Isolation of A Nocardiopsis Chromatogenes Strain That Degrades PLA (Polylactic Acid) From Pig Waste–Based Compost


 A new Nocardiopsis species that degrades polylactic acid (PLA) was isolated from pig dung–based compost from a municipal composting facility in Japan. To obtain strains capable of efficient PLA degradation, we minimized the effect of non-enzymatic degradation of PLA by maintaining the temperature at 37°C or below. After screening a total of 15 animal waste–based compost samples, consisting of pig dung, cow dung, horse dung, or chicken droppings, we found that compost derived from pig dung was most efficient for degradation of PLA film, and used it for isolation of PLA-degrading microorganisms. Screening for PLA-degrading microorganisms in compost was performed using an agar plate–based method; an emulsifier was omitted to avoid selection of strains that assimilated the emulsifier instead of PLA in the medium. After repeated enrichment, six strains were obtained. One strain that exhibited stable PLA degradation on agar plates was subjected to genomic analysis and identified as Nocardiopsis chromatogenes, an actinomycete.


Introduction
Plastic products contribute to many aspects of daily life, and the global community is harnessing their bene ts. Annual production of various types of plastics reached 368 million tons in 2019 [1]. With the rise in consumption, the risk of resource depletion and elevation of greenhouse gas (GHG) emissions due to increased use of resources and energy throughout the supply chain have become major issues. Furthermore, environmental burdens due to insu cient recovery of plastic products after use, in particular environmental pollution out owing into the ocean, has become a signi cant social issue around the world. To realize a resilient and sustainable economy, the idea of "reduce, reuse, and recycle" to focus on what is appropriate for each application, development of innovative technologies, and designs for social systems are required in order to enhance resource utilization and prevent out ow. These holistic concepts will be realized through effective collection and sorting after use, followed by mechanical recycling, chemical recycling, and energy and thermal recovery. At the same time, there is an urgent need to convert the economy from fossil resources to renewable resources [2]. Circular Economy is an integrated scheme involving resource circulation, carbon neutrality, and social systems. Future models of fossil-based plastics and bioplastics must encompass raw materials, plastic products, applications, and waste/byproduct management from the standpoint of Circular Economy [3]; this will be enhanced not only by reuse and recycling but also by maintenance, repair, and longer life, as well as sharing, throughout the value chain. This transition will be accelerated by digitalization and knowledge sharing such as trading platforms that identify the environmental impacts of individual materials and online databases that track the use of a material across its lifecycle [4]. Furthermore, this direction is expected to be enhanced by an introduction of a regulatory and legislative framework, collaboration with other industries such as agriculture, and incorporation of ambitious and innovative methods for carbon dioxide capture, utilization, and storage (CCUS).
To realize an essential and true Circular Economy for plastics, it is also necessary to take into account people's needs and pains, as well as the hidden struggles of society: convenience, enjoyment, nancial bene ts, and social prestige. This requires responsible efforts that utilize traceability throughout the value chain, rather than just part of the circulation of objects.
In Europe, composting to convert organic waste into fertilizer rather than placing it into a land ll is becoming more common [5]. In this context, organic wastes are being collected together with packaging and cutlery made of biodegradable or compostable materials, which are hard to separate after use; these materials are then composted, and the resultant compost is used as a fertilizer for plant growth. Furthermore, by using biomass instead of fossil resources as the raw material, it is possible to realize a carbon cycle and reduce GHG emissions, in addition to preventing out ow to the environment.
Among them, PLA, a bioplastic made from biomass, has been under development since the 90's [6,7] and has recently attracted renewed attention. PLA is a transparent plastic with high glass transition temperature (Tg) and high crystallinity.
These characteristics were widely exploited, and PLA is commercially manufactured and used in food packaging, containers, agriculture, hygiene, and durables [8,9].
