Biodegradation of bio-sourced and synthetic organic electronic materials towards green organic electronics

Ubiquitous use of electronic devices has led to an unprecedented increase in related waste as well as the worldwide depletion of reserves of key chemical elements required in their manufacturing. The use of biodegradable and abundant organic (carbon-based) electronic materials can contribute to alleviate the environmental impact of the electronic industry. The pigment eumelanin is a bio-sourced candidate for environmentally benign (green) organic electronics. The biodegradation of eumelanin extracted from cuttlefish ink is studied both at 25 °C (mesophilic conditions) and 58 °C (thermophilic conditions) following ASTM D5338 and comparatively evaluated with the biodegradation of two synthetic organic electronic materials, namely copper (II) phthalocyanine (Cu–Pc) and polyphenylene sulfide (PPS). Eumelanin biodegradation reaches 4.1% (25 °C) in 97 days and 37% (58 °C) in 98 days, and residual material is found to be without phytotoxic effects. The two synthetic materials, Cu–Pc and PPS, do not biodegrade; Cu–Pc brings about the inhibition of microbial respiration in the compost. PPS appears to be potentially phytotoxic. Finally, some considerations regarding the biodegradation test as well as the disambiguation of “biodegradability” and “bioresorbability” are highlighted.

Both weakly and strongly bound water can be present in Sepia Melanin after extraction 4,5 . Consequently, the dry solids (residual mass after heating the material at 105 °C until constant mass is reached) were quantified by means of a TGA. This measurement entailed a temperature ramp of 10 °C/min up to 105 °C, then an isothermal step at 105 °C, until the derivative (DTG) was less than 0.01 %/min in Ar atmosphere.
Thermogravimetric analyses provide insight into the thermal decomposition of a material. In particular, the minima of the DTG represent the completion of a thermogravimetric phenomenon and its maxima represent the maximum rate at which a thermogravimetric phenomenon takes place 5 Table 1) 5 follows. At 500 °C, the mass loss is 60% wt. in both cases. The hygroscopicity was studied comparing the TGAs at two relative humidity (RH) levels, 25% and 90%. The mass loss at the first DTG minimum gives an approximate but comparable estimation of the difference in the amount of weekly bound water absorbed at a certain RH level 5 . The thermogravimetric results suggest that SM-C retains more weakly bound water in its supramolecular structure than SM-E, since the observed water loss from SM-C is 5-6% more by weight (Supplementary Table 1).

Supplementary Figure 2.
Thermogravimetric analysis (TGA) of SM-E and SM-C (a) without any hydration treatment and (b) after 1 hour at 90% RH in inert N2 and Ar atmosphere, respectively. Briefly, the opening of each glass bioreactor is occupied by the above-mentioned CO2 trap, which is closed by a two-compartment electrolytic cell containing 25 mL of 1 N H2SO4 and a three-electrode cap (i.e. anode, cathode, and switch level). The compartment containing the anode is "exposed" to the pressure of the bioreactor's headspace and the other compartment containing the cathode is "exposed" to the pressure of the atmosphere. Noticeably, as the microorganisms present in the compost respire, oxygen (O2) in the bioreactor's headspace is being used, which causes a pressure drop; with respect to the external pressure it corresponds to ΔP.

Supplementary
This ΔP activates the electrolytic cell by means of a switch electrode, i.e. water electrolysis. Consequently, at the cathode H2 is produced and vented to the atmosphere, whereas at the anode O2 is produced in the bioreactor's headspace until ΔP = 0. This mode of operation allows a discrete measurement of the O2 consumed over time. Using Faraday's law, from the power provided to the electrolytic cell, the moles of O2 produced can be computed. Further details of the working principle of the electrolytic respirometers can be found in reference 7 . The software BI2000 (modified ad hoc for the National Research Council Canada) was used to collect the data.

