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Biodegradation kinetics in soil of a multi-constituent biodegradable plastic
Polymer Degradation and Stability 166 (2019) 213e218
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Polymer Degradation and Stability journal homepage: www.elsevier.com/locate/polydegstab
Biodegradation kinetics in soil of a multi-constituent biodegradable plastic Maurizio Tosin, Alessandro Pischedda, Francesco Degli-Innocenti* Novamont S.p.A., Via Fauser 8, 28100, Novara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 March 2019 Received in revised form 17 May 2019 Accepted 26 May 2019 Available online 29 May 2019
The biodegradation kinetics in soil of a biodegradable multi-constituent plastic material have been determined with a standard test method based on the measurement of the evolved CO2. Three different kinetics were identified, possibly corresponding to (i) the biodegradation of low molecular weight constituents, (ii) the self-degradation of biomass formed in the first phase, (iii) the biodegradation of the bulk polyesters. The relationship between surface area and mineralization rate was determined using regression analysis. The regression model suggests that if it were technically possible to test the plastic material in the form of nanoplastics (spheres of 100 nm diameter) it would take 15e20 days to reach full biodegradation, a time frame compatible with the OECD requirements for readily biodegradable chemicals. The specific mineralization rate of test material was estimated to be 0.003439 mg organic carbon/day/cm2. We put forward the testing approach applied in this work as a means to characterize biodegradable plastics and obtain constants relevant for eco-design and for environmental fate studies. © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Biodegradable Plastics Kinetics Mineralization rate Biodegradation in soil ASTM D 5988e12 Surface area
1. Introduction Under normal environmental conditions, most biodegradable plastics are water-insoluble solid materials [1]. They are made up of macromolecules, which due to their large size, do not pass through the cell membrane and therefore cannot be absorbed directly by microorganisms [2,3]. The first stage of biodegradation occurs outside the microorganisms and is caused by extracellular enzymes that erode the surfaces of solid materials [4]. The macromolecules are split up to the constituent elements, i.e. the monomers and oligomers, which pass through the cell membrane and are metabolized becoming part of the biochemistry of the micro-organisms and of the living mass (called “biomass”). Low molecular weight additives do not need the depolymerization phase to become available. The final degradation of the metabolites involves an oxidation process that requires oxygen and leads to the evolution of carbon dioxide [5]. Biodegradation can therefore be schematically represented in three stages: Stage 1: plastic / monomers/oligomers (depolymerization).
* Corresponding author. E-mail addresses: [email protected] (M. Tosin), alessandro. [email protected] (A. Pischedda), [email protected] (F. Degli-Innocenti).
Stage 2: monomers/oligomers / biomass (uptake and metabolism). Stage 3: biomass þ O2 / CO2 þ H2O (mineralization). The depolymerization (Stage 1) releases monomers that are assimilated by the surrounding microorganisms (the “central dogma” for biodegradation of polymers [6]). The enzymes and microbes in the liquid phase interact with the plastics' constituents at the surface of the solid phase. The available solid/liquid interphase is thus a potential limiting factor of the depolymerization and consequently of the overall biodegradation, including mineralization. Stage 1 is considered the limiting factor, while the subsequent assimilation of monomers by the microbes (Stage 2) is expected to be immediate [7]. Stage 3, i.e. the mineralization of organic carbon into CO2 and H2 O is fast in the early biodegradation phases. Afterwards, when there is no further plastic to biodegrade, the microbes are under starvation conditions, and mineralization affects the storage polymers and metabolites formed in Stage 2. This latter phase can be very long [8,9]. In a previous work [10], we showed quantitatively that the biodegradation rate of polybutylene sebacate (PBSe), a biodegradable polyester, is affected by the available surface area. Samples of PBSe with different granulometry were tested for biodegradation in soil. The relationship between the biodegradation rates and the respective available surface areas was linear when applying a
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double reciprocal regression model (i.e. the Lineweaver-Burk approach), making it possible to estimate the biodegradation kinetics using the same approach used by Michaelis-Menten for enzymatic reactions. This approach seems rather interesting for a better characterization of the biodegradability of plastics. Our previous work was carried out testing a neat polymer, i.e. a material formed by just one macromolecule. In this study, we wanted to verify whether the same analytical approach was applicable also to a commercial biodegradable plastic, i.e. a more complex material made up of a number of constituents (polymers and additives). 2. Materials and methods 2.1. Materials The test material used in this study is Mater-Bi HF03V1, a commercial biodegradable plastic produced by Novamont in the form of pellets. This plastic material is made with biodegradable polyesters (about 65%), starch (about 28%), and a natural plasticizer (about 6%). Polyesters are made with monomers that biodegrade in soil [11]. The plasticizer is a biobased biodegradable polyol. This substance is completely biodegraded within 28 days at ambient temperature, under aqueous aerobic conditions (Organic Waste Systems, Belgium, data not shown). The carbon content determined by elemental analysis of the test material was C ¼ 56.41% (analysis performed by Redox Snc, Monza, Italy). Density is 1.28 g/cm3. Powders were obtained from plastic granules by means of cryogenic grinding with liquid nitrogen. The powder was separated using a number of standard sieves (Endecotts, London), with mesh sizes of 500 mm, 355 mm, 210 mm, 180 mm, 125 mm, and 75 mm, in order to obtain the following three fractions: 355 mme500 mm, 180 mme210 mm, 75 mme125 mm. Pure micro-crystalline cellulose in powder (Merck) was used as reference material (C ¼ 44.44%). 2.2. Specific surface area determination The specific surface area (cm2/g) was determined for the pellets and for each powder class. The surface area of the pellet was determined considering this as a scalene ellipsoid. The dimensions of the three axes of 50 pellets were measured with the calibre. The approximate formula [Eq. (1)] was used to calculate their surface area:
1=p p p a b þ ap cp þ bp cp S cm2 4p 3
(Eq.1)
where a, b and c are the semi-axes and p ¼ 1.6075 (Knud Thomsen correction).
