Unveiling Gut Microbiota and Metabolic Functions Contributed to Polyvinyl Chloride Degradation in Spodoptera frugiperda Larvae

doi:10.21203/rs.3.rs-4686713/v1

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Unveiling Gut Microbiota and Metabolic Functions Contributed to Polyvinyl Chloride Degradation in Spodoptera frugiperda Larvae

https://doi.org/10.21203/rs.3.rs-4686713/v1

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Background:

The surge in synthetic plastic waste, especially polyvinyl chloride (PVC), poses severe environmental challenges. Recent discoveries in microbial biodegradation offer a hopeful approach to managing such pollutants. Notably, the gut microbiota of certain insect larvae have demonstrated potential for degrading PVC. However, due to challenges such as the significant variations in the microbial composition and low microbial biomass in the guts of insect larvae, there are currently no viable PVC-degrading biological resources derived from insect gut microbiota.

Results:

This research entailed sampling Spodoptera frugiperda larvae at various places and obtaining samples from multiple microbial habitats, employing 16S amplicon analysis to disclose the environmental influence on the larvae’s microbiota. Subsequently, we compared the changes in gut microbiota of S. frugiperda larvae and T. molitor larvae after consuming PVC, discovering that despite completely different microbial communities, they enriched similar functions. We then isolated Enterococcus casseliflavus EMBL-3, which was enriched in the gut of S. frugiperda larvae and verified that the NAD-dependent oxidoreductase it encodes can dechlorinate and degrade PVC.

Conclusions:

Besides emphasizing the stability of the microbiota in the midgut contents of S. frugiperda larvae in natural environments, this study leverages the gut microbiota resources of S. frugiperda larvae to discover an enzyme capable of degrading PVC, paving new paths for understanding the mechanisms of plastic biodegradation and advancing biotechnology to reduce global plastic pollution.

Polyvinyl Chloride

Gut microbiota

Biodegradation

Dechlorination

depolymerization

Spodoptera frugiperda

Enterococcus casseliflavus

The ever increasing of synthetic plastic waste has emerged as an urgent and widespread global environmental challenge. Since 1950, the total global production of plastics has soared to a staggering 400.3 million metric tons in 2022¹. However, a substantial 79% of plastics have accumulated in the natural environment², with polyvinyl chloride (PVC) comprising 13% of the overall plastics usage³. Since PVC releases toxic chlorine-based compounds during thermal processing, this poses challenges to PVC³ recycling. In light of these issues, alternative strategies like biological treatment and recycling or upcycling are needed as effective and environmentally friendly pathways for dealing with PVC plastic waste and mitigating its impacts⁴.

Recent studies found microorganisms that exist in nature present unique power in plastic biodegradation, like plastic polymers with C-O backbones the Polyethylene terephthalate (PET). Significant advances have been made in the biodegradation of PET, exemplified by the discovery of a hydrolase from T. fusca in 2005⁵. Subsequent studies identified PET-degrading enzymes from microorganisms such as Ideonella sakaiensis⁶ and Pseudomonas sp.⁷, and after characterizing and improving these enzymes, significant progress was made in large-scale PET biodegradation and recycling. However, currently for fossil fuel-based polymers with the C-C backbones⁸, there is only limited evidence suggesting that potential oxygenases might act randomly on the C-C backbone of polyethylene (PE), potentially leading to degradation. However, for polymers such as polypropylene (PP), polystyrene (PS), and PVC, there is no definitive evidence that they can be enzymatically degraded⁹. Numerous microbes found in nature, including fungi (e.g., White Rot Fungi¹⁰ and Phanerochaete chrysosporium¹¹) and bacteria (e.g., Pseudomonas citronellolis and Bacillus flexus^12,13), were reported to possess the potential to degrade PVC; however, enzymes characterized biochemically have not yet been reported.

The insect gut as a unique environment for microorganisms drew people’s interest for their role in the digestion of the fiber and exogenous substances¹⁴. In the meantime, studies reported that larvae like Zophobas atratus, Hermetia illucens and Tenebrio Molitor showed significant capacity of their gut microorganisms to biodegrade no-hydrolysis plastics^15–23. In our previous finding, the S. frugiperda larvae presented the ability to intake PVC and survive, additionally, their microbial composition was significantly changed²⁴. These findings indicate that insect guts and advanced sequencing technologies provide valuable resources for the study of plastic biodegradation, yet challenges remain. Firstly, the gut microbiota of S. frugiperda has recently attracted attention, but research on it is still insufficient. Currently, it is unclear which sampling site of the larvae best represents their gut microbial composition, which complicates our interpretation of the results. Secondly, when insects consume pollutants (plastics) as their only food source, the low biomass and high host content in the gut make shot-gun metagenomic sequencing challenging, and mixing numerous samples to meet sequencing requirements is not the ideal solution. Consequently, 16S sequencing has become the principal method for such studies, with extensive studies on the degradation of the same plastics by various insect larvae. However, the further discovery of biodegradation genes is complicated by the different sequencing regions of the 16S rRNA gene and the large variations among different insect species.

