Composting is a dynamic and robust process whose efficiency is well established for the treatment of a wide range of organic waste52. The biodegradation process that takes place in compost is primarily carried out by its microbiome, i.e., a community of microorganisms that includes mesophilic and thermophilic fungi, bacteria, and actinobacteria12. These microorganisms have evolved different enzymatic and non-enzymatic strategies52. The rate of biodegradation is related to abiotic factors, which include properties intrinsic to the chemical composition of the test material and its molecular and supramolecular structure, which will be discussed later. It also correlates to the environmental conditions specific to the composting process (e.g., aeration, temperature, pH, aw, particle surface area, and C/N ratio)12. Bio-sourced materials made of complex biopolymers such as lignocellulose or modified starch are biodegraded (composted) in a multi-step process. Initially, depolymerization takes place, i.e., enzymatic hydrolysis of cellulose or starch chains via, e.g., endo- and exo-glucanases53 and/or enzymatic oxidation of the lignin backbone via, e.g., manganese or lignin peroxidases54,55. Once the resulting oligomers, dimers, or monomers are released, they can be transported into the microorganisms56. Finally, mineralization follows, ending with the production of H2O and CO212.
Knowing that, in aerobic conditions, CO2 is the ultimate end product of the microbial respiration of organic substrates, the quantification of their level of biodegradation, once mixed or buried in active compost, can be directly assessed by measuring the total amount (cumulative) of CO2 respired over time45. To achieve this, several experimental procedures have been recognized by the scientific community57 and one of them has been adopted by ASTM58. ASTM D5338 is a detailed procedure that also describes the setup of the apparatus necessary for the rapid and reproducible determination of aerobic biodegradability under controlled composting conditions.
The results of our biodegradation tests on organic electronic materials carried out under mesophilic conditions at 25 °C revealed a mineralization rate of 4.1 ± 0.7% for Sepia Melanin, i.e., a rate 17× lower than 71.2 ± 0.2% for microcrystalline cellulose, the positive control (Fig. 2c and Table 1). Under thermophilic conditions (58 °C), the mineralization of Sepia Melanin reached a level 9× higher than that observed at 25 °C, still 2.6× lower than the 98 ± 6% mineralization level for cellulose (Fig. 3c and Table 1). Data show that the rate of melanin degradation increases with increasing incubation temperature (i.e., temperature coefficient Q10 = 1.9559), similarly to other biodegradation or bioremediation case studies. Further, they confirm the role of the thermophilic microbial community. Not only does the incubation temperature have an effect on the rate of melanin biodegradation because of its direct effect on the enzymes involved in the biodegradation but it also applies selective pressure on the microbiota of the compost that produces those enzymes. The two conditions used during the tests (25 °C and 58 °C) made it possible to select a community of mesophilic (optimal growth temperature is in the range 15–42 °C) or thermophilic (optimal growth temperature 42–70 °C) microorganisms60,61. Thermophilic microorganisms produce thermotolerant enzymes62,63 and exhibit higher respiration rates than mesophilic microorganisms, thus contributing to explain the significantly higher biodegradation rate observed at 58 °C. The fundamental difference between mesophilic and thermophilic organisms in terms of respiration rate at their respective optimal growth rate is well established in microbial ecology and it was noticeably confirmed by our observations of the blank compost incubated in those two temperature ranges (“Characterization of Blank Compost” in SI).
In order to explain the high degradation levels of cellulose (71% at 25 °C and 98% at 58 °C), it must be considered that the compost we used was from a facility dedicated to the biodegradation of lignocellulosic biomasses. On the other hand, the growth kinetics and the structure of the mesophilic microorganisms community compared to the thermophilic one as well as the intrinsic biodegradation-hindering factors of eumelanin (structural and chemical disorder, as will be discussed later) could explain its recalcitrance to biodegradation at 25 °C and its moderate biodegradation level at 58 °C (i.e., 4.1 and 37%, respectively). Further microbiological tests are needed, to assess the community structure of our compost. It is worth noting that the fungus Aspergillus fumigatus that biodegrades eumelanin at room temperature can tolerate environments of 50 °C and above64.
