Poly(1,4-butylene succinate), extended with 1,6 diisocyanatohexane (PBS, Aldrich Chemical Co., Japan) was used as received. Cellulose powder of thin-layer chromatography grade with a particle size of less than 20 μm (cellulose microcrystalline; Merck, Germany), soda talc (sodium hydroxide on support, granulated to about 1.6–3 mm; Merck, Germany), soda lime (sodium hydroxide on support, small granules of about 1.5–3 mm; Kanto Chemical, Japan) and sea sand (sand washed, 425–850 mm; Wako Pure Chemical, Japan) were used as received.

The PBS powder was prepared from polymer pellets (ca. 5 mm) [22,23]. The polymer pellets were crushed using a rotating mechanical mixer with titanium blades (10,000 rpm, 3 L) with cooling by dry ice. Crushing was done 15 times for 3 min each with a 5-min interval to prevent overheating of the motor in the mixer. After drying under reduced pressure at room temperature, the PBS powder was separated using sieves of 60 mesh (250 μm) and 120 mesh (125 μm). Standard sieves with a guarantee were used. These sieves with the crude polymer powders were placed on a sieve vibrator and vibrated for 15 min. The average size of the obtained PBS powders was 157.8 °m determined by the average of at least 100 particles in photographs by optical microscopy. This PBS is petroleum-based polymer. Therefore, the pMC of this PBS powders was 0% by AMS.

The controlled compost (YK-6, Hissan Trading Co., Ltd., Japan) for the Microbial Oxidative Degradation Analyzer (MODA) based on ISO 14855-2 was prepared as follows. The waste material of used wood block for growing mushrooms and chicken droppings was composted for seven months. During this period, a mature compost was prepared. After preparation, this obtained compost was sieved using a 4.7 mesh (4 mm), kept at room temperature and prevented from drying. The properties of this compost are shown in Table 1. This compost can presently be kept at room temperature for a long time, at least four years.

Before using this compost, an activation step (preincubation) was required to recover the biological activities for the respiration and biodegradation by the microorganisms. The controlled compost was prepared by mixing 144 g of compost (60 g total dry solids) and 320 g of sea sand and its water content was controlled to over 80 wt% (the weight of the volatile solids of this compost without sea sand). This amount was for one reaction vessel. Preincubation was done once for the total amount of blanks and samples using a large container (5 L). Sea sand was added to create good homogeneous conditions and a better aerobic environment inside the compost. This compost for the activation step was mixed once a day, and the water content was adjusted to 65 wt% for 7 days at 58 °C.

A biodegradation test was performed using the MODA apparatus (Hissan Trade Co., Ltd., Japan) in a controlled compost at 58 °C as shown in Figure 2. Two sample reactors were used in the same MODA apparatus test, one without Ba(OH)₂ trap bottle (to carry out a traditional ISO 14855-2 test), while the second sample reactor was equipped with the trap system for the percent modern carbon (pMC) determination by accelerator mass spectrometry (AMS) method as detailed in Section 2.3. The polymer sample powder (10 g) was well mixed in the activated compost (144 g) with sea sand (320 g) and transferred to each sample reaction vessel. Compost with no sample was used as a blank to determine the respiration activity of this compost during the test period under the same conditions as in the sample vessel. The biodegradation tests were performed at 58 °C and a 10 mL/min air (CO₂-free) flow rate for 74 days. The activated compost used in this study produced 84.6 and 89.5 mg CO₂ per gram of volatile solids over the first 10 days as determined for two blank vessels, respectively. In almost all cases, the number of experimental replicates of the blank or sample was two (duplicate). For the AMS measurement, only one sample vessel was used. The produced CO₂ amounts were measured once a day by measuring the weights of the absorption column for carbon dioxide and the absorption column for water. The percent of biodegradation was calculated from the produced CO₂ amount using the subtracted respiration CO₂ amount determined from a blank and the theoretically produced CO₂ amount of the added sample. For example, 10 g of PBS produced 20.47 g of CO₂ which was the theoretical amount for the 100% biodegradation. As recommended by ISO 14855-2, once a week, the sample and compost were well mixed, and the water content was controlled. When the absorbed CO₂ amounts for the absorption columns reached 40% of the theoretical absorption capacity, and the chemicals (soda lime and soda talc) inside the columns were changed.