To address this gap, this study initially collected S. frugiperda larvae from various places and used 16S rRNA gene sequencing to profile their microbiota. Results indicated the microbial compositions of midgut content more accurately represented the gut microbiota of S. frugiperda larvae. Subsequently, we integrated the dataset from the study on PVC biodegradation by T. Molitor¹⁵ and, building on our previous research on S. frugiperda²⁴ degradation of PVC, reanalyzed both datasets and conducted functional predictions. We found a NAD-dependent oxidoreductase encoded by Enterococcus casseliflavus EBML-3, can directly dechlorinate and biodegrade the PVC. This finding addresses the gap in the dichlorination process of PVC degradation and enriches current knowledge on the microbial and enzymatic resources available for PVC biodegradation.

The environment influences the microbiota composition in the excreted feces of S. frugiperda larvae

As a new invasive species, the gut microbiota of the S. frugiperda has not been well studied. To explore the impact of the environment on the microbiota of S. frugiperda larvae, as well as the representativeness of microbiota at different sampling places, we sampled from cornfields in four provinces (Hubei, Yunnan, Hainan, Henan) of China (Fig. 1a) at ecologically significant sites, including bulk soils (BS) in cornfields, the excreted feces (EF) from active 3rd -5th instar larvae, and gut content from the midgut. Within the midgut, we differentiated the microbiota into those within the gut contents and those attached to the gut mucosa, which we named gut feces (GF) and gut mucosa (GM) respectively.

The results showed that all bacteria found in the gut of the S. frugiperda can be detected in the soil. The environment surrounding the larvae provides the resource for their gut organisms. Specifically, the Sphingobacterium constitute 2.46% of the soil and 30.37% of the bacteria in the excreted soil (Fig. 1b, Figure S1). However, they make up only about 0.7% of the gut contents (GM and GF). This indicates the connection between the exposure of S. frugiperda larvae and host selection. Similarly, genera such as Flavobacterium, Empedobacter, and Acinetobacter are also in the same state, originating from the soil and enriching in the excreted feces, then disappearing in the gut contents. It is worth noting that these microbes are environmentally specific enrichments. For example, Sphingobacterium is enriched only in the larval feces from the Hubei samples (EF: 58.4 ± 17.4%, Figure S1a), Acinetobacter is enriched in samples from Henan (EF: 10.0 ± 6.84%, Figure S1b) and Yunnan (EF: 10.5 ± 2.54%, Figure S1c), and Empedobacter is enriched in the Hainan samples (EF: 58.4 ± 17.4%, Figure S1). Conversely, Klebsiella is enriched in the excreted feces (EF: 25.7%) but persists in the gut contents (GF: 25.7%, GM: 12.3%, Fig. 1b). The most interesting bacterium is Enterococcus, which comes from the soil (1.5%). However, it may have a strong survival ability in the gut content (GF: 50.4%, GM: 65.4%) and is not excreted (EF: 0.85%). The same phenomenon was also observed in the alpha diversity. Significant differences in alpha diversity were found between the soil and the feces excreted by insects from four sites (Figure S2 a-b), however, no significant differences in Shannon diversity were observed for the gut contents (GF and GM) (Figure S2c-d). This indicated the microbes in excreted feces are significantly influenced by the environments.

The host determines the stability of the microbiota in the gut contents of S. frugiperda larvae

Subsequently, to explore the stability and variation of different sampling sites, we conducted statistical tests on the microbial composition between four sampling sites and found significant differences between samples from different sites (Adonis: R² = 0.129, p = 1e-04, Fig. 1c). The microbial composition of BS and EF was particularly different among the four different locations (Adonis: R²_BS = 0.79, p_BS=1e-04, R²_EF = 0.72, p_EF = 1e-04, Figure S3a-b). While significant differences in microbial composition of the gut contents (GF, GM) remained across different sites (p_GF = 1e-04, p_GM = 1e-04), the their proportion of explained variance (R²) were considerably lower than BS and EF (R²_GF = 0.45, R²_GM = 0.45, Figure S3c-d). We then compared the differences between the microbiota of EF, GF, and GM from four places. We found significant differences in the similarities within the EF group at different sites compared to EF, GF, and GM (p < 2.22e-16, Fig. 1d), and the similarity within the GF group was significantly lower than within the EF group (p = 2e-05, Fig. 1d), and there were no differences in the similarities between GF and GM and within the GM group (p = 0.69, Fig. 1d). This further indicates that the microbiota in the excreted feces are significantly influenced by the environment than those in the gut contents, and there is almost no difference between the gut feces and gut mucosa-attached microbiota, which are much less affected by the environment, further highlighting the role of host selection. Therefore, we believe that, like other insects, the fecal microbiota of S. frugiperda cannot represent the microbiota colonizing within the gut; the microbial composition of the gut contents can represent the overall gut microbiota, and there is no difference between the microbiota in the gut feces and gut mucosa.