Next, the specific surface area (cm2/g) was calculated from the surface and weight averages of 50 pellets. The surface areas of all of the three powder fractions were determined using two approaches. In the first approach, the surface areas were obtained by mathematical calculation, assuming the particles to be spheres with a diameter equal to the median of each particle range (i.e. 100 mm, 195 mm, and 428 mm). Knowing the volume of a single sphere of each particle size, and that the density of the test material was equal to 1.28 g/cm3, the number of particles per gram of material was estimated and thus, so was the specific surface area for each powder fraction. In the second approach, the particle size of the sample fractions was analysed using a particle sizing instrument (Mastersizer 3000 equipped with a Hydro EV manual wet dispersion unit, Malvern Instruments Limited, UK). 2.3. Biodegradation test Biodegradation was determined by means of respirometric tests, in accordance with the ASTM D 5988e12 test method [12], based on the measurement of CO2 production. Soil was collected from an agricultural field at the Centro Sperimentazione ed Assistenza Agricola (CeRSAA), in Albenga (Italy). The soil that was routinely analysed by CeRSAA, had a C/N ratio of 10.38. Soil sieved to a 5-mm particle size was enriched with compost (40 g/Kg) and a salt solution (0.2 g KH2PO4; 0.1 g MgSO4; 0.4 g NaNO3; 0.2 g Urea; 0.4 g NH4Cl per Kg of soil). The water content was measured as weight loss (105 C), and the final soil moisture was adjusted to 14.6% (about 50% of the Water Holding Capacity). The final soil pH was determined in a mixture of soil and deionized water: ratio of 1:2.5 (w/v) [13]. The pH was 7.9. For each of the three test material powders and for the pellets, 1 g of test material was mixed with 200 g of soil in a 1000 ml hermetically-sealed glass jar. The test was set up with blank jars (without material) and with reference material jars (1 g of cellulose). Two replicates were carried out for each polymer particle size, for blank and for reference, and incubated in the dark at 28 ± 2 C. The test set-up is shown in Table 1. A 50 ml beaker filled with 30 ml of 0.5 M KOH, as a CO2 trapping solution, was placed in each jar. The amount of CO2 produced was measured by means of titration of the KOH solution, with 0.3 N HCl [14,15] with a Mettler Toledo (T50) potentiometric titrator. The measurement was made every 2e3 days during the first two weeks, when the mineralization rate was expected to be maximal, and weekly or biweekly thereafter. The moisture content was kept constant by adding deionized water throughout the biodegradation test, whenever the KOH solution was titrated and replaced with a fresh one. The net CO2 production evolved from the test materials was
Table 1 Biodegradation test set-up. Reactor
Test material
Sieving fraction
Total Amount of test material (g)
Soil (g)
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R15 R16
Blank Blank HF03V1 HF03V1 HF03V1 HF03V1 HF03V1 HF03V1 HF03V1 HF03V1 Microcrystalline cellulose Microcrystalline cellulose
e e 50e75 50e75 200e355 200e355 500e700 500e700 pellet pellet e e
e e 1.00903 1.00510 1.00375 1.00107 1.00223 1.00107 1.00193 1.00119 1.00297 1.00382
200 200 200 200 200 200 200 200 200 200 200 200
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Table 2 Specific surface area (cm2/g) of each fraction determined by theoretical approach (considering the particles as spheres, with a diameter corresponding to the median of the respective sieving range). Sieving range (mm)
Theoretical diameter of each sphere (mm)
Surface area of each sphere Volume of each sphere Mass of each (cm2) (cm3) sphere (g)
Number of particles Specific surface area per g (cm2/g)
355e500 180e210 75e125
428 195 100
5.75E-03 1.19E-03 3.14E-04
1.90 Eþ04 2.01 Eþ05 1.49 Eþ06
4.10E-05 3.88E-06 5.23E-07
5.25E-05 4.97E-06 6.70E-07
109.5 240.4 468.8
Table 3 Specific surface area (cm2/g) of each fraction determined using the three measurement approaches. Sieving range (mm)
Theoretical estimation
Particle sizing
Direct measurement of dimensions
pellets 355e500 180e210 75e125
e 109.6 240.4 468.8
e 102.2 228.6 754.7
15 e e e
calculated by subtracting the average amount of CO2 produced in the blank soils from the amount of CO2 produced in the test material jars. The biodegradation percentages were calculated from the ratio between the net CO2 production and the theoretical CO2 production (ThCO2) based on the carbon content. The regression analysis and data plotting were carried out using the statistical functions of Excel (Microsoft) and Statgraphics Centurion XVII. 3. Results 3.1. Determination of particle size Mater-Bi HF03V1, a biodegradable plastic material, was tested for soil biodegradation in its original form (pellets), and after milling and sieving. Three fractions were obtained, with the following particle size range classes: 355 mme500 mm, 180 mme210 mm, 75 mme125 mm. The surface area of 50 pellets was determined by measuring the dimensions and weight, and thereby determining the average specific area (cm2/g; see subsection 2.2). The surface area of the different sieved fractions was estimated, using a theoretical approach, by considering the particles of each fraction as spheres with a diameter equal to the median of the limits of each range (Table 2), and measured with a particle sizing instrument. The overall results are compared in Table 3. The values of the specific surface areas obtained by particle sizing were comparable to the theoretical estimates, with the exception of the 75 mme125 mm fraction.
Fig. 1. CO2 evolution courses of different reactors, including blank and reference material (cellulose). The numbers in parenthesis indicate the total surface area, in cm2, of the tested samples.