Although the biodegradation level under composting conditions (37% in 98 days) is significant, it does not meet the current industrial requirements for its labeling as biodegradable under composting conditions (ASTM D6400). As a matter of fact, even if we project the biodegradation level at the upper time limit suggested by ASTM D6400, 180 days, using the 0.26%/day mineralization rate observed during the incubation period corresponding to Δt35–98, Sepia Melanin would reach 58%, still not fulfilling the 90% threshold of biodegradability in composting conditions.
Our results suggest that the intrinsic characteristics of eumelanin, its molecular and supramolecular structure65 as well as its hygroscopicity21, affect the extent of its biodegradation by the compost microbiota. It is well established that eumelanin structure, rather than being composed of linear homopolymeric chains (e.g., cellulose and starch), is based on the oligomeric DHI and DHICA monomers (Fig. 1)65. The complexity of eumelanin structure (chemical disorder) also stems from the fact that oligomers differ from each other in the number of units, types of units (DHI vs. DHICA), and sites of polymerization (similarly to lignin whose supramolecular structure emerges from the assembly of three monomeric crosslinked monolignols: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol)66.
Eumelanin oligomers organize in a complex supramolecular structure: the oligomeric sheets form protoparticles via π–π stacking. The protoparticles arrange in an onion-like structure, densify into spherical particles (about 10 nm-sized) and eventually undergo aggregation into larger spherical particles (ca. 100 nm-sized)65. Eumelanin particles feature heterogeneity in supramolecular structure and size. In other words, the chemical disorder of eumelanin is paralleled by structural (physical) disorder22.
The chemical and structural disorder may limit the biodegradability of eumelanin, since not all oligomeric planes are exposed to the depolymerizing enzymes secreted by the microbiome and diffused in the compost. The monomer-monomer bonds may undergo enzymatic degradation only from the “external” oligomers (i.e., on the surface) of eumelanin particles. This might explain the observed low rate of biodegradation. It has been suggested that the “opening” of the supramolecular structure, i.e., the de-stacking of the oligomers, is a necessary step for biopigment degradation67.
As Sepia Melanin is a heterogeneous substrate, we hypothesize that its biodegradation requires several types of extracellular enzymes to achieve complete biodegradation, via a mechanism very similar to the enzymatic oxidation of lignin, based on manganese peroxidase (Mn–P) and lignin peroxidase (Li–P)54,68. Moreover, a type of enzyme able to act on one type of eumelanin oligomer may have a different affinity towards different oligomers33, whereas a non-biodegraded oligomer will limit the exposure to extracellular enzymes of all the oligomers stacking beneath it.
Furthermore, eumelanin is known to be a hygroscopic material21. The extracted Sepia Melanin absorbs 17 wt% of H2O in 1 h at 90% relative humidity (Supplementary Fig. 2, Supplementary Table 1). We already reported that in 24 h Sepia Melanin can absorb a water quantity equal to its weight21. Knowing that the compost used contained a significant amount of water, equivalent to 50% of its initial mass, it is reasonable to assume that hydration of Sepia Melanin took place in the bioreactor, which could have contributed to weakening the high chemical and physical stability level of melanin, thereby allowing its biodegradation. Indoles are indeed known to be microbially degraded with the indole ring cleavage following a pathway that ends with the formation of fumarate and pyruvate69. In addition, water would solubilize the resulting monomers or the low molecular weight biodegradation products, which could then diffuse into the microbial cells to be metabolized70. Consequently, biopigment hygroscopicity favors biodegradation.
Seedling emergence and plant biomass thresholds established for the terrestrial phytotoxicity tests were attained and surpassed by Sepia Melanin (Table 2). Thus, our ecotoxicological results show that the “residues” of the partially mineralized pigment are not phytotoxic. Although L-dopa, the precursor of melanin, shows phytotoxic effects due to the formation of reactive oxygen species and/or free-radical species during melanogenesis71, others report that fumarates resulting from the biodegradation of indoles69 would not be phytotoxic72.