PVC film is degraded by the microbiota in the larvae’s gut, rather than by the gut itself

Those ecological findings allowed us to understand the microbiota stability and representativeness of different sites of S. frugiperda larvae; therefore, based on the original experiment, we only included gut feces (GF) obtained from the midgut to represent the gut microbial composition. Previous studies measuring physiological indicators proved that larvae could survive on PVC film; however, there was no conclusive evidence of plastic degradation indicating whether the decomposition occurred due to the gut microbiota or the gut of the insect itself. Therefore, we collected plastic film fragments from the excreted feces, including those fed PVC film (PVC + Larvae), and PVC film with antibiotics (Anti + PVC), and set standard PVC film as control. Then we used Advanced Polymer Chromatography (APC, Waters, China) to measure changes in the number-average molecular weight (Mn) and the weight-average molecular weight (Mw). Compared to the control and PVC with antibiotics groups, the Mn of PVC film without antibiotics treatment group significantly increased (p < 0.01, Fig. 2a) by 24.6%. The Mw also increased but not significantly (p > 0.05, Fig. 2b), which can be explained by the degradation of short-chain polymers in the plastic. This result indicates that the S. frugiperda larvae gut cannot directly degrade PVC polymers, however, the microbiota living there can. Subsequently, we further detected the degradation products from the standard PVC film (PVC), the larvae fed only corn leaves (Corn), and PVC film fragments recovered after larvae were fed PVC (PVC + larvae) to distinguish whether they originated from the microbial metabolites, plastic itself or from the plasticizers. The GC-MS results showed that the PVC film in the larval gut was effectively degraded, producing smaller organic molecules. In the larvae fed with PVC without antibiotics treatment group (PVC + larvae), a total of 7 new substances were detected (labeled as compounds 1 to 7, Fig. 3c), which were not detected in the standard PVC film, including 2-ethyl-1-hexanol, 2-ethylhexanoic acid, 2-nonanol, adipic acid, 3-hydroxydodecanoic acid, hexadecenoic acid, and octadecenoic acid. Compounds 8 and 9 were identified as two known plasticizers, namely dioctyl adipate and dioctyl phthalate. Based on the structures of the compounds, compounds 1, 2, and 4 may originate from the degradation of plasticizers (compounds 8 or 9, Fig. 3c), and we observed a decrease in the abundance of compound 8 after digestion by the larval microbiota, indicating that this plasticizer can be utilized by the gut microbiota and may serve as a major nutritional source within the gut. Additionally, compounds 3, 5, 6, and 7 could be potential PVC degradation products. Overall, this indicates that the gut microbiota of the larvae can degrade PVC film through various microbial pathways, including the degradation of plasticizers and PVC itself, but the gut of the larvae itself does not play a role.

Feeding PVC leads to inconsistent changes in the gut microbiota of S. frugiperda and T. molitor

Having confirmed that the gut microbiota of S. frugiperda larvae can indeed degrade PVC film, we included a dataset from T. molitor that degrades pure PVC to broaden the data resources for PVC-degrading gut microbiota and to exclude the influence of degrading PVC plasticizers. Since the sequencing regions of the 16S rRNA gene and analysis pipelines of the two datasets were inconsistent, direct comparison was challenging. To solve that we applied the same pipeline and database to reanalyze and delve deeper into these two datasets (see Methods). Consistent with the results in T. molitor, we observed substantial differences between the PVC group and the Corn group in S. frugiperda, with the R² of 0.75 (Fig. 3a), higher than that observed in T. molitor (R² = 0.49, Fig. 3b). This suggests that the gut microbiota of insects undergoes significant changes after being fed PVC to adapt to this novel "food". Unlike S. frugiperda larvae collected from the wild, the guts of our lab-reared larvae, which were fed exclusively on corn leaves, were occupied by a kind of unclassified Enterobacteriaceae (Fig. 3c), suggesting that this bacterium is capable of efficiently degrading corn leaves.

