp\u003eConsistent with the approach used for shredded materials, the experimental period for films lasted up to 370 days, with tests terminated when biodegradation rates stabilized.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDuring the 370-day incubation period, only the PHBV-cellulose composite at ratios of 1/30 (93\u0026thinsp;\u0026plusmn;\u0026thinsp;8%) and 1/50 (108\u0026thinsp;\u0026plusmn;\u0026thinsp;15%), and PHBV at the 1/30 ratio (118\u0026thinsp;\u0026plusmn;\u0026thinsp;16%), achieved the 90% biodegradation threshold required by NF EN 13432:2000 standard [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], thus meeting the compostability requirements within one year. At the highest material-to-inoculum ratio (1/3), Kraft paper, PHBV, and the PHBV-cellulose composite reached biodegradation levels of 66\u0026thinsp;\u0026plusmn;\u0026thinsp;2%, 78\u0026thinsp;\u0026plusmn;\u0026thinsp;12%, and 76\u0026thinsp;\u0026plusmn;\u0026thinsp;7%, respectively, after 370 days of incubation. At the 1/50 ratio, PHBV and Kraft paper achieved biodegradation levels of 85\u0026thinsp;\u0026plusmn;\u0026thinsp;12% and 73\u0026thinsp;\u0026plusmn;\u0026thinsp;9%, respectively, after 266\u0026thinsp;\u0026plusmn;\u0026thinsp;13 days and 258\u0026thinsp;\u0026plusmn;\u0026thinsp;14 days. At the 1/30 ratio, Kraft paper achieved 89\u0026thinsp;\u0026plusmn;\u0026thinsp;7% in 242\u0026thinsp;\u0026plusmn;\u0026thinsp;0 days. Notably, visible fragments of PHBV and the PHBV-cellulose composite that were initially introduced at the 1/30 ratio remained in the reactors at the end of the experiment, despite reaching 100% biodegradation according to CO₂ measurements, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eComparatively to shredded material, for films conditions, the maximum biodegradation rate tends to increase as the material/inoculum ratio decreases (i.e. when less material is present in the inoculum). Regarding maximum biodegradation rates, two distinct patterns emerged depending on material type (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). For the PHBV-cellulose composite, a negative linear relationship was observed between the maximum biodegradation rate and the material-to-inoculum ratio: as the material/inoculum ratio decreased, the maximum biodegradation rate increased. Specifically, rates were measured at 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d, 0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d and 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d for ratios of 1/50, 1/30, and 1/3, respectively. In contrast, both PHBV and Kraft paper exhibited non-linear responses with the lower ratios (1/30 and 1/50) showing higher biodegradation rates than the higher ratio of 1/3. For PHBV, maximum rates were 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d, 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d, and 0.297\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001%/d for the ratios of 1/50, 1/30, and 1/3, respectively. For Kraft paper, maximum rates were 2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d, for 1/50 and 1/30 ratios but decreased dramatically at 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%/d at the highest material concentration (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This represents the most substantial difference observed, with Kraft paper exhibiting significantly higher biodegradation rates than both PHBV materials at 1/30 and 1/50 ratios. Notably, the 1/3 ratios consistently reduced biodegradation rates across all materials, meaning that independently of the material shape, the 1/3 ratios slow the biodegradation rate similarly.\u003c/p\u003e\u003cp\u003eFor film materials, maximum biodegradation rates correlated with ultimate biodegradation percentages: faster biodegradation rates were associated with higher maximum biodegradation percentages.\u003c/p\u003e\u003cp\u003eTo better capture the influence of material shape on biodegradation rates, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e compares all the tested materials and ratios using a 'shape effect', calculating as the relative increase in biodegradation rate between the shredded and film shapes of the same material. As expected, shredded materials exhibited significantly faster biodegradation rates under most conditions, with rates ranging from 1.6 to 3.2 times higher at material-to-inoculum ratios of 1/50 and 1/30. However, at the highest material concentration (1/3 ratio), this advantage was reduced: maximum biodegradation rates were either comparable for PHBV and PHBV-cellulose composite, or 1.3 times faster for Kraft paper.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of maximum biodegradation rate for each shape materials for the three tested material-to-inoculum ratio. The average and standard deviations were calculated based on 3 replicates. The shape effect indicates the proportional increase of the biodegradation rate between shredded and film shape (Rate \u003csub\u003eshredded\u003c/sub\u003e / rate \u003csub\u003efilm\u003c/sub\u003e). Standard deviations were deliberately rounded up to a hundredth to ensure robust conclusions and are indicated by an asterisk.