Plastic Degradation by the Gut Microbiome of Yellow Mealworms
Lummy Maria Oliveira Monteiro1,2, Jyoti Singh1,2, Ross Klauer1,2* (email@example.com), Harrison Hall3, Sankarganesh Krishnamoorthy4, Aaron Wright3, Mark Blenner1,2, and Kevin Solomon1,2
1Delaware Biotechnology Institute, 2University of Delaware; 3Baylor University; and 4Pacific Northwest National Laboratory
This project discovers and reconstructs the plastic degradation pathways distributed across the gut microbiome of yellow mealworms (larvae of Tenebrio molitor) to develop enhanced capabilities for biologically based polymer recycling.
Plastics, initially selected for their durability and environmental resiliency, pose a significant environmental challenge for modern economies. Polystyrene (PS), high- and low-density polyethylene (HDPE and LDPE), and polypropylene (PP) are produced at a rate of more than 228 million tonnes globally each year. However, none have robust infrastructures for mechanical or chemical recycling and ultimately become polluting waste streams. To address this need, the team will pursue biological strategies for plastics depolymerization. Researchers will focus on the microbiomes of insect larvae (colloquially called worms) as they degrade plastics more rapidly than microbial isolates and do not require clean plastics or pretreatment. In particular, the microbiome of yellow mealworms is unique in that its host does not appear to contribute to degradation of a wide range of plastics. While bacterial community members have been identified, the specific pathways responsible for biodegradation remain to be elucidated and the potential contributions of fungal members are unexamined. Additionally, emerging evidence suggests that nutrient supplementation enhances plastic metabolism up to 70% and gives rise to a gut community structure distinct from that without additional nutrients. However, it is unclear if nutrient supplementation induces microbes to participate in in the plastic degradation or if it supports an optimal community composition for function.
As a first step to address these gaps, researchers characterized the consumption rates of PS, LDPE, HDPE, and PP via T. molitor larvae in the presence and absence of co-fed oats as a nutritional supplement. The consumption rates of PS, LDPE, and HDPE were 20.4, 12, and 1.1 mg (100 larvae)-1d-1, respectively, in agreement with established studies. However, oat supplementation enhanced plastics consumption by ~160, 60, and 230%, respectively. These studies establish the use of oats as a potent supplement for enhancement of PS and LDPE consumption rates, up to double that obtained with established supplements, and validated HDPE consumption by T. molitor.
Worm-consumed plastics were chemically modified beyond simple mechanical degradation validating biological mechanisms for plastics depolymerization. Fourier transform infrared spectroscopy (FTIR) analysis of plastic extracted from the frass (excrement) of mealworms fed PS revealed incorporation of oxygen not found in untreated controls. Moreover, benzene ring cleavage was observed for treated PS samples. Similarly, FTIR spectra of extracted plastic from LDPE-fed mealworm frass revealed the incorporation of carbonyl and alcohol groups. Finally, gel permeation chromatography (GPC) of the ingested plastic from PS-fed T.molitor larvae confirmed a 40% decrease in polymer molecular weight while LDPE-fed worms were able to decrease the molecular weight by up to three orders of magnitude. Taken together, these results demonstrate that the plastics being ingested by the larvae are actively depolymerized and chemically modified.
Microbiome community analysis via 16s and ITS sequencing revealed a rich consortium of bacteria and fungi. The bacterial community was more diverse than the fungal community with observed taxa belonging to the bacterial phyla Firmicutes, Tenericutes, Proteobacteria, Actinobacteria, Spirochaetes, Bacteroidetes, and Fusobacteria, and fungal Ascomycota, Basidiomycota, and Mucoromycota. As expected, mealworm diet led to unique community structures adapted to degradation of the fed plastic substrate. However, oats co-supplementation frequently selected for taxa that were not observed in plastics-only or oats-only controls suggesting currently unrecognized interactions. Despite these unique community structures, microcosms of communities in planktonic culture selected for with LDPE, HDPE, PS, and PP diet were all able to grow on LDPE as a primary-carbon source. Finally, community analyses revealed many facultative and obligate anaerobic genera such as Spiroplasma associated with LDPE and PS degradation when supplemented with oats. Correspondingly, these communities were enriched with clusters of genes (COG) and protein family (pfam) for iron-dependent anaerobic oxidation enzymes and pathways, which may serve as novel oxygen-independent pathways for plastics depolymerization.
To determine microbes and active enzymes responsible for the degradation of PS, PE, and PP, researchers have developed a suite of photoreactive chemical probes that resemble oligomers of these polymers. These probes are fluorescently labeled, providing an avenue to selectively isolate microbes that take up these molecules via fluorescence-activated cell sorting and enable subsequent proteomic characterization of the proteins acting upon them. Team members have begun using these probes to characterize enzymes that bind them strongly in a series of microbial isolates and identify the taxonomy of cells capable of transporting these plastics in plastic-degrading worm gut microbiomes.
In summary, ongoing work has characterized plastic consumption rates in T. molitor microbiomes, revealing novel strategies to structure gut microbial populations for enhanced degradation. Plastics were noted to be metabolized and not only mechanically degraded by both bacterial and fungal communities that contribute to plastic degradation even independent of the host mealworm. Team members have also developed chemical probe analogs of common plastics to isolate plastic-binding microbes and proteins for study. Through these parallel efforts, researchers aim to generate systems-level insight into the metabolic pathways of plastic-degrading microbiomes and to develop consortia enriched in plastic degradation activity.
This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science Program under Award Number DE-SC0022018. A portion of this research will be performed under the Facilities Integrating Collaborations for User Science (FICUS) initiative and use resources at the DOE Joint Genome Institute and the Environmental Molecular Sciences Laboratory, which are DOE Office of Science User Facilities. Both facilities are sponsored by the Office of Biological and Environmental Research and operated under Contract Nos. DE-AC02-05CH11231 (JGI) and DE- AC05-76RL01830 (EMSL).