In contrast to T. molitor, the Shannon diversity significantly increased in the PVC group of S. frugiperda (p = 0.1, Figure S4a) but decreased in the PVC group of T. molitor (p = 0.17, Figure S4b). This might be explained by inconsistent feeding durations between the two experiments and the enrichment of Enterobacteriaceae in the Corn group of S. frugiperda, which inhibits the growth of other bacteria, with this inhibition disrupted in the PVC group. At the same time, we noted that the gut microbiota of both insects are partially similar, with numerous microbes belonging to Proteobacteria (primarily Enterobacteriaceae) and Firmicutes. In T. molitor, Actinobacteriota were not detected; instead, there were considerable numbers of Tenericutes (currently termed Mycoplasmatota) and Patescibacteria, though the microbes in this phylum disappeared after the PVC feeding (Fig. 3c-d). This likely indicates the differences in dietary preferences between the two insects, one eating corn leaves and the other eating bran. Following PVC feeding, the microbiota enriched in the two insects differed completely, with the most notable increase in S. frugiperda being Enterococcus, which surged from 4.7 ± 3.7% to 37.0 ± 19.8%. Additionally, there was enrichment of Ochrobactrum (from 0.1 ± 0.2% to 3.4 ± 0.7%), Klebsiella (from 1.4 ± 0.6% to 1.7 ± 0.4%), Falsochrobactrum (from 0% to 2.80 ± 1.40%), Microbacterium (from 0.03 ± 0.05% to 2.23 ± 1.55%), and Sphingobacterium (from 0.21 ± 0.30% to 2.55 ± 1.54%) (Fig. 3c). On the contrary, in T. molitor, significant enrichments were seen in Spiroplasma and Lactococcus, with Lactobacillus also showing notable enrichment in one of the parallel groups (Fig. 3d). These results indicate that the gut microbial compositions of the two insects are completely different, and the bacteria enriched after consuming PVC are also completely different.

Functional redundancy discovered in the gut microbiota of two PVC-fed insect larvae

Despite the distinct microbial compositions of the two insects, both of them are capable of degrading PVC, leading us to investigate further if they perform similar functions after feeding with PVC. Consequently, we performed PICRUSt2 to match the ASV sequences with the closest reference genomes to predict the metabolic functions of the gut microbiota in both insects. Through KEGG (Kyoto Encyclopedia of Genes and Genomes) database annotations, we identified 566 KEGG orthologs (KOs) significantly enriched in the PVC group of S. frugiperda (p < 0.01, Log2Foldchange > 2, Fig. 4a), mapping to 42 pathways. In T. molitor, 293 KOs were significantly enriched in the PVC group (p < 0.01, Log2Foldchange > 2, Fig. 4b), mapping to 39 pathways. We selected these pathways from the total identified and computed the overall enrichment across these pathways. Although the species of gut microbiota enriched in PVC differ between the two insects, the enriched metabolic functions show considerable redundancy. We discovered 23 enriched pathways common to both insects, with 19 pathways associated with metabolism (Fig. 4a-b). The enrichment of the other glycan degradation pathway might indicate the ability to degrade macromolecules.

To further identify the enzymes related to PVC degradation, we systematically summarized the plastic-degrading enzymes reported in the literature along with their Enzyme Commission (EC) numbers, and classified them into five categories: peroxidases, depolymerases, esterases, lipases, and cutinase. Subsequently, we used the same methodology above to predict the abundance of ECs in each sample, conducted the differential analysis using Deseq2, and extracted these five classes of enzymes based on text search. In S. frugiperda, we detected 32 enzymes, and for T. molitor, 33 enzymes were found. Carboxylesterase (EC 3.1.1.1) and Sialate O-acetylesterase (EC 3.1.1.53), NADH peroxidase (1.11.1.1), and Dye decolorizing peroxidase (1.11.1.19) were significantly enriched in the PVC group of S. frugiperda (Fig. 4c, p < 0.05). It is noteworthy that EC 1.11.1.1 and EC 1.11.1.19 were also observed to be enriched in T. molitor (Fig. 4d).

Since we mapped ASVs to the reference genome and estimated the gene abundance of the overall samples through the gene content of the reference genome, we traced back the microbes encoding these enzymes. We discovered that in S. frugiperda, EC 1.11.1.19 is primarily present in Microbacterium (ASV18) and Flavobacterium (ASV33), while EC 1.11.1.1 is mainly contributed by Enterococcus (ASV2, ASV4) (Dataset S2). Conversely, in T. molitor, these enzymes are mainly associated with ASVs belonging to Glutameicibacter and Lactobacillus (Dataset S3). Likewise, in the PVC group of S. frugiperda, there was a notable enrichment of Enterococcus ASVs (ASV2 and ASV4). The abundance of ASV2 rose from 2.59 ± 0.04% to 26.6 ± 0.69%, and ASV4 from 1.91 ± 0.01% to 10.3 ± 0.13% (Figure S5). The increase in abundance of ASV2 was twice that of ASV4, suggesting its greater potential for PVC degradation. Furthermore, in the genus Microbacterium, all eight ASVs showed an increase in abundance, especially ASV12, which increased from 0.2–0.4%. Similarly, in the genus Sphingobacterium, two branches showed an upward trend, with the most notable being ASV11, whose abundance increased from 0.1–2.8% (Figure S5). These results indicate the potential of these gut microbes and enzymes in the degradation of PVC.