3.2. Biodegradation The different samples, together with microcrystalline cellulose as a reference, were tested for soil biodegradation. The amount tested was 1 g per reactor. The total surface area was determined considering the specific surface area obtained by particle sizing and the mass of each sample. The cumulative CO2 production of the different reactors is shown in Fig. 1. The replicates (same symbol, with a continuous or dotted line in Fig. 1) showed courses that were superimposable in most cases. The average mineralization curves are shown in Fig. 2. The mineralization of cellulose was in line with the validity requirements (i.e. mineralization >70% after 6 months) of the standard test method ASTM D 5988e12 [12], showing the soil was active and the test valid. At the end of the test (day 276), the sample with a total surface
Fig. 2. Mineralization curves of samples with different initial surface areas (indicated as total surface area in cm2) and of reference material (cellulose). Each curve is the mean of two replicates.
area of 756 cm2 had reached a biodegradation level equal to 90% of that of cellulose. This is the minimum mineralization value to be reached in less than 2 years, for a material to be considered as suitable for biodegradable mulch films, according to the standard EN 17,033 [16]. All of the other samples (even if chemically identical) were still below that threshold.
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Fig. 3. Organic carbon (determined from evolved CeCO2), plotted as a function of time, of the samples with different surface areas (expressed in cm2). Each point is the mean of two replicates.
Fig. 4. Organic carbon (determined from evolved CeCO2) decrease of the samples with different surface area (expressed in cm2), in the first 10 days of testing (zooming of Fig. 3). Each point is the mean of two replicates.
The carbon evolved as net carbon dioxide (CeCO2) was subtracted from the amount of carbon originally present in the plastic sample at time 0, to obtain the plot of residual not mineralized carbon. This value includes both carbon still bound in the plastic constituents and carbon assimilated as biomass. For the sake of simplicity we will call this carbon “organic carbon”, that is, carbon that has not been converted into inorganic carbon, i.e. mineralized into CO2 yet (Fig. 3). The curves in Fig. 3 show the progressive decrease in carbon as a consequence of mineralization. Three different zones of the curves are recognised. (1) A first short, very active phase, from day 0 to about day 10 (Fig. 4); (2) a second phase, from day 10 to day 38 (Fig. 5); (3) a third phase, from day 52 onwards (Fig. 6). A linear regression analysis was carried out to determine the rates on three different zones of the curves. The regression analyses constants are reported in Table 4. All regressions had R-squared > 92%. Rates are reported as positive values, for convenience, but should be considered as negative, as they are depletion rates. The mineralization rates (k) of each phase were correlated with the corresponding surface areas, by regression analysis. The relationship between the mineralization rates from day 0 to day 10 and surface area is well described by the double reciprocal model Y ¼ 1/(a þ b/X). No significant correlation was found for the second set of data (from day 10 to day 38). The squared-Y model (i.e. Y ¼ sqrt (aþb X) best fits the data in the third data set. The regression coefficients are shown in Table 5. The specific mineralization rates (i.e. the rates expressed as mg organic carbon/day, with reference to surface area unit) relating to the last phase (from day 52 onwards), are reported in Table 6.
Fig. 5. Organic carbon (determined from evolved CeCO2) decrease of the samples with different surface area (expressed in cm2), in the period between day 10 and day 38 of testing (zooming of Fig. 3). Each point is the mean of two replicates.
4. Discussion 4.1. Mineralization kinetics The test material shows three main, different mineralization kinetics. An initial phase (from 0 to about 6 days) is characterized by a
Fig. 6. Organic carbon (determined from evolved CeCO2) decrease of the samples with different surface area (expressed in cm2), in the period from day 52 onwards of testing (zooming of Fig. 3). Each point is the mean of two replicates. Straight lines are linear regressions determined considering the period spanning between about 400 mg-C and 200 mg-C organic carbon.
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Table 4 Mineralization rates k (mineralized organic carbon, mg/day) as determined by regression analysis. Sample (total surface area,cm2)
Day 0 e day 6
Day 10 e day 38
Day 52 onwards
15 102 229 756
2.55 10.62 11.94 12.76
2.38 2.12 2.13 2.43
0.76 0.86 1.60 2.60
Table 5 Regression analysis of the mineralization rates and the surface areas, in the periods from day 0 to day 6, and from day 52 onwards. Period considered
Applied regression model
Intercept (a)
Slope (b)
R-squared
From day 0 to day 6 From day 52 onwards
double reciprocal Y ¼ 1/(a þ b/X) squared-Y Y ¼ sqrt (aþb X)
0.0605 0.008627
4.946 0.290
99.50 98.83
Table 6 Specific mineralization rates (k) per unit surface area (organic carbon, mg/day/cm2) from day 52 onwards. Sample (total surface area, cm2)
specific mineralization rate (k/cm2)
15 102 229 756
0.0506 0.008431 0.006987 0.003439
CO2 spike, a fast and short mineralization phase, which is very likely due to the release of soluble constituents leaking from the solid plastic particles by diffusion. The polyalcohol used as a plasticizer of starch is known to be a readily biodegradable substance, and thus is the best candidate. The samples reached the same mineralization level (of about 15%) with different rates, in good agreement with the respective surface area (Fig. 4). The mineralization is a function of the surface area, as shown by the regression analysis (Table 5). After that period, a slower biodegradation phase follows, where all samples display superimposable curves, independently of the surface area (Fig. 5). This second phase lasts until about day 50, and reaches a total mineralization level of 30%. The lack of correlation between the rate and surface area suggests that the ongoing mineralization is caused by the turnover (self-biodegradation induced by starvation) of the biomass formed in the first 10 days of biodegradation. This second phase seems to be a sort of lag phase, during which the conditions for the subsequent degradation phase are still to be established. A hypothesis is that this lag phase corresponds to the formation of a biofilm covering the solid particles needed for efficient enzymatic erosion [17]. More studies are needed to prove this hypothesis. Subsequently, in the third phase, which starts around day 50, the different samples diverge, with rates that are correlated with the available surface area, as shown by the regression analysis. This is probably the moment when the polymers are effectively eroded and the speed of the process is clearly affected by the available surface area. A squared-Y model describes the relationship between mineralization rates and surface areas in the third phase. The squared-Y model implies a progressive decrease in dependent variable Y (rate), with increasing values of independent variable X (surface area). This suggests that some factor progressively limits the mineralization rate with higher surface areas. This is in line with the behaviour found in the previous study with PBSe, where the best fit was a double reciprocal model, which enables the identification of a maximum rate reached at a given surface area.