There are various types of composts, based on the wastes from which they are derived: food waste, leaves, farm waste, animal manure, and sewage sludge. For animal waste-based compost, a previous study examined cow dung and pig dung-composts for the degradation of food waste [10]. We previously conducted a pilot-scale composting study of PLA products to con rm that there were no adverse effects on the composting process as well as the growth of plants cultivated using the obtained compost [11]. To date, studies have examined the e ciency of PLA degradation in compost at pilot and commercial scales [12][13][14][15][16].
The degradation of PLA in compost proceeds in two phases [6,8,[17][18][19][20]. First, PLA is disintegrated and fragmented via chemical or enzymatic hydrolysis. Second, the fragmented PLA is further hydrolyzed by microorganisms. Some studies, however, have reported that PLA degradation is mostly performed chemically, with only a limited contribution from microorganisms [21]. A previous study [22] concluded that degradation depended on temperature, not the presence of microbes. PLA sheet decomposition was demonstrated on a lab scale using yard waste compost; the results showed that an excessive amount of PLA input produced lactic acid via hydrolysis, resulting in toxicity to microorganisms [23]. Thus, PLA degradation mechanisms should be studied from the standpoints of chemistry, enzymes, and microorganisms, not only for scienti c analysis but also for e cient social implementation of composting.
After Tokiwa et al. isolated and identi ed Amycolatopsis, a genus of PLA-degrading actinomycetes, in 1997 [24], the same research group isolated a variety of PLA-degrading microorganisms from environmental sources, soil, and compost. Some of these microorganisms belong to the genera of Rhizobium, Bacillus, and Tuberibacillus, identi ed in 2008 [25]. Others belong to the family Pseudonocardiaceae and related genera such as Amycolatopsis, Lentzea, Kibdelosporangium, Streptoalloteichus, and Saccharothrix, described in 2006 [ 26]. These discoveries were subsequently reviewed in 2017 [27] and 2019 [28].
A study attempted to improve the e ciency of PLA degradation in the compost by spraying a mixture of potent PLAdegrading bacterial strains in 2016. In these experiments, a mixture of four strains, classi ed as species, Penicillium chrysogenum, Cladosporium sphaerospermum, Seratia marcescens, and Rhodotorula mucilaginosa, was sprayed onto compost made of vegetable waste, wood chips, and fruit peels. Addition of the bacterial cocktail facilitated PLA degradation and degraded 44 wt% of PLA in 30 days under laboratory conditions. The results demonstrated the importance of microorganisms in PLA degradation during composting [29].
The composition of composts varies depending on the types of waste and the facility where composting is performed. Further, even at the same composting facility, the degradability of PLA is expected to change if the composting conditions, including the microbiota, are different. Thus, in order to optimize degradation conditions, it is important to study the chemical and biological factors of the compost to be used in waste management. In this study, 15 different animal wastebased composts consisting of pig dung, cow dung, horse dung, or chicken droppings from nine different composting facilities in Japan were screened for e cient PLA degradation at a relatively low temperature, e.g., 37° C. In general, when the temperature of cow dung compost rises, ammonia is generated by the decomposition of proteins, and hydrolysis of PLA is accelerated. To focus on the isolation of microorganisms capable of degrading PLA, the temperature was regulated at 37°C to avoid promoting chemical decomposition. Emulsi ers such as those developed for PLA by Tokiwa et al. in 1997 [24] have been widely used to isolate PLA-degrading microorganisms. Emulsi ers were removed afterwards to avoid the possibility of selecting microorganisms that assimilate these compounds From the most e cient pig dung-based compost, we isolated a strain of Nocardiopsis chromatogenes. This is the rst study focusing on screening animal wastebased composts from farms and facilities leading to isolation of a strain of Nocardiopsis chromatogenes.

Compost
Fifteen different types of matured compost originating from nine municipal composting facilities in Japan were collected and used. Among 15 types of compost, eight were based on pig dung, four on cow dung, one on horse dung, one on chicken droppings, and one on a mixture of cow dung and chicken droppings.
λ PLA A PLA cast lm was used in the compost screening test; the size was 3 × 5 cm with a thickness of 20-30 µm. The lm was produced by lab-scale T-die extrusion using the PLA powder LACEA (produced by Mitsui Chemicals), which is a BPScerti ed Green Plastic (PL#40701). BPS was formerly the Biodegradable Plastics Society, but changed its name to the Japan Bioplastics Association (JBPA). Green Plastic is a brand name of biodegradable plastics. PLA powder (40-50 µm radius, Mw 120,000-150,000) was used to prepare PLA microspheres (1-5 µm radius).