NAD-dependent oxidoreductase isolated from E. casseliflavus EMBL-3 can dechlorinate and degrade PVC

The identified potential degradative enzymes and their encoding microbes leaded us to further verify these results in lab. Particularly interested in EC 1.11.1.1 encoded by Enterococcus ASV2, we isolated and cultivated microbes from the feces of S. frugiperda larvae and identified a strain of Enterococcus after several screenings (Figure S6a). By blasting the complete 16S rRNA gene sequence (Table S1) with the rRNA/ITS databases at NCBI, the isolate was confirmed to be Enterococcus casseliflavus (99.08%). Upon constructing a phylogenetic tree with our ASVs, we found that this bacterium was closer to ASV2 (Fig. 5a). This means that the bacterium we isolated had a low abundance in the gut of S. frugiperda fed on corn leaves, but its abundance significantly increased in the PVC group, and it was twice that of another Enterococcus strain, ASV4. However, the results of liquid culture experiments using PVC film (PVC and plasticizers) as the sole carbon source indicated that the strain E. casseliflavus did not solely exhibit significant degradative activity (Figure S6b).

To further investigate the gene content of this bacterium, we sequenced its whole genome using 2nd-generation sequencing and named this bacterium Enterococcus casseliflavus EMBL-3. By whole genome annotation using Prokka²⁵, we found a gene was annotated as EC 1.11.1.1 in E. casseliflavus EMBL-3 and extracted the corresponding sequence (Table S2). Through protein sequence blast in the uniprot database, we identified this protein as NAD-dependent Oxidoreductase (NDO). Then it was expressed and purified in vitro (Fig. S6c). The activity of the enzyme (9.8 U/mg_prot) was assessed by using peroxidase activity assay in accordance with the manufacturer’s instructions (Beijing Solarbio Science & Technology Co.,Ltd., Beijing, China). To validate the degradation ability of the enzyme on PVC, we set a 2 mL reaction system with 200 ug enzyme and 200 mg pure PVC powder (without plasticizer) and measured molecular weight change and dechlorination of the pure PVC powder (Method S7). The molecular weight of NDO-treated PVC decreased significantly than those untreated. The Mn decreased by 12.02% (from 79.34 kDa to 69.81 kDa, Fig. 5b) and the Mw decreased by 14.07% (from 164.04 kDa to 140.97 kDa, Fig. 5c). This result indicated a depolymerization of PVC polymer chains by NDO. A dechlorination of 0.74 mg/L was detected in the NDO-treatment system compared to the control system (Fig. 5d). NADH, which can be used as a reducing agent to provide hydrion and electron, may contribute to the dechlorination process. So we added NADH into the reaction system. When 2 mM NADH was added in combination with NDO, a distinct dechlorination activity was detected, with the dechlorination amount detected significantly increasing to 6.48 mg/L, while NADH alone did not have dechlorination activity on PVC (Figure S6d). This indicates that NDO works on PVC polymer by dechlorination. Although, we did not detect any degradation intermediates with a carbon chain shorter than 20 in the reaction system (Figure S7), the changes in polymer molecular weight due to dechlorination did not sufficiently account for the observed changes in the molecular mass of the polymer. Therefore, under several ideal assumptions, we performed a series of calculations on dechlorination amounts and changes in molecular weight (Method S8). Results demonstrated that 6.48 mg/L dechlorination had minimal impact on molecular weight, indicating that dechlorination cannot explain the molecular weight changes. The enzyme further cleaves long-chain PVC polymers beyond dechlorination, producing polymers with more than 20 carbons and resulting in decreased molecular weight of the PVC polymers. Overall, we demonstrated that this enzyme can act on pure PVC polymers, simultaneously dechlorinating and cutting long-chain PVC polymers.

This study, based on extensive sampling, explored the microbial composition of different habitats in the gut of S. frugiperda larvae, revealing that the microbiota of their feces is significantly influenced by the environment, while the microbiota of their gut contents is stable and representative of their gut microbes. From the PVC fragments recovered after S. frugiperda larvae, we detected degradation products of both the plasticizers in the PVC film and the PVC itself, produced by their gut microbiota. Further, by combining the dataset with T. molitor, we observed that two distinct insects enriched different bacteria upon consuming PVC, yet they performed similar functions. Guided by these results, we isolated E. casseliflavus EMBL-3 from S. frugiperda larvae and discovered its encoded NDO capable of dechlorinating and degrading pure PVC. This enzyme, being the first to directly dechlorinate polymers, signifies a breakthrough and invaluable advancement in devising effective plastic degradation strategies.