To determine the percent modern carbon (pMC) of evolved CO₂ produced during the biodegradation of PBS by accelerator mass spectrometry (AMS), the produced CO₂ was trapped as BaCO₃ in a Ba(OH)₂ aqueous solution (0.08 N, 500 mL in a 1 L gas-tight glass bottle with a bubbling tube) between the reaction vessel and the ammonia trap in the MODA apparatus as indicated in Figure 2 based on ISO 14855-2. The produced CO₂ from the reaction vessel including the sample and compost reacts with Ba(OH)₂ and is converted to BaCO₃. BaCO₃ is insoluble in the Ba(OH)₂ aqueous solution and precipitates. After the determined test period (3–14 days) for pMC (sample), the Ba(OH)₂ aqueous solution is changed to a fresh one. To determine the pMC (compost) of the produced CO₂ from the blank reaction vessel including no sample and the compost, BaCO₃ was collected using the Ba(OH)₂ trap bottle in the MODA apparatus during the 0–3-day test period. The precipitated BaCO₃ was well mixed by gently shaking the trap several times and collected by filtration under reduced pressure. The solution bottles were tightly closed before filtration, and the filtration step was quickly carried out to avoid any additional reaction with CO₂ in the room atmosphere. The collected BaCO₃ was freeze-dried under reduced pressure at room temperature and weighed. This dried BaCO₃ was used for the determination of pMC by AMS.

The sample preparation and measurements were done at the Institute of Accelerator Analysis, Ltd. (IAA), Japan. All carbon atoms of the collected BaCO₃ samples were transformed into graphite carbons through serial vaporization and reduction reactions using a gas-tight glass tube with a closing stopcock which can be connected to a vacuum manifold system as indicated in Figure 4 for the pMC measurement of the BaCO₃ powders by AMS. Liquid H₃PO₄ (5 mL) was poured into the left round-bottom portion of the gas-tight glass tube, and BaCO₃ (20 mg) was put into the right round-bottom section. This tube was connected to the vacuum manifold line under reduced pressure (<10⁻² mbar) for 24 hours at room temperature to remove any remaining water in the H₃PO₄ liquid. The H₃PO₄ liquid was slowly transferred to the right bottom including the BaCO₃ powders. Additional liquid was transferred if the reaction was not active. After all the liquid was moved, this tube was incubated in a hot water bath (ca. 90 °C) for 1 hour. Subsequently, the CO₂ and H₂O were cold-trapped into another tube using dry ice-ethanol (−76 °C) connected to the closed vacuum line system. The cold-trap step was repeated twice. Only pure CO₂ was cold-trapped in a quartz tube with pure ferrous powder, using liquid nitrogen (−196 °C) from the reactants such as CO₂, and H₂O in another tube under dry ice-ethanol. This CO₂ with hydrogen and the ferrous powder was reduced to graphite at 650 °C for 10 hours. After these processes, pure graphite with oxidized iron (1 mg) was transferred to a sample holder (small rod shape; 1 mm hole) as indicated in Figure 4 (right-bottom picture).

The measurement of the ratio of the three carbon isotopes (¹⁴C, ¹³C and ¹²C) using Accelerator Mass Spectroscopy (AMS) was performed at IAA as outlined in Figure 5. The carbon in graphite, transferred from the BaCO₃ samples, was ionized using a cesium cation beam. The anionized carbons were accelerated using a 3MV tandem accelerator (NEC Pelletron, 9SDH-2). The accelerated carbon isotopes were separated by an analyzing magnet based on the different atomic masses. The amounts of ¹²C and ¹³C were detected as a current using multi-Faraday cups. The ¹⁴C atoms were detected using a solid state detector with a semiconductor absorber. The ratio of ¹⁴C to ¹²C for the tested sample (¹⁴As) obtained from the analysis of the BaCO₃ powders was calculated from the measured amounts of ¹⁴C and ¹²C. The percent of modern carbon (pMC) for an oil-based carbon is 0%. The pMC for a biomass made by the fixation of CO₂ in the modern atmosphere through photosynthesis is 108–110%. A measurement of a product’s ¹⁴As(¹⁴C/¹²C) content is determined relative to the ratio of ¹⁴C to ¹²C for reference material (¹⁴Ar) such as the modern carbon-based oxalic acid radiocarbon (Standard Reference Material (SRM) 4990c, the National Institute of Standards and Technology, NIST, USA).

The pMC values for determining the CO₂ (respiration) were estimated by tertiary curve fitting for the measured pMC values as shown in Table 2.

The method used for the determination of the biodegradability of the PBS powders was based on the International Standard (ISO 14855-2) that measures the evolved CO₂ amount from both the blank vessel without a sample and the sample vessel including a 10 g PBS powder sample, 144 g mature compost, and 320 g sea sand.