Reagents
Plysurf A 210G, produced by Dai-ichi Kogyo Seiyaku or DKS, was used as an emulsi er for dispersion of PLA microspheres.

Screening of composts for PLA lm degradation [primary screening]
Fifteen composts from nine municipal composting facilities were placed in separate jars with PLA lms. Jars with PLA lms were placed in an incubator at 37°C for 3 months to minimize the effect of non-enzymatic degradation of PLA.
Two-thirds of the 5-cm PLA lm was inserted vertically into the jar with the compost (Figure 1).

Further evaluation of selected composts and collection of microorganisms [secondary screening]
One compost selected from the primary screen was subject to PLA lm degradation at 37°C for 6 months.
During test periods, the sample was occasionally sprayed with water to avoid drying.
Single-colony isolation of PLA-degrading microorganisms on agar plates For screening of the strains, glycerol/asparagine-based medium was used, as described previously [30].
Two-layer agar plates were prepared as follows.
(Top layer) One gram of PLA powder (40-50 µm radius, MW 120,000-150,000), as shown in Figure 2, was dissolved in 40 mL of methylene chloride. The resultant solution was emulsi ed with 1 L of 100 mg/L of emulsi er Plysurf A 210G. The emulsion was subjected to a warm bath and evaporator to evaporate the methylene chloride. The resultant PLA microsphere particles were collected by ltration, followed by repeated washing and ltration to remove the emulsi er. The collected PLA microsphere particles, as shown in Figure 2, were mixed with 1 L of the same glycerol/asparagine broth as for the bottom layer, and following the addition of 20 g of Bacto agar, the PLA-containing medium was autoclaved and poured onto the bottom layer of the petri dishes.
Isolation of strains from the agar plates After 6 months of secondary screening, degradation of PLA lm and growth of microorganisms on the degraded portion was observed. The microorganisms were collected with an inoculation loop and streaked on a two-layer agar plate of which the top layer contained PLA.
The plates were incubated at 37°C for about 2 weeks until clear zones were visible. The clear zone was scraped with an inoculation loop and streaked onto new agar plates to enrich and isolate single colonies. This process was repeated four times.

Characterization of isolated strains and phylogenetic analysis
Physiological characterization was conducted by Japan Food Research Laboratories.
To extract 16S rDNA, colonies on plates were collected and resuspended in TE buffer and disrupted by vortexing with glass beads. The debris was removed by centrifugation (7, Table 1 and Table 2.
One possible reason why the PLA lm was degraded in pig dung compost, but not in composts based on other material including cow dung, is as follows. Because these were mature composts and tests were conducted in small jars, it is probable that the temperature did not rise. In general, when the temperature of cow dung compost rises, e.g., to 58°C, the Tg of PLA, ammonia is generated by the decomposition of proteins and the hydrolysis of PLA is accelerated. However, in these trials, the temperature was controlled at 37°C, so chemical decomposition was not promoted and degradation by microorganisms did not occur. In other words, in this experiment, no microorganisms that degraded PLA at 37°C were present in compost derived from cow dung, but such organisms were present in compost derived from pig dung.
A previous report examined pig dung-and cow dung-based composts for degradation of food wastes [10]. The authors of that study compared the process of composting between cow dung/food waste and pig manure/food waste. The results revealed that the number of bacteria was about two orders of magnitude higher in the pig dung system than in the cow dung system, and the pH was also lower. This result relates to our assumptions regarding the comparison of cow and pig dung.
The standard test method for evaluating biodegradable plastic with compost (ISO14855) is performed at 58 ± 2°C. Many studies have the degradability of PLA products in a pilot scale compost, e.g., compost with ber, fat, and protein in animal fodder at 58°C [31]; compost with cow manure and wood waste at 60-65°C [12,13]; and compost with green yard waste at about 60°C [14]. In addition, in the PLA degradation test with our pilot scale compost [11], we used compost containing horse manure and plants. Because this was a fresh and large-scale compost with a total weight of 110 kg, the internal temperature was 70°C or higher at the beginning of the composting process. Despite the use of horse manure, degradation proceeded because hydrolysis was promoted at the early stage.
In our screening of the composts, one compost derived from pig dung (No. 2), which was provided by a municipal composting facility in Kita-hiroshima, Hokkaido, had the highest PLA degradation activity. Accordingly, we selected it for further study.