The regression model allows us to make some very interesting considerations. If it were possible to mill the test material to form 100 nm diameter spheres, we would get a total surface of 234,375 cm2/g, the density of the test material being 1.28 g/cm3. The mineralization rate that corresponds to 234,375 cm2 is about 45 mg C/day, and can be calculated by applying the model found with the regression analysis (Y ¼ sqrt (0.008627 þ 0.29014 X). With this mineralization rate, phase 3 would last only about 8 days. Phase 1 (mineralization of soluble constituents) would be reduced to about 3 days, with a mineralization rate of 16.4 mg C/day. It is difficult to predict the effect of very high surface areas on the lag phase (phase 2). In any case, the overall model predicts extremely fast biodegradation under these conditions, so that total mineralization could theoretically be reached in less than 10e20 days. The results confirm that the mineralization rate at the surface level, i.e. at the molecular level, is very fast, which is in agreement with the findings of our previous work carried out with PBSe.
4.2. Specific mineralization rates The plastic material tested in this work displays compliance with the EN 17,033 biodegradability requirement [16] in less than 1 year, when tested at 754.7 cm2/g, while the sample at 15 cm2/g is very slow and will probably fail the test after 2 years (Fig. 2). Thus, the same material is considered “quickly” biodegradable or “slowly” biodegradable, depending on the granulometry of the tested sample. The reason of this paradox is that the evolved CO2 is based on the biodegradation that effectively occurs at the surface of the plastic granules, but it is then related to the mass of the sample being tested, most of which is not involved in the biodegradation reaction. The evolved CO2 is divided by the theoretical CO2 (ThCO2), i.e. the CO2 evolved in case of total oxidation of the organic carbon present in the test material. If the mineralization rate value is to remain unaffected by the dimension of the tested sample, the evolved CO2 must be related to the surface area. The specific mineralization rates (k) per unit surface area (mg organic carbon/day/cm2) of the test material is estimated to be in the range 0.0506 to 0.0034 mg organic carbon/day/cm2. This value was calculated for the last and longest biodegradation phase, which presumably mainly involves the polymeric constituent. The value of the pellets is higher than the value of the milled particles. At this point in time, we do not have sufficient data to judge whether or not this difference is significant. The specific mineralization rate can be used to make an environmental fate prediction, and can be used in product design.
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5. Conclusions
References
The biodegradable plastics sector is developing fast, and inquiries for more detailed information on environmental characteristics are also on the increase. A relevant characteristic of biodegradable plastics is the biodegradation rate because this is a parameter that is necessary to predict the environmental fate. In this work, we tested the biodegradation in soil of a complex multiconstituent plastic product, identifying different kinetics that can be tentatively ascribed to the different constituents. A relationship between the biodegradation rate and the available surface area was recognised for the plastic product, and was similar to what had already been shown for a neat polymer [10]. The specific mineralization rate of the material tested was determined. This parameter can be used to compare different materials, to predict mineralization times, and perform risk assessment studies. The results show that the biodegradation process that occurs at the interface is very fast. If it were possible to mill the test material, to obtain nanoplastics, the biodegradation would be extremely fast, lasting 10e20 days at maximum, with a time frame similar to that required by the OECD for the classification of “readily biodegradable” chemicals.1 Therefore, the chemical permanence time of the test material is very short. On the other hand, the physical permanence time depends on the surface area, i.e. on the thickness of the plastic items. The results suggest that biodegradable plastics do not generate persistent microplastics, because as erosion increases the surface area, this in turn increases the biodegradation rate to levels similar to those required, by the OECD, for chemicals to be defined as readily biodegradable.
[1] G.M. Bohlmann, General characteristics, processability, industrial applications and market evolution of biodegradable polymers, in: C. Bastioli (Ed.), Handbook of Biodegradable Polymers, 2005, p. 183. [2] R.-J. Müller, Biodegradability of polymers: regulations and methods for testing, Biopolymers Online 10 (2005) 365e391, https://doi.org/10.1002/ 3527600035.bpola012. [3] F. Degli Innocenti, Biodegradable and bio-based polymers for the environment, in: F. Canepa P. Fava (Ed.), Production of Fuels, Specialty Chemicals and Biobased Products from Agro-Industrial Wastes and Surplus”, INCA, Venice, Italy, 2007. ISBN: 88-88214-18-6. [4] J. Boivin, J.W. Costerton, Biodeterioration of materials, in: Rossmoore H.W. Biodeterioration and Biodegradation 8, vols. 53e62, Elsevier Applied Science, 1991. ISBN 1-85166-626-5. [5] A.A. Shah, F. Hasan, A. Hameed, S. Ahmed, Biological degradation of plastics: a comprehensive review, Biotechnol. Adv. 26 (3) (2008) 246e265, 2008, https:// doi.org/10.1016/j.biotechadv.2007.12.005. ISSN 0734-9750. [6] D.J. Kaplan, J.M. Mayer, D. Ball, J. McMassie, A.L. Allen, P. Stenhouse, Fundamentals of biodegradable polymers, in: C. Ching, D.L. Kaplan, E.L. Thomas (Eds.), Biodegradable Polymers and Packaging, 1993, pp. 1e42. [7] S. Saponaro, E. Sezenna, F. Degli Innocenti, V. Mezzanotte, L. Bonomo, A screening model for fate and transport of biodegradable polyesters in soil, J. Environ. Manag. 