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMaterial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eShape\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e1/50 ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e1/30 ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e1/3 ratio\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eRate (%/d)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eShape effect\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eRate (%/d)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eShape effect\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eRate\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e(%/d)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eShape effect\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eBiodegradable control\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eCellulose\u003c/b\u003e powder\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eshredded\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eKraft paper\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003efilm\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003eTested material\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003ePHBV\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eshredded\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003efilm\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.297\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003ePHBV\u0026thinsp;+\u0026thinsp;20% cellulose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eshredded\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e3.2\u0026thinsp;+\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003efilm\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003e*: standard deviation rounded up to a hundredth\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThis study contributes to our understanding of the relationships between the material composition and shape, their loading rate in home composting environment, and their biodegradation performance (ultimate biodegradation percentage and maximum biodegradation rate).\u003c/p\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Material-to-inoculum ratio influences the biodegradation kinetics\u003c/h2\u003e\u003cp\u003eIn this study, we systematically evaluated three distinct material loading ratios: 1/3, 1/30, and 1/50, each representing different realistic loading scenarios. The 1/3 ratio simulates an extreme case where 100% of single-use plastic packaging is replaced by biodegradable alternatives and fully collected through biowaste systems in France (see supplementary material, section \u003cspan refid=\"Sec8\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The 1/30 ratio represents one-tenth of this scenario, to evaluate linear relationship between ratios, while the 1/50 ratio models more realistic conditions, such as 1.5 kg of apples packaged in biodegradable plastic bags typically available in French retail markets.\u003c/p\u003e\u003cp\u003eOur results demonstrate that material-to-inoculum ratios fundamentally control biodegradation kinetics rather than ultimate biodegradability, and this last one seems more impacted by the material shape. Each material exhibited optimal loading ratios, particularly pronounced in film configurations: ratios of 1/30 or below for PHBV and Kraft paper, and 1/50 or below for PHBV-cellulose composites. Most significantly, the highest ratio (1/3) induced substrate inhibition phenomenon well-established in microbiology but never demonstrated before in material biodegradability studies. Under these high-loading conditions, extrapolated biodegradation curves predicted extended degradation periods of 578\u0026thinsp;\u0026plusmn;\u0026thinsp;238 days for PHBV films and 613\u0026thinsp;\u0026plusmn;\u0026thinsp;169 days for PHBV-cellulose composite films to achieve\u0026thinsp;\u0026gt;\u0026thinsp;90% biodegradation as the ISO 13432-2 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] standard requires (see supplementary material, Figure S14). It let suppose that at optimal ratios, compost microbial community are able to process all available substrates including material while excessive material quantities (1/3 ratio) either saturate inoculum capacity or create inhibitory conditions. It lets suppose that at optimal ratios, compost microbial community are able to process all available substrates including material while excessive material quantities (1/3 ratio) either saturate inoculum capacity or create inhibitory conditions. This loading-dependent performance was consistently observed across both material shapes, though the magnitude of shape effects varied with ratio conditions. Under such high loading ratios, material fragments may remain in the mature compost, but they will likely continue to biodegrade once the compost is used and applied to the field, when the ratios will consequently decrease. Given that the ISO standard recommends a 1/6 ratio and our results show limitations at 1/3 ratios, adhering to recommended guidelines is crucial for proper biodegradation of these materials. The inhibitory effects observed at elevated ratios emphasize the importance of maintaining optimal conditions in domestic composting systems.\u003c/p\u003e\u003cp\u003eHowever, scaling these laboratory findings to home composting presents challenges, as domestic composters have limited control over the quantity of biodegradable plastics added to their bins. This necessitates awareness and moderation when disposing of biodegradable materials in home compost. Fortunately, exceeding optimal ratios would primarily extend decomposition times rather than prevent biodegradation entirely.