Further evaluation of selected composts and collection of microorganisms (secondary screening)
The selected compost (No. 2) was subject to further evaluation of PLA degradation. PLA lm was placed in the compost, and degradation was monitored.
After 6 months of incubation at 37°C, degradation of the lms was observed in the compost as shown in Figure 3. Notably, in our other experiment, the T-dye-extruded lm was more susceptible to degradation than the biaxial-oriented lm, which has higher crystallinity.
Isolation of the strain A cluster of microorganisms was observed on the degraded portions of the lms. The cluster was collected with an inoculation loop to isolate the strain responsible for the degradation of PLA lm.
The cluster was streaked on agar lm, and clear zones were spotted after 1.5 months. To avoid isolating microorganisms that assimilate and grow on the emulsi er, we removed the emulsi er during plate prep. As described in the Methods, after PLA powder was dissolved in CH 2 Cl 2 , the emulsi er was added, and the resultant emulsi ed solution was warmed to evaporate the CH 2 Cl 2 . PLA microspheres were collected by repeated ltration and washing to remove the emulsi er.
Conversion of PLA powder to microspheres was predicted to increase contact with the microorganisms.
To further purify the microorganisms, the clear zone was scraped and streaked onto a new plate (Figure 4). This process was repeated four times. Ultimately, six colonies were isolated that produced a clear zone on the plate. Because a large halo formed as shown in Figure 5, we speculated that enzymes were secreted. We chose one strain, MT-24107, for further analysis and identi cation. The edge of the white fungus that formed the halo shown in Figure 5 was observed by phasecontrast microscopy. The thread-like spreading pattern is characteristic of Actinomycetes ( Figure 6).
The reason for using the two-layer screening system in this experiment is as follows. Because it takes about 1 month to decompose PLA, it is di cult to detect the decomposition halo in a one-layer system in which PLA microspheres are dispersed over the entire agar medium. By adopting a two-layer system, when the upper microspheres decompose and halos are generated, it is easier for light to pass through, and detection is easier.
We employed PLA microspheres in our plate screen for PLA-degrading microorganisms in order to shorten the overall time required. Although PLA powder (40-50 µm radius) took 4 weeks to form a clear zone (i.e., halo), the process took only 2 weeks when PLA microspheres (1-5 µm radius) were used.
MT-20147 was suspended in medium with the same composition as during agar plate testing after secondary screening, except for the addition of Bacto agar, and the suspended liquid was poured onto a PLA lm placed in a Petri dish. After incubation for several days, the lm surface was washed and observed by electron microscopy. A characteristic degradation path was observed on the lm surface, as shown in Figure 7.