88 (4) (2008) 1078e1087. [8] P.W. Hill, J.F. Farrar, D.L. Jones, Decoupling of microbial glucose uptake and mineralization in soil, Soil Biol. Biochem. 40 (2008) 616e624. https://doi.org/ 10.1016/j.soilbio.2007.09.008. [9] E. Oburger, D.L. Jones, Substrate mineralization studies in the laboratory show different microbial C partitioning dynamics than in the field, Soil Biol. Biochem. 41 (2009) 1951e1956. https://doi.org/10.1016/j.soilbio.2009.06.020. [10] S. Chinaglia, M. Tosin, F. Degli Innocenti, Biodegradation rate of biodegradable plastics at molecular level, Polym. Degrad. Stabil. 147 (2008) 237e244. https://doi.org/10.1016/j.polymdegradstab.2017.12.011. [11] M. Siotto, M. Tosin, F. Degli Innocenti, V. Mezzanotte, Mineralization of monomeric components of biodegradable plastics in preconditioned and enriched sandy loam soil under laboratory conditions, Water Air Soil Pollut. 221 (2011) 245e254, https://doi.org/10.1007/s11270-011-0787-8. [12] ASTM D5988, Standard Test Method for Determining Aerobic Biodegradation of Plastic Materials in Soil, ASTM International, West Conshohocken, PA, 2012. [13] P. Violante, P. Adamo, II. Reazione, in: P. Violante (Ed.), in Metodi di Analisi Chimica del Suolo, Franco Angeli), Milano, 2000, pp. 1e13. [14] A. Modelli, B. Calcagno, M. Scandola, Kinetics of aerobic polymer degradation in soil by means of the ASTM D 5988-96 standard method, J. Environ. Polym. Degrad. 7 (1999) 109e116, https://doi.org/10.1023/A:1021864402395. [15] ASTM D7991, Standard Test Method for Determining Aerobic Biodegradation of Plastics Buried in Sandy Marine Sediment under Controlled Laboratory Conditions, ASTM International, West Conshohocken, PA, 2015. [16] EN 17033, Plastics - Biodegradable Mulch Films for Use in Agriculture and Horticulture - Requirements and Test Methods, European Committee for Standardization, 2018. [17] M. Tosin, F. Degli Innocenti, C. Bastioli, Effect of the composting substrate on biodegradation of solid materials under controlled composting conditions, J. Environ. Polym. Degrad. 4 (1996) 55e63, https://doi.org/10.1007/ BF02083883.
Acknowledgements Thanks to Paolo Magistrali and Selene Chinaglia for the support provided in the use of the particle sizing instrument. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.polymdegradstab.2019.05.034. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Biodegradation kinetics in soil of a multi-constituent biodegradable plastic Maurizio Tosin, Alessandro Pischedda, Francesco Degli-Innocenti* Novamont S.p.A., Via Fauser 8, 28100, Novara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 March 2019 Received in revised form 17 May 2019 Accepted 26 May 2019 Available online 29 May 2019
The biodegradation kinetics in soil of a biodegradable multi-constituent plastic material have been determined with a standard test method based on the measurement of the evolved CO2. Three different kinetics were identified, possibly corresponding to (i) the biodegradation of low molecular weight constituents, (ii) the self-degradation of biomass formed in the first phase, (iii) the biodegradation of the bulk polyesters. The relationship between surface area and mineralization rate was determined using regression analysis. The regression model suggests that if it were technically possible to test the plastic material in the form of nanoplastics (spheres of 100 nm diameter) it would take 15e20 days to reach full biodegradation, a time frame compatible with the OECD requirements for readily biodegradable chemicals. The specific mineralization rate of test material was estimated to be 0.003439 mg organic carbon/day/cm2. We put forward the testing approach applied in this work as a means to characterize biodegradable plastics and obtain constants relevant for eco-design and for environmental fate studies. © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Biodegradable Plastics Kinetics Mineralization rate Biodegradation in soil ASTM D 5988e12 Surface area
1. Introduction Under normal environmental conditions, most biodegradable plastics are water-insoluble solid materials [1]. They are made up of macromolecules, which due to their large size, do not pass through the cell membrane and therefore cannot be absorbed directly by microorganisms [2,3]. The first stage of biodegradation occurs outside the microorganisms and is caused by extracellular enzymes that erode the surfaces of solid materials [4]. The macromolecules are split up to the constituent elements, i.e. the monomers and oligomers, which pass through the cell membrane and are metabolized becoming part of the biochemistry of the micro-organisms and of the living mass (called “biomass”). Low molecular weight additives do not need the depolymerization phase to become available. The final degradation of the metabolites involves an oxidation process that requires oxygen and leads to the evolution of carbon dioxide [5]. Biodegradation can therefore be schematically represented in three stages: Stage 1: plastic / monomers/oligomers (depolymerization).
* Corresponding author. E-mail addresses: [email protected] (M. Tosin), alessandro. [email protected] (A. Pischedda), [email protected] (F. Degli-Innocenti).