\u003c/p\u003e\u003cp\u003eFuture research should validate these findings in real domestic composting conditions to provide practical guidelines for home composters.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Material shape effects within the context of loading ratio optimization\u003c/h2\u003e\u003cp\u003eWhile material-to-inoculum ratio serves as the primary determinant of biodegradation kinetics, material shape provides secondary but significant acceleration effects, particularly at intermediate loading ratios. Material particle size and biodegradation rates are interconnected through underlying mechanisms that become most apparent under optimal loading conditions. Previous studies demonstrate that reducing polymer granulometry enhances enzymatic hydrolysis and increases biodegradation rates, as observed in aqueous aerobic systems [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and in soil [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], though this finding was not confirmed by Yang et al. (2005) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] in industrial composting, highlighting behavioural diversity across different studies.\u003c/p\u003e\u003cp\u003eThe fragmentation of the material (film to shredded film shape) increased the surface area exposure, enabling enhanced microbial accessibility and enzymatic contact [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This enhancement stems from bypassing initial biodegradation stages devoted to material fragmentation under physicochemical phenomena, especially mechanical stress [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, the magnitude of these shape effects diminished under high loading conditions, particularly at the highest ratio, where substrate inhibition overshadowed surface area advantages. When biodegradable materials are reduced to sufficiently fine particles, and introduced at appropriate ratios, microorganisms can efficiently consume them within optimal composting timeframes. While these results suggest potential benefits of pre-treatment processes for post-use biodegradable materials, such processes require specific infrastructure and are difficult to implement and expensive [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor PHBV-cellulose composites, the interaction between loading ratio and shape became particularly complex. Mechanical shredding may release cellulose fractions separately from PHBV, and given cellulose's higher degradation rate, this separation potentially accelerates overall composite biodegradation, but only under appropriate loading conditions where microbial communities can effectively process the increased substrate availability.\u003c/p\u003e\u003cp\u003eThe \"shape effect\" (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) quantifies material shape impacts on biodegradation rates for identical materials (PHBV or PHBV\u0026thinsp;+\u0026thinsp;20 cellulose) under specific loading ratios in home composting. This provides realistic approaches for evaluating plastic waste fate and establishing PHBV material guidelines for composting applications. When biodegradable materials are reduced to sufficiently fine particles, and introduced at appropriate ratios, microorganisms can efficiently consume them within optimal composting timeframes.\u003c/p\u003e\u003cp\u003eFinally, this significant influence of material shape on biodegradation rates highlights the importance of using appropriate control shapes when applying NF EN ISO 14855-1 standard [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Control materials\u0026mdash;paper sheets, cellulose papers, or starch films\u0026mdash;should match tested polymer physical shapes and be evaluated under comparable loading ratios to ensure valid respirometry comparisons.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Limitations of respirometry analysis in presence of priming effect\u003c/h2\u003e\u003cp\u003eAssessment accuracy appeared to be influenced by specific material-to-inoculum ratio conditions. Nearly all shredded material conditions and two film conditions exhibited biodegradation percentages exceeding 100%, indicating CO₂ production greater than theoretically expected based on tested material carbon content in respirometry assays. This phenomenon was most evident at intermediate ratios, where optimal loading conditions appeared to maximize the \"priming effect\".\u003c/p\u003e\u003cp\u003eThe priming effect, first identified by L\u0026ouml;hnis (1926) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], refers to CO₂ overproduction from adding easily biodegradable organic matter to soil [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] or mature compost [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This addition stimulates microbial activity and enhances recalcitrant organic matter degradation, producing additional CO₂. Notably, for PHBV and PHBV-cellulose composite films at the 1/30 ratio, film fragments remained in compost despite an ultimate biodegradation percentage greater than 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), strongly supporting the priming effect theory and demonstrating the disconnect between CO₂ production and actual material disappearance. The magnitude of CO₂ overproduction varied systematically with material-to-inoculum ratios. Under high loading conditions, substrate inhibition appeared to suppress both material degradation and priming effects, resulting in more conservative CO₂ measurements. Conversely, optimal ratios maximized both material biodegradation and compost organic matter stimulation, leading to the highest CO₂ overproduction. Under shredded conditions across all ratios, visual identification of remaining materials became nearly impossible due to particle size, making material decomposition status assessment challenging regardless of loading conditions.