Identi cation of the strain
As a result of physiological characterization shown in Table 3, the strain was presumed to be Nocardiopsis, classi ed as an actinomycete because of its cell wall and quinone types. However, spore formation was not observed, and the genus could not be con rmed from this result alone.
Colonies of isolated strain MT-20147 was scraped and collected. The harvested cells were physically disrupted by glass beads and the genomic DNA was extracted.
Sequencing started with the primer 27f. Using the resultant sequence data, the new primers 519r, 357f, and 536f were designed. Sequence analysis was performed for the region read by these primers, and subsequent primers were designed in the same manner to cover a 1,489-bp fragment in the 16S rRNA gene by assembling contigs (Figure 8).
The sequence of the assembled contigs was analyzed using BLAST and exhibited 99.2% of similarity with Nocardiopsis chromatogenes, order Streptosporangiales, as summarized in Table 4. A phylogenetic tree was created with the MEGA5 software as shown in Figure 9.
Meanings and issues of compost treatment of PLA products after use PLA has been attracting attention as a representative commercially produced biodegradable plastic derived from renewable resources [8,9]. To achieve a Circular Economy, it is necessary to perform appropriate and e cient treatment of PLA products after use. Composting is the second-best approach for handling PLA composites; the best approach is recycling, as with fossil-based plastics [40]. In Europe, the role of composting is becoming more important as a means to treat organic wastes e ciently rather than become land ll [5], and it will be worthwhile to use compostable products such as food packaging and cutlery when it is di cult to separate and recover those products from organic waste such as food residues.
Because the resultant compost contributes to plant growth, it should be regarded as an effective use of waste and byproducts in the circular system. The CO 2 emissions are totally suppressed because the CO 2 generated by degradation is absorbed by the plant [40,41].
In regard to social implementation, we published a paper that describes degradation of PLA products in compost on a pilot scale. The results revealed that the presence or absence of PLA products does not adversely affect the degradation process or the quality and safety of the resultant compost [11]. In addition, pilot and commercial-scale composts, which contain cow dung as the main component [12][13][14] or green yard waste as the main component [15,16,42] were used to test the degradability of PLA products. In all these papers, the temperature condition of the compost was 58°C or higher.
The social implementation of composting has been widely studied. For examples, studies that elucidated the mechanism by which PLA is degraded in compost were published in in 1998 [22], 2001 [23], and 2014 [21]. Papers describing the bacteria and enzymes that promote degradation were published in 2004 [33], 2005 [43], and 2006 [26]. A review of microbial degradation of polymers was published in 2017 [27].
To construct a feasible framework for PLA waste management, it is important to further study the factors and mechanisms affecting biodegradability, for example, types of composts, microorganisms involved in degradation, physical properties of PLA, temperature, pH, and time.