From samples collected at four sites, we conjecture that the feces expelled by S. frugiperda reflect the microbes absorbed from the environment. Many microbes in its excreted feces, such as Klebsiella varicolla²⁴, perform cellulose degradation functions; because they are abundant in both feces and the gut, they were easily isolated in a previous study. Additionally, Ochrobactrum was enriched in samples from Hainan Province. This bacterium is reported to degrade herbicides²⁶ as well as PE and PET²⁷, possibly reflecting the use of herbicides in the region and its potential to degrade PVC. We found Enterococcus has a strong ability to colonize because it can form biofilm microcolonies²⁸ in the gut. Thus, it is enriched in the gut contents of S. frugiperda across all regions. There appear to be two different types of Enterococcus; one is the isolated E. casseliflavus, the other unknown. Both might play a crucial role in the degradation of cellulose²⁹ and xenobiotic substances. E. casseliflavus has been reported to degrade multiple substances including chlorantraniliprole, decabromodiphenyl ether, and a sulphonated azo-dye^30–32 Interestingly, feeding S. frugiperda larvae solely on corn leaves in the lab resulted in a gut microbiota distinct from those in the wild, dominated by an unidentified Enterobacteriaceae, suggesting its capacity to break down cellulose. In larvae that consumed PVC, there was a rapid shift in the gut microbiota, with Enterococcus swiftly regaining dominance. These results indicate that the gut of S. frugiperda larvae is relatively simple yet remarkably adaptive, able to quickly respond to different food sources. This adaptability may reflect their unique capability as a rapidly invading species, and targeting their gut microbiota could potentially diminish this ability.

Our research noted that PVC plastic films (including plasticizers) consumed by S. frugiperda were broken down by gut microbes, resulting in specific degradation products of the plasticizers and possible degradation products of PVC itself. However, using only our isolated strain E. casseliflavus EMBL-3, we did not observe any degradation products of the plastic film. This could indicate that EMBL-3 does not function independently, or the complex makeup of the plastic film, including the plasticizers, might be impeding its effectiveness. However, the NDO isolated and purified from E. casseliflavus EMBL-3 still marks the first discovery of an enzyme capable of dechlorinating and degrading PVC polymers. Microbial reductive dichlorination, a main route for biodegradation of chlorinated compounds, involves replacing chlorine atoms with hydrogen atoms, requiring electron donors and energy^33,34. Some bacteria in the environment, such as Dehalococcoides and Dehalogenimonas spp., were reported to have the capability of reductive vinyl chloride (VC) dechlorination to nontoxic ethene^34–38. This study presumed the reductive dechlorination as the major biotransformation pathway of NDO working on PVC, leading to increased free chloride. However, the degradation of PVC is a complicated process, in which the key steps, such as depolymerization, remain unrevealed. Although the molecular weight was observed to decrease, the solid degradation product (Carbon atoms less than 20) was not detected. This may be due to the limited detection range of the instrument. therefore, whether the long-chain polymer was degraded into smaller molecules require further verification through more studies.

We found that the microbiota in gut contents of S. frugiperda larvae are highly stable, with key colonizing microbes present, which may reveal their rapid invasion capabilities and potential pest control strategies. We described the ability of the gut microbiota of S. frugiperda larvae to degrade PVC. By comparing the composition and function of the microbial community, we further confirmed the ability of an NDO encoded by E. cassiliflavus EMBL-3 to dechlorinate and depolymerize PVC, marking the first enzyme to dechlorinate a polymer. Additionally, our results lay the foundation for the discovery of pollutant-degrading microbial strains and enzymes in the guts of insect larvae with low microbial biomass and varying microbial compositions, paving the way for the development of innovative biodegradation strategies.

Field sampling and processing of S. frugiperda specimen

According to the geographic spreading routes of S. frugiperda in China³⁹, eight cornfields in eight cities of four provinces, i.e., Yunnan Province, Hainan Province, Hubei Province, and Henan Province, were selected for larvae and bulk soil sampling from during August 5th and October 19th. Earlier studies on the gut microbes of the larvae of Noctuidae showed that the composition of gut microbes is relatively stable during the 3–5 instar larvae, such as the study of the gut microbial composition of the third and fifth instar larvae of Spodoptera exigua in the Noctuidae family through the sequencing of the V4 segment of 16S rRNA gene^40,41. Therefore, the 3rd -5th instar larvae of S. frugiperda were selected for field and indoor experiments in this study. For each cornfield, 50 pieces of 3rd -5th instar larvae specimens of S. frugiperda were collected from the corn fields (Fig. 1a). For details on the experimental design plan for sample collection, see Method S1. The collected larvae were aseptically dissected in the evening, and the midgut of each larva was collected. Then the midgut was separated from the gut mucosa and gut contents by shaking and washing. The processing steps are described in Method S2. Samples name and places could be found in Dataset S1. The excreted feces of the larvae were collected from 4 places, including Yunnan (YN_EF), Hainan (HN_EF), Hubei (HB_EF) and Henan (HE_EF). Accordingly, the obtained four gut mucosa (GM) samples and four gut feces (GF) samples were respectively named: YN_GF, HN_GF, HB_GF and HE_GF; YN_GM, HN_GM, HB_GM and HE_GM, and the four samples of the bulk soil (BS) were named as YN_BS, HN_BS, HB_BS and HE_BS. All collected samples were stored in dry ice and sent back to the laboratory in 48 h, and then stored at -80°C for further experiments. The undissected larvae specimens were stored at room temperature and fed with corn leaves.