\u003c/p\u003e\u003cp\u003eIn the literature, similar CO₂ overproduction occurred during biodegradation of diverse materials, including biodegradable (PHA, PBS) and hydrolytically degradable polymer (poly (lactic acid)) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and cellulose/starch [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This CO₂ overproduction may stem from additional metabolic processes within mature compost, particularly nitrogen cycle processes [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Since tested polymers lack nitrogen, microorganisms must biodegrade native compost organic matter to acquire nitrogen, simultaneously inducing CO₂ production through surrounding organic matter degradation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, considering that compost remained unstirred for one-year, anaerobic pockets could form near film materials due to their barrier properties, creating favourable conditions for anaerobic community proliferation and CO₂ production through denitrification.\u003c/p\u003e\u003cp\u003eThese possible metabolic process accumulations suggest needs for more precise CO\u003csub\u003e2\u003c/sub\u003e quantification during our respirometry tests. However, in this study, distinguishing CO₂ from material versus compost biodegradation was impossible. Moreover, CO\u003csub\u003e2\u003c/sub\u003e monitoring provides indirect biodegradation quantification that may introduce measurement errors. The standard addresses this limitation through 90% thresholds accounting for potential errors.\u003c/p\u003e\u003cp\u003eTo address indirect quantification issues and improve respirometry accuracy, the literature suggests several differentiation approaches: \u0026sup1;⁴C isotope labelling for CO\u003csub\u003e2\u003c/sub\u003e distribution quantification [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], biomass growth quantification through insoluble protein content measurement [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The NF EN ISO 14855-1 standard specifies that vermiculite addition could also limit material CO\u003csub\u003e2\u003c/sub\u003e overproduction, but, unfortunately, it will not prove that all CO\u003csub\u003e2\u003c/sub\u003e derives solely from material.\u003c/p\u003e\u003cp\u003eRespirometry approaches such as those presented in the current study and by NF EN ISO 14855-1 focus on the use of mature compost with consistent characteristics (C/N ratio, pH, organic matter content). However, extrapolating results to broader applications like home composting presents challenges when materials must be processed alongside easily biodegradable sources such as biowaste. A key question remains whether microorganisms preferentially degrade biowaste or biodegradable materials.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion and perspective","content":"\u003cp\u003eThis research investigated the critical role of material-to-inoculum ratio in biodegradation kinetics under home composting conditions using ISO 14855-1 respirometry protocols. The material-to-inoculum ratio emerged as the primary determinant of biodegradation performance, with each material exhibiting distinct optimal loading ranges: 1/50 or below for PHBV-cellulose composites, and 1/30 to 1/50 for PHBV and Kraft paper films. Most significantly, this study revealed that high loading ratios (1/3) induce substrate inhibition\u0026mdash;a novel phenomenon in polymer biodegradability research\u0026mdash;dramatically extending degradation times to 578\u0026ndash;613 days for PHBV-based film materials. This loading-dependent behaviour was consistently observed across both material shapes, though shape effects provided secondary acceleration under optimal ratio conditions.\u003c/p\u003e\u003cp\u003eRespirometry data showed CO2 levels exceeding theoretical values through priming effects within the composting environment, highlighting the importance of further research into these ratios and priming mechanisms to refine biodegradation quantification approaches. These findings emphasize the critical need for controlled material loading in composting applications, improved assessment methodologies for reliable polymer biodegradation evaluation, and careful consideration of material introduction rates in waste management policies. Reassuringly, excess ratios would predominantly delay decomposition timelines without blocking the biodegradation pathway entirely.\u003c/p\u003e\u003cp\u003eDuring the preparation of this work the authors used Claude AI (Anthropic, 2024) to improve the readability and language of the manuscript. After using this tool/service, the authors reviewed and edited the content as needed and takes full responsibility for the content of the published article.