Isolation of N. chromatogenes
In this study, we isolated and identi ed PLA-degrading microorganisms using animal-based composts (pig, cow, horse, and chicken). Multiple reports have described the isolation of PLA-degrading microorganisms, but to date no study has identi ed N. chromatogenes as a PLA-degrading microbe.
In the 90's, actinomycetes isolated from the natural environment (e.g., soil) were identi ed as PLA-degrading microorganisms, and multiple actinomycetes have been described not only in soil but also in compost. Furthermore, Bacillus subtilis and fungi capable of degrading PLA have also been reported.
PLA-degrading microorganisms were reviewed as follows based on the classi cation of the cited references. We expect to convey that the species we identi ed had not been previously reported in the literature.
In 1997, Tokiwa et al. isolated and identi ed Amycolatopsis, a genus of actinomycete whose members are capable of degrading PLA, from 45 types of soil samples in Tsukuba City, Japan [24]. This bacterium also degrades silk, as reported in 1999 [44]. Using actinomycetes obtained from public institutions, Kibdelosporangium aridum [45] and Saccharothrix waywayandensis [46] were shown to degrade PLA in 2003, and Amycolatopsis orientalis was shown to produce PLAdegrading enzymes in 2006 [17]. Furthermore, in 2004, Tokiwa et al. reviewed the importance of actinomycetes as PLAdegrading microorganisms in conjunction with active enzymes [33]. In 2001, the same type of microorganism, Amycolatopsis sp., was isolated from 300 soil samples [32].
In addition to actinomycetes, Laceyella sacchari isolated from forest soil in 2014 [36] and strains of Pseudomonas and Bacillus, both isolated from sludge in 2017 [20], are capable of PLA degradation. In 2016, a paper showed that three out of four microbes isolated from 300 soil samples from various sources were fungi: Penicillium chrysogenum sp., Cladosporium sphaerospermum sp., and Rhodotorula mucilaginosa sp.; the exception was Serratia marcescens [29].
PLA-degrading microorganisms in compost are diverse. The raw materials used in the compost test for isolation and identi cation of PLA-degrading microorganisms are mainly animal feed, food waste, and plant residue, and animal manure has been used in only a few cases. Bacillus smithii of the order Bacillales was obtained from a garbage fermentor, and the PLA-degrading enzyme was identi ed as a serine protease in 2001 [47]. In 2008, Bacillus licheniformis of the order Bacillales was isolated from compost made from animal fodder and identi ed as a PLA-degrading microorganism [34].
Microorganisms that form bio lms were shown to degrade PLA in compost in 2015 [48]. These organisms were of the genera Acidovorax, Aeromonas, Arthrobacter, and Chryseobacterium. All four are Bacteria, and Arthrobacter is a type of actinomycete. Thermopolyspora exuosa, another actinomycete, was identi ed from lab-scale compost in 2014 [21].
As reviewed above, it is clear that PLA-degrading microorganisms range from prokaryotes such as actinomycetes to eukaryotes such as fungi.
A comprehensive review on PLA-degrading microorganisms was published in 2019 [27]. That paper described ve families, eleven genera, and twenty-ve species for which enzyme classi cation was also available. Furthermore, enzymes related to PLA-degrading microbes were reviewed in a paper from 2006 [25]. A comprehensive review article of microorganisms that degrade fossil-based plastics as well as biodegradable plastics, including PLA, was published in 2017 [26].
In the long history of research on PLA-degrading microorganisms, Nocardiopsis chromatogenes has not been identi ed.
Recent studies have examined microorganisms that degrade PET have been conducted. Among them, Nocardiopsis chromatogenes was shown to be a PET-degrading species in 2018 [49]. Although PET is aromatic and PLA is aliphatic, and the two compounds have different chemical structures, it is noteworthy that microorganisms from the same genus are involved in the degradation of both polyesters.
As previously described, the PLA degradation mechanism consists of hydrolysis, enzymatic degradation, microbial degradation, and combinations thereof. To carry out the degradation in compost more e ciently and economically, future work should elucidate the mechanisms, microbes, and enzymes secreted during the process of degradation.

Conclusions
In this study, we used two speci c approaches to isolate PLA-speci c degrading microorganisms. First, we attempted to rule out the possibility of PLA degradation by ammonia, which promotes proteolysis at high temperatures, by limiting the temperature below 37°C in compost derived from various types of livestock manure. Normally, the composting of organic waste by cow dung compost is performed at high temperature. Because we suppressed the temperature at or below 37°C, hydrolysis at the initial stage of degradation did not proceed and PLA was not degraded, i.e., PLA-degrading microorganisms could not be obtained. On the other hand, PLA-degrading microbes were obtained only from pig dung compost. Furthermore, to avoid the possibility of selecting microbes that assimilate the emulsi er, it was removed after the screening process.
As a result, a microorganism capable of degrading PLA was isolated from pig dung-based compost. Based on the properties of the microorganism and a genetic analysis, it was identi ed as Nocardiopsis chromatogenes, an actinomycete of order Streptosporangiales. This is the rst time that N. chromatogenes has been shown to degrade PLA. Further research is needed to characterize the microbe we isolated, the enzymes it secretes, and the mechanism of degradation.   PLA lm degradation test with the compost in a jar.  A photo of PLA lm before and after the degradation test. PLA lms: Control before the testing (left), which is hard to see because of its transparency, and the lm after 7 weeks in the compost (right).

Figure 4
Photo of six strains isolated from pig dung-compost. Six strains isolated from the pig dung compost that exhibited haloforming ability. White indicates a colony of microorganisms, and the transparent part is the halo.
Page 21/23 Figure 5 Photo of halo formation on a PLA agar plate by strain MT-20147. It took 2-3 weeks for strain MT-20147 to form a halo on a PLA agar plate. This image was acquired 1.5 months after cultivation started.

Figure 6
Phase-contrast microscopy of strain MT-20147.  Phylogenetic tree analysis of strain MT-20147.