Indoor breeding of S. frugiperda and physicochemical characterization of PVC film

The larva of S. frugiperda were bred in an artificial climate room (temperature 22°C and humidity 55%) to explore the larval ability to live on PVC film, the specific experimental method can be found in our previous article²⁴. The experimental design was briefly summarized as follows: 130 pieces of 4th-instar larvae with the same growth status were divided into four groups: 1) the control group (starvation, 15 pcs), 2) the Corn group (fed with corn leaves, 35 pcs), 3) the PVC group (fed with PVC film, 50 pcs), and 4) the Antibiotic group (fed with gentamicin sulfate antibiotic-soaked (100:3, w/w) corn leaves for 1 day before PVC film feeding).

The gut feces (GF) and excreted feces (EF) samples collected from the PVC and Corn groups were labelled PVC_GF, Corn_GF, PVC_EF and Corn_EF. Total DNA extraction and sequencing strategies were as previously described²⁴. Fragments of PVC film in the excreted feces of the PVC groups (PVC_EF) were collected. APC is used to detect the changes in the molecular weight of PVC films. For sample preparation and detection methods, see Method S3.

To obtain the products of PVC film biodegradation, PVC fraction in excreted feces (EF) in the Corn and PVC groups were collected for Gas Chromatography Mass Spectrometry (GC-MS) and named as Corn and PVC + Larvae, Control. Meanwhile, a control group (PVC film) was carried out and named as Control. PVC fraction weighing 0.1 g was mixed with 10 mL of tetrahydrofuran, and the mixture was ultrasonicated for 30 min at room temperature. The extract was concentrated to 0.5 ml by drying with nitrogen gas and mixed with 1 mL of N-hexane to obtain some possible products by vortexing and ultrasonication for 10 min. The samples were filtered using a 0.22 µm PTFE syringe filter for subsequent steps^15,42. The sample was injected at an initial temperature of 40 ℃ (hold, 4 min), which was progressively increased at 10°C per minute and held at 280°C (hold, 5 min). Moreover, the detector conditions, i.e., the transfer line temperature, ion source temperature, ionization mode electron impact and scan time, were maintained at 250°C, 280°C, 70 eV and 0.3 s, respectively.

DNA extraction, PCR amplification and sequencing

Total DNA was extracted from gut feces samples collected using a QIAamp Fast DNA Stool Mini Kit following the manufacturer’s recommendations (QIAGEN GmbH, Germany). Then, the hypervariable V4-V5 regions of the prokaryotic 16S rRNA gene were amplified using 515F (5’-GTGYCAGCMGCCGCGGTAA-3’) and 926R (5’-CCGYCAATTYMTTTRAGTTT-3’). The modified 515F/926R double-ended primer was selected as the primer to amplify the V4/V5 segment. The amplicon products of each sample were evenly mixed and sequenced using a paired-end sequencing strategy (PE250) on the Illumina HiSeq2500 at Guangdong MAGIGene Technology Co., Ltd

Bioinformatics, statistics and data visualization

Bioinformatics analysis

FastQC (Version 0.11.9) was used to check the quality of the obtained sequencing data. The double-ended data was imported through the software package Quantitative Insight into Microbial Ecology (QIIME2-2020.6), and DADA2 (1.14)⁴³, which has higher sensitivity, more accurate clustering, and fewer false positives, was employed in construction of Amplicon Sequence Variation (ASVs). The qiime2 built-in package, the feature-classifier classify-sklearn machine learning method, was used for taxonomic classification using the SILVA SSU138⁴⁴ as the reference database. To compare the larvae gut microbiota profile between this and prior studies, the SRA Toolkit (2.10.5) tool was used to download the 16S rRNA gene sequencing data of PVC degradation by T. molitor in the SRA database (download No. SRP186213)¹⁵. The experimental data of T. molitor includes the PVC group (6 replicates) and the Bran group (4 replicates). The data of indoor S. frugiperda larvae ingested the PVC film and the experimental data of T. molitor were processed by the same analysis method mentioned above.

Statistical analysis and visualization

Processed data from Qiime2⁴³ was imported into Rstudio Version 1.1.414 (R version 4.0.3). The phyloseq (1.32.0)⁴⁵, MicrobiotaProcess⁴⁶, and ggplot2 (3.3.3)⁴⁷ were used to do further analysis and visualization. The MAFFT⁴⁸and FastTree⁴⁹ were used to build the phylogenetic tree for the top 60 ASVs, default parameters were applied. The volcano plots were displayed with the R package Enhanced Volcano. PICRUST2⁵⁰ was used to do microbial community function prediction. The abundances of genes and metabolic pathways were used for further analysis. Firstly, we performed differential analysis on genes, and differentially enriched genes (padj < 0.01, Log₂Foldchange > 2) were further mapped to pathways. Meanwhile, differential analysis was performed in all pathways, but only those differential genes mapped pathways were presented and considered to be the PVC-enriched pathways in this study. All the above differential gene and pathway analysis was performed in Deseq2⁵¹.