Stage 2: monomers/oligomers / biomass (uptake and metabolism). Stage 3: biomass þ O2 / CO2 þ H2O (mineralization). The depolymerization (Stage 1) releases monomers that are assimilated by the surrounding microorganisms (the “central dogma” for biodegradation of polymers [6]). The enzymes and microbes in the liquid phase interact with the plastics' constituents at the surface of the solid phase. The available solid/liquid interphase is thus a potential limiting factor of the depolymerization and consequently of the overall biodegradation, including mineralization. Stage 1 is considered the limiting factor, while the subsequent assimilation of monomers by the microbes (Stage 2) is expected to be immediate [7]. Stage 3, i.e. the mineralization of organic carbon into CO2 and H2 O is fast in the early biodegradation phases. Afterwards, when there is no further plastic to biodegrade, the microbes are under starvation conditions, and mineralization affects the storage polymers and metabolites formed in Stage 2. This latter phase can be very long [8,9]. In a previous work [10], we showed quantitatively that the biodegradation rate of polybutylene sebacate (PBSe), a biodegradable polyester, is affected by the available surface area. Samples of PBSe with different granulometry were tested for biodegradation in soil. The relationship between the biodegradation rates and the respective available surface areas was linear when applying a
https://doi.org/10.1016/j.polymdegradstab.2019.05.034 0141-3910/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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double reciprocal regression model (i.e. the Lineweaver-Burk approach), making it possible to estimate the biodegradation kinetics using the same approach used by Michaelis-Menten for enzymatic reactions. This approach seems rather interesting for a better characterization of the biodegradability of plastics. Our previous work was carried out testing a neat polymer, i.e. a material formed by just one macromolecule. In this study, we wanted to verify whether the same analytical approach was applicable also to a commercial biodegradable plastic, i.e. a more complex material made up of a number of constituents (polymers and additives). 2. Materials and methods 2.1. Materials The test material used in this study is Mater-Bi HF03V1, a commercial biodegradable plastic produced by Novamont in the form of pellets. This plastic material is made with biodegradable polyesters (about 65%), starch (about 28%), and a natural plasticizer (about 6%). Polyesters are made with monomers that biodegrade in soil [11]. The plasticizer is a biobased biodegradable polyol. This substance is completely biodegraded within 28 days at ambient temperature, under aqueous aerobic conditions (Organic Waste Systems, Belgium, data not shown). The carbon content determined by elemental analysis of the test material was C ¼ 56.41% (analysis performed by Redox Snc, Monza, Italy). Density is 1.28 g/cm3. Powders were obtained from plastic granules by means of cryogenic grinding with liquid nitrogen. The powder was separated using a number of standard sieves (Endecotts, London), with mesh sizes of 500 mm, 355 mm, 210 mm, 180 mm, 125 mm, and 75 mm, in order to obtain the following three fractions: 355 mme500 mm, 180 mme210 mm, 75 mme125 mm. Pure micro-crystalline cellulose in powder (Merck) was used as reference material (C ¼ 44.44%). 2.2. Specific surface area determination The specific surface area (cm2/g) was determined for the pellets and for each powder class. The surface area of the pellet was determined considering this as a scalene ellipsoid. The dimensions of the three axes of 50 pellets were measured with the calibre. The approximate formula [Eq. (1)] was used to calculate their surface area:
1=p p p a b þ ap cp þ bp cp S cm2 4p 3
(Eq.1)
where a, b and c are the semi-axes and p ¼ 1.6075 (Knud Thomsen correction).
Next, the specific surface area (cm2/g) was calculated from the surface and weight averages of 50 pellets. The surface areas of all of the three powder fractions were determined using two approaches. In the first approach, the surface areas were obtained by mathematical calculation, assuming the particles to be spheres with a diameter equal to the median of each particle range (i.e. 100 mm, 195 mm, and 428 mm). Knowing the volume of a single sphere of each particle size, and that the density of the test material was equal to 1.28 g/cm3, the number of particles per gram of material was estimated and thus, so was the specific surface area for each powder fraction. In the second approach, the particle size of the sample fractions was analysed using a particle sizing instrument (Mastersizer 3000 equipped with a Hydro EV manual wet dispersion unit, Malvern Instruments Limited, UK). 2.3. Biodegradation test Biodegradation was determined by means of respirometric tests, in accordance with the ASTM D 5988e12 test method [12], based on the measurement of CO2 production. Soil was collected from an agricultural field at the Centro Sperimentazione ed Assistenza Agricola (CeRSAA), in Albenga (Italy). The soil that was routinely analysed by CeRSAA, had a C/N ratio of 10.38. Soil sieved to a 5-mm particle size was enriched with compost (40 g/Kg) and a salt solution (0.2 g KH2PO4; 0.1 g MgSO4; 0.4 g NaNO3; 0.2 g Urea; 0.4 g NH4Cl per Kg of soil). The water content was measured as weight loss (105 C), and the final soil moisture was adjusted to 14.6% (about 50% of the Water Holding Capacity). The final soil pH was determined in a mixture of soil and deionized water: ratio of 1:2.5 (w/v) [13]. The pH was 7.9. For each of the three test material powders and for the pellets, 1 g of test material was mixed with 200 g of soil in a 1000 ml hermetically-sealed glass jar. The test was set up with blank jars (without material) and with reference material jars (1 g of cellulose). Two replicates were carried out for each polymer particle size, for blank and for reference, and incubated in the dark at 28 ± 2 C. The test set-up is shown in Table 1. A 50 ml beaker filled with 30 ml of 0.5 M KOH, as a CO2 trapping solution, was placed in each jar. The amount of CO2 produced was measured by means of titration of the KOH solution, with 0.3 N HCl [14,15] with a Mettler Toledo (T50) potentiometric titrator. The measurement was made every 2e3 days during the first two weeks, when the mineralization rate was expected to be maximal, and weekly or biweekly thereafter. The moisture content was kept constant by adding deionized water throughout the biodegradation test, whenever the KOH solution was titrated and replaced with a fresh one. The net CO2 production evolved from the test materials was
Table 1 Biodegradation test set-up. Reactor
Test material
Sieving fraction
Total Amount of test material (g)
Soil (g)
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R15 R16
Blank Blank HF03V1 HF03V1 HF03V1 HF03V1 HF03V1 HF03V1 HF03V1 HF03V1 Microcrystalline cellulose Microcrystalline cellulose
e e 50e75 50e75 200e355 200e355 500e700 500e700 pellet pellet e e
e e 1.00903 1.00510 1.00375 1.00107 1.00223 1.00107 1.00193 1.00119 1.00297 1.00382
200 200 200 200 200 200 200 200 200 200 200 200
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Table 2 Specific surface area (cm2/g) of each fraction determined by theoretical approach (considering the particles as spheres, with a diameter corresponding to the median of the respective sieving range). Sieving range (mm)
Theoretical diameter of each sphere (mm)
Surface area of each sphere Volume of each sphere Mass of each (cm2) (cm3) sphere (g)
Number of particles Specific surface area per g (cm2/g)
355e500 180e210 75e125
428 195 100
5.75E-03 1.19E-03 3.14E-04
1.90 Eþ04 2.01 Eþ05 1.49 Eþ06
4.10E-05 3.88E-06 5.23E-07
5.25E-05 4.97E-06 6.70E-07
109.5 240.4 468.8
Table 3 Specific surface area (cm2/g) of each fraction determined using the three measurement approaches. Sieving range (mm)
Theoretical estimation
Particle sizing
Direct measurement of dimensions
pellets 355e500 180e210 75e125
e 109.6 240.4 468.8
e 102.2 228.6 754.7
15 e e e
calculated by subtracting the average amount of CO2 produced in the blank soils from the amount of CO2 produced in the test material jars. The biodegradation percentages were calculated from the ratio between the net CO2 production and the theoretical CO2 production (ThCO2) based on the carbon content. The regression analysis and data plotting were carried out using the statistical functions of Excel (Microsoft) and Statgraphics Centurion XVII. 3. Results 3.1. Determination of particle size Mater-Bi HF03V1, a biodegradable plastic material, was tested for soil biodegradation in its original form (pellets), and after milling and sieving. Three fractions were obtained, with the following particle size range classes: 355 mme500 mm, 180 mme210 mm, 75 mme125 mm. The surface area of 50 pellets was determined by measuring the dimensions and weight, and thereby determining the average specific area (cm2/g; see subsection 2.2). The surface area of the different sieved fractions was estimated, using a theoretical approach, by considering the particles of each fraction as spheres with a diameter equal to the median of the limits of each range (Table 2), and measured with a particle sizing instrument. The overall results are compared in Table 3. The values of the specific surface areas obtained by particle sizing were comparable to the theoretical estimates, with the exception of the 75 mme125 mm fraction.