The newly developed biodegradation measurement system using the MODA apparatus with the absorption columns is shown in Figure 2. This evaluation system for the biodegradation uses the CO₂ trap system with CO₂ absorption columns. This MODA mechanism is as follows. First, room air is passed into the carbon dioxide trap to remove the CO₂ in the air as shown in Figure 2. This air is moisturized and passed into the reaction vessel controlled at 58 °C using a thermosensor and ribbon heater. The air with the produced CO₂ from the biodegradation of the samples and respiration of the microorganisms in the compost is passed into the ammonia trap to remove the produced ammonia from the compost to obtain an accurate carbon balance using a gravimetric measurement. The air with its CO₂ is passed into dehumidifying traps to remove the moisture from the air stream for an accurate carbon weight balance and then passed into an absorption column for the carbon dioxide and an absorption column for water. In these two columns with soda lime (NaOH immobilized in flaked lime) and soda talc (NaOH immobilized in talc), the produced CO₂ is completely absorbed by the reactions indicated by Equation (2): (2) CO 2 + 2 NaOH → Na 2 CO 3 + H 2 O

The H₂O produced by this reaction is simultaneously trapped in these two columns. According to this reaction, the weight of these two columns is increased by the same amount as the weight of the produced CO₂. In this way, the produced CO₂ amount is easily obtained by a gravimetric measurement. Once a day, the weights of these two columns are measured. From the increasing weight of these two columns for the sample and blank, and the theoretical CO₂ amount, the percent of biodegradation can be calculated.

The PBS powders were almost degraded in the controlled compost at 58 °C using the MODA apparatus. The cumulative evolved CO₂ amounts of the blank and sample vessels were 11.96 and 28.88 g after a 74-day incubation time, respectively. Figure 6 shows the evolved CO₂ amounts from the blank and sample vessels. From the beginning of the test period, the difference in the evolved CO₂ amounts from the blank and sample vessels was very small indicating that PBS was not actively biodegraded during the initial period. During the first 20 days, the difference between the CO₂ from the blank and that from sample was 3.8 g, indicating that 19% of the PBS was biodegraded. The theoretical evolved CO₂ from 10 g of PBS is 20.47 g. The determined biodegradability of PBS at 74 days was 82.27% [= (28.88 – 11.96)/20.47 × 100]. Figure 8 shows the calculated biodegradability of PBS determined by the ISO and AMS methods. In this way, the PBS powder was gradually biodegraded in the controlled compost at 58 °C according to the ISO method. PBS is a biodegradable polyesters, that dose not exist in the natural environment and is an artificial copolyester with the ABAB structure (an alternating copolyester). Active degradation enzymes for PBS are available as commercial isolated enzyme [21], filamentous fungus [24] or yeast [25]. However, under general composting conditions, there is no special biodegradation enzyme for PBS as in the soil environment [26]. In this way, PBS is gradually biodegraded by general lipases and changes to CO₂ or metabolites via an oligomer or monomer such as 1,4-butanediol and succinic acid of PBS.

For the biodegradability determination of a sample based on the International Standard, the evolved CO₂ amount from a blank vessel including the compost inoculum is necessary, as already described, because the amount of evolved CO₂ from the sample vessel includes the respired CO₂ and CO₂ biodegraded from the PBS sample. To calculate only the CO₂ amount from the PBS biodegradation, the evolved CO₂ amount from the blank is subtracted from that of the sample vessel. Therefore, the respiration activity in the reaction vessel in the presence of the sample is speculated to be the same as that of the blank reaction vessel without the sample.