The apparent respiration activity of Cu–Pc blended with compost initially (Δt0–50) falls within the range of the blank compost, but then, after 50 days, it becomes lower, which implies that there was no net CO2 production with respect to the blank compost and, consequently, no biodegradation (Fig. 4). This trend could be explained with a partial release of Cu ions by the synthetic molecule: such cations are able to inhibit microbial activity73. The molecular structure of Cu–Pc presents a planar aromatic macrocycle with the metal cation at its center74 (Fig. 1). Such a molecular structure is based on indoles, like that of eumelanin. However, in Cu–Pc, the four indole units do not include catechol groups and are not directly chemically bound to each other74 (Fig. 1). The absence of biodegradation of Cu–Pc could also be due to inhibition of microbial metabolism by the released copper cations and to resistance to enzymatic hydrolysis of the nitrogen-including bonds between the indole units73. The π–π stacking of the molecules of Cu–Pc and their insolubility in water may represent additional biodegradation-hindering factors74. Prior to this study, no phytotoxicity test had been conducted with Cu–Pc, although the phytotoxicity of several other phthalocyanines has been evaluated75. Following the biodegradation test, the “residual” Cu–Pc in the compost shows no phytotoxic effects (Table 2).
The apparent respiration rate of PPS blended with compost falls within the range of the blank compost over the entire incubation period (Δt0–98) (Table 1 and Fig. 4). Consequently, no mineralization took place. The chemical bond between a sulfur atom and a benzene ring in a synthetic polymer appears to be resistant to the action of numerous extracellular hydrolytic enzymes present in the compost under thermophilic conditions (Fig. 1). PPS does not pass the thresholds for two phytotoxicity tests; it limits both the emergence of seedlings and the growth of plants. This points to the potential phytotoxicity of the polymer (Table 2).
We wish to emphasize that “bioresorbability” does not equate to “biodegradability”. The concept of bioresorbability applies to transformations in an aqueous physiological environment (mainly in vivo), while biodegradability refers to biotic processes catalyzed by enzymatic reactions within communities of microorganisms active in their environment or ecosystem48,76. For example, Bettinger et al. report that synthetic melanin implants were almost completely resorbed after 56 days of in vivo exposure31, which differs significantly from the experimental ecosystem we have used to observe the biodegradation of Sepia Melanin in the compost after 98 days at 58 °C. Although incomplete, our mineralization result (i.e., 37%) reveals an average specific degradation rate of 0.63 g melanin per day per kg dry weight compost.
Abundant and biodegradable organic (carbon-based) electronic materials and devices represent one of the viable ways of reducing the environmental footprint of the electronic sector. At present, no guidelines for testing the biodegradability of organic electronic materials and devices are available. Green organic electronics have access only to a test protocol specifically dedicated to the certification of compostable plastic materials. Our work is the first of its kind in the development of new guidelines and test protocols.
We found that eumelanin, an organic electronic bio-sourced material, attained a mineralization level of 37% after 97 days under composting conditions (at 58 °C); plant seedling and germination tests revealed that the residual material at the end-point of the composting test did not exhibit phytotoxic effects. We also tested the biodegradability in composting conditions of two non-bio-sourced (synthetic) organic electronic materials, namely copper (II) phthalocyanine, Cu–Pc, and poly(1–4)phenylene sulfide, PPS. The conjugated molecular structures of Cu–Pc and PPS feature C=C bonds (for Cu–Pc and PPS) and C=N (for Cu–Pc) bonds as well as the absence of oxygen atoms, characteristics that help explain the negligible mineralization levels observed. Finally, Cu–Pc inhibited the respiration of the microorganisms while PPS showed potential phytotoxicity. These differences suggest that bio-sourced materials constitute a viable option for the eco-design of organic electronic devices.
We wish to point out that CO2 monitoring is an “acceptable” analytic method for assessing the biodegradation of a given material buried in a substrate as complex as compost. Nevertheless, the method does not provide information about the material’s half-life and the identification of the microbes responsible for biodegradation. A series of tests including radio-respirometry, extraction of the residual test material, and in vitro tests using commercially available enzymes or isolated microbes are deemed necessary to properly describe the mechanism of biodegradation. Such tests are not part of ASTM D5338.
Further studies exploring other types of compost, e.g., from manure or garden waste, that feature different microbial communities, or other types of compost facilities, e.g., home composting for which different standards exist, are needed to identify the most suitable conditions for eumelanin’s biodegradability. In addition, to accelerate the degradation process inoculation and biostimulation are envisaged and can be adopted since microbial populations can evolve and derive their energy from an array of chemicals of different origins77. Work is in progress towards the evaluation of the biodegradability of organic electronic devices, beyond the constituent materials.