Fig. 1. CO2 evolution courses of different reactors, including blank and reference material (cellulose). The numbers in parenthesis indicate the total surface area, in cm2, of the tested samples.
3.2. Biodegradation The different samples, together with microcrystalline cellulose as a reference, were tested for soil biodegradation. The amount tested was 1 g per reactor. The total surface area was determined considering the specific surface area obtained by particle sizing and the mass of each sample. The cumulative CO2 production of the different reactors is shown in Fig. 1. The replicates (same symbol, with a continuous or dotted line in Fig. 1) showed courses that were superimposable in most cases. The average mineralization curves are shown in Fig. 2. The mineralization of cellulose was in line with the validity requirements (i.e. mineralization >70% after 6 months) of the standard test method ASTM D 5988e12 [12], showing the soil was active and the test valid. At the end of the test (day 276), the sample with a total surface
Fig. 2. Mineralization curves of samples with different initial surface areas (indicated as total surface area in cm2) and of reference material (cellulose). Each curve is the mean of two replicates.
area of 756 cm2 had reached a biodegradation level equal to 90% of that of cellulose. This is the minimum mineralization value to be reached in less than 2 years, for a material to be considered as suitable for biodegradable mulch films, according to the standard EN 17,033 [16]. All of the other samples (even if chemically identical) were still below that threshold.
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Fig. 3. Organic carbon (determined from evolved CeCO2), plotted as a function of time, of the samples with different surface areas (expressed in cm2). Each point is the mean of two replicates.
Fig. 4. Organic carbon (determined from evolved CeCO2) decrease of the samples with different surface area (expressed in cm2), in the first 10 days of testing (zooming of Fig. 3). Each point is the mean of two replicates.
The carbon evolved as net carbon dioxide (CeCO2) was subtracted from the amount of carbon originally present in the plastic sample at time 0, to obtain the plot of residual not mineralized carbon. This value includes both carbon still bound in the plastic constituents and carbon assimilated as biomass. For the sake of simplicity we will call this carbon “organic carbon”, that is, carbon that has not been converted into inorganic carbon, i.e. mineralized into CO2 yet (Fig. 3). The curves in Fig. 3 show the progressive decrease in carbon as a consequence of mineralization. Three different zones of the curves are recognised. (1) A first short, very active phase, from day 0 to about day 10 (Fig. 4); (2) a second phase, from day 10 to day 38 (Fig. 5); (3) a third phase, from day 52 onwards (Fig. 6). A linear regression analysis was carried out to determine the rates on three different zones of the curves. The regression analyses constants are reported in Table 4. All regressions had R-squared > 92%. Rates are reported as positive values, for convenience, but should be considered as negative, as they are depletion rates. The mineralization rates (k) of each phase were correlated with the corresponding surface areas, by regression analysis. The relationship between the mineralization rates from day 0 to day 10 and surface area is well described by the double reciprocal model Y ¼ 1/(a þ b/X). No significant correlation was found for the second set of data (from day 10 to day 38). The squared-Y model (i.e. Y ¼ sqrt (aþb X) best fits the data in the third data set. The regression coefficients are shown in Table 5. The specific mineralization rates (i.e. the rates expressed as mg organic carbon/day, with reference to surface area unit) relating to the last phase (from day 52 onwards), are reported in Table 6.
Fig. 5. Organic carbon (determined from evolved CeCO2) decrease of the samples with different surface area (expressed in cm2), in the period between day 10 and day 38 of testing (zooming of Fig. 3). Each point is the mean of two replicates.
4. Discussion 4.1. Mineralization kinetics The test material shows three main, different mineralization kinetics. An initial phase (from 0 to about 6 days) is characterized by a
Fig. 6. Organic carbon (determined from evolved CeCO2) decrease of the samples with different surface area (expressed in cm2), in the period from day 52 onwards of testing (zooming of Fig. 3). Each point is the mean of two replicates. Straight lines are linear regressions determined considering the period spanning between about 400 mg-C and 200 mg-C organic carbon.