\u003c/p\u003e\u003cp\u003eThe 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.\u003c/p\u003e\u003cp\u003eThe authors thank the PLANET facility (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15454/1.5572338990609338E12\u003c/span\u003e\u003cspan address=\"10.15454/1.5572338990609338E12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) run by the IATE joint research unit for providing process experiment supports: granulometer Beckman Coulter, Nabertherm ash oven and RITEC sifter.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was funded by the French National Research Agency (ANR) under the ANR-21-CE43 project BioCyPlast.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.B: Conceptualization, formal analysis, investigation, methodology, writing - original draftV.G: Conceptualization, Supervision, writing - review \u0026amp; editingN.G: Conceptualization, Project administration, Supervision, writing - review \u0026amp; editingL.C: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Validation, Writing - review \u0026amp; editing\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData are provided at this link: https://entrepot.recherche.data.gouv.fr/privateurl.xhtml?token=ff8b2c30-670c-4f29-9902-faf4babc9d1b\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRitchie, Hannah, Samborska, Veronika, Roser, Max. 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Priming effects induced by degradable microplastics in agricultural soils. Soil Biol Biochem. 2023;180:109006. \u003c/li\u003e\n\u003cli\u003eJung E-J, Shin P-K, Bae H-K. Effects of temperature and compost conditions on the biodegradation of degradable polymers. J Microbiol Biotechnol. 1999;9:464‑8. \u003c/li\u003e\n\u003cli\u003eDlamini JC, Chadwick D, Hawkins JMB, Martinez J, Scholefield D, Ma Y, et al. Evaluating the potential of different carbon sources to promote denitrification. J Agric Sci. 2020;158:194‑205. \u003c/li\u003e\n\u003cli\u003eTuomela M, Hatakka A, Karjomaa S, It\u0026auml;vaara M. Priming effect as determined by adding 14 C-glucose to modified controlled composting test. Biodegradation. 2002;13:131‑40. \u003c/li\u003e\n\u003cli\u003eFritz I, Hergolitsch L, Dalnodar D, Zerobin A. Quantifying the difference between CO2-release and carbon conversion in aerobic aquatic biodegradation tests. bioRxiv. 2025;2025‑03. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Plastic biodegradation, Loading ratio, PHBV, Home composting","lastPublishedDoi":"10.21203/rs.3.rs-7491274/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7491274/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the context of growing plastic pollution in the packaging sector, biodegradable polymer alternatives present viable solutions, necessitating comprehensive understanding of biodegradation in accessible systems such as home composting. This research aimed to clarify the little-studied impact of PHBV-based material loading ratios on biodegradation kinetics in home composting environments, examining both film and shredded materials shape.\u003c/p\u003e\u003cp\u003eThe materials were tested according to three usage scenarios (high, medium and low) corresponding to material-to-compost ratios of 1/3, 1/30 and 1/50, under home composting conditions using ISO 14855-1 respirometry protocols. For both material forms, loading ratios has a stronger influence on the maximum biodegradation rate rather than on the ultimate biodegradation percentage. For shredded materials, the biodegradation rate linearly decreases with the increase of the ratio, whereas no direct correlation was shown for the material in film form. This suggests a material form effect on the polymer biodegradation rate regarding its loading ratio in the compost. For films, material residue persisting after one year suggested priming effects and revealed that PHBV biodegradation in our home composting setup occurs over extended timeframes beyond those captured by standard respirometry measurements. The study revealed that high loading ratios (1/3) induced substrate inhibition across all tested materials\u0026mdash;a previously unreported phenomenon in PHBV biodegradability research. This inhibition extended the PHBV biodegradation time required to achieve 90% ultimate biodegradation from a maximum of 365 days prescribed by the standard to 578\u0026ndash;613 days. These results establish the existence of a maximum optimal material loading ratio, above which biodegradation becomes suboptimal. Thanks to such a threshold, a careful consideration of material introduction rates would significantly benefit to the current waste management strategies and regulatory frameworks.\u003c/p\u003e","manuscriptTitle":"Loading ratio is key for biodegradation PHBV-based materials in home- composting conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 09:20:49","doi":"10.21203/rs.3.rs-7491274/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"62523568-b980-406e-a776-75e667ea77f0","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-20T12:23:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-11 09:20:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7491274","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7491274","identity":"rs-7491274","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}