Screening and functional verification of potential PVC-degrading strains and enzymes

The bioinformatics analysis results showed us that the bacteria of the genera Microbacterium, Enterococcus, and Ochrobactrum were predicted to be related to PVC degradation. In order to further verify whether the bacteria of these genera have the activity of degrading PVC, we will adopt the method of isolation and screening to obtain and verify them. It is difficult to isolate and screen target strains directly from midgut microorganisms, so the Known Media Database (KOMODO)⁵² was used to select appropriate screening medium type for potential PVC-degrading strains. Based on the results of KOMODO predictions, a purposeful screening of bacteria was conducted. First, the gut feces obtained from the PVC group of S. frugiperda were diluted step by step (10^− 1 to 10^− 8) by the serial dilution method, and three solutions of 10^− 5 to 10^− 7 concentrations were selected for plating culture. All treatments were placed in a 30°C incubator for static culture. According to the basic characteristics of the target microorganism, some single colonies were selected from the plate for continuous culture until a single colony was obtained.

Based on the appearance and morphology of the strains, 16S rRNA gene sequencing method was used to amplify the full-length sequence for strain identification. The full-length sequence of the 16S rRNA gene of the strain was amplified with 27F and 1492R universal primers⁵³, and then the sequences were aligned against the NCBI’s 16S rRNA sequences (Bacteria and Archaea) database using BlastN tool.

The obtained target strains were tested for the degradation activity of the self-made PVC film (Method S4). The verification system is an MSM liquid medium system with self-prepared pure PVC film as the only carbon source and energy source. All treatments were cultured at 30°C and 150 rpm for 42d, and the MSM medium was changed at 21d to maintain nutrient requirements for strain growth. Finally, the degradation activity of the strains was evaluated through the change of the molecular weight of the PVC film on 42d (Method S3).

Expression, purification and functional verification of potential PVC-degrading enzyme

The bioinformatics analysis results showed us information about potential PVC-degrading enzymes. NDO was analyzed to be a potential PVC-degrading enzyme. Based on the genomic information of EMBL-3 (Enterococcus sp.), We extracted separately the gene information of NDO from the genome of Enterococcus sp. strain and then expressed and purified this enzyme in vitro by prokaryotic expression and purification method. The method of expression and purification of protein in vitro (Method S5) was used to obtain the pure enzyme for further enzyme activity assay (Method S6). Moreover, the degradation detection method ¹⁸ was used to validate the PVC-degrading activity (Method S6).

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Funding

Author Contribution

F.J. obtained funding and supervised this study. F.J., H.P., Z.Z. and X.K conceptualized this study. Z.Z., H.P., D.Y., and J.Z. contributed to the collection of field larvae samples. H.P. performed metagenomics data analysis and strain analysis. Z.Z. and H.Z. performed strain culture. X.K and Y.X.W performed enzyme purification and degradation validation. Y.J. W. provided design and improvement suggestions to the enzyme verification experiment. H.P., Z.Z, X.K. and F.J. drafted the manuscript. Y.G.Z reviewed and edited the manuscript. F.J. edited and finalized the manuscript. All authors read and approved the final version of the manuscript.

Acknowledgement

This work was supported by the National Science Foundation of China (Grant No. 22241603 to JF), the Westlake Center for Synthetic Biology and Integrated Bioengineering (Grant No. WU2022A009 to JF), the Research Center for Industries of the Future (Grant No. WU2022C030 to JF), and the National Natural Science Foundation of China (grant no.: 32100091 to ZZ). JF also acknowledged support from the HRHI program 202309010 of Westlake Laboratory of Life Sciences. We thank Dr. Yinjuan Chen and Ke Wang from the Instrumentation and Service Center for Molecular Sciences at Westlake University for their assistance and discussion during the experiment. We also thank Dr. Xiaoyuan Zhao, Dr. Guoping Li, Dr. Shiyou Yang and Haixia Tian in different provinces for support and assistance during the field sampling.

Availability of data and materials

The 16S rRNA gene sequencing data generated in this study has been uploaded to the NCBI under the accession numbers PRJNA877394. The E. casseliflavus EMBL-3 whole genome sequencing data is deposited in the China National GeneBank DataBase (CNP0004265). The code used to analyse the data can be found at https://github.com/EMBLab-Westlake-U/S.frugiperda_PVC_Project.

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No competing interests reported.

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Unveiling Gut Microbiota and Metabolic Functions Contributed to Polyvinyl Chloride Degradation in Spodoptera frugiperda Larvae

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Introduction

Results

Functional redundancy discovered in the gut microbiota of two PVC-fed insect larvae

Discussion

Conclusions

Materials and Methods

DNA extraction, PCR amplification and sequencing

Bioinformatics, statistics and data visualization

Bioinformatics analysis

Statistical analysis and visualization

Screening and functional verification of potential PVC-degrading strains and enzymes

Expression, purification and functional verification of potential PVC-degrading enzyme

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Ethics approval and consent to participate

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