Monitoring CO2 Evolution Using Wet Scrubbers
To monitor the microbial respiration of the tested materials, the CO2 evolved under thermophilic conditions was monitored using an indirect method, cumulative measurement respirometry. All CO2 produced, regardless of origin, was captured. Throughout the entire duration of the experiment, by means of an air compressor, 50 mL per minute of humidified air was "pushed" into each bioreactor and circulated within the compost to bring O2 to the aerobic microorganisms and to release respired CO2. Three traps filled with a Ba(OH)2 solution were placed in a series after each bioreactor. As air came into the bioreactor, the same volume of air came out, not to the atmosphere but to a wet scrubber unit (i.e. a series of three 1-L bottles each one filled with 900 mL of 0.12 M Ba(OH)2 solution), to precipitate all CO2 respired.
The solution is able to capture CO2 as the reaction (S1) takes place: At regular intervals, liquid samples taken from the 72 scrubbers [i.e. 8 bioreactors x 3 scrubbers/bioreactor x 3 (triplicate)] were titrated to calculate respired CO2. During the titration, the remaining Ba(OH)2 that has not reacted with CO2 is neutralized with HCl 0.1 M: From the molar ratios of equation (S2), at a certain incubation time t, it can be inferred that: Once a week, all scrubbers were replenished with freshly prepared Ba(OH)2 solution to ensure full efficiency. The bioreactors (two replicates) containing the active compost were run specifically to monitor the background respiration level: essentially, [CO2 production] -[CO2 assimilation] = actual or observed CO2 production. The background respiration was then subtracted from the material's respiration data to obtain the net respiration level for each test material. Consequently, this procedure, based on the equation [test] -[background] = net [CO2 production], allowed us to calculate the biodegradation level based on its carbon (C) content.

Characterization of Blank Compost
The compost, organic fraction of municipal solid waste, was kindly provided by the company GSI Environnement, subsidiary of Englobe (Québec, Canada). Its characteristics satisfied the requirements of ASTM D5338: • sieved with a 10-mm mesh, • ash content 51.4% over dry weight, • dry solids 46 ± 4 % over wet weight, • C/N ratio 24.3 with %C = 50% organic matter over dry weight, and • water content 50% over wet weight.
The figure of merit that indicates respiration activity of the compost falls within the acceptable range (i.e. fresh compost) is represented by the specific respiration rate, i.e. CO2 evolved per g of volatile solids per day, mg CO2/(g volatile solids • d). Consequently, the specific respiration rate of the compost was measured both under mesophilic (25 °C) and thermophilic (58 °C) conditions with ad hoc tests, before the main tests that involved the materials of interest.
Under thermophilic conditions, the specific respiration rate was assessed deploying 6 bioreactors filled with 1 kg of blank wet compost and incubated for 11 days at 58 °C. The CO2 evolved was followed using wet scrubbers (working mechanism in "Monitoring CO2 Evolution Using Wet Scrubbers"). The result was 10.5 ± 0.1 mg CO2/(g • d), and, consequently, fell within the acceptable range set by ASTM D5338, i.e. 5 -15 mg CO2 evolved per g volatile solids per day, in the first 10 days.
Under mesophilic conditions, the specific respiration rate was measured deploying 7 bioreactors of 1.2 L filled with 100 g of blank wet compost and incubated for 7 days at 25 °C. As described above, the O2 consumed was monitored by means of seven electrolytic respirometers. Then, CO2 evolved was computed using the O2 consumed data, assuming the respiratory quotient CO2 respired / O2 consumed to be 1.0 mol/mol 11 (working mechanism in "Monitoring O2 Consumption using Electrolytic Respirometers").
Therefore, the average respiration rate was 1.4 ± 0.1 mg CO2/(g • d). This respiration rate, 8x lower than the rate measured under thermophilic conditions, is in agreement with the difference in the respiration rate between the two temperature ranges already reported in the literature 12 .

Biodegradability Test Under Mesophilic Conditions
For the biodegradability test under mesophilic conditions (25 °C), bioreactors of 1.2 L were filled with 100 g of wet compost. A total of 7 bioreactors were used: two of plain compost (blank), two with cellulose blended with compost (positive control) and three with Sepia Melanin blended with compost. The weight ratio of material to dry compost was 1:6, as for the biodegradability test in composting conditions (ASTM D5338). Electrolytic respirometers (designed by Young et al. 6 , model BI-2000, Bioscience Inc.) were used to follow the O2 consumption (working mechanism in "Monitoring O2 Consumption using Electrolytic Respirometers"). The CO2 evolved was computed using the O2 consumed data, assuming the respiratory quotient CO2 respired / O2 consumed to be 1.0 mol/mol 11 .