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Table 4 Mineralization rates k (mineralized organic carbon, mg/day) as determined by regression analysis. Sample (total surface area,cm2)
Day 0 e day 6
Day 10 e day 38
Day 52 onwards
15 102 229 756
2.55 10.62 11.94 12.76
2.38 2.12 2.13 2.43
0.76 0.86 1.60 2.60
Table 5 Regression analysis of the mineralization rates and the surface areas, in the periods from day 0 to day 6, and from day 52 onwards. Period considered
Applied regression model
Intercept (a)
Slope (b)
R-squared
From day 0 to day 6 From day 52 onwards
double reciprocal Y ¼ 1/(a þ b/X) squared-Y Y ¼ sqrt (aþb X)
0.0605 0.008627
4.946 0.290
99.50 98.83
Table 6 Specific mineralization rates (k) per unit surface area (organic carbon, mg/day/cm2) from day 52 onwards. Sample (total surface area, cm2)
specific mineralization rate (k/cm2)
15 102 229 756
0.0506 0.008431 0.006987 0.003439
CO2 spike, a fast and short mineralization phase, which is very likely due to the release of soluble constituents leaking from the solid plastic particles by diffusion. The polyalcohol used as a plasticizer of starch is known to be a readily biodegradable substance, and thus is the best candidate. The samples reached the same mineralization level (of about 15%) with different rates, in good agreement with the respective surface area (Fig. 4). The mineralization is a function of the surface area, as shown by the regression analysis (Table 5). After that period, a slower biodegradation phase follows, where all samples display superimposable curves, independently of the surface area (Fig. 5). This second phase lasts until about day 50, and reaches a total mineralization level of 30%. The lack of correlation between the rate and surface area suggests that the ongoing mineralization is caused by the turnover (self-biodegradation induced by starvation) of the biomass formed in the first 10 days of biodegradation. This second phase seems to be a sort of lag phase, during which the conditions for the subsequent degradation phase are still to be established. A hypothesis is that this lag phase corresponds to the formation of a biofilm covering the solid particles needed for efficient enzymatic erosion [17]. More studies are needed to prove this hypothesis. Subsequently, in the third phase, which starts around day 50, the different samples diverge, with rates that are correlated with the available surface area, as shown by the regression analysis. This is probably the moment when the polymers are effectively eroded and the speed of the process is clearly affected by the available surface area. A squared-Y model describes the relationship between mineralization rates and surface areas in the third phase. The squared-Y model implies a progressive decrease in dependent variable Y (rate), with increasing values of independent variable X (surface area). This suggests that some factor progressively limits the mineralization rate with higher surface areas. This is in line with the behaviour found in the previous study with PBSe, where the best fit was a double reciprocal model, which enables the identification of a maximum rate reached at a given surface area.
The regression model allows us to make some very interesting considerations. If it were possible to mill the test material to form 100 nm diameter spheres, we would get a total surface of 234,375 cm2/g, the density of the test material being 1.28 g/cm3. The mineralization rate that corresponds to 234,375 cm2 is about 45 mg C/day, and can be calculated by applying the model found with the regression analysis (Y ¼ sqrt (0.008627 þ 0.29014 X). With this mineralization rate, phase 3 would last only about 8 days. Phase 1 (mineralization of soluble constituents) would be reduced to about 3 days, with a mineralization rate of 16.4 mg C/day. It is difficult to predict the effect of very high surface areas on the lag phase (phase 2). In any case, the overall model predicts extremely fast biodegradation under these conditions, so that total mineralization could theoretically be reached in less than 10e20 days. The results confirm that the mineralization rate at the surface level, i.e. at the molecular level, is very fast, which is in agreement with the findings of our previous work carried out with PBSe.
4.2. Specific mineralization rates The plastic material tested in this work displays compliance with the EN 17,033 biodegradability requirement [16] in less than 1 year, when tested at 754.7 cm2/g, while the sample at 15 cm2/g is very slow and will probably fail the test after 2 years (Fig. 2). Thus, the same material is considered “quickly” biodegradable or “slowly” biodegradable, depending on the granulometry of the tested sample. The reason of this paradox is that the evolved CO2 is based on the biodegradation that effectively occurs at the surface of the plastic granules, but it is then related to the mass of the sample being tested, most of which is not involved in the biodegradation reaction. The evolved CO2 is divided by the theoretical CO2 (ThCO2), i.e. the CO2 evolved in case of total oxidation of the organic carbon present in the test material. If the mineralization rate value is to remain unaffected by the dimension of the tested sample, the evolved CO2 must be related to the surface area. The specific mineralization rates (k) per unit surface area (mg organic carbon/day/cm2) of the test material is estimated to be in the range 0.0506 to 0.0034 mg organic carbon/day/cm2. This value was calculated for the last and longest biodegradation phase, which presumably mainly involves the polymeric constituent. The value of the pellets is higher than the value of the milled particles. At this point in time, we do not have sufficient data to judge whether or not this difference is significant. The specific mineralization rate can be used to make an environmental fate prediction, and can be used in product design.
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5. Conclusions
References
The biodegradable plastics sector is developing fast, and inquiries for more detailed information on environmental characteristics are also on the increase. A relevant characteristic of biodegradable plastics is the biodegradation rate because this is a parameter that is necessary to predict the environmental fate. In this work, we tested the biodegradation in soil of a complex multiconstituent plastic product, identifying different kinetics that can be tentatively ascribed to the different constituents. A relationship between the biodegradation rate and the available surface area was recognised for the plastic product, and was similar to what had already been shown for a neat polymer [10]. The specific mineralization rate of the material tested was determined. This parameter can be used to compare different materials, to predict mineralization times, and perform risk assessment studies. The results show that the biodegradation process that occurs at the interface is very fast. If it were possible to mill the test material, to obtain nanoplastics, the biodegradation would be extremely fast, lasting 10e20 days at maximum, with a time frame similar to that required by the OECD for the classification of “readily biodegradable” chemicals.1 Therefore, the chemical permanence time of the test material is very short. On the other hand, the physical permanence time depends on the surface area, i.e. on the thickness of the plastic items. The results suggest that biodegradable plastics do not generate persistent microplastics, because as erosion increases the surface area, this in turn increases the biodegradation rate to levels similar to those required, by the OECD, for chemicals to be defined as readily biodegradable.
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Acknowledgements Thanks to Paolo Magistrali and Selene Chinaglia for the support provided in the use of the particle sizing instrument. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.polymdegradstab.2019.05.034. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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