There is a method of determining the ratio of CO₂ from the compost which is biomass and that biodegraded from PBS which is produced from petroleum during the evaluation period of the biodegradation. Biomass carbon, such as the compost produced from agricultural waste, includes ¹⁴C radio carbon atoms which have been photosynthesized from CO₂ in the modern atmosphere. The percent modern carbon (pMC) is relative to the ¹⁴C concentration based on oxalic acid as a standard reference material (SRM 4990c) measured by AMS as indicated by Equations (3) and (4). From the pMC value of evolved CO₂, the ratio of the evolved CO₂ from the compost including ¹⁴C, which is at the biomass level (ca. 1 × 10⁻¹²), and that from PBS, which is at the petroleum level (no ¹⁴C), can be determined. The pMC of PBS used in this paper was 0%. (3) Δ 14 C = [ ( 14 As − 14 Ar / 14 Ar ] × 1000     ( ‰ ) (4) pMC = Δ 14 C / 10 + 100                                                         ( % )where ¹⁴As and ¹⁴Ar are the ratio of the ¹⁴C to ¹²C for the sample and reference (SRM 4990c), respectively. Table 2 indicates the evolved CO₂ amounts from one vessel, and the pMC of the trapped CO₂ in BaCO₃ measured by AMS. In the AMS method, the total evolved CO₂ amount is the sum of the CO₂ amount in BaCO₃ and the trapped CO₂ in the absorption columns after the Ba(OH)₂ trap bottle as indicated in Figure 2. The CO₂ (respiration) and CO₂ (biodegradation) values were calculated as follows: (5) CO 2   ( respiration )   value   =   total   evolved   CO 2 × pMC   ( sample ) pMC   ( compost ) (6) CO 2   ( biodegradation )   value   =   total   evolved   CO 2   × pMC   ( compost ) − pMC   ( sample ) pMC   ( compost )

The pMC (compost) value was 109.87 measured by AMS for the trapped BaCO₃ from different blank vessels for 0–3 days. Indeed, this value is constant when using the same compost, and that of the ordinary compost is around 108, which is the biomass value. In these AMS methods, the measurement of pMC (compost) is not necessary if the same compost is used as the inoculum. These respiration and biodegradation CO₂ values can be calculated from only one reaction vessel. The evolved CO₂ value from the sample for the ISO method and the total CO₂ value for the AMS method were measured under the same compost conditions using a CO₂ trap apparatus which included the absorption columns and a Ba(OH)₂ bottle. At the end of the test period (74 days), the total evolved CO₂ amount in the ISO method was 28.88 g, and the total CO₂ amount in the AMS method was 28.82 g. Based on these results, the evolved CO₂ was completely trapped without any loss in the AMS method.

In this experiment during the PBS biodegradation, the measured pMC of the trapped BaCO₃ gradually decreased to 42.98% after a 49-day incubation time from 109.87% [pMC (compost)]. The measured pMC value of the trapped BaCO₃ during 60–74 days had still not recovered to the value of the compost, when the biodegradation was almost finished based on the ISO method. In addition, the total CO₂ (respiration) amount was 17.32 g. This value was higher than that (11.96 g) of the blank vessels based on the ISO method as indicated in Figure 6. For the PCL biodegradation in the controlled compost at 58 °C, 80% of the PCL was well-degraded after 20 days [11]. The pMC values of the evolved CO₂ from the sample vessel with the PCL ranged from 20 to 30 in this period. However, for the PBS in this experiment, a 37% of the evolved CO₂ was still the respiration during the 38–60 days. This may be due to not only respiration, but also metabolism of the microorganisms in the compost.

PBS is biodegraded slower than PCL. In addition, the intermediate materials during the PBS biodegradation were 1,4-butanediol and succinic acid monomer which are components or related metabolites in the TCA cycle. Figure 7 shows the metabolic pathways of the PCL and PBS biodegradations by microorganisms living in the controlled compost. For the PBS biodegradation, changing from the already present metabolite to the newly produced metabolite from PBS occurs in microorganisms in compost. The old metabolites are then metabolized to CO₂ which is evolved with ¹⁴C at the natural level during the PBS biodegradation without ¹⁴C. Part of the carbon from PBS is changed to CO₂, while others are changed to biomass (metabolites), when PBS was biodegraded to organic compounds in the compost. For the PCL, the biodegraded monomer unit is hydroxycaproic acid. This material may be easily incorporated into the β-oxidation cycle. Therefore, acetyl-CoA may be actively produced. CO₂ is actively evolved from PCL via the TCA cycle from acetyl-CoA.

Figure 8 shows the biodegradabilities of PBS based for the ISO and AMS methods. For the ISO method, the biodegradabilities were calculated by Equation (1) using the data in Figure 6. For the AMS method, the biodegradabilities were calculated from the CO₂ (biodegradation) and theoretical evolved CO₂ from 10 g of PBS in each test period as shown in Table 2. It was found that the biodegradabilities determined by the CO₂ amounts from PBS in the sample vessel were 30% lower than those based on the total CO₂ amounts from the sample and blank vessels. These differences between the ISO and AMS methods are caused by the carbons from PBS that are changed to metabolites by the microorganisms in the compost, and still not changed to CO₂. Instead, the already present metabolites inside the microorganisms are changed to CO₂. This CO₂ amounts are not counted by the AMS method.