Biodegradability Test in Composting Conditions
The standards ASTM D6400 and ASTM D5338-2015 define the terminology and the procedure to assess aerobic biodegradability of a plastic material in composting conditions 13,14 .
In accordance with ASTM D5338, the respired CO2 was monitored for 98 days. The Sepia Melanin blended with compost and the blank compost were tested using duplicates. The Cu-Pc, PPS, microcrystalline cellulose (positive control) and polyethylene (PE, negative control) blended or buried in compost were tested using triplicates. In each of the 16x 6-L bioreactors, 250 g of wet compost was present; and the materials were added to the compost in a dry weight ratio of 1:6. All bioreactors were kept in darkness at 58 °C in a controlled temperature chamber (Caron Stability Chamber) and continuously fed with air (flow of 32 ± 5 mL/min). The air passed through a stainless-steel aerator installed at the bottom of a 50-cm-high water column to be humidified before reaching the bioreactors.
Starting on day 47, the 16 bioreactors were opened once a week. Each of them was weighed and the difference between this mass and their respective initial mass was attributed to water loss. Consequently, DI water was added to keep the dry weight at or slightly below 50% wt. On average, the DI water added weekly was approximately 30 mL. While adding water to the compost, it was carefully mixed by hand. Then, each of them was reconnected to the bioreactors' air inlet and outlet.
The respired CO2 was determined using an indirect measurement method, i.e. cumulative measurement respirometry (working mechanism detailed in the paragraph "Monitoring CO2 Evolution using Wet

Mineralization
The percent of carbon by weight used to compute the mineralization levels (denominator of equation (2) of the main text) can be inferred from the molecular formula of the material, with the assumption that the tested materials are pure (absence of water or inorganic matter). In practice, the extraction process could leave water or inorganic matter (i.e. salts) as residues. Water could also be absorbed in the storage environment.
Consequently, we validated such theoretical computations experimentally. The dry solids, the ash content Where: • smat is the standard deviation of the average measured mass of CO2 evolved from the bioreactors containing the test material blended with the compost (average CO2 (material) of equation (2) of the main text); n1 is the number of such bioreactors, • sblank is the standard deviation of the average measured masses of CO2 evolved from the bioreactors containing blank compost (average CO2 (blank compost) of equation (2) of the main text); n2 is the number of bioreactors containing blank compost.
• CO2 (total) is the theoretical mass of CO2 that would evolve if all the test material were completely respired by the microorganisms (equation (3) of the main text). Note :

Supplementary
The amount of Cu-Pc added was chosen taking into consideration that the dye content in the material as received was 90% by weight, with the remaining 10% being inorganic, as stated by Sigma-Aldrich. N/A, not available; N/D, not determined.
Prior to seeding the sandy soil, its water content was adjusted to 8.3% wt., which is 75% of its water holding capacity. Water content of compost samples, which were recovered from bioreactors at the end-point (98 d) of the biodegradability test under thermophilic conditions (58 °C), was adjusted to 40% wt. Secondly, 77 g of the wet sandy soil was mixed with approximately 23 g of compost + test material, that is a 3.3:1 weight ratio. Twenty (20) seeds of ryegrass (Lolium perenne) were sown in 15-cm-wide dishes (TC dish, Sarstedt #83-3903) containing 100 g of wet sandy soil with compost + test material. Such TC dishes were incubated in sealed plastic bags to ensure maintenance of the soil moisture throughout the duration of the germination test.
The phytotoxicity test was conducted on six samples collected at the end of the composting tests and on two subsamples of wet sandy soil, refrigerator stored at 5 °C. The six compost samples are: (i) blank compost, (ii) compost + PE, (iii) compost + cellulose, (iv) compost + Sepia Melanin, (v) compost + PPS, and (vi) compost + Cu-Pc. The two soil samples are: a 100-g soil sample and a 220-g soil sample. The result of an inter-experiment control test showed that the use of 100 g or 200 g of a control soil did not influence the end results of a seedling emergence test as we report 93% seedling emergence compared with our previous works 16 . These results are statistically equivalent, therefore a 100-g soil sample can be taken as the reference for the other samples.

Statistical analysis
The analysis of variance (ANOVA) test was carried out to compare the means of • X is an individual observation, • ̅ is the sample mean of the j th group, • ̅ is the overall sample mean, • k is the number of groups, • N is the total number of observations.

O2 consumed
• k=3; N=7 F > F critical value, so the null hypothesis is rejected.